WO1985000503A1 - Monoclonal antibodies to the h-y antigen, methods of production, applications, and products of manufacture - Google Patents

Abstract

Methods for identification of live bovine embroys by means of H-Y antibody. The antibodies are produced by clones and have particularly high titres. They are also identifiable by other immunological characteristics. Biological compositions of live male or female embryos are also disclosed, the gender of which is identified. The invention also provides a method and compositions wherein the gender has been identified without affecting the embryos.

Description

MONOCLONAL ANTIBODIES TO THE H-Y ANTIGEN, METHODS OF PRODUCTION, APPLICATIONS, AND PRODUCTS OF MANUFACTURE

This invention relates to monoclonal antibodies to the H-Y antigen and to their uses in differentiating between males and females. The monoclonal antibodies of the invention are of special interest in determining the presence of the H-Y antigen on the cell surface or in serum of mammals or in growth media of embryos.

The invention also relates to hybridoma cell lines which produce antibody to the H-Y antigen and the monoclonal anti H-Y antibodies which are raised by said cell lines.

The invention also relates to methods of use of these H-Y antibodies to differentiate and separate embryos by sex, to determine the presence of H-Y antigen in serum of vertebrates including various species of mammals and birds, to assign H-Y phenotype and to diagnose and clarify the nature of aberrant sexual development.

A particular noteworthy aspect of the invention is that viable (live) male and female embryos are identified rather than embryos which are not viable anymore because of the selection process to which they have been subjected.

The invention also relates to live embryos which are identified by an indicia (indicator) which provides information or is gender-specific, and to compositions comprising such embryos.

Another particular noteworthy aspect of the invention is the unusually high activity of the H-Y antibodies (as measured by traditionally accepted tests) used in this invention.

Other embodiments of the invention are described and will become apparent hereinafter. Embryo transfer, and especially bovine embryo transfer has become an important commercial technique in the food industry. Embryo transfer refers to placing an embryo into the lumen of the oviduct or uterus. In a broad sense, however, embryo transfer has come to mean the sequence of steps for moving embryos from one female to another, including superovulation, embryo recovery and storage of embryos in vitro. The donor is the genetic mother from which embryos are recovered; the host, or recipient, is the surrogate mother that receives the embryos after storage. An informative discussion of bovine embryo transfer is found in Superovulation and Embryo Transfer in Cattle, by George E. Seidel Jr., 211, Science 351 (1981) which is herein incorporated by reference.

Sex identification of embryos before transfer has several applications, especially in the cattle industry. Because of artificial insemination, the number of bulls required for breeding is greatly reduced and the value of the few very good bulls is greatly increased. Thus male calves will be more valuable than female calves in a few instances. In the great majority of cases, however, females are considerably more valuable than males. It is financially risky if one must count on at least five females from ten pregnancies for profitability. For example, in seven natural births, the probability of seven offspring of one sex is 0.0078125 if the true sex ratio is 50:50. In those cases where one sex is preferred, for example, to replace breeding stock or dairy stock or for fattening, it would be advantageous to transfer only embryos of the desired sex. In order for embryo sex selection to be commercially feasible the technology involved must be fast, simple, inexpensive, reliable and, very importantly, non-damaging to the embryo, i.e. the embryo should retain its viability. Mechanical methods of increasing the percentage of mammalian offspring of either sex are disclosed in the following U.S. Patents No. 4,083,957, Lang, Process for the Alteration of the Sex Ratio of Mammals; No. 4,092,229, Bhattacharya, Thermal Convection Counter Streaming Sedimentation and Forced Convection Galvanization Method for Controlling the Sex of Mammalian Offspring; No. 4,155,831, Bhattacharya, Thermal Convection Counter Streaming Sedimentation and Forced Convection Galvanization Method and Apparatus for Controlling the Sex of Mammalian Offspring; No. 4,327,177, Shrimpton, Method and Means for Controlling the Sex of Mammalian Offspring and Product Therefor; No. 4,362,246, Adair, Method of Treating Collected Mammal Semen and Separating Sperm into X Y Components. In the disclosed method, mammal semen is treated with an anti-coagulant, such as mammal saliva. Such methods are known, however, to cause reduced sperm survivability and reduced viability of the surviving sperm or have other drawbacks.

In most animals with a backbone, sex is determined by the sex chromosomes. In warm-blooded mammals including humans, for instance, embryos with an X and a Y chromosome (XY) become males, and embryos with two X chromosomes (XX) become females. There are exceptions to this rule but sex-reversal can be explained by abnormalitites of Y chromosome function, as in XY females, or abnormal retention of Y chromosome function, as in XX males.

The Y chromosome is responsible for production of a cell surface molecule or group of molecules called H-Y antigen. Since there is no Y chromosome in normal females, H-Y is a male specific antigen, and can be used to produce a state of immunity in females. Thus, H-Y, meaning histocompatibility-Y, antigen was discovered with the observation that among highly inbred populations of mice, skin grafts from males (H-Y⁺) are rejected by females (H-Y-), whereas skin grafts exchanged among the other three sex combinations (male-to-male; female-to-female; female-to-male) are accepted. Therefore H-Y antigen can be used as a marker for identification of male cells.

Female mice that have been exposed to male tissues, as in skin grafts or inoculations of cells in suspension, produce antibodies (called H-Y antibodies) that can be used to identify male cells in serological systems. The antibodies specifically recognize and physically combine with H-Y antigen. Although there are statements in the literature that the molecule or group of molecules recognized by H-Y antibody is not the same as the molecule which causes rejection of male skin grafts, for the purposes of this invention, the term H-Y antigen shall refer to the male-specific cell surface antigen or antigens identified by and capable of combining with H-Y antibody.

Male cells (H-Y⁺) can be identified by any of several assays. The following procedures are illustrative.

a. Cytotoxic and labeling assays. H-Y antibody is used for applications of H-Y serology in the sperm cytotoxicity test. In the test male cells are killed or labeled in such a way as to facilitate visual scoring or identification. An example of the killing technique is the complement-mediated epidermal cell cytotoxicity test, in which only male epidermal cells are killed. Scheid M, Boyse EA, Carswell EA, Old LJ, Serologically Demonstratable Alloantigens of Mouse Epidermal Cells, 135 J. Exp. Med. 938-955 (1972). In that test, dead cells are visualized with the aid of a blue stain (and see Goldberg EH, Boyse EA, Bennett D, Scheid M, Carswell EA, Serological Demonstration of H-Y (Male) Antigen on Mouse Sperm, 232 Nature 478 (1971)). An example of the labeling technique is the fluorescent test in which only male cells are illuminated under ultraviolet light (Galbraith MP, Galbraith RM, Faulk WP, Wachtel SS, Detection of H-Y Antigen by Fluorescence Microscopy, 26 Transplantation 25-27 (1978)).

b. Absorption assays. In certain cases it is helpful to identify male cells indirectly, by measuring their ability or the ability of their secretions to inhibit reaction of H-Y antibody with a secondary male target cell. For example, when white blood cells from a human male are suspended in a batch of H-Y antiserum (which contains H-Y antibodies), the white blood cells absorb the H-Y antibodies. When those cells are removed, the relevant antibodies (the H-Y antibodies) are also removed, and the antiserum loses its ability to react with H-Y target cells in cytotoxicity and labeling assays. Female cells do not absorb H-Y antibodies. See Wachtel SS, H-Y Antigen and the Biology of Sex Determination, Grune and Stratton, N. Y. 1983.

Other techniques for identification of male cells (H-Y⁺) are reviewed in Wachtel, 1983, supra. They are useful techniques which can be used in the practice of this invention and such techniques are incorporated herein by reference.

It has been reported that H-Y antibodies of the mouse recognize male cells of rat, guinea pig, rabbit and man. See Wachtel SS, Koo GC, Zuckerman EE et al., Serological Crossreactivity Between H-Y [Male] Antigens of Mouse and Man, 71 Proc. Nat. Acad Sci. U.S.A. 1215-1218 (1974). It has also been reported that H-Y antibodies of the mouse recognize male cells of the leopard frog and female cells of the chicken, Wachtel SS, Koo GC, Boyse EA, Evolutionary Conservation of H-Y ('Male') Antigen, 254 Nature 270-272 (1975). In the chicken, the female has the XY chromosome constitution and the male has the XX pair. Mouse H-Y antibodies also recognize male cells of the goat (Wachtel SS, Basrur P, Koo GC, Recessive Male-Determining Genes, 15 Cell 279 (1978)), horse (Sharp AJ, Wachtel SS, Benirschke K, H-Y antigen in a fertile XY female horse, 58 J. Reprod. Fert. 157 (1980)), and bovine (Wachtel SS, Koo GC, Ohno s, "H-Y Antigen and Male Development", The Testis in Normal and Infertile Man, Troen P and Nankin HR, ed., Raven Press, New York 1977)). Thus, H-Y antigen is widespread among the vertebrates and it was concluded that H-Y antibodies of the mouse or rat can be used to distinguish between male and female cells of any vertebrate species.

