AU5238586A - Improvements relating to viral isolates and their use - Google Patents
Improvements relating to viral isolates and their useInfo
- Publication number
- AU5238586A AU5238586A AU52385/86A AU5238586A AU5238586A AU 5238586 A AU5238586 A AU 5238586A AU 52385/86 A AU52385/86 A AU 52385/86A AU 5238586 A AU5238586 A AU 5238586A AU 5238586 A AU5238586 A AU 5238586A
- Authority
- AU
- Australia
- Prior art keywords
- retrovirus
- human
- cbl
- globulin
- htlv
- 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.)
- Granted
Links
- 230000003612 virological effect Effects 0.000 title description 10
- 239000000427 antigen Substances 0.000 claims description 83
- 102000036639 antigens Human genes 0.000 claims description 83
- 108091007433 antigens Proteins 0.000 claims description 83
- 238000012360 testing method Methods 0.000 claims description 56
- 241001430294 unidentified retrovirus Species 0.000 claims description 54
- 210000002966 serum Anatomy 0.000 claims description 43
- 239000007790 solid phase Substances 0.000 claims description 41
- 102000006395 Globulins Human genes 0.000 claims description 30
- 108010044091 Globulins Proteins 0.000 claims description 30
- 238000003556 assay Methods 0.000 claims description 30
- 208000030507 AIDS Diseases 0.000 claims description 21
- 210000004369 blood Anatomy 0.000 claims description 20
- 239000008280 blood Substances 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 18
- 108060003951 Immunoglobulin Proteins 0.000 claims description 12
- 102000018358 immunoglobulin Human genes 0.000 claims description 12
- 241000598436 Human T-cell lymphotropic virus Species 0.000 claims description 11
- 102000004190 Enzymes Human genes 0.000 claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 10
- 239000007791 liquid phase Substances 0.000 claims description 7
- 239000012472 biological sample Substances 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 208000032839 leukemia Diseases 0.000 claims description 5
- 230000002285 radioactive effect Effects 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 241000714260 Human T-lymphotropic virus 1 Species 0.000 claims description 3
- 241000714259 Human T-lymphotropic virus 2 Species 0.000 claims description 3
- 206010020460 Human T-cell lymphotropic virus type I infection Diseases 0.000 claims description 2
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 241000700605 Viruses Species 0.000 description 41
- 210000004027 cell Anatomy 0.000 description 38
- 239000003153 chemical reaction reagent Substances 0.000 description 17
- 238000002965 ELISA Methods 0.000 description 16
- 238000010790 dilution Methods 0.000 description 15
- 239000012895 dilution Substances 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 15
- 238000011534 incubation Methods 0.000 description 13
- 230000009257 reactivity Effects 0.000 description 12
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 11
- 239000004793 Polystyrene Substances 0.000 description 11
- 238000012875 competitive assay Methods 0.000 description 11
- 229920002223 polystyrene Polymers 0.000 description 11
- 239000000463 material Substances 0.000 description 10
- 230000003287 optical effect Effects 0.000 description 9
- 238000003127 radioimmunoassay Methods 0.000 description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 8
- 230000008901 benefit Effects 0.000 description 8
- 229940098773 bovine serum albumin Drugs 0.000 description 8
- 239000011248 coating agent Substances 0.000 description 8
- 238000000576 coating method Methods 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 230000002860 competitive effect Effects 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 7
- 210000004698 lymphocyte Anatomy 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 7
- 206010028980 Neoplasm Diseases 0.000 description 6
- 229920001213 Polysorbate 20 Polymers 0.000 description 6
- VEZXCJBBBCKRPI-UHFFFAOYSA-N beta-propiolactone Chemical compound O=C1CCO1 VEZXCJBBBCKRPI-UHFFFAOYSA-N 0.000 description 6
- 238000010166 immunofluorescence Methods 0.000 description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 6
- 229960000380 propiolactone Drugs 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 238000001574 biopsy Methods 0.000 description 5
- 210000001165 lymph node Anatomy 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000003018 immunoassay Methods 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 229920000136 polysorbate Polymers 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 102000000588 Interleukin-2 Human genes 0.000 description 3
- 108010002350 Interleukin-2 Proteins 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000001117 sulphuric acid Substances 0.000 description 3
- 235000011149 sulphuric acid Nutrition 0.000 description 3
- 208000011580 syndromic disease Diseases 0.000 description 3
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 2
- 206010001513 AIDS related complex Diseases 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 206010053574 Immunoblastic lymphoma Diseases 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 201000006747 infectious mononucleosis Diseases 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 238000011176 pooling Methods 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 102000001690 Factor VIII Human genes 0.000 description 1
- 108010054218 Factor VIII Proteins 0.000 description 1
- 101710177291 Gag polyprotein Proteins 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 208000008771 Lymphadenopathy Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 101150111781 PGL1 gene Proteins 0.000 description 1
- 241000125945 Protoparvovirus Species 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 241000712907 Retroviridae Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241000473945 Theria <moth genus> Species 0.000 description 1
- 208000010094 Visna Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000001064 anti-interferon Effects 0.000 description 1
- 230000000781 anti-lymphocytic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229960000301 factor viii Drugs 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 230000000652 homosexual effect Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000007373 indentation Methods 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 208000018555 lymphatic system disease Diseases 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000013102 re-test Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 238000009666 routine test Methods 0.000 description 1
- 201000005404 rubella Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 238000002764 solid phase assay Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000006379 syphilis Diseases 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- SRVJKTDHMYAMHA-WUXMJOGZSA-N thioacetazone Chemical compound CC(=O)NC1=CC=C(\C=N\NC(N)=S)C=C1 SRVJKTDHMYAMHA-WUXMJOGZSA-N 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
