GB1576981A - Dane anti-core reagents and assays - Google Patents

Description

(54) DANE ANTI-CORE REAGENTS AND ASSAYS (71) We, ABBOTT LABORATORIES, a corporation organised and existing under the laws of the State of Illinois, United States of America, of 14th Street and Sheridan Road, North Chicago, County of Lake, State of Illinois, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed to be particularly described in and by the following statement: This invention relates to immunoassays for the detection of anti-HBc and to reagents for use therein.

Hepatitis B surface antigen (HBsAg) in blood serum has been a consistent marker for the presence of an infectious agent, as reported by Alter et al, Am. J. Med. Sci., 270:329-334, 1975 or Goldfield et al, Am. J. Med. Sci., 270:335-342, 1975. The HBsAg resembles a virus morphologically, but there has been no consistent evidence for infectivity or nucleic acid associated directly with this particle (See Gerin et al, J. Virol 7:569-576, 1971). The HBsAg is immunogenic since anti-HBs (antibody) is normally found in patients and animals during recovery and convalescence. The presence of anti-HBs is associated with protective immunity and this suggests that HBsAg is a structural component of the infectious virion. Thus, the association of HBsAg as a marker for infectivity of blood and blood products, and anti-HBs as a marker for immunity has stimulated wide spread diagnostic and epidemiologic studies of hepatitis type B. Highly sensitive and specific immuno-logic assay systems for antigen and antibody have been necessary and useful in such studies. One such assay system is described in United States Patent Specification 3,867,517.

Awareness of multiple transmission routes and the use of highly sensitive immunologic test procedures for HBsAg have been reported to reduce significantly transfusion-associated hepatitis type B (See Hollinger et al, N. Eng. J. Med. 290: 1104-1109, 1974). However, in careful follow-ups of recipients of blood negative for HBsAg, Goldfield et al, Am. J. Med.

Sci., 270:335-342, 1975 has reported evidence of exposure to the antigen in 7 of 465 patients.

This suggests that further studies of other immunologic systems associated with the hepatitis B virus could lead to more complete diagnosis of disease and detection of the infectious agent.

The Dane particle, Dane et al, Lancet, 1:695-698, 1970, with its associated core, has led to identification of core antigen (HBcAg) and its antibody (anti-core or anti-HBc) as a second immunologic system. The finding that the surface of the Dane particle contains HBsAg and the internal core particle contains DNA and DNA polymerase have strengthened the proposal for this particle as a candidate for the infectious virus. However, until tissue culture or convenient laboratory animal models are developed, immunochemical, biochemical or electron microscopic techniques are necessary for assessing the presence and possible infectivity of Dane particles.

It has been reported in the literature that the presence of antibodies to the core of Dane particles (anti-HBc) in human blood could be an indication of Hepatitis B virus infection or of a carrier state of this virus. Methods for detecting anti-HBc as reported in the literature include complement fixation, immune adherence hemagglutination and radioimmunoassay.

Hoofnagle et al, N. Eng. J. Med. 290:1336-1340 (1974); Tsuda et al, J. Immunol. 115:834838 (1976); Greenman et al, Vox Sang. 29:77-80 (1975); Moritsugu et al, J. Immunol.

114:17921798(1975); Purcell et al, Intervirol. 2:231-243 (1973/74); Vyas et al, Transfusion 16:536-537 (1976).

It seems clear that analysis of anti-HBc can have epidemiologic and diagnostic significance.

Some possible interpretations of the presence of anti-HBc and absence of HBsAg or anti-HBs are: 1. Early convalescence from disease with undetectable levels of replicating virus and before the appearance of detectable anti-HBs.

2. A carrier state of HB virus in which the presence of HBsAg is below detectable levels while anti-HBe is continuously stimulated.

3. Restimulation of the immune system in previously immune individuals where antisurface has decreased to undetectable levels.

