FR2554127A1 - METHOD FOR DIAGNOSING INFECTIONS - Google Patents

Abstract

THE OBJECT OF THE INVENTION IS A SEROLOGICAL DIAGNOSIS AMONG LIVESTOCK. A TEST PLATE 38 SHOWING A SERIES OF WELLS 40 IS PROVIDED; AT LEAST SOME OF WELLS 42 ARE COATED WITH A BOVINE LEUKEMIA VIRUS ANTIGEN; AN ELISA BUFFER CONTAINING A BLOCKING AGENT TO RESIST NON-SPECIFIC BINDING IS INTRODUCED INTO THE WELLS; THE TESTED SERUMS ARE INTRODUCED INTO THE WELLS DIRECTLY OR AFTER THE MIXING WITH THE BLOCKING SOLUTION; AN ENZYMATIC ANALYTE IS INTRODUCED IN THE WELLS AND THEN A CHROMAGEN SUBSTRATE; READ OR STOP THE REACTION; BY VISUAL DETERMINATION OF THE COLOR OF THE WELLS, WITH THE NAKED EYE OR BY MEANS OF AN APPROPRIATE APPARATUS, A DETERMINATION IS MADE; INDICATOR WELLS CAN BE PROVIDED; INTERMEDIATE WASHING AND INCUBATION STAGES ARE USED; THE PREFERRED BLOCKING AGENTS ARE CHOSEN FROM GOAT, CHICKEN, COBAYE AND SHEEP SERUMS. DIAGNOSIS OF BOVINE LEUKEMIA VIRUS INFECTION.

Description

1 2554127

  The present invention relates to a method of

  diagnosis of infections and it concerns more specifically

  First, an enzyme-linked immunosorbent assay for serological diagnosis of bovine leukemia virus infection in cattle. Direct methods for diagnosing Bovine Leukemia Virus infection in cattle are already known. These methods generally include the isolation and culture of lymphocytes. This way of approaching

  problem is rather complicated and long and the use of

  The regular practice of such a process is limited.

  In addition, it has generally been found that such methods

  are less sensitive than serological methods.

  It has been found that infection with bovine leukemia virus (VLV) in cattle results in the production of viral specific antibodies for a glycoprotein (gp51) which is present in the viral surface and for

  the main protein of nuclear virions (p52 or p24).

  Since these antibodies provide an accurate indication of BSE infection, their detection by serological testing is an effective way of identifying cattle infected with the virus.

of bovine leukemia.

  A known serological procedure for this purpose is the immunodiffusion test of agar gel (AGID). In such a test, an antigen composition, which is generally predominantly gp51, is introduced into a well formed in a thin layer of agar gel. We place in

  another well a sample of serum that must be tested.

  If antibodies to the bovine leukemia virus are present

  serum, they will combine with the anti-

  VLB gp gene and form a visible line in the agar.

  agar between the wells. It is known, however, that this test is less sensitive than other tests especially during the early stages of infection and that, in some cases, false negative reactions are obtained. On the other hand, the reactions of a weak precipitation give equivocal results. In addition, since the gp antigen used in such assays is a crude composition, it tends to contain fetal calf serum proteins in high concentrations. These proteins give lines of precipitin with bovine serum containing isoantibodies. Such antibodies are common among cattle immunized with vaccines containing bovine proteins, among the

  pre-immunized cattle against babesiosis and piroplas-

  mose by transfusion of bovine blood and also among multiparous cows. In a normal way, the lines of

  precipitin due to bovine protein in the

  gp51 antigen events should give non-identity or partial identity responses to adjoining lines of control precipitin. However, under the conditions of a routine test, such reactions can be very difficult to recognize or can

  be subject to errors attributable to "writing".

  This is particularly so in cases where precipitin lines are weak or scattered, which occurs frequently. As a result, a test result is obtained which is subjective and unreliable. The only sera that can be accurately titrated are therefore

  sera giving distinct precipitin lines.