Classifications
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56988—HIV or HTLV
-
- 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
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
-
- 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
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
- C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
-
- 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
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/81—Packaged device 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
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/974—Aids related test
-
- 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
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/975—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
- Y10S436/804—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
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/809—Multifield plates or multicontainer arrays
-
- 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
- Y10S436/81—Tube, bottle, or dipstick
-
- 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
- Y10S436/815—Test for named compound or class of compounds
-
- 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
-
- 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
- Y10S436/823—Immunogenic carrier or carrier per se
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Virology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Urology & Nephrology (AREA)
- AIDS & HIV (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Oncology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
DESCRIPTION
TITLE: IMPROVEMENTS RELATING TO VIRAL ISOLATES AND THEIR USE
THIS INVENTION relates to a new assay for antibody to retroviruses and to new viral isolates for use in the method, and more specifically to the isolation of a virus related to human T-lymphotropic virus and to its use and the use of other retroviruses in the assay of sera to determine the presence of antibodies to retroviruses, particularly those associated with acquired immune deficiency syndrome (AIDS) .
The likelihood of acquired immune deficiency syndrome (AIDS) being caused by an infectious agent has been apparent for some years. Symptoms of this disease have been restricted to certain well-defined risk groups in a pattern that strongly suggests an agent transmissible by sexual or blood contact. While the disease was initially detected in the United States of America, there is an increasing prevalence of the disease elsewhere including Europe.
One method by which the disease is believed to be spread is by the transfusion of blood that has been donated by donors who are themselves infected with Aids virus. The pooling of donated blood means that if blood
given by a donor carrying Aids virus is pooled with samples of blood from other donors, then all of the blood and other products derived from that pool may be contaminated, however large or small the sample. Many blood donors who are carrying Aids virus are unaware of their infection, particularly in the initial phases of the infection, and other infected donors, even if they are aware of their infection, may be reluctant to reveal either that they have the disease or that they belong to a high risk group. There is therefore an increasing demand for relatively simple but completely reliable tests by which samples of blood donated for transfusion purposes can be routinely tested for the presence of Aids virus or antibodies to Aids virus. Blood donations that show up positive in the test can be rejected for transfusion purposes and the contamination of other blood samples by pooling with the contaminated sample can be avoided.
Research into the identification and isolation of the causative agent for Aids has been conducted actively for about 2 years but there is still disagreement between various research workers in the field as to the precise identity and nomenclature of it. A so-called Aids virus isolate was first reported in 1983 by Montagnier and his colleagues in France who named the material "Lymphadenopathy Associated Virus One" (LAV-1) . Almost one year later, Gallo and his
colleagues in the United States published details of the isolation of another so-called Aids virus which they named "Human T-lymphotropic Virus Type III" (HTLV-III) .
We have now been able to isolate, from a lymph node tumour, a human T-lymphotropic retrovirus related to HTLV-III and LAV which we have designated CBL-1. Our CBL-1 material is a stable isolate which can be cultivated in a host cell-line and can be used in an assay for the detection of antibody to Aids virus in serum samples. Accordingly, the present invention provides human T-lymphotropic retrovirus CBL-1 etiologically related to AIDS.
We have found that CBL-1 can be maintained for prolonged periods of time in a leukaemic T-cell-line designated CCRF-CEM and described by Foley e_t
Cancer 18, 522-529 (1965) and a further feature of the present invention comprises CCRF-CEM cells harbouring our CBL-1 virus.
For confirmation purposes, we have deposited samples of CCRF-CEM cells harbouring our CBL-1 virus with the National (now European) Collection of Animal Cell Cultures (NCACC) at the Public Health Laboratory Service Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, SP4 OJG England. The NCACC has been designated an International Depository Authority under the BudapestTreaty 1977. Our deposit was made on 11th January 1985 and has been given the Deposit Number 85 01 1101.
Our CBL-1 material was isolated from lymphocytes cultivated from a lymph node tumour biopsy of a British patient undergoing treatment for immunoblastic lymphoma associated with AIDS. Fragments of the biopsy were maintained in a culture medium to which T-cell growth factor was added and after about 2 days, the CCRF-CEM cells were added to the culture which was maintained under conditions such that the primary lymphocytes disappear over the course of about a month while the CCRF-CEM cells proliferate and become chronically infected with the CBL-1 virus.
CBL-1 virus was shown to be related to the previous described isolates of HTLV-III in possessing
(a) reverse transcriptase activity with a preference
++ for Mg cations;
(b) by the morphology of the virus particles visualised by electronmicroscopy;
(c) by indirect immunofluorescence specific for viral antigens in CBL-1 infected CCRF-CEM cells with sera prepared from Aids patients;
(d) by comparison in solid-phase radioimmunoassay with HTLV-III and LAV-1; and thereby is included in the taxanomic group of Retroviridae containing human T-cell lymphotropic viruses.
