CA1100037A - Hb.sub.c ag coated on solid phase - Google Patents
Hb.sub.c ag coated on solid phaseInfo
- Publication number
- CA1100037A CA1100037A CA296,740A CA296740A CA1100037A CA 1100037 A CA1100037 A CA 1100037A CA 296740 A CA296740 A CA 296740A CA 1100037 A CA1100037 A CA 1100037A
- Authority
- CA
- Canada
- Prior art keywords
- hbc
- dane
- solid phase
- core
- bead
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Organic Chemistry (AREA)
- Communicable Diseases (AREA)
- Food Science & Technology (AREA)
- Virology (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Abstract of the Disclosure Dane core antibody (anti-HBc) is detected in an unknown sample, e.g., serum, by reagent and assays based upon purified dane cores.
Description
llO~Q37 Background of the Invention ffepatitis 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 H~ Ag resembles a virus morpholo~ically, 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 H~SAg is immunogenic since anti-HBS (antibody) is normally found in patients and animals during recovery and convalescence. The presence of anti-~Bs is associated with protective immunity and this sugqests that HBsAg is a structural component of the infectious virion.
Thus, the asociation of HBsAg as a marker for infectivity of blood and blood products, and anti-ff~s 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 U.S. Patent 3,867,517 issued February 18, 1975.
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 ~lood negative for ffBsAg, Goldfield et al, Am. J. Med. Sci., 270:335-342, 1975 has reported evidence of exposure to the an~igen in 7 o~ 465 patients. This suggests that further
Virol 7:569-576, 1971). The H~SAg is immunogenic since anti-HBS (antibody) is normally found in patients and animals during recovery and convalescence. The presence of anti-~Bs is associated with protective immunity and this sugqests that HBsAg is a structural component of the infectious virion.
Thus, the asociation of HBsAg as a marker for infectivity of blood and blood products, and anti-ff~s 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 U.S. Patent 3,867,517 issued February 18, 1975.
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 ~lood negative for ffBsAg, Goldfield et al, Am. J. Med. Sci., 270:335-342, 1975 has reported evidence of exposure to the an~igen in 7 o~ 465 patients. This suggests that further
-2-110~037 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 o~ core antigen (HBcAg) and its antibody (anti-core or anti-HBc) as a second immunoloqic 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 micro-scopic techniques are necessary for assessing the presence and possible infectivity of Dane particles.
lS 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. 115:834-838 (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); ~yas et al, Transfusion 16:536-~37 (1976).
110~037 It seems clear that analysis of anti-HBc can have epidemiologic and diagnostic significance. Some possible inter-pretations of the presence of anti-HBc and absence of HBsAg or anti-HB~ are:
l. Early convalescence from disease with undetectable levels of replicating virus and before the appearance of detectable anti-HB8 2. A carrier state of HB virus in which the presence of HBsAg is below detecta~le levels while anti-HBc is continuously stimulated.
lS 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. 115:834-838 (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); ~yas et al, Transfusion 16:536-~37 (1976).
110~037 It seems clear that analysis of anti-HBc can have epidemiologic and diagnostic significance. Some possible inter-pretations of the presence of anti-HBc and absence of HBsAg or anti-HB~ are:
l. Early convalescence from disease with undetectable levels of replicating virus and before the appearance of detectable anti-HB8 2. A carrier state of HB virus in which the presence of HBsAg is below detecta~le levels while anti-HBc is continuously stimulated.
3. Restimulation of the immune system in previously immune individuals where anti-surface has decreased to undetectable levels.
4. Persistence of anti-HBc, in some cases, for periods where anti-HBs has decreased to undetectable level.
Briefly, 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 poten-tially infective blood in comparison to the detection of surface antigen (HBsAg)~ using the methods of U.S. Patent 3,867,517. For example, samples have been examined wherein no surface antigen (HBSAg) is detected using the method of the 3,867,517 patent but anti-HBc was found present using the method of this invention.
11~00:~7 SUMMARY OF THE INVENTION
In one particular aspect the present invention provides a reagent suitable for use in immunoassay methods for the detection of antibodies evidencing exposure to heptatitis B
virus, said reagent comprising a solid phase coated with HBCAg-In another particular aspect the present inventionprovides in an immunoassay for the detection in blood or blood constituents of antibodies evidencing exposure to hepatitis B virus, the improvement comprising the use of a binding reagent comprising a solid phase coated with HBCAg.