  Another disadvantage of this test is that it is not suitable for large-scale work and is not easy to automate and in addition it requires a relatively long incubation period, that is to say

of the order of 48 hours.

  It is also known to use a radioimmunoprecipitation titration with purified gp51 labeled with radioactive iodine as a marker (RIAgp). We know

  that this test is more reliable than the AGID test in parti-

3 2554127

  when antibody titres are not very high. One of the problems with this test is the need for special equipment such as

  as a gamma counter, in order to implement the test.

  It is also necessary to have special equipment to purify the antigen to a state of homogeneity. In addition, this test requires a certain qualification to perform it, as well as the need to use

  appropriate safety techniques when handling

the radioactive material.

  Another known type of assay is enzyme-linked immunosorbent titration (ELISA) which has the advantage of being practical and inexpensive as

  AGID test, while giving results that are acceptable

  tables. Moreover, it does not imply some of the

  problems encountered with the RIA gp test. We can refer for example to Ressang et ai. "Enzyme Linked Immunosorbent Assay for the Detection of Antibodies to Bovine Leukosis Virus", Ann. Rech. Vet. 1978, 9 (4) pp. 663-666; Behrens et al., Comparative Studies on

  the Enzyme Linked Immuno Sorbent Assay and Immunodiffu-

  Test and Diagnosis of Enzootic Bovine Leukosis, Fourth International Symposium on Bovine Leukosis, pp. 173-184, Todd et al., "Use of Controlled Antigen to Improve the Enzyme Linked Immunosorbent Assay for Enzootic Bovine Leukosis Antibodies, Veterinary Record (1982) 110, pp. 307-308; Poli et al., "Microplate Enzyme-Linked Immunosorbent Assay for Bovine Leukemia Virus Antibody", Journal of Clinal Microbiology, January 1981, pp. 46-48; Serious et al, "Comparison of Enzyme Linked Immunosorbent Assay with Polykaryocytosis Inhibition Assay and the Agar-Gel Immunodiffusion Assay for the Detection of Antibodies to Bovine Leukemia

  Virus ", Am., J. Vet Res, Volume 43, Number 6, pp. 960-

  966; Biancifiori et al., "Elisa Tests for Detection

25541Z?

  of Antibodies to Enzootic Bovine Leukosis Virus ",

  3rd International Symposium on Bovine Leucosis,

  pp. 167-172 and Todd et al., "An Enzyme-Linked Immuno-

  Sorbent Assay for Enzootic Bovine Leukosis Virus Anti-

  bodies ", Vet Rec 1980, 107 pp. 124-126 One of the serious problems with

  The known ELISA tests are that sera from certain

  some animals, including possible VLB infection

  is not certain, give false positive reactions

  tives. These false positive reactions seem to come from the binding of the immunoglobulin with the receptacles

  plastic material used for the ELISA test.

  According to the ELISA method used, approximately 1 to 30% of serum of cattle not affected by VLB will give

  false positive reactions, which is an absolute rate

  unacceptable. This problem can be minimized to some extent by comparing the reactions of a

  serum tested in containers with and without antigen.

  However, if the reaction in the container without

  gene is too high, it is impossible to identify the sample. Due to the apparent frequency of false positive reactions in known techniques, there is a definite need to improve the ELISA test with respect to specificity, sensitivity and practicality, so that this test is superior to known ELISA tests. and gives effective results from the

same way as the RIA gp test.

  The present invention has made it possible to solve this problem

  by performing an ELISA test in which a test plate with a series of wells is used. At least some of the wells are coated with an antigen to the bovine leukemia virus. After incubation and washing, blocking of nonspecific binding sites can be achieved by applying a solution of a suitable animal serum. The sera of the test can be mixed with the solution of the animal blocking serum or it can be introduced directly into the wells. After this operation, an additional incubation and washing is carried out. An antibody-bound enzyme ("enzymatic analyte") capable of recognizing bovine immunoglobulin is introduced into the wells, followed by additional incubation and washing to remove the non-combined analyte. A chromagene-containing substrate is introduced into the wells and an additional stage of incubation is performed. The result of the reaction is then read out or stopped and can be determined visually, with the naked eye or through a suitable optical instrument such as a

  spectrophotometer, the presence or absence of an infection

  bovine leukemia virus. The materials used are

  and the preferred operating parameters.