In light of the phylogenetic conservation of H-Y antigen, and its identity or shared specificity or close similarity among the various vertebrate species, the invention is applicable by use of or with conventional or monoclonal H-Y antibodies produced in other vertebrate species, such as the rabbit or rat. Application involves the use of the same procedures as described in this specification.

The ontogeny of the appearance of H-Y antigen in embryos is not known. The presence of H-Y antigen in bovine embryos of 150 to 175 days has been demonstrated (Ohno S, Christian LC, Wachtel SS, Koo GC, Hormone-like role of H-Y antigen in bovine freemartin gonad, 261 Nature 597 (1976); Wachtel SS, Hall JL, Müller U, Chaganti RSK, Serum-Borne H-Y Antigen in the Fetal Bovine Freemartin, 21 Cell 917 (1980)). The presence of H-Y antigen in 8-cell embryos of the mouse has also been demonstrated (Krco CJ and Goldberg EH, Detection of H-Y (Male) Antigen On Eight-cell Mouse Embryos, 193 Science 1134-1135 (1976)).

The presence of H-Y antigen is also helpful in the study of abnormal sexual development. Thus, the presence of H-Y antigen has been demonstrated in the cells of XX males (Wachtel SS, Ohno S, Koo GC, Boyse EA, Possible role for H-Y antigen in the primary determination of sex, 257 Nature 235 (1975)) and in XYY males (Wachtel SS, Koo GC, Breg WR, Elias S, Boyse EA and Miller OJ, Expression of H-Y Antigen in Human Males with Two Y Chromosomes, 293 New England J. Med. 1070 (1975)). The areas of H-Y research have been hampered by the lack of reliable H-Y antiserum. Antiserum is conventionally produced by injecting the antigen of interest into an immunologically responsive laboratory animal such as a mouse or rat, and subsequently preparing antiserum from the blood of the animal, which will contain a mixture of antibodies developed against the antigen together with other antibody substances. Conventional mouse antibody not only is usually low-titered but contains naturally occurring heteroantibodies reactive with cell surface components of other species. The heteroantibodies would react indiscriminately with male and female embryos of other species.

For example, an attempt to solve the problem of unwanted (or contaminant) antibodies is shown by Bryant, U.S. Patent No. 4,191,749, March 4, 1980, which discloses a separation scheme for male- and female-determining spermatozoa utilizing a male-specific antibody. The antibody of this reference is prepared from the serum of female rabbits hyperimmunized with male rabbit epidermal cells. The antiserum, which must be complement inactivated, is "purified" by being repeatedly absorbed with washed female rabbit spleen cells and fractionated by agarose gel filtration to obtain the Immunoglobulin G (IgG) antiserum fraction.

Theoretically, X-bearing sperm will elute out of a column in which Bryant's Immunoglobulin G antiserum is coupled with solid phase immunosorbent material, while the Y-bearing sperm attach to the antiserum on the column. The Y-bearing sperm are then eluted out of the column separately with more antiserum solution in accordance with the principles of competitive binding. Such conventionally-produced H-Y antiserum, although it contains H-Y antibody substances, is usually low-titered and contaminated with heteroantibody and autoantibody which will react with male and female cells of other species due to species-specific cell surface components which are not related to the H-Y antigen. Therefore assays using the H-Y antiserum to detect the H-Y antigen on male cells or aid in separating male embryos and Y-bearing spermatozoa from female embryos and X-bearing spermatozoa will often yield ambiguous and inaccurate results in embryo transfer and fertilization experiments.

This ambiguity in anti-H-Y antiserum is also a serious problem when used in serologic procedures for the detection of H-Y antigen in patients (both animal and human) that demonstrate ambiguous sexual development (hermaphroditic differentiation) or other physical conditions caused by genetic aberrancies in the sex chromosomes. Hermaphroditic differentiation in XX subjects is believed to be caused by the presence of Y-chromosome material as an intact Y chromosome in a mosaic cell line, or as a minute part of extra Y- material attached to an X chromosome or an autosome. See Wachtel et al, Serological Detection of a Y-Linked Gene in XX Males and XX True Hermaphrodites, 295 New England J. Med. 750-754 (1976). Because the chromosome segments and genes which form them are so small, the usual cytological assays cannot generally detect them. Serological detection of H-Y antigen expression is considered a useful adjunct to other techniques in clarification of these genotypic abnormalities. However, the serological assays are only as precise in clarifying the genetic abnormality as is the anti-H-Y antiserum available for use.

There is therefore a serious need for highly specific monoclonal H-Y antibody, H-Y antiserum and other such components as will be discussed further hereinafter. It is known that hybridomas produced by fusion of a selected antibody-producing cell with a tumor cell (for example, a myeloma cell) give rise to clones of cells, all of which produce the same antibody. This antibody, called monoclonal antibody, has the advantage of purity, specificity and potency, over conventional antibody, which consists, as discussed above, of a heterogeneous collection of relevant and irrelevant, or contaminant, antibodies. Characteristically, monoclonal antibodies can be used in highly dilute form. The broad applications of monoclonal antibody techniques and the potential uses for these reagents are discussed in Monoclonal Antibodies, Roger H. Kennett, Thomas J. McKearn, and Kathleen B. Bechtol, eds., Plenum Press, New York, 1980, which is herein incorporated as reference.

In 1975, Koehler and Milstein demonstrated that individual clones of normal antibody-secreting cells could be immortalized by fusion with myeloma (256 Nature 495 (1975)). Hybrid cells derived by fusion of a murine myeloma with spleen cells from appropriately immunized donors were shown to secrete antibodies against the immunogen used. These hybrid cells (hybridomas) could be grown in tissue culture,, producing antibodies of defined specificity in vitro; alternatively, antibody secretion could be obtained in vivo by inoculation of the hybridoma cells subcutaneously or intraperitoneally into syngeneic recipients. This approach thus offered the possibility of the production of monoclonal antibody of defined specificity by selective cloning procedures, avoiding the need for highly purified immunogen or elaborate antibody purification procedures.

Although it is now established that the fusion of mouse myeloma cells and antibody-secreting splenic lymphocytes is an effective means of producing homogeneous antibody of defined specificity, a number of technical variables remain. The same basic principles are applicable to many systems, but fairly extensive preliminary investigation is required to define the optimal conditions, particularly in relation to the choice of immunization schedule and donor species used (70 Meth. in Enzymology 135-142, incorporated herein by reference).

Spontaneous fusion of cells is usually rare with some exceptions, i.e., that of sperm and egg. Nevertheless two cells can be made to fuse by the addition of a fusing agent such as polyethylene glycol. The fusion of the plasma membranes results in the formation of heterokaryons which possess two or more nuclei. At the next division the nuclei fuse and a hybrid cell results.

The hybrid cells produce a single antibody and all descendants (the monoclone) of the hybrid cell produce the original, single antibody. Each monoclonal antibody is a single chemical entity, a protein with an amino acid sequence that can generally be determined. The cells that produce the antibody may be permanently maintained. The hybrid cells give pure antibody in response to not only a pure antigen but also in response to a complex stimulus such as a whole virus or a whole cell.

Lymphocytes removed from the spleen of an animal previously injected with the selected antigen of interest are allowed to fuse with myeloma cells in the presence of polyethylene glycol or equivalent known fusogen. Thousands of "hybrid" myeloma cells are produced from the fusion. The supernatant from growth of each "hybridoma" cell culture is tested for the presence of the desired antibody activity. When such activity is found in the supernatant of one cell culture, it is cloned by limiting dilutions, and the clones produced are individually assayed for supernatant activity. Once the active cells have been identified they are cloned to reduce the chance of being overgrown by irrelevant cells. Once the hybridoma cells have been successfully cloned, they may be grown in bulk.

The antibodies formed by these cells may then be purified using a variety of techniques including affinity chromatography on protein A-Sepharose. However, not all immunoglobulins bind to protein A since binding depends on the species and subclass of the antibody. In these cases the antibody may be purified by ion exchange on DEAE columns or affinity chromatography on anti-Ig-agarose. A discussion of hybridoma production and purification is contained in Goding JW, Antibody Production by Hybridomas, 39 J. Immun. Meth. 285-308 (1980), incorporated herein by reference.

To date, monoclonal antibody methodologies have produced monoclonal anti-H-Y antibodies of widely varying specificities and physiological characteristics. An H-Y antibody less than highly specific, however, will not provide an accurate detection of solely the H-Y antigen cell surface molecule (or component) which is necessary to enable identification and separation of male from female cells and accurate prediction of the results of sex immunoselection techniques, or locate fragments of Y chromosomes in cells of patients with abnormal sex chromosome constitutions, which are objects of this invention.