Preliminary DNA sequence analysis indicates that the sequence of the CBL-1 virus, while closely related to
that of the other human T-lymphotropic retroviruses so far described, is not identical to that of the previously described materials but exhibits some sequence variation at various locations. The infected cells that we have deposited with the ECACC have been shown to continue to synthesise virus and viral antigens over a period of at least 6 months. While we have found CCRF-CEM cells to be an ideal host for our CBL-1 virus, we have found that the cells could also be used to harbour similar virus that we have isolated from the peripheral blood of the same patient from whom the lymph node tumour biopsy was taken and similar viruses isolated from other patients infected with Aids related viruses. Thus, we have shown that this cell-line can provide a useful host for the iji vitro cultivation of not only our CBL-1 material but other related materials.
According to a further feature of the present invention we provide a method of assaying a biological sample for antibody to retroviruses which comprises bringing the biological sample into contact with
(1) an insolubilised antigen comprising retrovirus antigens bound to globulin, the globulin itself being bound to an inert solid support; and (2) an immunoglobulin which contains specific antibody to the retrovirus antigens and which is labelled
with a non-radioactive revealing label, and separating the liquid phase from the solid phase and determining the quantity of revealing label associated with either the liquid or the solid phase. In a still further feature of the invention we provide a test kit comprising
(1) a first component which is an insolubilised antigen comprising retrovirus antigens bound to globulin, the globulin itself being bound to an inert solid support and,
(2) a second component which is an iπtmunoglobulin which contains specific antibody to the retrovirus antigens and which is labelled with a non-radioactive revealing label. As indicated above, a major use of CBL-1 is as a component in a test system for the routine testing of transfusion blood to see if it comes from a subject infected with Aids virus. We have developed a system based upon a simultaneous competitive assay using, immobilised CBL-1 antigens or other retrovirus antigens as the solid phase, and the test serum as the liquid phase.
More specifically, our simultaneous competitive assay is based upon the immobilisation of the CBL-1 antigen or other retrovirus antigen by binding it to a solid phase, via a ligand e.g. globulin or other antibody and allowing the binding sites on this immobilised antigen
to be competed for by a mixture of test serum antibody and known antibody to the Aids virus which can be identified by the revealing agent. We have found that the most satisfactory results can be achieved when the revealing agent is an enzyme label. The use of an enzyme label rather than a radio-label has advantages in removal of the radiation biohazard and an unexpected consequence is that it allows the assay immune incubation to be reduced to 1 hour or less. A further alternative is to use immuno- fluorescence to reveal the presence of the known antibody bound to the immobilised antigen.
As indicated above, we have found that the best results are obtained when the antigen is attached to the solid phase through a ligand which is an antibody. One technique is to utilise a high titre human serum taken from someone asymptomatically infected with Aids virus. The ideal serum will have a high titre of antibodies to Aids virus but will come from a patient whose immunoglobulin levels are in the normal range. Another approach is to use monoclonal antibody raised against the retrovirus. If this is raised conventionally using mice, then certain advantages can be secured as will be described in more detail below. The solid phase material is conveniently polystyrene which can be used in the form of tubes, beads
or preferably plates with a plurality of surface wells or other surface indentations. Gamma-globulin prepared from the selected serum or other antibody is then coated onto the polystyrene and the antigen is then bound to the globulin-coated polystyrene to form the first component of the test. This component can be stored wet or dry for several months.
The second component of the test comprises the IgG fraction isolated from the test serum and, when revealing is to be done by means of an enzyme label, this IgG fraction can be conjugated with for example horseradish peroxidase (HRPO) according to known techniques.
The test kit of the invention can be used by mixing the labelled IgG component with the test serum and then bringing the mixture of IgG and test serum into contact with the insolubilised antigen, separating the solid and liquid phases from one another and determining the extent to which the IgG has become bound to the antigen. In accordance with simultaneous competitive assays, the greater the extent to which the IgG component of the liquid phase is bound to the solid phase, the smaller the amount of antibody or antigen in the test serum. By selection of appropriate control values, it is therefore possible to have a routine test where, if HRPO is used as the revealing agent, the test serum can be
regarded as free from Aids antibody or antigen provided that the optical density (OD) on the test sample reaches a pre-determined minimum level. Any sample of test serum which causes an OD count below the predetermined level to be recorded can therefore be rejected so avoiding transfusion with a blood donation possibly contaminated with Aids virus or antibody.
As an alternative to CBL-1 as the retrovirus antigen in the assay method and diagnostic test kit of the invention, use may be made of other retroviruses. These may be other human retroviruses etiologically related to AIDS such as human T-lymphotropic retrovirus HTLV-III or other human retroviruses such as human T-leukaemia virus, HTLV-II or HTLV-I or animal retroviruses such as Maedi-Visna or bovine leukosis virus.
As will be described in more detail below, the indirect binding of human retrovirus antigens to the solid phase via the globulin provides a certain amplification of results and the test format permits a simple, quick and accurate determination of the presence or absence of contamination in blood samples by relatively unskilled and inexpe ienced technicians.