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 in cesium chloride. DNA polymerase (E.C.2.7.7.7), 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 posi~ive plasmas; one banded in sucrose at a density of about 1.28 gm/cm, and another at about 1.25 gm/cm;
jl/ ; -5-ll~QQ3~
the latter band was the major band of the two, but relative quantities of the two populations in terms of DNA polymerase activities varied f~om batch to batch of plasma pools. The light and heavy peaks were selected and rebanded in cesium chloride. In electron micrographs, no morphological diff-erence 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 volu~es of genetron 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 llOS. Electron microscopy showed that 27 nm core particles co-sedimented with the poly-merase. Only trace amounts of B5Ag were detected in the sucrose gradient by radioimmunoassay.
ilO0~37 ~mmunogenicity and Antigenicity of Dane Particle Cores Guinea pigs and rabbits were immunizied by injection with the sucrose gradient purified core preparation mixed with equal volume of Freund's adjuvant. The serums from these ~mmunized animals were reactive with purified Dane cores and were routinely examined for anti-HBc titres by a direct radio-immunoassay described hereinafter. The hyper-immune anti-HBc serums were passed through immunoabsorbent columns of purified H~ Ag 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 U.S. Patent 3,867,S17 and as measured by the product sold under the trademark AUSAB. When the anti-HBcanimal serum was incubated with highly purified 22 nanometers (nm) HBsAg particles the anti-H~c antibody formed a complex with the HBsAg particles but when the HBsAg particles were first digested by pepsin, the H~ Ag particles lost the binding ability to anti-HBc but retained full H~ Ag activity, i.e., binding ability to anti~
It has therefore been concluded that on the 22 nm H~ Ag particle there are antigenic sites similar to or the same as the Dane particle core. This unexpected property of ~ Ag particles can be used in various forms of immunoassays for the detection of anti-H~ .
110003~
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-lO0 or Nonidet~P-40, in a concentration of about l.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, O.OlM Tris-HCl, pH 7.1, in physiological saline solution containing O.OOlM EDTA. The solution is used immediatel~
to coat the solid surface of objects such as beads, tubes or wells made of plastic or glass. Dane cores prepared in this manner are very n 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-~Bc 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 ~hat in the coating solution, the detergent concentration should be very low (preferably lower than 0.005~), 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-coatinq with anti-HBc ~l~OQ37 is the adaptability of the reagent for use in a direct sandwich type RIA for anti-HBc.
Preparation of Radioactive Anti-HBc Immuno-globulins 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 I 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 I-anti-HBc is then added to the reaction mixture to bind to the available ~BcAg-like sites.
After this reaction time, the bead is washed and the amount of I-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 I-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 I-anti-HBc may be pre-mixed before reacting with the bead, or a washing step may be incorporated between the two _g_ incubation periods of the bead with the sample and with the I-anti-HBc.
Example 2 Solid-phase Competitive RIA Double-anti-bodY sYstem _ This procedure is similar to the single antibody system described above, except that the anti-HBc (guinea piq 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 Cate~ory B Reagents:
Example 3 Solid-phase Competitive RIA
An aliquot of labeled human immune globulin contain-ing anti-HBc 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-8Bc negative sample is assayed in the same manner as a control.
A sample is positive for anti-HBc 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:
ll~QQ37 (l) The Dane core coated beads are first incubated with the sample before the addition of labeled anti-HBC; (2) same as (l), except the bead is washed before the addition of labeled anti-HBc.
Example 4 Solid-phase Direct Assay: Ho~ologous 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 signifi-cantly higher count rate or other measurement than control beads which were assayed with anti-8Bc negative samples.
ExamPle S
Solid-phase Direct Assay - Heterologous Sandwich Tvpe Dane core coated beads are first incubated in an aliquo~ of properly diluted human serum. If the sample contains anti-~Bc, the anti-~Bc 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-~Bc 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 ~requency Distribution of Anti-Core A collection of serums from consecutive plasma-pheresis donors (paid donors) were analyzed for anti-HBc using the methods of the present invention, and for HBSAg (using I-anti-HBs), and for anti-HBs (using 125I-HBSA~) using the method of U.S. Patent 3,867,517. Correlation of the findings with regard to the Hepatitis a surface and core systems using category A reagents is summarized in Table I. About 81% of the HBsAg positive specimens also contained anti-HBc. About 15% of the specimens containing anti-HBs also contained anti-HBc.
This suggests that the persistence of anti-HBc in these donors may be of shorter duration than persistence of anti-HBs. 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
Briefly, 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 poten-tially infective blood in comparison to the detection of surface antigen (HBsAg)~ using the methods of U.S. Patent 3,867,517. For example, samples have been examined wherein no surface antigen (HBSAg) is detected using the method of the 3,867,517 patent but anti-HBc was found present using the method of this invention.