Also, monoclonal antibodies of the invention were found not to be too specific to the H-Y antigen inasmuch as they recognized the antigen, whereas they could also have failed to detect it for undue specificity. The production of a pure H-Y antibody which recognizes the H-Y antigen is thus highly desirable. Antibodies with higher titers would thereby be obtained and unwanted reaction caused by contaminants eliminated.

Accordingly, it is a primary object of the invention to provide a source of pure, high-titer H-Y antibody.

It is also an object of the invention to provide a method for the identification of male and female embryos, particularly viable or live embryos.

Another object of the invention is a method for sex identification of bovine embryos and the products of such identification and selection.

Another object of the invention is a method for sex identification of bovine embryos and the separation of live male embryos from live female embryos or vice versa.

Another object of the invention is to provide a method for the assignment of H-Y phenotype in male, female and intersexual patients with chromosomal, genital, or endocrinological disorders.

There are numerous other objects of the invention all of which will become apparent hereinafter.

The invention provides pure, high-titer H-Y antibodies for various uses in the medical and veterinary fields of particular interest for use in testing and differentiation between sexes of embryos, for instance in cattle, and in other applications where such recognition is necessary. The invention provides for monoclonal antibodies to H-Y antigen and for methods and applications by which these antibodies are used in the medical and veterinary fields.

An important finding made in conjunction with this invention is the presence of H-Y antigen in male bovine embryos when they have reached a certain stage of development, generally from about 6 to about 12, preferably 8, days of gestation. A conclusion drawn in conjunction with the invention is that H-Y antigen is present in all vertebrate XY embryos from the 8 cell stage to the late blastocyst stage. These are the stages at which the embryos can be used in accordance with the invention for identification of gender.

It is noteworthy that the H-Y antigen in bovine embryos is present at the time of embryos transfer. Many antigens have a transient expression and it is known that proteins which are involved in the biological cycle will appear and disappear. (M. Polackova, Ontogenetic Development of the Male-Specific Antigen in Mice, 16 Folia Biologica (Praha) 12-19 (1970)). The H-Y antigen is not present at the 2-4 cell stage, though it appears later. Thus, the timely presence of the H-Y antigen when the embryos is transferred is an unexpected and yet very important aspect of the invention.

In conjunction with this invention it has been found that H-Y antigen is present in male bovine embryos during the entire period of the embryos' development during which a surgical or nonsurgical embryonic transfer can be performed without loss of viability of the embryo.

Other features and advantages of the invention will appear from the examples which follow and by referring to the appended drawings in which: Figure 1 shows the Inhibition of Reaction between TS, testis supernatant containing soluble H-Y antigen, and anti H-Y antibody (_― ) by supernatant fluid of male (O) and female (X) lymphocytes.

Figure 2 shows Optical Density Scores which show the ratio of reading in the presence of lymphocyte supernatant to reading in presence of Hanks buffer alone. Each point represents the mean of duplicate scores from a single donor.

Figure 3 shows Inhibition of uptake of labeled H-Y antigen by male and female serum.

Figure 4 is a flow chart showing methodology for two-dimensional gel electrophoresis for H-Y antigen.

Figure 5 shows a computer graphic printout of immunoprecipitation of H-Y antigen with H-Y antiserum.

To achieve the foregoing objects and advantages, there is provided as one aspect of the invention novel hybridomas producing novel antibodies to the H-Y antigen, the antibodies themselves and diagnostic and therapeutic methods which employ the antibodies.

The hybridomas were prepared generally by the immunization of highly inbred female mice with spleen cells from male mice of the same highly inbred population. The spleen cells from the immunized female mice were fused with cells from a mouse myeloma line and the resultant hybridomas were screened for supernatants containing antibody to the H-Y antigen. The desired hybridomas were subsequently cloned and characterized. The antibodies are used to identify the sex of individual embryos and as a diagnostic tool where aberrant sexual development is known or to be determined. In accordance with the invention, the embryos of the selected mammal are exposed to the H-Y antibodies when the mammalian embryo has reached the stage at which there has developed H-Y antigen. Generally, in bovine species, this stage is reached when the cell division has adequately progressed and prior to the fetus stage. The embryos can be approximately 6 to 12 days old if best results are to be obtained. It may vary from mammalian species to mammalian species.

The monoclonal antibodies thus produced in accordance with the invention are then used in the identification of male and female embryos. The identification of each embryo may be directly or indirectly accomplished. In a direct method (see Examples 1 and 2, infra) the monoclonal antibody binds to the male embryo but not the female embryo. A secondary reagent (such as Protein A or Protein A plus a visual or, when appropriate, a radioactive label) is then added which binds to the antibody to form a complex which may be readily differentiated or separated from non-bound embryos. In an indirect method (see Example 10, infra), a single embryo is cultured for a suitable amount of time in a microtiter plate. The embryo is then removed to a separate nutrient medium and the first supernatant liquid is tested for the presence or absence of soluble H-Y antigen, thereby identifying the gender of the embryo.

In the case where the monoclonal antibody is of the IgM type, the secondary reagent added should be of the IgG type which can in turn react with Protein A or an antibody which itself has a label. The embryo from either means whose sex has been determined is then transferred to a pseudo-pregnant female (a female that has been set up hormonally) by standard procedures, for example but not limited to those described in Seidel, supra, for bovines. In this manner, up to about 85% of calves whose sex had been correctly identified are born at term and healthy. It is within the scope of the invention that methods may be used to identify and provide live embryos of predetermined sex of those mammal vertebrates which produce H-Y antigen, more especially presexed embryos of bovines, equines, porcines, ovines and caprines.

It is also within the scope of the invention that there are produced full-term, healthy offspring of predetermined gender of said bovines, equines, porcines, ovines and caprines. An especially interesting class of mammals are the domestic animals.

In accordance with the invention, there are provided new hybridoma clones and H-Y antibodies. The hybridoma clones are designated as murine hybridoma clones No. 2-4/3; gw 9/8; 16-3/6; 109-6/2; and 113. Specifically, these were produced by the following procedure. Panels of C57BL/6J female mice were immunized with five weekly injections of spleen cells from C57BL/6J males. The females were bled and the serum was separated and evaluated for the presence of H-Y antibodies.

Determination was carried out as follows: Epididymalsperm cell suspensions were prepared from male mice of the highly inbred BALB strain essentially according to Goldberg et al, Nature (1971), supra. The epididymis was removed and cut into several pieces in phosphate-buffered saline (PBS) (pH 7.0) containing 0.5% fructose and 2% fetal calf serum (previously heat-inactivated at 56°C for 30 minutes). After 5 minutes the sperm cells were pipetted off and placed in the PBS, and suspension maintained at room temperature until use. Equal volumes of 0.05 ml of (a) putative H-Y antibody to be tested (serially diluted 1/2, 1/4 and 1/8); (b) sperm suspension (about 5 x 10⁶ cells/ml); and (c) selected absorbed rabbit complement (diluted 1/20) were incubated at 37°C for 40 minutes in a rocking waterbath. A freshly prepared solution of trypan blue dye was added during the last 7 minutes of incubation to stain dead sperm. At the end of the incubation period, the suspensions were placed on ice and live and dead sperm enumerated in a hemacytometer.

Specificity of the reaction (for male cells) was established by serologic absorption. Before each test, antiserum from a particular female was diluted 1/2 and divided into 3 parts: one part was unabsorbed; one part was absorbed with male cells; and one part with the corresponding female cells. As an example, 20 x 10⁶ male (or female) spleen cells) were suspended in particular portions of putative H-Y antibody for 30 minutes on ice with frequent stirring. At the end of that period the three portions of antiserum (unabsorbed, male absorbed, female absorbed) were centrifuged; the serum (supernatant) retained for the cytotoxicity test, and the absorbing cells (pellets) discarded. Positive absorption by male cells was manifested as a fall in cytotoxicity - i.e. as a reduced capacity to kill sperm. This fact indicated that the serum was H-Y antiserum because male and female cells were taken from male and female members of inbred strains in which the only genetic difference between male and female is presence of the Y chromosome in the male.

Table 1 shows an example of the sperm cytotoxicity test with ascites fluid using monoclonal H-Y antibody 16-3/6.

Table 2 shows the results of two sperm cytotoxicity tests with male-absorbed and female-absorbed monoclonal H-Y antibody gw 9/8.

Cells from females whose sera produced cytotoxic titers 30% above background were used for production of hybridomas as follows: The females were killed and their spleen cells were fused with murine myeloma cells of cell line P3/NS1/1-Ag 4-1, according to the method of Koehler and Milstein (1975, supra), the standard reference for the production of hybridomas.