Description of the Drawings
Figure 1 shows an electron-micrograph at 81,000 times magnification showing CBL-1 particles budding from and located at the boundary of the CCRF-CEM host cells.
Figure 2 shows the performance of the solid-phase ELISA for anti-HTLV III. The figure shows the optical densities derived from testing a number of clinical sera, including those reactive and unreactive for anti-HTLV III, results previously known by radioimmunoassay.
Figure 3 shows data similar to that of Fig. 2 but this time for anti-HTLV I, determined in a similar one step simultaneous competitive ELISA.
The following Examples are given to illustrate the invention.
EXAMPLE 1
ISOLATION AND CHARACTERISATION OF CBL-1
Virus CBL-1 was isolated from lymphocytes cultivated from a lymph node tumour biopsy of a British patient undergoing treatment for immunoblastic lymphoma associated with acquired immune deficiency syndrome (AIDS) . The lymph node biopsy was cut into small fragments and placed in culture medium composed of RPMI-1640 basal medium supplemented with 10% foetal calf serum and 10 ug/ml phytohaemagglutinin. After 24 hours conditioned medium containing T-cell growth factor harvested from mixed cultures of tonsillar lymphocytes was added, and phytohaemagglutinin removed. At 48 hours after initiation of the culture antiserum to interferon alpha was added. At 48 hours CCRF-CEM cells (hereafter called
CEM cells) were also added. CEM cells are a permanently growing leukaemic T-cell line described by Foley e_t al (1965) Cancer 18: 522-9.
Under the conditions of cocultivation the primary lymphocytes disappeared over the course of 4 weeks culture while the CEM cells proliferated exponentially. The CBL-1 virus produced by the primary lymphocytes infected the CEM cells and after 8 weeks in culture CEM cells were chronically infected with the CBL-1 virus. Maintenance of the CBL-1 infected CEM cells did not require T-cell growth factor or anti-interferon alpha. These CBL-1 infected CEM cells were deposited as indicated above, with the NCACC under the Deposit Number 85 01 11 01 CBL-1 virus was shown -to be related to previously described isolates of HTLV-III/LAV in possessing (a) reverse transc iptase activity with a preference for Mg 2+ cations, by the morphology of the virus particles visualised by electromicroscopy (see
Figure 1) and (b) by indirect immunofluorescence specific for viral antigens in CBL-1 infected CEM cells with sera prepared from Aids patients and by comparison with other
Aids virus isolate in solid-phase RIA. CEM/CBL-1 cells have continued to synthesise virus and viral antigens over a period of 6 months.
EXAMPLE 2
1. Antigen Preparation
A crude freeze-thaw cell lysate was prepared in the following way. HT-H9 cells infected with HTLV-IIIB were allowed to grow to maximum density in suspension culture. Cultures were cooled to 4°C and 0.25% v/v β-propiolactone was added for 2 hours. Cells were then spun out of culture fluid and pelletted at low speed. The cell pellet was resuspended in distilled water and subjected to three freeze/thaw cycles. At each cycle the residue of the cell pellet was carefully resuspended. After the third cycle, the extract was made to 0.25% vol/vol with β-propiolactone and kept at 4 C for 2 hours; it was then warmed to 37 C for half an hour. During this time the pH was maintained in the region of 7.4. The crude lysate was clarified and stored at -20°C. Prior to use it was diluted in TRIS Buffer supplemented with 0.1% BSA and detergent. The choice of detergent and its concentration was important and the use of Tween 20 at 0.1% concentration was found to be optimum, enhancing the activity of the antigen preparation three-fold or sometimes more so. After dilution in TRIS BSA/Tween 20, the antigen was incubated for 30 minutes at 37°C. It is during this step that antigen enhancement occurs.
Antigen preparation in this way may be used to monitor the expression of HTLV-III antigen under various defined culture conditions. The antigenicity may be quantified in solid-phase RIA employing solid-phase anti-HTLV-III and a second reagent, comprising 125-I anti-HTLV-III. In the final analysis, antigen was used at a dilution which allows a P:N ratio of 10:1 with 1-2% of label binding in the presence of negative sera (see below under assay method) . There was a variable shedding of viral antigens into the supernatant fluid of the cells but in relative terms the bulk of detectable antigen was left in the cell pellet. This was true for CBL-1 and for other isolates of HTLV-III carried in CEM cells or HT/H9 cells. There was no constraint on antigen purity (see below) and it was appropriate to maximise antigen expression in the cells. The antigen preparation used here was easy to manufacture, likely to be of a higher yield per volume of cell culture than antigen prepared from supernatant fluid and appears stable at -20 C. It can readily be inactivated by β-propiolactone. The use of Tween 20 would also be expected greatly to reduce the titre of any infectious virus. 2. Antisera
Reagent for the assay was prepared -from high-titre human sera taken from persons asymptomatically
infected with AIDS retrovirus. The selection of sera for reagent preparation was important. The serum had a high titre of anti-HTLV-III by RIA but came from a patient whose immunoglobulin levels was in the normal range. In practice it is usually necessary to prepare reagents from a small number of sera meeting the above criteria and test the performance of the reagents in the assay. In order to render these reagents non-infectious the starting sera were treated by heating at 56°C for 30 minutes. (a) Preparation of anti-HTLV-IIIB globulin. This material was used to purify _irι situ on a solid phase HTLV-IIIB antigen. There were considerable benefits in this strategy. Globulin from heat treated serum was precipitated twice with 40% saturated ammonium sulphate. This was the simplest reagent for globulin coating though it was possible to use either whole serum or purified IgG. The globulin was used at an 'optimum dilution which has to be determined by individual titration of the reagent. The solid-phase material was polystyrene in the form of wells and was coated with globulin and spare binding sites were then quenched with an inert protein - in this case 0.1% bovine serum albumin (BSA) . The coated and quenched solid phase was stored wet at 4°C for months; drying gave a more stable product. For coating the solid phase,. HT-H9/HTLV-IIIB lysate described above was diluted in
TRIS/BSA/Tween buffer to a dilution which, in subsequent testing, allowed 1-2% of the label to bind with a negative serum. Coating was most efficiently achieved by a 2-day incubation of antigen at room temperature in the solid phase followed by storage, wet, at 4°C until use.