  Accordingly, the main objects of the present invention are to provide: - a method of serologically diagnosing bovine leukemia virus, which is highly specific, has the desired sensitivity and is practical for routine use to obtain the results in a few hours;

  - an ELISA test which effectively reduces the reactions

  VLB antibody-free sera at an insignificant level; an ELISA test which does not require specialized equipment or radioactive materials: an ELISA test whose implementation is inexpensive;

  - an ELISA test that does not require special personnel

cialized;

  - an ELISA test which reduces the non-specific binding

  than serum containing the VLB antibody; an ELISA test well adapted to a visual determination with the naked eye of the results obtained; and - other objectives that will emerge from reading

  of the description below with reference to the attached drawings

  in which: Figure 1 is a general diagram of a preferred embodiment of the invention; Figure 2 is a view from above of a plate usable in the present invention; Figure 3 is a view from above of a well in the plate shown in Figure 2; and FIG. 4 is a cross-section of the well of

  Figure 3 through line 4-4 thereof.

  In this memo, when we speak of a

  visual termination by "the naked eye", we include the deter-

  by a person who wears or uses long

  corrective measures of any type, but

  the exclusion of equipment such as a spectrophotometer.

  Among the many features that contribute to the success of the ELISA test of the present invention, it is appropriate to mention the use of an animal serum such as

  blocking agent for the non-specific binding of the immune

  bovine noglobulin without significantly interfering with specific reactions. Also of interest are the remarkable washing techniques

  used after the first and second stages of incu-

  proval. The chromagen solution as a substrate

  also to a certain extent to the effective obtaining of a good result because of its high sensitivity,

  low toxicity and good stability.

  In the preferred embodiment of the present invention,

  In this invention, the following sequence of operations is performed. Referring to Figure 1, the process will be summarized as details about each stage

  will be given later. A suitable plate is provided

  with a series of wells and

  a number of samples. (For convenience of nomenclature, the stages shown in Figure 1 have been designated by numbers). About half of the wells are coated with VLB antigen, after which the wells are incubated at 4 and washed at 6 to remove the non-combined antigen. The plate can then be stored, if desired (8). An ELISA buffer which comprises (a) a blocking serum to block non-specific binding of bovine immunoglobulins to the plastic container in (b) a buffer of high ionic strength, is then introduced into the wells 14 and the test sera are also introduced into the wells 14, after which they are incubated at 16 and washed at 18 to remove the non-combined antibody. The ELISA buffer and serum can be pre-mixed and then the mixture introduced into the wells, after which the incubation and washing steps 16 are performed.

  18. A modified but less advantageous technique

  would introduce the ELISA buffer into the wells, incubate, wash to remove excess material and then introduce the tested sera into the wells containing the serum-free ELISA buffer, then incubate and wash the assembly. The sera tested should be added to at least one well with antigen and one well that does not contain antigen. The enzymatic analyte is then added to the wells, incubated in

  22 and washed at 24 to remove the non-combined analyte.

  One can then stop the reaction by 30 or read its result. In the plate 38 shown in FIG. 2, a series of 96 wells are formed which are preferably

  substantially cylindrical. The plate can be an ele--

  plastic unit, the wells being of substantially equal size and having a capacity

  from about 100 to 300 microliters. The plate can be

  any suitable material such as, for example,

  polystyrene or polyvinyl chloride. In the example shown, the then are arranged in a series of columns 1 to 12 and rows A to H. In general,

  prefers to alternate the rows when performing the

  antigen production, to facilitate reading

  results. For example, you can smear with

  gene the wells of rows A, C, E and G and leave antigen-free wells in rows B, D, F and H. Wells 40 and 42 can serve as wells for the low positive control serum with the antigen and wells

  44 and 46 can be used as positive control serum wells

low tif without antigen.

