US3896218A - Radiommunoassay determining the hepatitis associated antigen content of blood - Google Patents

Radiommunoassay determining the hepatitis associated antigen content of blood Download PDF

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US3896218A
US3896218A US271452A US27145272A US3896218A US 3896218 A US3896218 A US 3896218A US 271452 A US271452 A US 271452A US 27145272 A US27145272 A US 27145272A US 3896218 A US3896218 A US 3896218A
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haa
bhab
antibody
complex
washing
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Stanley E Charm
Bing Lou Wong
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Research Corp
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Research Corp
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Priority to US271452A priority Critical patent/US3896218A/en
Priority to CH973873A priority patent/CH595104A5/xx
Priority to GB3258773A priority patent/GB1430461A/en
Priority to DE19732335145 priority patent/DE2335145A1/de
Priority to FR7325355A priority patent/FR2263781B1/fr
Priority to NL7309699A priority patent/NL7309699A/xx
Priority to JP48079722A priority patent/JPS4962626A/ja
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/804—Radioisotope, e.g. radioimmunoassay
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y10S436/808—Automated or kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/82—Hepatitis associated antigens and antibodies

Definitions

  • ABSTRACT Antibodies to serum hepatitis are coupled to carbohydrate macro particles which are then contacted with blood fluids, suitably plasma, which are believed to contain serum hepatitis antigen.
  • the serum hepatitis antigen is thus complexed with the bound antibody and the particles bearing both the antibody and the antigen are removed from the blood fluid.
  • the antigen is then cleaved from the antibody by alkaline treatment, and may, if desired, be itself isolated, suitably by dialysis.
  • the antibody/agarose complex is then available for recycling in a further batch of contaminated blood fluids.
  • the amount of serum hepatitis antigen particles present in most blood fluids such as plasma varies widely. however, such fluids generally contain between It) and I0 particles per ml.
  • Many methods of detecting serum hepatitis are known. however, in view of the practical problems caused by the presence of small amounts of serum hepatitis antigen particles much research effort has been directed to detecting trace amounts of antigen.
  • IEOP irnmunoelectro-osmophoresis
  • a further problem associated with the techniques of study of serum hepatitis is the difficulty of obtaining pure serum hepatitis antibody.
  • the techniques of antibody production are well known, however, if the antibody producing system is infused with proteinaceous material of different immunological characteristics it may produce antibodies corresponding to each of those immunological characteristics. Thus, a mode of isolation of high purity antigen is clearly to be considered a highly desirable goal.
  • serum hepatitis antibody (goat) is coupled to agarose. suitably Sepharose 28. by the cyanogen bromide method (See Borath. ct al. Nature, 2l5. l49l (1967)).
  • the thus bound antibody. (hereinafter BHAB) is then mixed with a blood fluid. suitably plasma. and agitated for up to 30 hours.
  • the plasma is separated from the BHABI HAA complex by filtration.
  • the complex is dissociated by washing with saline at pH 9-12, suitably 10.5-1 l .5 and the saline wash containing the HAA concentrated by dialysis.
  • the BHAB is then washed again and utilized for the removal of HAA from a further batch of plasma.
  • the BHAB may be utilized to test the presence of HAA down to a level of IO to 10 particles per ml.
  • BHAB is incubated with the test plasma at about 37 for about 3 hours and a similar sample similarly incu bated in saline to serve as control.
  • the BHAB is separated from the test plasma suitably by filtration or centrifugation and incubated for 1 hour with "I-labeled hepatitis antigen.
  • the control BHAB is of course similarly treated.
  • the radio activity associated with the test and control samples of BHAB is then measured and the ratio compared to a plot of a similar ratio against the number of antigen particles in a test sample set forth on a standard curve produced by serial dilution methods.
  • filter paper may be used as a support for the test procedure.
  • HAA removal may be extended to the preparation of hepatitis free fibrinogen, a clotting factor of great utility in the treatment of the hemophilia and similar conditions.
  • plasma is treated with BHAB as hereinabove, and further treated with agarose to which antifibrinogen has been coupled by similar means.
  • the fibrinogen is then cleaved from the agarose/antifibrinogen complex and is available for use as a hepatitis-free product.
  • FIG. I is a graphic plot of HAA particles per ml. of standardized fluid on the linear X axis plotted against the ratio of radio activity of a BHAB/HAA/labeled HAA sample to the radio activity of a BHAB/labeled HAA sample plotted on a logarithmic Y axis.