Preliminary experiments indicated that the solid phase was stable in this form for several months. However, it can also be dried without significant loss of potency and remains stable for several months. (b) Labelling of globulin
The IgG described in 2(a) above was conjugated with HRPO using heterobifunctional derivatives in the usual way to give a molar substitution ratio of 1:3. This material was used in association with the solid phase described in 2(a) above in an enzyme immunoassay for the detection of antibody to AIDS retroviruses in samples of body fluid.
For comparison purposes, a further sample of the IgG was labelled with 125-I to a specific activity of 15 uCi per ig protein.
EXAMPLE 3
One step competitive ELISA for anti-CEM/CBL 1 Serum reagents
A human serum containing a high titre of anti-HTLV III was used for the preparation of coating globulin as previously described in Example 2.2. Polystyrene wells with a round bottom configuration were coated with an optimum dilution of globulin and then quenched with an inert protein, in this case 0.1% BSA. The same serum was used as a source of immunoglobulin, which was purified by ion-exchange chromatography on DE 52 as previously described. IgG was coupled with horseraddish peroxidase at an approximate substitution ratio of 3 molecules of horseradish peroxidase per 1 molecule of IgG.
The performance of the conjugate was determined by incubation over wells previously coated via an indirect antibody with virus antigens derived from HTLV III infected cells and with antigens uninfected cells. The
optimum dilution of conjugate was taken as that which gave maximum colour binding with the infected cell antigen and minimum colour production with the uninfected cells. CEM/CBL 1 antigen Cells persistently infected with CEM/CBL 1 virus were grown up to maximum density in non-adherent stirrer cultures. Stirrer cultures were cooled to 4°C and then brought to 0.25% volume with β-propiolactone. After 2 hours incubation at 4°C, the cell pellet was harvested and subjected to freeze and thaw cycles, and then clarified as previously by centrifugation. The tissue culture extract was then treated to a further 2 hour incubation at 4°C with 0.25% β-propiolactone and then hydrolised as previously. In this case the diluent for cell disruption and freeze thaw cycling was phosphate buffered saline, supplemented with 0.1% tween 20. Antigen preparations were subsequently titrated on a solid phase coated with high-titre anti-HTLV III globulin, and that dilution which gave an OD 450nm between 1.0 and 1.2 under conditions defined below was used as a working dilution for subsequent testing. Optimisation and format of testing
Wells coated with high-titre globulin and subsequently quenched, were incubated with a-100ul of optimum dilution of CEM/CBL 1 antigen. After an overnight
incubation at room temperature, the antigen extract was washed from the wells which were then dried under conditions which were found to promote stability of immobilised CEM/CBL 1 virus antigens. For testing 25ul of antibody positive and antibody negative sera were placed in separate wells, and 75^_1 of ELISA conjugate in detergent-supplemented distilled water added to the wells. The mixture of test serum and conjugate was incubated (immune incubation) in the wells for variable times under different conditions. The wells were then washed three times with saline tween (0.8% sodium chloride solution supplemented with 0.1% tween 20). After three washes the wells were filled with saline tween and allowed to soak for 2 minutes at room temperature, after which the washing procedure was repeated with three cycles of washing.
Following this 100^1 of chromogen substrate, in this case 3,3' ,5,5' tetramethyl-benzidine (TMB) , were added. After a 20 minute incubation at room temperature the chromogen release was terminated by the addition of 50^pl of 4 normal sulphuric acid. The colour release was measured spectrophotometrically at 450nm. The working dilution of any particular CEM/CBL 1 antigen was considered as that dilution which reliably gave an optical density of between 1 and 1.2 with negative serum when used in th'e ELISA under the optimum conditions, defined as an immune incubation of 1 hour at 45°C.
Results
In a comparison between results obtained in a conventional RIA using the solid phase and 125-I labelled
IgG described in Example 2 and the ELISA results obtained in this Example, two unexpected advantages were demonstrated in favour of the ELISA. Firstly, sub-optimum antigen preparations which gave mediocre results in RIA were able to produce,at the same titre, good ELISA results. Secondly, illustrated in the Table, with the RIA it was not possible to reduce incubation times below 3 hours. There is a need for a rapid assay, i.e. 1-2 hours in total, in the transfusion setting and this was only possible with the ELISA.