  In order to prevent an unwanted flow of material from one well to an adjacent well, one can (see FIGS. 3 and 4) form wells in the plate which are

  surrounded by a continuous annular barrier

tied up 48.

  In addition to the wells that will be treated in the way

  with reference to FIG. 1, it is preferred to provide certain

  some additional wells to contain control serum and also to serve as control wells. The nature and exact function of the control wells will be explained

further.

Example 1

  A plate which may be a polystyrene plate for microtitration of the type described above is used. Half of the wells were initially coated with Bovine Leukemia Virus Antigen. This can be accomplished by collecting the VLB producing cell supernatant fluid, removing large debris by centrifugation and subjecting VLB to centrifugation in the clarified supernatant on a dense layer of sucrose. We can get the cells

  VLB from any suitable source, such as

  will take the specialists, but we prefer to use a

  single-layer cell culture producing continuous VLB

  and substantially free of other viruses and accidental agents. An example of such known means is to use VLB infected bat lung cells, as taught by Graves and Ferrer in Cancer Research, Vol 36, pp 4152-4159, 1976. Another suitable source is constituted by VLB infected lamb kidney kidney cells as it is

  taught by Van der Maaten et al, Bibliotheca Haemato-

logica, Vol 43, pp 360-362 (1976).

  We recover the viruses and we set about 5 to

  20 μg / ml with phosphate or saline tris

  buffered (0.15 M NaCl, 0.05 PO4 buffer, pH 7.0-7.4) ("PBS"). The antigen solution is added to the wells in amounts of about 200 microliters per well in alternating rows. The plate is then incubated at about 3 to 25 ° C for about

  1 to 5 pm After this incubation, the anti-

  gene not combined by a first wash with a solution

  isotonic suitable for example a phosphate solution

  or a tris-buffered saline solution.

  At this stage, the plants can be

  and store them dry at a temperature of

  4 C or lower for long periods (can reach more than 10 months if desired)

before use in the test.

  In the preferred test, as seen in the paper

  1, about 100 to 200 microliters of the ELISA buffer are added to the wells into which

  at 20 microliters of the bovine sera tested. (We pre-

  that the ratio of sera tested to ELISA buffer is about 1: 5 to 1:10). The ELISA buffer preferably contains about 2 to 20% of the animal blocking serum (serum to block non-specific binding of

  bovine immunoglobulin to the plastic container

  tick). It is preferred that the animal serum be selected from the group consisting of goat serum, chicken serum, guinea pig serum and sheep serum, with a preference for goat serum. It is also desirable to add to the solution about 0.25 to 2, OM and, preferably, about 0.5 to 1.0M NaCl and

  0.01 to 0.2M phosphate or tris-buffer at pH 6 to 8.

  The wells are then incubated at about 4 to 25 ° C for about 1 to 16 hours. An optional stage of washing 12

remove all excess.

  A also preferred variant consists in mixing

  administer the ELISA buffer with the test sera and introduce

  mix in wells. A less preferred technique is to introduce the ELISA buffer into the wells and then incubate at about 4 to 25 ° C for about 1 to 16 hours. A washing step can then be performed to remove excess material. The plates thus treated can be stored in a dry state at a temperature of 4 ° C. for more than 3 months. If the ELISA buffer is not removed before storage, the serum

  test may be introduced directly into the wells.

  If the ELISA buffer has been removed prior to storage, the test serum can be diluted and

  well or introduce it into wells that contain

  a suitable diluent such as an ELISA buffer or

  an ELISA buffer without blocking serum.