TABLE 1

Sperm Cytotoxicity Test with Ascites Fluid Monoclonal H-Y Antibody 16-3/6

Cytotoxic index is given by the formula (a-b/100-b) x 100 where a = % dead at dilution and b = % dead in suspensions containing complement absorbed rabbit serum and sperm but no antiserum. TABLE 2

Sperm Cytotoxicity Test With Male-absorbed and Female-absorbed Monoclonal H-Y Antibody gw 9/8 (serum + ascites)

* Cytotoxic index.

Antiserum diluted and divided into 3 parts (A, B, C) .

Parts B and C absorbed with 20 x 10⁶ spleen cells from inbred female and male mice. Two ml of myeloma cells were placed in a tissue culture flask and 23 ml RPMI added together with 1% Azaguane.

After one week of culture the cells were transferred to petri dishes and cultured for 4 days. Then suspensions of cells were prepared from the spleens of selected females. The myeloma and spleen cells were counted and washed twice separately, and then combined in a ratio of 1:4 to 1:10 = myeloma: spleen, and washed again. Next polyethylene glycol (PEG) was added to the combined pellet at 1.0 ml for every 200 x 10⁶ spleen cells, and the pellet gently disrupted and stirred for 1 minute at 37°C. Medium RPMI and hypoxanthine aminopterin thymidine (HAT) were then added to the PEG-cell suspension drop by drop, taking great care with the first 2 ml.

The suspensions were then plated onto 96-well feeder layers. After 4 days RPMI + HAT were added as necessary to each well and the medium changed twice weekly thereafter. After 2 weeks the medium was changed to RPMI + TH. Feeder layers were then prepared in 24 well plates.

Initial screening for antibody (using supernatant from wells exhibiting good growth of hybridoma cells) was performed in the sperm cytotoxicity test as described above. The hybridoma supernatants were used at 1/2 and 1/4 dilution; 0.05 ml of dilution were reacted with equal volumes of sperm cell suspension and rabbit complement. Cells from wells showing positive reactivity (cytotoxicity 30% above background) were transferred to 24 well feeder layer plates using 0.5 ml RPMI + TH for transfer and RPMI there-after. After 1 week initial screening technique was repeated, and cells giving positive reaction used to make feeder layers in 96 well plates. When the clones came up (after about 2 weeks) the supernatant was screened again, and positive clones transferred to 24 well feeder layer plates and then, after growth, to 96 well feeder layer plates. On growth of secondary clones, intial screening was repeated and positive clones transferred to 24 well plates. The clones were then dispersed into 4 wells each, and then to flasks.

Selected clones were now injected into female C57BL/6J mice. The clones yielded ascites tumors that secreted malespecific H-Y antibodies. Secondary screening was now performed by absorbing the serum and ascites fluid with male or female spleen cells and scoring the serum in the sperm cytotoxicity and epidermal cell cytotoxicity tests (the latter being performed in essentially the same way as the sperm cell cytotoxicity test except that male and female epidermal cells are used as targets); male epidermal cells are killed; female cells are not; male cells absorbed the antibodies in inhibition tests; female cells did not.

Five clones producing male-specific H-Y antibodies were thereby identified. These were clones 16-3/6; 109-6/2; 2-4/3, gw 9/8 and 113. These can be reproduced and generated using the procedure described above. The specific titers of the antibodies ranged from 1/10,000 to 1/100,000 in the two assays (Table 3). It was further found that clone 109-6/2 produces IgG antibodies, 16-3/6 produces IgG antibodies and clones 2-4/3, gw 9/8 and 113 produce IgM antibodies. The ascites fluid in which the IgG was found contained the prominant fractions IgG₂a and IgG ₁.

It is noteworthy that the H-Y antibodies of the invention are remarkable in their significantly higher titer. This suggests that the instant antibodies are constituted of a unique sequence of amino acids. Clones 16-3/6 and 109-6/2 are further identified in that they produce antibodies of the IgG type rather than of the IgM type. Another feature is that they also show affinity for Protein A. These specific attributes of the clones further identify and distinguish them from others.

The differences in types of antibodies (IgM, IgG, etc.) is discussed in Joseph Bellanti, Immunology, W. B. Saunders Company, 1978, Philadelphia, Pa., pp. 115-137, herein incorporated by reference.

In accordance with the invention, it has also been found that antibodies from clones gw 9/8 and 16-3/6 are capable of immunoprecipitating molecules of 15,000 MW and 30,000 MW in sodium dodecyl sulfate polyacrylamide gel electrophoresis (see Fig. 4). The procedure followed was that of Hall JL and Wachtel SS, Primary Sex Determination: Genetics and Biochemistry, 33 Molec. Cell Biochem. 49-66 (1980).

Monoclonal H-Y antibodies of the invention may be purified and concentrated by the following method. The serum proteins are precipitated with 43% ammonium sulfate. Then they are purified by using PA-Sepharose 4B (Pharmacia). The IgG molecules are adsorbed to the PA Sepharose at room temperature for 10-15 minutes. The reaction mixture is washed thoroughly in PBS and the IgG molecules (H-Y antibodies) are then eluted off the PA Sepharose with 0.1M glycine-HCl acidic buffer (pH 3.5). The solubilized IgG antibody is dialyzed against PBS or borate buffer. The purified IgG antibody is then ready for use and may be preserved in 0.1% azide.

One of the important applications of the monoclonal antibodies of the invention is in the identification, selection and separation of a live embryo of an undetermined gender, i.e. male or female. The monoclonal antibodies to the H-Y antigen may be used in several ways to select and obtain embryos of a desired sex. Illustrative examples, which are not to be construed as limiting the invention in any manner whatever, are given below.

Example 1

Selective labeling of male embryos. Male and female bovine embryos 6-12 days old are flushed and recovered in a complete tissue culture medium (Dulbecco's PBS and 10% FCS and antibiotics) (PBS = phosphate-buffered saline, a standard solution; FCS= fetal calf serum, used for the protection and nutrition of embryos). The embryos are then washed in PBS and IPT (immune-precipitin tested) (FCS) three times prior to transfer to microtiter wells for further treatment. The embryos are then incubated in a concentrated monoclonal H-Y antiserum obtained in the manner previously described for 30 minutes at 22°C. The monoclonal antiserum is diluted in PBS with a 2.5% concentrationof IPT. After incubation, the embryos are washed 3 times in PBS with 5% IPT, and transferred to a solution of FITC-PA in PBS at a 1:20 dilution for 30 minutes. FITC (fluorescein-isothiocyanate) fluoresces under UV light. PA is a component of the cell wall of Staphylocoecus aureus that binds to the Fc portion of antibody molecules, especially IgG antibody molecules. Monoclonal H-Y antibodies of IgG type stick to male (H-Y⁺) cells. FITC-PA selectively adheres to the H-Y antigen-antibody complexes. Thus cells of male embryos but not female embryos fluoresce under UV light.

With the bovine embryos used, about 45% fluorescence versus about 45% non-fluorescence was observed, and about 10% was uncertain. Thus, by this method, in accordance with the invention, it is possible to detect and then select male embryos, or female embryos, as desired. The male embryos are separated from the female embryos by physical separation of the fluorescent from the non-fluorescent embryos.

Once the gender of the embryo has been identified, the embryo may be implanted immediately or it may be stored in any of the ways well known to those skilled in the art until transfer implantation can take place. Examples of the storage procedures include but are not limited to embryo culture at ambient temperature, storage at 0 to 10°C and storage at -196°C in liquid nitrogen.

The live embryo of the desired gender is then implanted in the uterus of a pseudo-pregnant bovine by a nonsurgical procedure known to one skilled in the art of embryo transfer. After the necessary gestation period the calf of preselected gender is born. In this manner, female and male calves can be obtained as are preselected.

Alternatively, the desired embryo is implanted by a surgical means wherein a midline incision is made in the flank of a locally anesthetized pseudo-pregnant bovine and one of the uterine tubes is exposed. The live embryo is inserted directly into the uterus. Subsequently a calf of predetermined sex is born full term and healthy. This procedure is followed for the male and female calf except that in the selection of the type of embryo, whether it be male or female, the procedure is different as described above. Example 2

a. Plating is another suitable method of selection. Male and female embryos (6-12 days old) are reacted for 20 minutes with monoclonal H-Y antibody. The antibody is fixed on the H-Y antigen as follows.