11~00:~7 SUMMARY OF THE INVENTION
In one particular aspect the present invention provides a reagent suitable for use in immunoassay methods for the detection of antibodies evidencing exposure to heptatitis B
virus, said reagent comprising a solid phase coated with HBCAg-In another particular aspect the present inventionprovides in an immunoassay for the detection in blood or blood constituents of antibodies evidencing exposure to hepatitis B virus, the improvement comprising the use of a binding reagent comprising a solid phase coated with HBCAg.
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 in cesium chloride. DNA polymerase (E.C.2.7.7.7), 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 posi~ive plasmas; one banded in sucrose at a density of about 1.28 gm/cm, and another at about 1.25 gm/cm;
jl/ ; -5-ll~QQ3~
the latter band was the major band of the two, but relative quantities of the two populations in terms of DNA polymerase activities varied f~om batch to batch of plasma pools. The light and heavy peaks were selected and rebanded in cesium chloride. In electron micrographs, no morphological diff-erence 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 volu~es of genetron 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 llOS. Electron microscopy showed that 27 nm core particles co-sedimented with the poly-merase. Only trace amounts of B5Ag were detected in the sucrose gradient by radioimmunoassay.
ilO0~37 ~mmunogenicity and Antigenicity of Dane Particle Cores Guinea pigs and rabbits were immunizied by injection with the sucrose gradient purified core preparation mixed with equal volume of Freund's adjuvant. The serums from these ~mmunized animals were reactive with purified Dane cores and were routinely examined for anti-HBc titres by a direct radio-immunoassay described hereinafter. The hyper-immune anti-HBc serums were passed through immunoabsorbent columns of purified H~ Ag 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 U.S. Patent 3,867,S17 and as measured by the product sold under the trademark AUSAB. When the anti-HBcanimal serum was incubated with highly purified 22 nanometers (nm) HBsAg particles the anti-H~c antibody formed a complex with the HBsAg particles but when the HBsAg particles were first digested by pepsin, the H~ Ag particles lost the binding ability to anti-HBc but retained full H~ Ag activity, i.e., binding ability to anti~
It has therefore been concluded that on the 22 nm H~ Ag particle there are antigenic sites similar to or the same as the Dane particle core. This unexpected property of ~ Ag particles can be used in various forms of immunoassays for the detection of anti-H~ .
110003~
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-lO0 or Nonidet~P-40, in a concentration of about l.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, O.OlM Tris-HCl, pH 7.1, in physiological saline solution containing O.OOlM EDTA. The solution is used immediatel~
to coat the solid surface of objects such as beads, tubes or wells made of plastic or glass. Dane cores prepared in this manner are very n 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-~Bc 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 ~hat in the coating solution, the detergent concentration should be very low (preferably lower than 0.005~), 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-coatinq with anti-HBc ~l~OQ37 is the adaptability of the reagent for use in a direct sandwich type RIA for anti-HBc.
Preparation of Radioactive Anti-HBc Immuno-globulins 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 I 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 I-anti-HBc is then added to the reaction mixture to bind to the available ~BcAg-like sites.
After this reaction time, the bead is washed and the amount of I-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 I-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 I-anti-HBc may be pre-mixed before reacting with the bead, or a washing step may be incorporated between the two _g_ incubation periods of the bead with the sample and with the I-anti-HBc.
Example 2 Solid-phase Competitive RIA Double-anti-bodY sYstem _ This procedure is similar to the single antibody system described above, except that the anti-HBc (guinea piq 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 Cate~ory B Reagents:
Example 3 Solid-phase Competitive RIA
An aliquot of labeled human immune globulin contain-ing anti-HBc 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-8Bc negative sample is assayed in the same manner as a control.
A sample is positive for anti-HBc 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:
ll~QQ37 (l) The Dane core coated beads are first incubated with the sample before the addition of labeled anti-HBC; (2) same as (l), except the bead is washed before the addition of labeled anti-HBc.
Example 4 Solid-phase Direct Assay: Ho~ologous 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 signifi-cantly higher count rate or other measurement than control beads which were assayed with anti-8Bc negative samples.