4. Persistence of anti-HBc, in some cases, for periods where anti-HBs has decreased to undetectable level.

According to the present invention there is provided a method for detecting antibodies to the Dane core antigen in an unknown sample as evidence of exposure to hepatitis B virus, comprising: A. placing the unknown sample in contact with immobilized Dane core antigen whereby any Dane core antibody present in the sample will bind to the Dane core antigen; B. washing the immobilized Dane core antigen to remove any unbound unknown sample; C. adding a solution of labeled Dane core antigen to the washed immobilized Dane core antigen whereby the labeled Dane core antigen will bond to any Dane core antibody bonded to the immobilized Dane core antigen to thereby produce a labeled complex; D. washing the labeled complex to remove any unbonded labeled Dane core antigen; and E. examining the complex for the presence of labeled Dane core antigen.

Also according to the invention there is provided a method for detecting antibodies to the Dane core antigen in an unknown sample as evidence of exposure to hepatitis B virus, comprising: A. placing the unknown sample in contact with immobilized Dane core antigen and labeled human immune globulin containing antibody to the Dane core antigen to produce a mixture; B. incubating the mixture to permit the labeled material to bond to the immobilized Dane core antigen; C. washing the immobilized Dane core antigen; and D. examining the immobilized Dane core antigen to determine the amount of labeled material attached thereto.

The invention also provides a reagent suitable for use in immunoassay methods for the detection of anti-HBc in an unknown sample as evidence of exposure to hepatitis B virus comprising a solid-phase coated with Dane particle cores.

The present invention provides improved reagents and methods for the presence of anti-HBc (antibody). Detection of anti-HBc (antibody) using the present invention provides a significant increase in detectability for potentially infective blood in comparison to the detection of surface antigen (HBsAg), using the methods of United States Patent Specification No. 3 867 517. For example, samples have been examined wherein no surface antigen (HBsAg) is detected using the method of United States Patent Specification No. 3 867 517 but anti-HBc was found present using the method of this invention.

In the various immunoassays described herein two categories of reagents are useful, e.g., radio immunoassays, for the detection of anti-HBc. Category A consists of a hyper-immune serum produced in guinea pigs, rabbits or some other such animal by immunizing the animals with highly purified Dane particle cores; an l25I-labeled anti-guinea pig immune globulin antibody solution; or 1251-labeled guinea pig anti-HBc solution, and a solid-phase coated with HBsAG. Category B consists of a solid-phase coated with Dane particle core; an l25I-labeled human anti-HBc immune globulin, an '25I-labeled antibody solution against human immunoglobulins or an 1251-labeled particle core solution. The above mentioned l25I-label may be replaced by other markers such as other radioactive isotopes, fluorescent compounds or enzymes.

Detailed Description of Reagents Purification of Dane Particles Plasmas positive for HBsAg and containing Dane particles were pooled and used as the source of Dane particles. The purification procedure for Dane particles consisted of isopycnic banding in sucrose gradients and a single isopycnic banding is cesium chloride. DNA polymerase, an enzyme associated with HBcAg was used as an indicator of Dane particles throughout the purification procedure. Two populations of Dane particles with different buoyant densities were usually present in HBsAg positive plasmas; one banded in sucrose at a density of about 1.28 gm/cm, and another at about 1.25 gm/cm; the latter band was the major band of the two, but relative quantities of the two populations in terms of DNA polymerase activities varied from batch to batch of plasma pools. The light and heavy peaks were selected and rebanded in cesium chloride. In electron micorgraphs, no morphological difference was observed between these two populations of Dane particles with different densities.

Purification of Dane Particle Cores Highly purified Dane particles from either heavy or light fraction were treated with a non-ionic detergent designated Nonidet P-40 and 2-mercaptoethanol to remove surface components. The preparations were then mixed with equal volumes of genetron (dichlorodifluoromethane) and shaken vigorously to liberate the free cores. DNA polymerase remained in the aqueous phase. This genetron treatment step is not essential and may be omitted. However, its use contributes to a purer product. Further purification of the aqueous DNA polymerase activity in linear sucrose gradients gave a homogeneous band with a sedimentation rate of approximately 110S. Electron microscopy showed that 27 nm core particles co-sedimented with the polymerase. Only trace amounts of HBsAg were detected in the sucrose gradient by radioimmunoassay.

Immunogenicity and Antigenicity of Dane Particle Cores Guinea pigs and rabbits were immunized by injection with the sucrose gradient purified core preparation mixed with equal volume of Freund's adjuvant. The serums from these immunized animals were reactive with purified Dane cores and were routinely examined for anti-HBc titres by a direct radioimmunoassay described hereinafter. The hyper-immune anti-HBc serums were passed through immunoabsorbent columns of purified HBsAg and normal human serum proteins to remove possible antibodies induced by these components.