Bacteriological polystyrene plates are coated with rabbit anti-mouse affinity purified antibodies at a dilution of 1:10 to facilitate embryo removal after selection (1 rabbit antimouse:10 normal rabbit IgG). Bacteriological plates are used instead of tissue culture quality plates because treatment of the plate in this system could lead to non-specific adherence of the embryo; this does not occur with bacteriological plates. The antibodies are diluted in 0.05 M Tris pH 9.5 buffer, poured onto the plate, swirled and kept at room temperature for 40 minutes for attachment. Next the plates are shed with PBS followed by PBS containing 1.0% FCS. The embryos are then added to the plates and the plates incubated for 2 hours at room temperature. The fluid is gently poured off to remove female embryos. Five washes are performed. For positive selection, PBS with 1% FCS is added and vigorous pipetting (Pasteur) performed to dislodge the male embryos.

b. Protein-A batch settling method: Twenty embryos (male and female bovine, 6 to 12 days old) are exposed to antibodies as above. A suspension of PA Sepharose (commercially available) is prepared in a bacteriological plate or Buchner funnel. The embryos are added to the PA Sepharose and gently swirled or rotated for 20-25 minutes and allowed to settle for two hours at room temperature. At the end of that period the supernatant, containing the female embryos, is gently aspirated A solution containing concentrated normal IgG is now added with gentle swirling of the plate or funnel. This competes with male-embryo-bound monoclonal H-Y antibody for available PA, thereby liberating the bound embryos.

The embryos of the desired sex, in this case the male embryos, are implanted in the surrogate mother as in Example 1 to produce full term healthy male calves. With female calves the same procedure is followed.

Example 3

Cytotoxicity is another test for selecting the desired antibody. Embryos recovered as in Example 1 above are exposed to H-Y antibody and absorbed complement as in the cytotoxicity tests described above, under conditions that kill male (H-Y⁺) cells. Dead embryos are identified visually by uptake of trypan blue dye, or by noting lysis of the cells.

Rabbit serum is used as a source of complement, which is required for lysis in this system. The rabbit serum is previously selected for low toxicity to mouse thymocytes and for high complement content, indicated by its ability to support a cytotoxic reaction. The complement is absorbed to remove heteroantibody which might be expected to kill bovine cells indiscriminately. That procedure is accomplished by twice reacting the undiluted serum with suspensions of bovine fetal thymus, spleen, and testis cells in the presence of ethylenediamine tetraacetate (EDTA) which chelates divalent cations. Naturally occurring anti-bovine antibodies (heteroantibodies) are thereby absorbed from the serum, but the complement system is not absorbed, being inactivated in the absence of Ca⁺⁺. After the absorption, free Ca⁺⁺ is restored by addition of calcium chloride. The procedure allows use of a more concentrated complement source without the complicating effects of heteroantibody. The term "complement", as is known, refers to a complex group of proteins in body fluids that, working together with antibodies or other factors, play an important role as mediators of immune, allergic, immunochemical and/or immunopathological reactions. The reactions in which complement participates take place in blood serum or in other body fluids, and hence are considered to be humoral reactions.

With regard to human blood, there are at present more than 20 proteins in the complement system consisting of the so-called classical and alternative pathways. A more detailed discussion of the complement system and its biochemical, biological and pathological role in the body processes can be found in U.S. Patent 4,357,326 to Nair et al, in particular in columns 1 and 2 and the references cited there, which columns are incorporated herein by reference.

Example 4

a. Embryos obtained from a naturally or artificially inseminated female (or females) can each be physically dissociated into single living cells.

One or more of the separated cells can be taken from each embryo, and the sex of that cell (or cells) determined by (i) visual examination of chromosomes; (ii) serological assay for H-Y antigen; and (iii) physical methods such as sedimentation at unit gravity, gradient density centrifugation, and migration in an electric field; or (iv) by other methods deemed necessary or appropriate as, for example, labeling of male cells with the radioactive Bkm probe. Satellite DNA from the sex-determining W chromosome of the banded krait, a venomous snake of southern Asia, contains nucleotide sequences common to the Y chromosome of the mammal. The snake DNA (called Bkm for branded krait minor satellite DNA) thus can be used as a probe for Y chromosome-specific DNA by allowing it to hybridize in situ with enzyme dissociated DNA in male cells according to the techniques outlined by Jones FW and Singh L, in Conserved Repeated DNA Sequences in Vertebrate Sex Chromosomes, 58 Hum Genet 46-53 (1981) (see also Singh L, Jones KW, Sex Reversal in the Mouse [Mus musculus] is Caused by a Recurrent Nonreciprocal Crossover Involving the X and an Aberrant Y Chromosome, 28 Cell 205-216 (1982)). According to this technique, which does not involve the use of H-Y antibodies, DNA molecules from male and female cells are digested with the restriction endonucleases Alu I and Hae III (these enzymes cause the DNA strands to "unwind"). Then the Bkm probe, which has been radiolabeled by replication in medium containing tritiated thymidine (³H) ( for example) is added to the preparation. The ³H-Bkm hybridizes in situ with complementary DNA and thereby preferentially labels male cells (which carry the corresponding sequences in DNA of the Y chromosome). Hybridization of the ³H-Bkm probe with Y chromosome specific

DNA can be scored visually by allowing the chromosome preparation to expose a photographic plate (autoradiography). The resulting concentration of silver grains over the small Y chromosome readily identifies male cells. Alternatively (after digestion of DNA with Hae III endonuclease) the polynucleotides can be transferred to nitrocellulose and then hybridized with radioactive Bkm probes and run in a one-dimensional gel electrophoresis for detailed analysis of male specific banding patterns. As above, the gels are used to expose photographic plates. The different DNA fragments migrate in the gels according to mass and charge, and thus form a series of distinct bands. Of the various hybridization bands that result, several are male-specific. In this manner male cells are identifiable by comparison with banding patterns obtained with DNA from adult male and female cells of the same species.

b. Modification using Biotin-labeled cDNA to Bkm probe for detecting Bkm-like sequences by in situ hybridization with cells dissociated from embryo.

Biotin-labeled deoxyuridine triphosphate (Bio-dUTP) complexes are produced according to the method of Ward (Longer et al, 78 PNAS 6633 (1981)), and these are incorporated into cDNA by nick-translation using E. coli polymerase I (Rigby et al, 113 J.M.B. 237 (1972)) and the Bkm probe (Singh and Jones, 28 Cell 208 (1982)) as template. Then cells dissociated from bovine embryos are disrupted, and in situ hybridization performed with the biotinylated DNA probe. After reaction with enzyme (HRP), or fluorescin-conjugated avidin, or anti-biotin antibody, the ELISA test can be performed, or fluorescence determined by mucroscopy.

The result is a collection of dissociated early embryos lacking one or more than one cell whose sex is known. Reassociation of the cells can then be carried out in culture according to whichever of the following (or other) constitutions is desired:

(i) reassociation of the remaining cells of each embryo; (ii) reassociation of the remaining cells of each embryo, supplemented by a cell or cells of another embryo, of the same ascertained sex, to restore the original number of cells; (iii) reassociation of any number of cells of the same sex, less or more than the original number. It is also contemplated that a single cell can be used. Sexed embryos (i.e. in which sex characteristics have been so modified) can be stored frozen and used when desired.

Of course, it is also contemplated to be within the scope of the invention to obtain sex-identified cells in any vertebrate species characterized by heterogametic (XY) sex-specific expression of H-Y antigen. The methods are similar to those described for bovine embryos in examples above, which can be readily followed by one skilled in the art and adapted if necessary.

Example 5

Selective labeling of H-Y⁺(XY) cells. This selection can be accomplished by fluorescence as in Example 1 above, or by any technique which attaches a visual marker to a cell bearing H-Y antigen.

For example H-Y antibody is allowed to engage H-Y antigen at the cell surface; and a marker is allowed to engage the antibody. Examples of markers which recognize IgG monoclonal antibody are protein A and goat antimouse IgG, the latter being a goat antibody directed against mouse IgG antibody, which is, in this case, an antigen.

Thus, selective identification of XY cells can be accomplished by the following tests: IFA; MHA-HA; PA-SRBC; PAP; indirect radiobinding with ¹²⁵I-PA; ELISA; and other similar assays. Typical assays are described hereinbelow.

a. Immune fluorescent assay (IFA). Target cells are first exposed to H-Y antiserum (IgG) and then to a fluorescein isothiocyanate (FITC) labeled goat mouse IgG conjugate, or alternatively to FITC-PA as described in Example 1 above. Cells are incubated for 30 minutes with the FITC-goatanti-mouse or FITC-PA and then washed gently in PBS (pH 7.0) before fluorescence microscopy (Galbraith et al. Transplantation, 1978, supra).

b. Mixed hemadsorption-hybrid antibody (MHA-HA). Cells are reacted first with H-Y antibody, and then with a rabbit hybrid antibody of the specificity anti-mouse Ig:: antisheep red blood cells (SRBC). (Wachtel et al, 1974, supra).

Hammerling, Stackpole, and Koo, Hybrid Antibodies for Labeling Cell Surface Antigens, 9 Meth. Cancer Res. 255-282 (1973) used hybrid antibody for such a purpose, ferritin or another visual electron microscopic marker being bound to cell surface antigen by the linkage: antigen - antibody - hybrid antibody (anti-Ig:: anti marker) - marker.