  • FIG. 2 is a multiple plot of the number of HAA particles shown to remain in a fluid containing said particles after treatment by the removal method of the present invention and recovered, by standard radio tracer methods, IEOP and the radio immuno assay method of the present invention plotted on a logarithmic Y axis against contact time of the removal agent with the HAA contaminated fluid, plotted on a linear X axis.
  • FIG. 3 is a flow diagram of the HAA removal method of the present invention.
  • FIG. 4 is a flow diagram of the HAA detection method of the present method.
  • serum hepati tis antibody (suitably but not critically goat antibody) is coupled to agarose particles, preferably Sepharose 28 by a modification of the cyanogen bromide method.
  • agarose has been found particularly suitable as a support phase (hereinafter BHAB-A) the invention is not limited thereto.
  • Physically stable supports containing carbohydrate groups may be employed.
  • cellusosic materials such as cotton cloth have been found operative, and, indeed high grade filter paper is the carrier of choice in the testing modification of the invention.
  • cyanogen bromide is agitated with the carrier, suitably agarose and the pH increases to between 10 to 12, suitably to about pH II, and maintained at this level for from about 5 to about 20 minutes, suitably for from about 8 to about l0 min utes.
  • the aqueous supernate is removed, suitably by filtration and the suspension washed at reduced temperatures suitably between about 0C and 10C, preferably at about 4C at pH from about 7 to about 8.5, suitably about 7.8.
  • To this suspension of activated agarose is added antibody.
  • the amount of antibody utilized will of course depend upon the titer of the antibody used. However, it has been found suitable to utilize approximately I50 milligrams of antibody to l0 ml.
  • the antibody/carrier complex suitably antibody/sepharose complex (hereinafter BHAB) is then thoroughly washed with a slightly alkaline wash, suitably 0.1 M sodium bicarbonate at pH 8, therafter with an acetate buffer at about pH 4 and finally with a tris buffer at pH 7.4.
  • a slightly alkaline wash suitably 0.1 M sodium bicarbonate at pH 8, therafter with an acetate buffer at about pH 4 and finally with a tris buffer at pH 7.4.
  • this last wash contains a trace suitably about 0.2% of bovine serum albumin. All steps are carried out in the temperarange of 0 to 10C, suitably at about 8C.
  • the BHAB in the removal of serum hepatitis antigen (hereinafter HAA) the BHAB, suitably BHAB-A, in con tacted with the plasma and the mixture agitated.
  • the agitation is suitably carried out by means of gentle shaking.
  • the contacting between the BHABA and the plasma containing the HAA may take place at any temperature between about 0C and 45C.
  • the tempera ture decided upon will be as a result of a compromise between two competing factors.
  • the higher the temperature the more rapid the reaction between the BHAB-A and the HAA.
  • side reac tions and deterioration of the plasma is less likely to occur ar lower temperatures.
  • the BHAB-A/HAA complex is then separated from the blood fluid.
  • the blood fluid such as the plasma. either by centrifugation or filtration. Either method is entirely satisfactory.
  • a filter of polypropylene cloth is especially preferred.
  • the BHAB-A/HAA complex is then dissociated.
  • the conditions of dissociation have been found to be critical where it is desired to recycle the BHAB-A thus produced.
  • the BHAB-A/HAA complex is washed with alkaline saline, suitably made alkaline with aqueous ammonia.
  • a pH range of pH 9 to pH 12 is operative. however. at pH 12 the sepharose support swells and while the antibody can be regenerated and reused at this pH such a high pH value is undesirable for purposes of multiple recycling.
  • pH 10.5 the dissociation rate is rather slow while the most satisfactory dissociation rate, that is to say, rapid without swelling of the se pharose occurs at pH 1 1.
  • the BHAB-A is subjected to several washes with the saline suitably about 4 washes are employed utilizing about volumes of saline for each volume of BHAB-A.
  • the contact time in each instance being from about 15 minutes to 1 hour.
  • the saline containing the HAA is separated by filtration and the BHAB-A is then washed, suitably with substantially neutral saline, for example, saline at about pH 7.2, the wash separated and the regenerated BHAB-A reutilized in treating further samples of, say, plasma in accordance with the methods set forth hereinabove.
  • substantially neutral saline for example, saline at about pH 7.2
  • the aforementioned saline wash containing the HAA is then dialyzed, suitably in dialysis tubing surrounded by polyethylene glycol.
  • the concentration of HAA is increased l0O-fold from the wash.
  • the alkaline saline is removed thru the dialysis membrane.
  • the ph of the HAA concentrate is about 7.0.
  • the HAA tested by immunoelectrophoresis is found to be free of the plasma proteins it was originally associated with. HAA in the concentrated condition may have l0 to l0 particles/ml.
  • HAA particles down to a level of at least l0" particles per ml. may be carried out.
  • BHAB is incubated with test plasma, at the same time, a similar sample is incubated with neutral saline as a control.