ExamPle S
Solid-phase Direct Assay - Heterologous Sandwich Tvpe Dane core coated beads are first incubated in an aliquo~ of properly diluted human serum. If the sample contains anti-~Bc, the anti-~Bc 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-~Bc 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 ~requency Distribution of Anti-Core A collection of serums from consecutive plasma-pheresis donors (paid donors) were analyzed for anti-HBc using the methods of the present invention, and for HBSAg (using I-anti-HBs), and for anti-HBs (using 125I-HBSA~) using the method of U.S. Patent 3,867,517. Correlation of the findings with regard to the Hepatitis a surface and core systems using category A reagents is summarized in Table I. About 81% of the HBsAg positive specimens also contained anti-HBc. About 15% of the specimens containing anti-HBs also contained anti-HBc.
This suggests that the persistence of anti-HBc in these donors may be of shorter duration than persistence of anti-HBs. 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-H~s 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 ~B~Ag positive serums from carriers, 106 of 113 (94%) were positive for anti-HBc~
2S In the above studies anti-HBc was found in 29~ (7.3%) of the 3929 plasmapheresis donors, and 34 (3.2%) of 1049 volun- ~`
teer blood donors. There were 145 and 19 donors, respectively, where an~i-HBc was the only detectable marker for hepatitis 8 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 per-centage 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 H~ Ag carriers by complement fixation with HBCAg from infected chimpanzee liver hepatocytes. Anti-HBc was found in 8 of 100 com-mercial 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 ad-herence hemagglutination with purified plasma Dane particle cores for the analyses of the serum from 215 healthy blood donors. There were 36 positive for anti-H~c These were composed of 2 of 2 HB Ag positives, 28 of 31 anti-HBs positives and 6 from the 192 surface-negative group.
~100~37 C~
CO
.~ I dPI ~ ~
~ U~ l ~ ~; ~1 o I o a) c I ~; m ~
.
~ Z ~
Q~
~ C ~
~ o m ~ oz ~ +
O ~ ~ ~
o ~Y 5 Z
~ O ~
a I ED ~ ~t C
~1 0 D I t I ~J O
u~ ~: u~ o u~ O a) w ~: ~ m ~ ~ ~ ~
o c~; m 3 c ~: 0 ~1 ~ ~ ol C~l m~
t ~1 o z C~ ,~ ,# ..
+ m I z _~
~: -,, ~
~ q ~ ~ JJ
m c m o x o ~ .s O Z ~ X ¦ ~
~ + _ _ _ ~ O T ~ _ _ O
R c ~ 1~ ~0 ~
Zo 3 ,~
~C
9 O~
m ~ m E~ .,,, c o;3 ~ C~
~ + I _ _ _ _~
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_ h ~: a~ ~
O J z E 0~ t~ ~ 1 1 o 3 U
+
h + I Z
S ~ I i---~5-
About 2% of the surface antigen and antibody negative donors were positive for anti-core. In a collection of ~B~Ag positive serums from carriers, 106 of 113 (94%) were positive for anti-HBc~
2S In the above studies anti-HBc was found in 29~ (7.3%) of the 3929 plasmapheresis donors, and 34 (3.2%) of 1049 volun- ~`
teer blood donors. There were 145 and 19 donors, respectively, where an~i-HBc was the only detectable marker for hepatitis 8 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 per-centage 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 H~ Ag carriers by complement fixation with HBCAg from infected chimpanzee liver hepatocytes. Anti-HBc was found in 8 of 100 com-mercial 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 ad-herence hemagglutination with purified plasma Dane particle cores for the analyses of the serum from 215 healthy blood donors. There were 36 positive for anti-H~c These were composed of 2 of 2 HB Ag positives, 28 of 31 anti-HBs positives and 6 from the 192 surface-negative group.
~100~37 C~
CO
.~ I dPI ~ ~
~ U~ l ~ ~; ~1 o I o a) c I ~; m ~
.
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u~ ~: u~ o u~ O a) w ~: ~ m ~ ~ ~ ~
o c~; m 3 c ~: 0 ~1 ~ ~ ol C~l m~
t ~1 o z C~ ,~ ,# ..
+ m I z _~
~: -,, ~
~ q ~ ~ JJ
m c m o x o ~ .s O Z ~ X ¦ ~
~ + _ _ _ ~ O T ~ _ _ O
R c ~ 1~ ~0 ~
Zo 3 ,~
~C
9 O~
m ~ m E~ .,,, c o;3 ~ C~
~ + I _ _ _ _~
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S ~ I i---~5-
Claims (6)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A reagent suitable for use in immunoassay methods for the detection of antibodies evidencing exposure to hepatitis B virus, said reagent comprising a solid phase coated with HBcAg.
2. A reagent according to Claim 1, wherein said solid phase is a bead, tube or well of plastic or glass.
3. A reagent according to Claim 1, wherein said solid phase is a polystyrene bead.
4. In an immunoassay for the detection in blood or blood constituents of antibodies evidencing exposure to hepatitis B virus, the improvement comprising the use of a binding reagent comprising a solid phase coated with HBcAg.