Animals immunized with highly purified Dane particle cores produced a high titer of anti-HBc, while anti-HBs titer was comparatively low or undetectable by solid phase radioimmunoassay, as described in United States Patent Specification No. 3 867 517 and as measured by the product sold under the trade mark AUSAB. Highly purified 22 nanometres (nm) HBsAg particles were prepared as described in J. Virol, 4, 763 (1969), Gerin et al and J.

Immunol. 109, No 4 (1972) Ling et al. When the anti-HBc animal serum was incubated with highly purified 22 nanometres (nm) HBsAg particles, the anti-HBe antibody formed a complex with the HBsAg particles but when the HBsAg particles were first digested by pepsin, the HBsAg particles lost the binding ability to anti-HBc but retained full HBsAg activity, i.e, binding ability to anti-HB5. It has therefore been concluded that on the 22 nm HBsAg particles there are antigenic sites similar to or the same as the Dane particle core. This unexpected property of HBsAg particles can be used in various forms of immunoassays for the detection of anti-HBc.

Coating of solid surface with the cores of Dane particles: Dane particle preparations of various stages of purity are treated with 2-mercaptoethanol (0.30-0.75%) and a non-ionic detergent, such as Triton X-100 or Nonidet P-40, in a concentration of about 1.0 to 2.5 % at 37 C. for one hour. The purpose of this treatment is to remove the lipoprotein coat and to release the core antigen of the Dane particles. The above conditions for the treatment are preferred, but they may be varied without adverse effect.

After the treatment, the mixture is appropriately diluted with a buffer solution; for example, 0.01M Tris-HCl, pH 7.1, in physiological saline solution containing 0.001M EDTA. The solution is used immediately to coat the solid surface of objects such as beads, tubes or wells made of plastics or glass. Dane cores prepared in this manner are very "sticky", and easily attach to solid surfaces by absorption. When preparations of Dane particles are grossly impure and contain high levels of extraneous proteins, it is necessary to pre-coat the solid objects with anti-HBe before reacting with Dane cores, as above. However, with our preparations, when polystyrene beads were incubated with the Dane core solution for 24 to 72 hours, there was more Dane core on plain beads than on anti-HBcpre-coated beads, especially when very low concentrations of Dane core were used. It has been discovered that in the coating solution, the detergent concentration should be very low (preferably lower than 0.0050/0), if Dane cores are coated directly on solid surfaces. Another advantage of coating solid objects with Dane core directly on the surface rather than first pre-coating with anti-HBc is the adaptability of the reagent for use in a direct sandwich type RIA for anti-HBc.

Preparation of Radioactive Anti-HBc Immunoglobulins for Radioimmunoassay Serum containing anti-HBc activity was fractionated on DEAE-cellulose columns to obtain the immune globulin fraction. The fraction was then labeled with l25I or other marker labels by conventional techniques.

Radioimmunoassay (RIA) Methodology for Anti-HBc Detection: Use of Category A Reagents: Example 1 Solid-phase Competitive RIA - Single-Antibody System HBsAg coated beads are incubated with an aliquot of a serum sample for a length of time to allow the anti-HBc in the sample, if present, to react with the HBcAg-like sites on the beads.

An aliquot of '25I-anti-HBc is then added to the reaction mixture to bind to the available HBcAg-like sites. After this reaction time, the bead is washed and the amount of l25I-anti- HBc bound to the bead is determined. A normal serum known to be negative for anti-HBc is run parallel with the test sample. The presence of anti-HBc in the test sample is signified by a decrease of l25I-anti-HBc uptake by the bead, compared to the controls.

The above procedure may be modified for some specific purposes. For example, in the above assay procedure, the sample and l25I-anti-HBc may be pre-mixed before reacting with the bead, or a washing step may be incorporated between the two incubation periods of the bead with the sample and with the l25I-anti-HBc.