  As a precaution, it is generally preferred to

  kill the titration of sera tested in duplicate. The plate is then incubated at a temperature of about 3 ° to 37 ° C. for about 1 to 16 hours. The wells are washed in a solution of distilled water containing about 0.01 to

  0.2% of a suitable surfactant or detergent. An over-

  suitable facer is polyoxyethylene monolaurate

  sorbitan sold under the trademark "Tween 20" in

  a concentration of about 0.05%, for the intended use.

  There could be five or more quick washings but applicants prefer four

  1 minute incubations in the wash solution.

  The enzymatic analyte is then diluted in the drum.

  ELISA (serum-free) at about 50 to 70 ng / ml and preferably about 50 ng / ml. It is added to the wells in amounts of about 200 μl per well. The preferred material for this purpose is the prepared anti-serum

  in goats or rabbits that react to immunity

  bovine noglobulin and chemically coupled to horseradish peroxidase. It is preferred that the anti-serum be purified so as to contain only antibodies which

  react with bovine immunoglobulin, which is

  also shovels "affinity purification". After the incubation

  For about 15 to 60, and preferably about 20 to 37 ° C, the wells are washed with distilled water to remove the non-combined bovine enzyme analyte. Several quick washes can be done, but applicants prefer four washes

of a minute in the water.

  Subsequently, a chromagene substrate is introduced into the wells at a rate of about 200 microliters per well. This solution may consist of H 2 O 2 in a suitable buffered solution and a chromagene

  appropriate. Chromagene is a substance that is

  stains until it gives electrons in reaction with peroxidase, after which it develops an intense color. A preferred solution comprises about 0.2 microliter of 30% H 2 O 2 per ml of a solution (0.11 mg / ml 0.05 M citrate buffer pH 4.0) of 2,2'-azino acid. -di- (3-ethylbenzothiazoline) sulfonic acid. This material is available from several manufacturers, including Sigma Chemical Company. Wells containing the substrate-chromagene are then submitted

  incubation at about 15 to 39 C for 5 to 60 minutes.

  and preferably about 20 to 30 minutes. The reaction is then read or stopped by the introduction into the wells of a suitable acid. A material suitable for stopping the reaction is about 50 to 100 microliters of hydrofluoric acid in a concentration

  from about 0.1 to 0.5 N and preferably about 0.2N.

  It is preferred that the pH of the acid be from about 3.0 to 3.5.

  A visual inspection by a technique which will be described later, allows the determination of whether the infection with the bovine leukemia virus is or

  is not present in the tested serum.

  In order to provide a more effective test, we use

  preferably some witnesses, in the present invention.

  tion. In the preferred embodiment, at least four wells are used in each plate for each test serum and four control wells to ensure that the titration has been carried out under suitable conditions.

  to facilitate the estimation of results.

  Each test serum is incubated, diluted as described

  higher in the ELISA buffer, in two wells not

  in two wells previously coated with VLB antigen. While the test can be performed in individual wells as opposed to double wells, ie two of each type, this provides additional assurance and facilitates visual estimation. The antigen-free control determines the degree to which the serum in question binds nonspecifically

  at the well. The sensitivity and specificity of the test depends on

  the difference in binding with antigen-coated wells and uncoated wells. Ideally, with a serum containing the VLB antibody, strong binding was observed with the antigen-coated wells but no significant binding with the uncoated wells. The degree of bonding results in a color intensity,

  that is, a strong bond gives a decisive color

  finished like a dark green while the lack of

bond remains colorless.

  To determine whether the titration is of optimal sensitivity, two uncoated wells and two wells coated with VLB antigen are incubated with a weak positive serum. The title of this low positive serum is adjusted to

  give a minimal coloring reaction that is considered

  will derive as positive. With a plate containing 96 wells the use of four of these wells for the low positive control serum leaves 92 wells for the test. this allows

  the test in two copies of 23 sera.

  One of the advantages of the process of the present invention

  However, the precise determination of the test can be done visually by comparing the color developed in VLB-coated wells with the color of the control wells. This determination may be made with the naked eye or with the aid of suitable equipment such as, for example,

example, a spectrophotometer.