Finally, the cells are reacted with SRBC, which bind to the H-Y antigen-positive (male) cell, forming a rosette. To ensure that all major classes of mouse antibody are detected in the MHA-HA test, the specificities of the anti Ig arm of the hybrid reagent include at least anti , anti , and anti .

Test cells are processed in the MHA-HA test by centrifugation through a discontinuous density gradient containing the reagents in appropriate sequence, a method developed to obviate the need for repeated centrifugation (see Koo GC, Boyse EA, Wachtel SS, "Immunogenetic Techniques and Approaches in the Study of Sperm and Testicular Cell Antigens", in Immunobiology of Gametes, M. Edidin and MH Johnson ed., Alden Press, Oxford, 73-84 (1977)). Gradients of the immune reagents, interspersed with wash solution, are established in narrow tubes made from 1 ml disposable pipettes (internal diameter, 2.5 mm). Heat-inactivated fetal bovine serum is added to each layer in concentrations which prevent intermixing of layers during subsequent centrifugations of the cells (range: 20% top layer, to 45% bottom layer). The wash layers may be visually distinguished by phenol red. Gradients are prepared and maintained at 4°C, and used within one to two hours after preparation. The gradients comprise (from top to bottom): (a) sensitized test cells, (b) wash layer, (c) hybrid antibody, (d) wash layer, (e) SRBC. After centrifugation, the tubes are allowed to stand for a minimum of 30 minutes. Each tube is cut just above the pellet, which is then gently resuspended and the labeled and non labeled cells (embryos) scored microscopically.

c. Protein A - sheep red blood cell test (PA-SRBC). This test, described by Koo GC and Goldberg CL (A Simplified Technique for H-Y Typing, 23 J. Immunol. Meth. 197-201 (1978)) is based on the observation of Goldberg EH, Arrington T, Tokuda S (Detection of H-Y antigen on human male leukocytes, 23 J. Immunol. Meth. 23 203-206, (1978)) that Staphylococcus can be used to label human male leukocytes. Target cells are exposed to H-Y antiserum and then reacted with a suspension of sheep red blood cells that have been coated with Protein-A, a cell wall polypeptide which binds immunoglobulins of the IgG class. Male cells thus form rosettes.

The method proceeds as follows: PA-SRBC are prepared according to the method of Goding JW (The Chromic Chloride Method of Coupling Antigens to Erythrocytes: Definition of Some Important Parameters, 10 J. Immunol. Meth. 61-66 (1976)): A solution of Protein-A purified from the cell wall of SA (Pharmacia Co.) is added to a suspension of SRBC. To this mixture a solution of 0.01% of CrCl₃ is added. The suspension is allowed to stand for five minutes at room temperature. The PA-SRBC are washed and stored as a 10% stock solution containing 0.01% sodium azide.

The assay for H-Y antigen is a modification of the MHA-HA test. Target cells are reacted with H-Y antiserum and then washed and resuspended in PA-SRBC. The mixture is spun slowly and the pellet allowed to stand at room temperature for 30 minutes before reading in a hemacytometer. Any cell with three or more SRBC is scored as a 'rosette'.

d. Peroxidase anti perioxidase method (PAP). The ABC variation of the PAP method by Hsu (29 J. Histochem. Cytochem. 557 (1981)) is reported to be 8 to 40 times more sensitive than the PAP method of Sternberger. The technique may be used to stain H-Y⁺ embryos (males). Our variation consists of a primary monoclonal H-Y antibody, a biotinylated goat (affinity purified), secondary antibody and an avidin-biotinylated horseradish peroxidase complex (which consists of many biotinylated HRP molecules crosslinked by avidin into a 3-dimensional array (commercially available from Vector Labs, Burlingame, CA as

Vectastain^TM ABC kit).

The primary monoclonal antibody is reacted with the embryo or with male cells for 30 minutes. Then the second biotinylated antibody is reacted for 30 minutes. Finally the avidin DH:Biotinylated HPR H complex is added; the substrate is added; and color is produced which can be scored.

e. Radiobinding with iodinated protein A (¹²⁵I-PA).

The radiobinding assay exploits the specific reaction of protein A with the Fc portion of mouse IgG. Accordingly radio-iodinated protein A (I-PA) can be used to label H-Y antibody, thereby identifying antigen-positive cells. Soluble H-Y antigen from the supernatant of the mouse testicular cell preparations of Daudi cell cultures (see Wachtel supra, 1983) is diluted with phosphate buffered saline (PBS); 0.05 ml of the dilution is placed in each of several wells in a microtiter plate. The plate is incubated for several hours in dry heat to evaporate the PBS; the wells are thereby coated with a residue of H-Y antigen. After refrigeration overnight, the plate is washed, and 0.05 ml of (diluted) H-Y antiserum is added to each well. The plate is then refrigerated for one hour and washed again. Protein A (available commercially) that has been labeled with radioactive ¹²⁵iodine (¹²⁵I-PA) is added to each well and the reaction mixture is incubated for one hour.

Then the plate is washed several times and radioactivity scored in a gamma counter.

The techniques can be used as an indirect assay by absorbing portions of H-Y antisera with male and female cells and then using the absorbed portions with soluble H-Y antigen, or as a direct assay, by using unabsorbed H-Y antisera directly with target cells in place of soluble H-Y antigen.

Radio-iodination of PA is performed according to the techniques of Dorval G, Welsh KI, Wigzell H (Labeled Staphylococcal Protein A as an Immunological probe in the Analysis of Cell Surface Markers, 3 Scand. J. Immunol. 405-411 (1974)) as modified by Thorley-Lawson DA (Characterization of CrossReacting Antigens on the Epstein-Barr Virus Envelope and Plasma Membranes of Producer Cells, 16 Cell 33-42 (1979)): 200 g of protein A in 50 of 0.5 M phosphate buffer (pH 7.5) are reacted first with 5mCi of ¹²⁵I (50 in NaOH) (Amersham) and then with 60 g of Chloramine T (20 at 3 mg/ml aqueous).

After 15 minutes on ice, the reaction is quenched by addition of 80 g of sodium metabisulfite (20 at 4 mg/ml aqueous). Unincorporated ¹²⁵I is removed by passage over a 7 ml

Sephadex G-25 column that has been washed and equilibrated with TEN buffer (0,02 M Tris, pH 7.6; 0.001 M EDTA; 0.1 M NaCl). 0.5 ml fractions are collected into tubes containing 50 TEN buffer with 20 mg/ml BSA. Five of each fraction are scored in a gamma counter, and peak fractions containing I-PA are pooled.

f. Enzyme-linked immunosorbent assay (ELISA). This is described in detail in Example 11 below. The ELISA can be used to detect cell bound or soluble H-Y antigen, and as a direct or indirect test. The indirect test is accomplished by first absorbing H-Y antibody with male, female or test cells, and then applying the antibody as in a direct test.

Example 6

Physical separation. The separation is accomplished as in Example 2 above. The means of separation may also include sedimentation at unit gravity, gradient density centrifugation, and migration in electric field.

Example 7

Absorption. The cells of a tissue or organism to be typed for H-Y antigen are suspended in aliquots of H-Y antibody, which is then used in cytotoxicity tests with sperm or epidermal cells, or in any assay for H-Y. The loss of reactivity indicates absorption of H-Y antibody and thus expression of H-Y antigen in the absorbing cells.

Example 8

Cytotoxicity. This is accomplished as in Example 3 above. The method may also include absorption and indirect cytotoxicity as discussed in Example 7. Example 9

Direct radiobinding. The H-Y antibody itself is labeled with a radioactive isotope such as ¹²⁵I. Attachment of the radiolabeled H-Y antibody to a particular (H-Y⁺) cell is monitored by scoring cell bound radioactive counts per minute (cpm) in a standard scintillation counter.

Example 10

H-Y antigen is shed or secreted by male cells in culture, and this occurs as a soluble constituent of the nutrient medium in which male cells are cultivated. Shalev et al (1980) reported that 'free' H-Y was present in the medium of cultured skin fibroblasts of the goat. It has been reported (Wachtel et al, 1980) that H-Y may occur as a soluble component of (male) fetal calf serum.

It has been discovered that soluble H-Y in supernatant medium of bovine male embryos can be detected. In this way male and female embryos can be identified in solution without directly manipulating the embryos themselves. Embryos are cultured for as long as from 24 to about 36 hours without loss of viability. Fluid from individually cultured embryos may then be used in inhibition assays.

The procedure is as follows: H-Y antiserum is reacted with H-Y⁺ target cells; H-Y antiserum that has been first exposed to soluble H-Y is inhibited from reacting with H-Y⁺ target cells, because available molecules of H-Y antibody which are bound to soluble H-Y antigen have been specifically removed by male supernatant.