Example 2 Solid-phase Competitive RIA Double-antibody System This procedure is similar to the single antibody system described above, except that the anti-HBc (guinea pig for example) used is not radio- or otherwise- labeled. After washing to remove this excess reagent, another incubation step with labeled antibody to the anti-HBc immune globulin (guinea pig, for example) prepared in another animal species (rabbit, for example) is added to make the anti-HBc on the bead "visible" by radioactivity, or other marker.

Use of Category B Reagents: Example 3 Solid-phase Competitive RIA An aliquot of labeled human immune globulin containing anti-HBe and the solution to be assayed are incubated with a Dane core coated bead. After thorough washing the bead is counted for the amount of label marker attached. An anti-HBe negative sample is assayed in the same manner as a control. A sample is positive for anti-HBe when the count rate or other measure of the sample is significantly lower than that of the negative control sample.

Alternatively, variants of the above procedure can be used: (1) The Dane core coated beads are first incubated with the sample before the addition of labeled anti-Hl3; (2) same as (1), except the bead is washed before the addition of labeled anti-HBc.

Example 4 Solid-phase Direct Assay: Homologous Sandwith Type Dane core coated beads in conjunction with labeled HBcAg can be used in direct RIA of the sandwich type. An aliquot of a sample is incubated with a Dane core coated bead, tube or object. The object is then washed and further incubated with a solution of labeled HBcAg. If anti-HBc is present in the sample, a sandwich structure of HBcAg-anti-HBc-labeled HBcAg is formed. After washing, the bead will show significantly higher count rate or other measurement than control beads which were assayed with anti-HBc negative samples.

Example 5 Solid-phase Direct Assay - Heterologous Sandwich Type Dane core coated beads are first incubated in an aliquot of properly diluted human serum.

If the sample contains anti-HBc, the anti-HBc will specifically attach to the Dane core on the beads. After washing the beads with water or a buffer solution, a labeled anti-human immunoglobulin solution is added to the beads to react with the anti-HBc previously attached.

This method can distinguish anti-HBc being IgG or IgM, if labeled specific anti-human IgG or IgM is used.

Findings Using the Assay Procedures Frequency Distribution of Anti-Core A collection of serums from consecutive plasmapheresis donors (paid donors) were analyzed for anti-HBc using the methods of the present invention, and for HBsAg (using 125it anti-HBs), and for anti-HBs (using '25I-HB-sAg) using the method of U.S. Patent 3,867,517.

Correlation of the findings with regard to the Hepatitis B surface and core systems using category A reagents is summarized in Table I. About 81% of the HBsAg positive specimens also contained anti-HB. About 15% of the specimens containing anti-HBs also contained anti-HBc. This suggests that the persistence of anti-HBe in these donors may be of shorter duration than persistence of anti-HB5. A third category of 3105 donors showed no evidence of prior exposure to the hepatitis B virus, based on negative results with tests for surface antigen and antibody. However, almost 5 % of this group were positive for anti-core. Similar results were observed with a group of 1049 volunteer blood donors. The HBsAg positive serums were not available for testing. About 21% of the anti-HBs specimens were also positive for anti-core. About 2% of the surface antigen and antibody negative donors were positive for anti-core. In a collection of HBsAg positive serums from carriers, 106 of 113 (94%) were positive for anti-HB.

In the above studies anti-HBe was found in 290 (7.3%) of the 3929 plasmapheresis donors, and 34 (3.2 ito) of 1049 volunteer blood donors. There were 145 and 19 donors, respectively, where anti-HBe was the only detectable marker for hepatitis B virus exposure.

In another population study. anti-HBc was determined by using the category B reagents, comparable results were obtained (Table II) except a higher percentage of anti-HBs positive samples were also found to be anti-HBc positive.

These results agree qualitatively with two other studies of the occurrence of anti-HBc in healthy populations. Hoofnagle et al, Lancet 2:869-873, 1973 and N. Eng. J. Med.

290:1336-1340, 1974 analyzed serums from commercial donors, volunteer donors and HBsAg carriers by complement fixation with HBcAg from infected chimpanzee liver hepatocytes. Anti-HBcwas found in 8 of 100 commercial donor serums and 2 of 200 volunteer donor serums. There were 5 and 1 donors, respectively, where anti-HBc was the only evidence of exposure to the hepatitis B virus. Tsuda et al, J. Immunol. 115:834-838 1975 used immune adherence hemagglutination with purified plasma Dane particle cores for the analyses of the serum from 215 healthy blood donors. There were 36 positive for anti-HBc. These were composed of 2 of 2 HB Ag positives, 28 of 31 anti-HBs positives and 6 from the 192 surface-negative group.