  For visual determination using an instrument such as a spectrophotometer, an absorbency at about 405 to 415 nm of each well can be determined using an ELISA spectrophotometer of a type appropriate commercially available. The OD values of the VLB antibody-free sera are generally below 0.4 units and the differences in OD values given by the same serum in the wells containing the VLB antigen and antigen-containing wells - control are generally less than 0.05 unit.

  In order to provide additional information concerning

  the preferred embodiment of the invention,

2 5 5 4 12 7

  we will give after another example.

Example 2

  Returning to Figure 2 for general guidance,

  the alternating rows are provided with a micro-wave plate.

  polystyrene titration having 96 substantially cylindrical wells of 200 microliters, BLV antigen purified with a sucrose cushion at about 10 μg / ml in a phosphate buffer solution. The stored rows (untreated) do not undergo any treatment. We incubate

  then the plate at 4 C for 16 hours. After the incu-

  tion, the non-combined antigen is removed by washing with

  a phosphate buffer solution. We add 200 micro-

  1 liter of ELISA buffer in all wells and then 20 microliters of either weak positive serum or test serum are added to two wells coated with VLB antigen and two adjacent wells not coated with VLB antigen. We

  test all sera in wells

  VLB antigen only in wells containing the anti-

  control gene. As is preferred, all assays of the tested sera are run in duplicate. After two hours of incubation at room temperature, the wells are washed

  four times with a 0.05% solution of a detergent

  suitable gent such as the "Tween 20" (monolaurate of

  polyoxoéthylène sorbitan). Each wash is an incu-

  for one minute in the wash solution.

  Subsequently, the affinity purified rabbit anti-bovine immunoglobulin chemically coupled to the conjugated horseradish peroxidase is diluted to about ng / ml in the ELISA buffer and added to the wells (200 μl per well) which is then incubated with test sera and bovine-control sera. After incubation for 30 minutes at approximately 25 ° C., the wells are washed with distilled water four times

  to remove the non-combined enzymatic analyte. This la-

  Vage contributes to a further reduction of non-specific reactivity. Then add about 200 microliters

  of a substrate-chromagen solution having a concentration

  0.11 mg of 2,2'-azino-di- (3-ethylbenzoic acid)

  thiazoline) sulfonic acid and 0.2 μl of 30% H 2 O 2 per ml of citrate buffer at pH 4.0. If it is desired to read the results spectrophotometrically or to preserve the results, the reaction is stopped after about 25 minutes by adding 50 microliters of 0.2N hydrofluoric acid at pH 3.3. The results of the test are then visually determined either with the naked eye or with the aid of a

  instrument such as a spectrophotometer.

Example 3

  Referring again to Figure 2, we will de-

  provide a further example assuming that a specific sample of test serum has been introduced into wells 13, A4, B3, B4 and that wells A1, A2, B1, B2 function as low positive control wells and that perform a direct reading. Row A has wells containing antigen then

  that the wells in row B do not contain any anti-

  gene. After the completion of the tests, the A1 and A2 wells are of a darker (green) color representing the minimum intensity of color that can be considered as a positive test result. While wells B1 and B2 remain clear, control wells of negative antigen B3 and B4 are also clear. A dark color (green) in wells A3 and A4 at color level in wells A1, A2 or at a higher level will be considered a positive test result. If wells A3 and A4 are clear or of a less intense color than wells A1 and A2, the result of the test is negative. It is understood that the test could be performed if A1 and B1 serve as a weak positive control and wells A3 and B3 are test wells. We prefer to use

  two wells for each function to give

  confirmation of accuracy.

  The method of the invention contributes to a better specificity by reducing the background color to a level

  negligible in the antigen control wells. The speci-

  The test is such that even weak serums

  can be identified precisely in terms of

  its the virtual absence of an undesirable background in the control wells. The test of the invention is also

  practice in that a staff without special qualifications

  and without any special equipment can treat a

  large number of samples in a relatively short time.