The culture fluid is reacted with monoclonal H-Y antiserum in any of several assays including (i) the enzyme-linked immunosorbent assay (ELISA), (ii) the immune fluorescent assay (IFA), (iii) cytotoxicity, (iv) the Protein-A sheep red blood cell (PA-SRBC) assay, or (v) the radiobinding assay.

Lack of inhibition which is observed signifies that the culture fluid did not contain H-Y and thus originated from a female embryo. Inhibition of reaction signifies that the culture fluid contained H-Y from a male embryo.

An embryo of the desired sex, a male, or female, as appropriate, is then transferred to a female and a full term, healthy offspring is born.

Though monoclonal H-Y antiserum is by far preferred, conventional H-Y antiserum might be useful in certain circumstances.

Examples of systems to detect soluble H-Y antigen in a culture fluid follow below:

Example 11

Soluble H-Y can also be detected in the enzyme-linked immunosorbent assay (ELISA). Polystyrene tubes or microtiter plates are coated with H-Y monoclonal antibody in mouse ascites fluid. The antibody is diluted in 0.1 M bicarbonate buffer. The mixture is incubated for 3 hours at 37°C and then stored at 4°C until use. The tubes or plates are washed several times with NaCl solution. Now the antigen source is added in PBS containing 0.05% Tween 20, and the mixture is agitated gently for several hours at room temperature to promote formation of H-Y antigen-antibody complexes. The mixture is washed again with NaCl-Tween 20 to remove non-adherent molecules. Monoclonal H-Y antibody conjugated with alkaline phosphatase (diluted in PBS-Tween) is then added, and the mixture is allowed to stand overnight at room temperature. After washing, nitrophenylphosphate (NPP) is added. This reacts with alkaline phosphatase giving a yellow color to the solution. The optical density of the solution is determined with a spectrophotometer at 400nm. The amount of yellow color present is a function of the amount of alkaline phosphate present. That is a function of the amount of H-Y antigen that has complexed to the solid phase.

An alternative procedure of ELISA consists in measuring the inhibition of the reaction between monoclonal H-Y antibody and mouse testis supernatant brought about by addition of supernatants from male or female lymphocytes. Mouse testis supernatant (TS), a known source of soluble H-Y, is prepared according to Wachtel SS, Hall JL (H-Y Binding in the Gonad: Inhibition by a Supernatant of the Fetal Ovary, 17 Cell 327-329

(1979)) which is incorporated by reference. The lymphocyte supernatant is prepared by incubating 10 x 10⁶ lymphocytes of each subject at 4°C overnight in Hank's buffer. Serial dilutions of TS are prepared in 0.1 M bicarbonate buffer, pH 9.6, at an estimated total protein concentration of 100 g/ml to 0.12 g/ml. One hundred fifty lambdas of each dilution are placed in wells of a polystyrene microtiter plate, and the plate is incubated at 37°C for 3 hours. After incubation, the plate is washed in 0.15 M NaCl containing 0.02% Tween 80; 90 of a 1:60 dilution of the monoclonal conjugate are added to each well. Next, 50 of Hank's solution without phenol red are added in one series of wells, and 50 of filtered lymphocyte supernatant from male or female controls respectively are added in two other series of wells. The plate is then incubated for another 3 hours at room temperature and washed. A substrate of alkaline phosphatase-para-nitrophenyl phosphate is added and the amount of released yellow para-nitrophenol measured in an ELISA reader. The tests are performed in replicate samples. Whereas female lymphocyte supernatant has no effect on the reaction of TS with the monoclonal antibody, readings are 30-40 percent lower than those obtained with TS alone when male lymphocyte supernatant is used (See Figure 1).

During development of this assay lymphocytes from 10 normal men and 10 normal women were isolated from the blood, the concentration was adjusted to 10 x 10⁶ cells/ml Hank's, and the assay run as above using the TS at 1:30 dilution. In this case introduction of male lymphocyte supernatant gave readings 69-78 percent of those obtained with TS, (mean 75 percent). The results are shown in Figure 2. The scores are expressed as a ratio of the reading obtained when TS alone was allowed to react with H-Y antibody. In this manner it was possible to identify the male lymphocytes from the female ones.

This is shown in Figure 2.

It is yet another embodiment of the invention and also within its scope that monoclonal antibodies to the H-Y antigen be used for the assignment of H-Y phenotype in male, female and intersexual patients with chromosomal, genital or endocrinological disorders. The assignment of cell surface H-Y phenotypes is accomplished by any of the cell surface techniques described in Examples 1, 2, and 3.

In a further embodiment of the invention it has been discovered that H-Y antigen is a component of the serum. The serum-borne molecule or family of molecules can be detected and assigned the proper H-Y phenotype using the following procedure.

Monoclonal H-Y antibody is diluted in 0.1 M bicarbonated buffer, and portions of the diluted serum are placed in each of several wells of a microtiter plate. Plating efficiency is improved by addition of poly-L-lysine. The plate is incubated for three hours in dry heat (37°C) to coat the wells with antibody. After refrigeration overnight, the plate is washed several times, and human sera from male or female controls or an unknown sample are added to each well. The plate is allowed to stand at 37°C, 22°C and 4°C for twenty minutes at each temperature. After repeated washing, a small portion of triple-labeled (³H-leu, ³H-ala, ³H-val) H-Y antigen is added to each well. The H-Y antigen is prepared and partially purified according to Hall JL and Wachtel SS, Primary Sex Determination: Genetics and Biochemistry, 33 Mol. Cell Biochem. 49-66 (1980). After incubation, the plate is washed again and allowed to dry. The bottoms of the wells are punched out with a manual cam punch press and dissolved in scintillation fluid for enumeration of cpm.

In this system, soluble H-Y antigen in male serum reacts with the plated antibody. The reaction blocks subsequent binding of the labeled antigen, and the cpm is decreased relative to the cpm scored for wells containing female serum (H-Y).

Figure 3 shows the results of a preliminary assay run with conventional H-Y antibody. At the optimal dilutions of 1/8 and 1/16, human male serum readily blocked solid phase uptake of labeled H-Y antigen, but human female serum did not. The horizontal dotted line represents standard cpm in plate containing buffer, antibody and labeled antigen. The same procedure is applicable to monoclonal H-Y antibody.

In accordance with the invention, there is provided a method by which mutant forms of H-Y antigen may also be detected. Biochemical systems that detect H-Y antigen offer the advantage that information about the nature of the molecule itself is gained in the same operation that determines its presence or absence. Thus mutant H-Y antigens could be detected as shown in the schematic Figure 4.

Solubilized membrane fragments, from cells of the subject under study, are dispersed in cylindrical gels according to the technique of iso-electric focusing. See Anderson NG, Anderson HL, Analytical Techniques for Cell Fractions. XXI. Two-dimensional Analysis of Serum and Tissue Proteins: Multiple Isolectric Focusing, 85 Analyt. Biochem. 331-340 (1979), which is incorporated herein by reference.

Ampholines are added to a polyacrylamide matrix prepared in a glass cylinder. Under the influence of an electric charge, the ampholines distribute themselves so as to establish pH gradient in the gel. The solubilized membrane preparation is then added, and a current of 10,000-12,000 volt hours applied to induce migration of individual peptides. Under those conditions, the peptides migrate to their isoelectric points, where they attain a zero net charge. To insure accurate estimation of isoelectric point, proteins of known isoelectric point are focused in parallel gels. The cylindrical gels are removed and equilibrated in a second SDS buffer. Secondary application of SDS is necessary because SDS, which is negatively charged, focuses on its own, causing dissociation of SDS-peptide complexes. The cylindrical gels, containing the focused peptides, are now run at right angles to the initial migration, according to the technique of SDS slab gel electrophoresis, in a polyacrylamide gradient of 10-20% acrylamide. The cylindrical gels are placed on top of slab gels and electrophoresis carried out at 100 ma per gel until the bromophenol blue tracking dye migrates to the end of the preparation. The gels are fixed by soaking in a series of diluted alcohol and acetic acid washes. Polymers such as polyacrylamide provide a molecular sieve through which protein molecules migrate at a rate proportional to the size of the SDS-polypeptide complex. Relative molecular weight is determined by reference to migration patterns of standard protein markers. The peptides are thus distributed according to isoelectric point and molecular weight, and it remains to visualize them. This can be accomplished by staining with Coomassie Blue dye or by radioactive labeling of the proteins followed by auto radiography.

Alternatively, visualization can be accomplished by using the commercially available silver staining technique developed by Sammons DW, Adams LD, Nishizawa EE, Ultrasensitive Silver-Based Color Staining of Polypeptides in Polyacrylamide Gels, 2 Electrophoresis 135-140 (1981) incorporated herein by reference. This method allows a third dimension of resolution based on the formation of colored polypeptide-silver complexes.