TABLE
Time/temperature experiments In RIA with 125-I labelled
IgG.
Time/hrs. Temperature Contol Sera
Negative Cutoff Positive
*
18 1.13 0.28 0.08
3 } RT 0.37 0.19 0.1
2 0.30 0.18 0.09
1 0.34 0.15 0.1
3 0.43 0.15 0.11
2 } 37°C 0.43 0.19 0.09
1 0.28 0.18 0.12
3 0.45 0.14 0.06
2 } 45°c 0.40 0.14 0.07
1 0.27 0.13 0.05
* % binding of input level.
In Figure 2, the results of testing by ELISA 56 clinical sera, in this case derived from haemophiliacs treated with commercial and non-commercial Factor VIII concentrates and homosexuals are shown. As can be seen in the Figure, there was clear separation between sera reactive for anti-CEM/CBL 1 and sera unreactive. In this particular example, the mean optical density of 16 sera reactive for CEM/CBL 1 antibody was 0.085 (0.037 to 0.327) and the mean optical density for 40 sera unreactive for this antibody was 1.16 (0.736 to 1.352). Serum a was from a terminally ill AIDS patient and was weakly reactive in IF and direct binding assays. Sera b and c were unreactive on retesting.
Exactly the same format has been used in a survey of Blood Transfusion sera in the United Kingdom. At the present time over 12,000 sera from donors have been tested. 3 sera gave screen test reactions however, on retesting, only one single donor has been found to be reactive. The serum was repeatably reactive, was titratable, and demonstrated strong reactivity in irmunofluorescence against infected but not uninfected cells. The random reactivity of sera which was unrelated to CEM/CBL 1 infection was therefore extremely low. Subsequent experiments on titrating sera reactive for
anti-HTLV III demonstrated that the titres were broadly the same when tested in both the CEM/CBL 1 ELISA and the direct assays. This has been confirmed by subsequent examination of sera taken from patients undergoing acute seroconversion following primary HTLV III infection.
Again there was broadly no difference in the level or time of initial reactivity in either the direct binding assay nor the competitive ELISA. Using the same assay, sera from patients with oligo and pan-reactive anti-lymphocyte antibodies, and sera from patients with auto immune disease were unreactive in the competitive ELISA. Taken together, these data indicate that the specificity and sensitivity of this competitive ELISA for antibody to the AIDS related retrovirus are high. EXAMPLE 4
One Step Competitive ELISA for anti-HTLV I
Sera and reagents were selected and prepared in same way as for the CEM/CBL 1 immunoassay of Example 3 with the exception that cells infected with HTLV I were used for antigen preparation, and sera from patients infected with HTLV I were used as a source of reagents. In Figure 3 the optical density given by 32 clinical sera, including 3 known weak positive controls is shown. Clear differentiation occurred between reactive and unreactive sera, with the mean optical density of 5 sera reactive for
this antibody being 0.099 (0.060 to 0.129) and the mean optical density for 24 sera unreactive for this antibody being 1.64 (1.48 to 1.83). The test was robust and the conditions could be varied but in practice it was decided to optimise on an immune incubation of 1 hour at 45°C. This was in parallel for those conditions for CEM/CBL 1 antibody detection.
This test has been used in the same format to screen over 1000 unselected British blood donors and no reactive sera were identified. In contrast, selective screening of some 75 African and Carribean origin donors identified a single seropositive first-time donor.
The procedure described above was repeated but using sera from patients infected with HTLV II as a source of coating globulin and as a source of IgG. Cells infected with HTLV II were used as an antigen source. These reagents were selected and prepared in the same manner as described previously in Example 2 for the anti-HTLV III RIA.
EXAMPLE 5 Production and use of test kit.
1. Reagents
A polystyrene multiwell plate containing 96 wells was coated on at least the internal surface of the wells with purified heat-treated human antibody to CBL-1. The antibody came from a heat-treated serum from a human infected with the Aids retrovirus. After coating the polystyrene with the antibody, spare binding sites were quenched with an inert protein, in this case bovine serum albumin. The antigen used for insolubilisation on the polystyrene plates was CBL-1. This was first treated with the viricide beta-propiolactone and a suspension of the inactivated CBL-1 in TRIS/BSA/Tween buffer was incubated with the treated polystyrene for about 2 days at room temperature. In this way, the CBL-1 became insolubilised by indirect binding to the polystyrene support through the antibody. This component was the first component of the test kit.
The second component of the test kit was prepared by labelling the same human antibody that was used to coat the polystyrene as described above. The antibody was labelled by conjugation with horseradish peroxidase (HRPO) using conventional conjugation techniques. This HRPO labelled antibody was stored in a
freeze-dried condition and was reconstituted immediately prior to use using a suspension in an aqueous diluent. In addition to the two components mentioned above, it was desirable to include in the test kit ancillary reagents so that all the necessary revealing reagents and control reagents were available. For this kit, using HRPO as the label, it was convenient to use 3,3' ,5,5'-tetramethylbenzidine (TMB) as the substrate for the enzyme and the TMB was supplied in tablet form, each tablet providing sufficient substrate for 96 wells.