  In addition, the test is of economical use and the plates as well as the associated materials have a duration

  acceptable conservation. We can make a deter-

  accurate visual mination either with the naked eye or using

appropriate equipment.

  Whereas for the sake of clarity of description

  A 96 well microtiter plate has been developed, it is conceivable that other types of plates, different numbers of wells, and other means could be used to establish a group of containers for single or multiple tests. multiple, for example a series of separate tubes or other containers. For the sake of convenience, the term "sink" includes all

  variants of the containers that can be used.

  It goes without saying that we can make various modifications

  cations to the modes of implementation which have been described

  without departing from the scope of the invention.

Claims (11)

  1. Method for diagnosing an infection,   characterized by providing a test plate (38) having a series of wells (40); coating at least some of these wells (42) with an antigen; to perform a first incubation of the plate; to perform a first wash of the plate to remove   uncombined antigen; to block non-specific sites   binders by applying a solution of a blocking serum to the wells; to add test sera to   at least some wells; to perform a second incu-   bation of the plate; perform a second wash to remove uncombined components; introducing an enzymatic analyte into the wells; to perform a   third incubation of the plate; to perform a third   second wash to remove non-combined test serum; introducing a chromagen substrate into the wells; to perform a fourth incubation of the plate; and   to visually determine by color whether the   body to infection are present or not in the serum test.   2. Method according to claim 1, characterized   in that said test serum is introduced into the solution   blocking before admitting this blocking solution in the wells.   3. Method according to claim 1, characterized in that after the introduction of the serum blocking solution in said wells (40) and before said second incubation, the test sera are introduced into the   well and mixed with the blocking serum.   4. Method according to claim 3, characterized in that the blocking serum solution is prepared in the form of a buffered serum solution of a high ionic strength, and said buffered serum solution   contains about 2 to 20% of serum.   The method of claim 1, wherein at least one control well containing negative antigen (44) is provided for each serum sample tested; antigen is not introduced into the negative antigen pools; and introducing the blocking serum solution and said test serum into   control wells containing negative antigen.   6. Method according to claim 5, characterized   in that the reaction is stopped after the 4th incubation.   7. Method according to claim 5, characterized in that at least one control well (42) minimum positive serum; the minimum serum concentrations of antibodies which are presumed to be positive are introduced into said wells, so that the results of the sera tested can be compared with said well.   minimum positive serum control; and we determine visual-   if the test serum is positive by comparing the difference in its color reaction in the antigen coated wells and the negative antigen wells with the differences in the color responses of the control serum positive minimum.   8. Process according to claim 1, characterized in that the blocking serum solution contains a material chosen from goat, chicken, and other serums. guinea pig and sheep.   9. Method according to claim 4, characterized   in that said buffered serum solution is approximately   5 to 10% of this serum in a saline solution   phosphate; in that said blocking solution contains about 0.25 M to 2.0 M NaCl and about 0.01 M to 0.2 M phosphate buffer having a pH of about 6 to 8; and again in that we mix   test sera with the blocking solution before   depositing the sera in the wells, said test sera being diluted in a ratio of about 1: 5 to 1:10 with the buffered serum solution; we introduce   the test serum in a first series of wells con-   holding the antigen and a second set of wells   holding no antigen; a low titre control serum of the said antibodies is provided and the control serum is introduced into at least one well containing the antigen and   at least one well that does not contain the antigen.   10. Process according to claim 1, characterized in that the enzymatic analyte used is an affinity purified goat anti-bovine immunoglobulin or purified rabbit immunoglobulin which is coupled to said horseradish peroxidase-affinity immunoglobulin and is incorporated as a of chromagen substrate a titrant material peroxidase.   11. Process according to any one of the claims   previous, characterized in that the diagnosis is a serological diagnosis of the infection with the Bovine leukemia.