Membranes from male and female cells are prepared by solubilization in urea or detergent as above, and the membrane fraction subjected to immunoprecipitation with H-Y antibody and PA-Sepharose according to the two step procedure. In the first step, monoclonal H-Y antibody of class IgG is reacted with protein A-coated Sepharose beads. The protein A binds the Fc portion of IgG; those molecules are attached to the beads so that their antigen reactive sites are exposed. After washing, in a second step super natants containing H-Y antigen are added, and the mixture of beads and supernatant is agitated for one hour. This is designed to promote formation of antibodyantigen complexes at the surfaces of the beads. Material that is bound nonspecifically is removed by repeated washing. Then the suspension is layered over a solution of sucrose in PBS and the beads are centrifuged through the solution to remove all but tightly bound material. The bound antibody-antigen complexes are dissociated and eluted by resuspending the beads in SDS sample buffer and heating to 100°C for 3 minutes. Sepharose is removed by centrifugation. The method is also described in Hall JL, Bushkin Y, Wachtel SS, Immunoprecipitation of Human H-Y Antigen, 58 Hum. Genet. 34-36 (1981) which is incorporated herein by reference.

The supernatant is now run in the two-dimensional system. Male specific proteins identified in the two dimensional system correlate with peptides recovered with monoclonal H-Y antibody. An electrophoretic profile obtained with the Protein A technique is represented in Figure 5. A prominent peak corresponding to 15,000 molecular weight was present after immunoprecipitation with H-Y antibody.

The method described here in accordance with the invention permits ready identification of a certain percentage of H-Y mutants incorporating single amino acid substitutions or simple deletions, in addition to providing typing information.

Thus direct information concerning presence or absence of H-Y becomes available (presence or absence of a spot or spots), whereas occurrence of a novel but adjacent spot is an indication of a mutant species. The fact is that each spot is characteristic of a particular peptide, and thus is an adequate and sensitive indicator of differentiation among the alternative peptide species.

It is also possible to evaluate the H-Y receptor. The H-Y receptor phenotype is assigned in the following manner. The gonadal H-Y receptor is present in the sexually indifferent embryonic gonad in XX and XY embryos. Usually H-Y is present only in the XY gonad? and so it is usually the XY gonad that becomes a testis. Presence or absence of the H-Y receptor can be tested by adding soluble H-Y antigen to cells of the gonad in question, and then by determining whether or not the H-Y has reacted positively, i.e. is attached. This is accomplished by testing the ability of H-Y-antigen-exposed gonadal cells to bind H-Y antibody in any of the tests described herein above; or alternatively by measuring radioactive uptake in gonadal target cells exposed sources of radiolabeled H-Y antigen.

Labeled H-Y is prepared as follows: 150 x 10⁶ exponentially growing Daudi cells in RPMI 1640 with 10% FCS are suspended in RPMI 1640 with 2% FCS in which ³H-leucine,

³H-alanine and ³H-valine (100 uci/ml each) have been substituted for "cold" leucine, alanine and valine. The cells are, pelleted and resuspended in RPMI 1640 with 10% FCS for a recovery period of 24 hours. During the next 16 hours the cells are incubated in RPMI 1640 with 2% FCS, containing cold leucine, alanine and valine. The second supernatant, which contains slow turnover proteins including labeled H-Y, is centrifuged at 100,000 x g and stored frozen.

Other applications contemplated by the invention include several areas in H-Y serology. For instance an area contemplated by the invention is in gene mapping. As another example analysis of H-Y phenotype in cases of XY gonadal dysgenesis (defective embryonic development of the gonad) may provide valuable clues as to the risk of gonadal malignancy in that condition, with implications for etiology, as discussed further below.

Various assays for H-Y have provided information concerning presence or absence of testis determining genes, and information concerning the location and function of those genes. Thus H-Y antigen can be useful in diagnosis of the existence of such genes. Another area of interest is in the diagnosis of sexual abnormality. Detection of H-Y in a female is almost always a sign of abnormality, and H-Y⁺ phenotypes in white blood cells or cultured skin fibroblasts from phenotypic females are correlated in general with aberrant development of the gonad. Thus, it is contemplated that H-Y antigen serology be used in conjunction with the endocrinologic assays in the diagnosis of XX true hermaphroditism, i.e. sexual ambiguity caused by concurrent development of testis and ovary in the same individual.

Another area of application of this invention is in etiology. Clinical H-Y typing is expected to clarify the nature of aberrant sexual development. Absence of H-Y in an XY female is expected to indicate loss of testis determining genes, for instance. The presence of H-Y in an XY female is expected to indicate failure of engagement of the inducer and its gonadal receptor. It is within the contemplation of the invention to differentiate between that alternative and that failure attributable to mutation of the receptor or of the H-Y inducer.

Another area of application of the invention is in the prediction of risk of malignancy. The syndrome of XY gonadal dysgenesis is characterized by retarded puberty and amenorrhea in females whose ovaries fail to develop properly. The gonad is usually represented by a fibrous streak of ovarian-like tissue, but germ cells and follicles are absent and endocrine function is abnormal. There is high risk of malignancy in the gonads of females with a Y chromosome. Among some 46 patients with XY gonadal dysgenesis that were surveyed, seventeen (37%) had at least one gonadal tumor. Serological analysis reveals two classes of XY gonadal dysgenesis: one is H-Y- and one is H-Y⁺. It is believed that the former is due to absence or mutation of H-Y antigen; the latter to absence or mutation of the H-Y antigen receptor. Of the 46 patients described above ten (22%) were typed H-Y- and thirty-six (78%) were typed H-Y⁺. But no tumor was reported in a patient with H-Y- phenotype (Wachtel, 1983).

The invention, therefore contemplates an early diagnosis of gonal malignancy.

While a select and representative number of hybridoma producing monoclonal H-Y antibody are described herein, it is contemplated that the present invention encompasses other monoclonal H-Y antibodies which exhibit the same or equivalent serological and immunological characteristics as shown herein.

In accordance with the invention, any embryo of a vertebrate (e.g. warm-blooded mammal) in which there is present H-Y antigen can be identified or typed for gender. For bovine selection, and for highest yield of live embryos, it has been found desirable to test the embryos when they are from about 6 to 12 days old. A lesser number of viable embryos are likely to be obtained. Identification of gender can nonetheless be obtained when the treatment is performed earlier or later.

Description of Drawings

Figure 1 shows the inhibition of reaction between TS and anti H-Y antibody (_) by supernatant fluid of male (0) and female (X) lymphocytes.

Figure 2 is the optical density scores showing ratio of reading in presence of lymphocyte supernatant to reading in presence of Hanks buffer alone. Each point represents the mean of duplicate scores from a single donor.

Figure 3 shows the inhibition of uptake of labeled H-Y antigen by male and female serum.

Figure 4 is a diagram of the two-dimensional gel electrophoresis of H-Y antigen.

Figure 5 is a computer graphic printout of immunoprecipitation of H-Y antigen with H-Y antiserum.

Claims

1. The method for identifying a live bovine embryo of predetermined sex which comprises contacting a mixture of male and female gender live bovine embryos, the male embryo having H-Y antigen, with a monoclonal antibody to the H-Y antigen, fixing the H-Y antibody to the H-Y antigen of the male embryos, thereby identifying the male embryos, the monoclonal antibody having a titer of from about 1/10,000 to about 1/100,000.

2. The process of claim 1 which comprises the additional step of separating the live identified embryos from, the unidentified embryos, thereby collecting the live male embryos.

3. The process of claim 1 which comprises the additional step of separating the identified embryos from the live unidentified embryos, thereby collecting the live female embryos.

4. The process of. claim 2 which comprises implanting the identified, live male embryo in a surrogate mother cow.

5. The process of claim 3 which comprises implanting the live female embryo in a surrogate mother cow.

6. A method for identifying male or female bovine embryos which comprises treating bovine embryos of undetermined gender with a monoclonal H-Y antibody, affixing the H-Y antibody to the H-Y antigen of the male embryos, and identifying the gender of the embryo, the monoclonal antibody having a titer of about 1/10,000 to about 1/100,000.

7. A method for identifying male or female bovine embryos which comprises culture medium which comprises H-Y antigen from bovine embryos with monoclonal H-Y antibody, affixing the H-Y antibody to H-Y antigen of the male embryos and identifying the male embryos, the monoclonal antibody having a titer of about 1/10,000 to about 1/100,000.

8. Hybridoma cell lines which produce monoclonal H-Y antibody to the H-Y antigen, wherein the titers of the antibody range from about 1/10,000 to about 1/100,000.

9. Monoclonal H-Y antibody which is raised by hybridoma cell lines and has a titer from about 1/10,000 to about 1/100,000.

10. The antibody of claim 9 which is raised by clone 2-4/3, clone gw 9/8, clone 16-3/6, clone 109-6/2 or clone 113.