5. An immunoassay according to Claim 4, wherein said solid phase is a bead, tube or well of plastic or glass.
6. An immunoassay according to Claim 5, wherein said solid phase is a polystyrene bead.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US77728377A | 1977-03-11 | 1977-03-11 | |
| US777,283 | 1977-03-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1100037A true CA1100037A (en) | 1981-04-28 |
Family
ID=25109817
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA296,740A Expired CA1100037A (en) | 1977-03-11 | 1978-02-10 | Hb.sub.c ag coated on solid phase |
Country Status (8)
| Country | Link |
|---|---|
| JP (1) | JPS53113009A (en) |
| AU (1) | AU3332278A (en) |
| CA (1) | CA1100037A (en) |
| DE (1) | DE2810506A1 (en) |
| FR (1) | FR2383446A1 (en) |
| GB (1) | GB1576981A (en) |
| NL (1) | NL7802259A (en) |
| SE (1) | SE7802461L (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4241175A (en) * | 1978-12-18 | 1980-12-23 | Merck & Co., Inc. | Assay for hepatitis B core antibody |
| CA1148859A (en) * | 1979-06-14 | 1983-06-28 | Lacy R. Overby | Simultaneous assay of two hepatitis viruses using a solid phase |
| US4748109A (en) * | 1983-07-01 | 1988-05-31 | Baird Phillip J | Assay method and reagent to determine antibodies to papillomavirus virions |
| FR2560890B1 (en) * | 1984-03-07 | 1987-10-16 | Grp Genie Genetique | COMPOSITION USEFUL FOR THE MANUFACTURE OF VACCINES CONTAINING PARTICLES CARRYING THE SURFACE ANTIGEN OF HEPATITIS B VIRUS AND THE POLYMERIZED HUMAN SERUM ALBUMIN RECEPTOR, ANIMAL CELLS CAPABLE OF PRODUCING SUCH PARTICLES |
| JPH073427B2 (en) * | 1985-03-14 | 1995-01-18 | 財団法人化学及血清療法研究所 | Method for measuring HBC antibody |
| ES2059601T3 (en) * | 1988-05-11 | 1994-11-16 | Abbott Lab | AN IMMUNO ASSAY FOR THE DETECTION OF ANTI-HBC IN A BIOLOGICAL SAMPLE. |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BE787014A (en) * | 1971-07-31 | 1973-01-31 | Pfizer | METHOD AND REAGENT FOR DETECTING THE PRESENCE OF THE AUSTRALIAN ANTIGEN |
| US3867517A (en) * | 1971-12-21 | 1975-02-18 | Abbott Lab | Direct radioimmunoassay for antigens and their antibodies |
| DE2424465C3 (en) * | 1974-05-20 | 1978-03-23 | Biotest-Serum-Institut Gmbh, 6000 Frankfurt | Method for the simultaneous detection of antigens and their antibodies in the solid-state radioimmuno test |
| US4038378A (en) * | 1975-12-22 | 1977-07-26 | Gyaneshwar Prasad Khare | Radioimmunoassay for hepatitis b antigen |
| AT343822B (en) * | 1976-08-20 | 1978-06-26 | Immuno Ag | RADIOIMMUNOLOGICAL METHOD AND EQUIPMENT FOR DETERMINING ANTIGENES |
-
1978
- 1978-02-10 CA CA296,740A patent/CA1100037A/en not_active Expired
- 1978-02-15 AU AU33322/78A patent/AU3332278A/en active Pending
- 1978-03-01 NL NL7802259A patent/NL7802259A/en not_active Application Discontinuation
- 1978-03-03 SE SE7802461A patent/SE7802461L/en unknown
- 1978-03-08 JP JP2554278A patent/JPS53113009A/en active Pending
- 1978-03-09 FR FR7806854A patent/FR2383446A1/en active Granted
- 1978-03-10 GB GB9634/78A patent/GB1576981A/en not_active Expired
- 1978-03-10 DE DE19782810506 patent/DE2810506A1/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| JPS53113009A (en) | 1978-10-03 |
| SE7802461L (en) | 1978-09-12 |
| FR2383446A1 (en) | 1978-10-06 |
| GB1576981A (en) | 1980-10-15 |
| FR2383446B1 (en) | 1982-10-29 |
| DE2810506A1 (en) | 1978-09-14 |
| AU3332278A (en) | 1979-08-23 |
| NL7802259A (en) | 1978-09-13 |
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