  • BHAB-P antibody/filter paper complex
  • filter paper of about 2,-50 sq.mm. in area. Whatman No. 3 chromatographic paper (manufactured by W&R Balston l.td., Maidenstone, England) has been found especially suitable. While the ratio of paper to antibody is not critical, it has been found satisfactory to suspend 70 mg. of antibody in a l0 ml. solution of ().l M sodium bicarbonate containing about 200 pieces of filter paper of the foregoing dimensions.
  • the incubation is suitably carried out at between about and suitably about 37C for from about l to about 4, preferably for about 3 hours.
  • the BHAB is then separated by filtration or contrifugation and washed, suitably twice, with neutral saline.
  • HAA Prior to the assay, HAA is labeled with or similar radioisotope compatable with and attachable to proteins by methods unknown in the art.
  • Approximately l0 pl. of antigen (HAA), having a radio activity count of from about 2,000 to about 8,000 preferably about 5.000 counts/min. in 0.5 ml. of neutral saline are incubated with the reacted BHAB-A or BHAB-P of the pre vious step.
  • the incubation is carried on for from about 30 to about 40, suitably about 37 for from about 30 minutes to about 2 hours, suitably for about l hour, with slow stirring.
  • the BHAB-A or BHAB-P carrying HAA from the test plasma and the "'l labeled HAA upon it, is then separated by centrifugation or, preferably, filtration and washed, suitably three times, with neutral saline until no further radio activity is noted in the wash.
  • the radio activity of the control carrier is then read by means known to the art. This count is designated as B.
  • the control BHAB-A (or BHAB-P) utilized in the first stage of the present process is similarly treated with labeled antigen (HAA) and washed and the radio activity associated with said control is counted and designated as B,,.
  • the actual number of particles of HAA per unit volume in the test plasma may be determined by, for example, by a method of direct calculation or by determination from a graph.
  • l I The number of particles of HAA per unit volume in a standard sample ("l I is determined by known methods, such as lEOP which are not as sensitive as the method of the present invention. Serial dilutions of this test sample are then prepared using bovine serum albumin in neutral saline to produce a series of test samples of known fivalue, some of these diluted standards will of course be beyond the sensitivity of the [EOF test.
  • the B value is determined for each of these diluted standards and designated "B units of radio activity.
  • the value of B is of course the same for any sample taken from a given batch of BHAB.
  • BHAB-A was washed twice with 10 times volume of 0.1 M NaHCO;, pH 8, twice with 10 times volume 0.1 M acetate buffer, pH 4, and twice with 10 times volume of 0.1 M tris buffer, Ph 7.4, containing 0.2% of bovine sermum albumin (all steps at 4C).
  • EXAMPLE 11 Plasma Extraction/Purification Antibody bound to Sepharose 2B (BHAB-A), l ml. (decanted volume with mg. protein) is mixed with plasma (about 8 ml.) containing HAA (obtained from Massachusetts Red Cross) at 89C for 26 hours. Mixing is carried out with gentle stirring or shaking. Plasma is separated from the BHAB-A/HAA complex by filtration through Whatman No. 1 filter paper. Small sam ples are withdrawn at various times and tested for HAA.
  • BHAB-A Plasma Extraction/Purification Antibody bound to Sepharose 2B
  • HAA obtained from Massachusetts Red Cross
  • FIG. I shows a plot of the ratio of HAA particles in BHAB-A treated HAA containing plasma to the number of particles in the original sample of plasma, indicating the present limits of detectability in the standard radio tracer, lEOP and modified radio immuno assay technique of the present invention.
  • the regeneration efficiency of this system was mea sured by reacting about 8 ml. of HAA containing plasma with a batch of BHAB-A originally containing 20.17 mg. of serum hepatitis antibody at 37.
  • the regenerated material was contacted with a fresh sample of plasma from the same bach, and the time required for a negative HAA reading noted using the IEOP technique.
  • the BHAB-P papers are each separated and washed with saline three times (5 ml. each time, 25C).
  • Step (I) incubating a predetermined portion of the support phase of Step (I) designated as BHAB using the same quantity thereof as utilized in Step (II) under the same physical conditions of incubation using neutral saline as the incubation medium,
  • Step (Vlll) Incubating the product of Step (Vlll) with radio active HAA selected from the sample used in Step (IV) for the same time under the same conditions of temperature, pressure and pH.
  • a process according to claim 1 additionally comprising repeating the procedure of Step (Xll) thereof utilizing a plurality of solutions containing predetermined quantities of HAA particles per unit value to obtain thereby a series of values for the quantity B corresponding to predetermined values of F 4.
  • a process according to claim 2 additionally comprising the steps of calculating an an rage value for fT/B and determining the value of H in accordance with the formula