Immediately prior to use, the TMB tablets were dissolved in an aqueous solution containing trisodium citrate and hydrogen peroxide. It was also desirable to provide positive and negative control sera. The negative control serum was normal human serum non-reactive for antibody to CBL-1 in this test. The positive control serum heat-treated human serum which was reactive for antibody to CBL-1 in the test procedure. Additionally, it was desirable to provide, as a cut-off control serum, heat- treated human serum reactive (at the cut-off level in the test) for antibody to CBL-1, and also a wash fluid comprising saline and Tween 20.
2. Use of test kit
Immediately prior to use, the HRPO labelled antibody was reconstituted. The test was carried out using 25 microlitre samples of serum per well. Two wells were supplied with negative control serum and one with positive control serum, three with cut-off control serum and the test samples introduced into the remaining wells. 75 Microlitre portions of the reconstituted HRPO labelled antibody were introduced into each well and the well plate was then covered and incubated on a heating block at 45 C for one hour or alternatively, at 20 to 25 C overnight for at least a 16 hour incubation. At the end of the incubation period, the plate was washed and 100 microlitre portions of substrate solution added to each well. The plate was then incubated for 20 minutes at 18 to 25°C after which period a blue colour developed in the wells containing the negative samples. 50 Microlitre portions of 2M sulphuric acid were then added to each well, the blue colour then changing to yellow. The absorbance of each well at 450nm was then read, within 30 minutes of the addition of the sulphuric acid, using a microwell plate reader.
3 . Results
The results were assessed in the following way: The mean absorbance of the three cut-off control sera was calculated. Any sample then with an absorbance equal to or greater than that of the mean cut-off control value was regarded as negative for antibody to AIDS retrovirus within the limits of the test system. Any samples with an absorbance below that of the mean cut-off control or up to 10% above the mean cut-off control were retested. Any sera repeatedly shown as positive in this test were then retested for confirmation purposes using immuno-fluorescence or Western blotting.
The procedure described above was tested with 1600 routine blood-bank donor samples with negative results. However, when other clinical specimens were tested from patients having a clinical diagnosis of Aids, Aids-related complex or other diseases, positive results were detected and in virtually every case, confirmed by immunofluorescence testing and by a different enzyme immunoassay. The following results were obtained.
TABLE
Reactivity of Sera from blood donors and patients with AIDS, AIDS associated conditions and other diseases.
Clinical Number Antibody Confirmed Group of Positive by Antibody
Samples Test Kit Positive3
Blood Donors 1600 0
AIDS 4 4 4
AIDS Related Complex 39 39 39
High Risk Groups ' 7 7 7
Other Diseases
Infectious Mononucleosis 48 0
Hepatitis B 25
Syphilis 117
Rheumatoid Arthritis 9 0
Other infections 37 0
aConfirmatory tests included immunofluorescence and an alternative enzyme immunoassay
Only positive samples confirmed
'One sample positive on 2 of 4 test occasions; confirmed negative
Includes Rubella (18 cases), Parvovirus (9), Cytomegalovirus (5) , Toxoplas osis (5) .
The principle of using a human anti-serum to bind viral antigen to the solid phase gives a solid phase which itself reflects the pattern of viral antigens best recognised humans. This may be advantageous since there will then be a good "fit" between the mixture of captured antigens and the profile of human antibodies. Further, the mild conditions us for antigen preparation may allow the preservation of delicate conformational epitopes unrepresented in the gradient purified and disrupted preparations used by other solid-phase assays. is now possible to adjust the mixture of solid-phase antigens by coating the solid-phase with murine monoclonal antibody of defined specificity so that the immobilised antigens will be o a predetermined specificity.
Data shown here demonstrate the superior nature of the test, when a selected monoclonal antibody reacting with p25 (a 25 K Dalton protein) is used on the solid phase. Advantages are i terms of a smaller quantity of the antigen required for a suitable assay (Table A) and in the increased sensitivity of resulting test, see Table B which shows that the end point dilutions (i.e. lower OD reading) of individual sera give greater inhibition in the monoclonal compared to the polyclon test. The increase in sensitivity has made the test more effective in detecting early antibody production when compare with many other assays. In addition the use of a urine antib on the solid phase avoids the potential problem of rheumatoid-like factors cross-linking between solid phase hum IgG and the human IgG conjugate in the homologous assay. Practically, a few sera have been identified which show consistently a greater degree of inhibition in the monoclonal-based assay compared with the polyclonal-based ass This phenomenon is apparently due to low level and low titre reactivity which binds conjugate to the solid phase in a non-specific manner in the homologous assay only. This or a similar phenomenon may also explain another unexpected findi that the distribution of optical density reactivity of normal sera is much tighter in the monoclonal-based assay than in a simultaneously performed polyclonal assay. Since it has proved simple to use a monoclonal antibody to a retroviral gag gene product, essentially provid
a competitive assay for the human anti-HTLV-III core response it is likely that use of antibodies to defined env products w enable the development of analogous assays for antibodies to selected envelope antigens. This will allow a much more prec investigation of the human response to different virus antige which may be of importance in clinical, prognostic and infect control terms.