Table 1 Correlation of Anti-HBc with Hepatitis B Surface Antigen and Antibody in Donor Populations * PLASMAPHERESIS DONORS VOLUNTEER DONORS HB Ag CARRIERS No Anti- No. Anti- No. Anti Sample HBc + % Sample HBc + % Sample HBc + % HBsAg + 31 25 80.6 3 NA ** 113 106 93.8 Anti-HBs + 793 120 15.1 72 15 20.8 HBs Neg. 3105 145 4.6 974 19 1.9 Total 3929 290 7.3 1049 34 3.2 113 106 93.8 * Anti-HBc was determined using Category A reagents, Example 2.

** Not available for testing.

Table 2 Correlation of Anti-HBc with Hepatitis B Surface Antigen and Antibody in Donor Populations * PLASMAPHERESIS DONORS PAID BLOOD DONORS VOLUNTEER BLOOD DONORS HBsAg CARRIERS No. Anti-HBc + No. Anti-HBc + No. Anti-HBc + No. Angi-HBc + Sample (%) Sample (%) Sample (%) Sample (%) HBsAg + 30 26 (86.7) 5 4 (80.0) 830 798 (96.1) Anti-HBs + 573 414 (72.2) 144 131 (91.0) 15 7 (46.6) HBs Neg. 1043 112 (07.9) 776 14 (01.8) 775 6 (00.7) Total 2006 552 (27.5) 88 149 (16.8) 790 13 (01.6) 830 798 (96.1) *Anti-HBc was determined using Category B reagents, Example 3.

Claims (10)

WHAT WE CLAIM IS:

1. A reagent suitable for use in immunoassay methods for the detection of anti-HBe in an unknown sample as evidence of exposure to hepatitis B virus comprising a solid-phase coated with Dane particle cores.

2. A reagent as claimed in Claim 1 in which the Dane particle cores are coated directly onto the solid without pre-coating with antibodies.

3. A reagent as claimed in Claim 1 in which the solid phase comprises plastics beads.

4. A reagent as claimed in Claim 1 for use in immunoassay methods for the detection of anti-HBc in an unknown sample as evidence of exposure to hepatitis B virus, substantially as herein described with reference to any one of Examples 3 to 5.

5. A method for detecting antibodies to the Dane core antigen in an unknown sample as evidence of exposure to hepatitis B virus, comprising: A. placing the unknown sample in contact with immobilized Dane core antigen whereby any Dane core antibody present in the sample will bind to the Dane core antigen; B. washing the immobilized Dane core antigen to remove any unbound unknown sample; C. adding a solution of labeled Dane core antigen to the washed immobilized Dane core antigen whereby the labeled Dane core antigen will bond to any Dane core antibody bonded to the immobilized Dane core antigen to thereby produce a labeled complex; D. washing the labeled complex to remove any unbound labeled Dane core antigen; and E. examining the complex for the presence of labeled Dane core antigen.

6. A method for detecting antibodies to the Dane core antigen in an unknown sample as evidence of exposure to hepatitis B virus comprising: A. placing the unknown sample in contact with immobilized Dane core antigen and labeled human immune globulin containing antibody to the Dane core antigen to produce a mixture; B. incubating the mixture to permit the labeled material to bond to the immobilized Dane core antigen; C. washing the immobilized Dane core antigen; and D. examining the immobilized Dane core antigen to determine the amount of labeled material attached thereto.

7. A method as claimed in Claim 5 or Claim 6 in which the Dane core antigen is coated on a solid object.

8. A method as claimed in Claim 7 in which the labeled Dane core antigen is labeled with a radioactive isotope.

9. A method as claimed in Claim 8 in which the radioactive isotope is iodine 125.

10. A method of detecting antibodies to the Dane core antigen in an unknown sample as evidence of exposure to hepatitis B virus substantially as herein described with reference to any one of Examples 3 to 5.