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US271452A 1972-07-13 1972-07-13 Radiommunoassay determining the hepatitis associated antigen content of blood Expired - Lifetime US3896218A (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
US271452A US3896218A (en) 1972-07-13 1972-07-13 Radiommunoassay determining the hepatitis associated antigen content of blood
CH973873A CH595104A5 (fr) 1972-07-13 1973-07-04
GB3258773A GB1430461A (en) 1972-07-13 1973-07-09 Purification of blood
DE19732335145 DE2335145A1 (de) 1972-07-13 1973-07-11 Verfahren zum entfernen von hepatitisantigen aus blutfluessigkeiten
FR7325355A FR2263781B1 (fr) 1972-07-13 1973-07-11
NL7309699A NL7309699A (fr) 1972-07-13 1973-07-12
JP48079722A JPS4962626A (fr) 1972-07-13 1973-07-13

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US271452A US3896218A (en) 1972-07-13 1972-07-13 Radiommunoassay determining the hepatitis associated antigen content of blood

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JP (1) JPS4962626A (fr)
CH (1) CH595104A5 (fr)
FR (1) FR2263781B1 (fr)
NL (1) NL7309699A (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4038378A (en) * 1975-12-22 1977-07-26 Gyaneshwar Prasad Khare Radioimmunoassay for hepatitis b antigen
US4292296A (en) * 1978-09-12 1981-09-29 Baxter Travenol Laboratories, Inc. Diagnostic method
US4474878A (en) * 1975-09-29 1984-10-02 Cordis Laboratories, Inc. Sandwich EIA for antigen associated with hepatitis
US4642285A (en) * 1975-09-29 1987-02-10 Diamedix Corporation Sandwich EIA for antigen
USRE32696E (en) * 1975-09-04 1988-06-14 Akzona Incorporated Enzymatic immunological method for determination of antigens and antibodies
KR100421763B1 (ko) * 1995-02-09 2004-05-10 아벤티스 베링 게엠베하 한외여과에 의한 단백질용액으로부터의 바이러스의 제거방법

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2335237A1 (fr) * 1975-12-19 1977-07-15 Pasteur Institut Vaccin contre l'hepatite virale de type b et procede de preparation de ce vaccin
NL7602422A (nl) * 1976-03-08 1977-09-12 Leuven Res & Dev Vzw Trombosetest.
SE420977B (sv) * 1976-03-18 1981-11-16 Kabi Ab Forfarande for rening och isolering av hepatitvirus for vaccinframstellning
FR2486802B1 (fr) * 1980-07-21 1985-08-09 Merck & Co Inc Purification de l'antigene de surface de l'hepatite b (hbsag)
AT403477B (de) * 1996-04-30 1998-02-25 Immuno Ag Biologisches material frei von viralen und molekularen pathogenen und verfahren zur herstellung

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3555143A (en) * 1966-06-02 1971-01-12 Pharmacia Ab Method for the determination of proteins and polypeptides
US3645852A (en) * 1967-05-23 1972-02-29 Pharmacia Ab Method of binding water-soluble proteins and water-soluble peptides to water-insoluble polymers using cyanogen halide

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3555143A (en) * 1966-06-02 1971-01-12 Pharmacia Ab Method for the determination of proteins and polypeptides
US3645852A (en) * 1967-05-23 1972-02-29 Pharmacia Ab Method of binding water-soluble proteins and water-soluble peptides to water-insoluble polymers using cyanogen halide

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE32696E (en) * 1975-09-04 1988-06-14 Akzona Incorporated Enzymatic immunological method for determination of antigens and antibodies
US4474878A (en) * 1975-09-29 1984-10-02 Cordis Laboratories, Inc. Sandwich EIA for antigen associated with hepatitis
US4642285A (en) * 1975-09-29 1987-02-10 Diamedix Corporation Sandwich EIA for antigen
US4038378A (en) * 1975-12-22 1977-07-26 Gyaneshwar Prasad Khare Radioimmunoassay for hepatitis b antigen
US4292296A (en) * 1978-09-12 1981-09-29 Baxter Travenol Laboratories, Inc. Diagnostic method
KR100421763B1 (ko) * 1995-02-09 2004-05-10 아벤티스 베링 게엠베하 한외여과에 의한 단백질용액으로부터의 바이러스의 제거방법

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FR2263781B1 (fr) 1977-04-08
NL7309699A (fr) 1974-01-15
FR2263781A1 (fr) 1975-10-10
CH595104A5 (fr) 1978-01-31
JPS4962626A (fr) 1974-06-18

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