TABLE A
Titration of CEM/CBL-I Antigen on polyclonal and mono- clonal solid phases.
Solid phase coat
Antigen dilution Polyclonal Monoclonal
*
Neg. cut off Positive Neg.cut-off Positive
**
10 1.34 0.69 0.05 NT NT NT
*** 20 1.14 0.45 0.03 1.85 0.86 0.11
30 0.82 0.42 0.03 NT NT NT
40 NT NT NT 1.50 0.37 0.09
60 NT NT NT 1.36 0.38 0.08
80 NT NT NT 1.23 0.28 0.07
***
100 NT NT NT 1.18 0.27 0.07
* Cut-off - weak positive control
**
OD at 450 nm in standard condition test for anti-CEM/CBL-I
***
"working" titre of antigen preparation
NT = not tested
TABLE B
OD 450 nm of dilutions of 19 sera titrated to 50% inhibitory end point,tested on polyclonal and monoclonal solid phase.
Solid-phase coat Serum
Polyclonal Monoclonal
Control positive 0.08 0.09 cut off 0.76 0.55 Negative 1.39 1.42
Haemophiliac 1 1.08 0.86 2 0.95 0.77 3 1.01 0.77 4 1.04 0.74
Aids 1 0.63 0.48 2 0.93 0.71 3 0.85 0.52 4 0.70 0.56
PGL 1 0.84 0.62 (persistent 2 0.84 0.58 general 3 0.73 0.56 lymphadeno- 4 0.60 0.48 pathy)
Drug Addict 1 0.50 0.44 2 0.66 0.50 3 0.95 0.73 4 0.85 0.63
Healthy 1 0.50 0.41 2 0.56 0.42 3 0.69 0.49
In the Examples above, the assay is conducted by a determination of label associated with the solid phase. However, since the label level associated with the starting sera is or can be determined, it is also possible to monitor
the reduction in radioactive count or enzyme level associated with the liquid phase after the known labelled sera and the test sera have been brought into contact with the solid-phase antigen and the reduction in label level of the liquid phase used as a measure of the antibody content of the test sera. The advantages of the simultaneous competitive assay can be summarised under the following headings. Antigen Preparation
Although Example 2 described the use of purified HTLV-III viruses, the enhanced binding resulting from use of an immobilised antibody allows the use of a crude cell lysate preparation for coating when by itself, such an antigen will not bind directly in any useful way to a solid phase. This step removes the constraint of needing highly purified antigen for production of diagnostic tests and will make their manufacture easier. It is an advantage which is not applicable to direct assays where a whole human globulin coat would give an unavoidable signal with the final anti-human globulin label. Format
The use of a volume of undiluted serum offers a significant advantage to blood transfusion centres where a need to make an initial dilution would increase the work load. The ratio between serum and enzyme-label volumes can be varied through the range 1:1 to 1:10 with only
little alteration in test performance. The optimum is 1:3, employing 25 ul of serum and 75 ul of enzyme-label and gives good differentiation between positive and negative sera. Assay times can be shortened to 1 hour or less by the use of an Elisa technique.
Sensitivity
One measure of the sensitivity of any assay for Aids antibody is given by the proportion of sera taken from terminally ill patients with Aids which remain positive. With direct assays it seems that a variable number of patients lose antibody reactivity. This does not happen with the competitive assay, suggesting that the sensitivity of the competitive assay is as good as or better than the other assays currently used.
Specificity
A competitive assay would not be expected to have major problems of false-positive reactivity. Inclusion of lymphocyte membrane antigens into the envelope of HTLV virions certainly leads to false reactivity, as does the presence of aggregates of IgG which stick nonspecifically to the solid phase. Such phenomenon do not give rise to reactivity in the competitive assay. Non-specific affects of variable protein concentration can be minimised by selection of an appropriate serum/label ratio (1:3, see above) and by the
use of a cut-off of greater than 50% inhibition. Sera which produce an inhibition of label binding equal to or greater than that given by an internal control serum are considered screen-test positive but the testing should be repeated. In view of the low level of false reactivity, expected to be less than 1 in 5,000, it is unlikely that false-positive reactions in the competitive ELISA will give such a numerically large problem as they seem to do in the direct assays. This is particularly so where direct assays are performed without a control antigen. It is anticipated that this will be the case in transfusion centres as the use of a control antigen would double the requirement for reagents in the direct assay.
Summary The combination of simple format, high sensitivity and high specificity makes the competitive assay ideal for use in screening of blood donations and for diagnostic use. In addition, where centres use a direct assay for anti-HTLV-III it may prove better to confirm serum reactivity by re-testing in competitive assay rather than subjecting sera to the more laborious and less sensitive Western Blotting that may be recommended by the FDA in the United States.
Claims (21)
1. A method of assaying a biological sample for antibody to retroviruses which comprises bringing the biological sample into contact with
(1) an insolubilised antigen comprising retrovirus antigens bound to globulin, the globulin itself being bound to an inert solid support; and
(2) an immunoglobulin which contains specific antibody to the retrovirus antigens and which is labelled with a non-radioactive revealing label, and separating the liquid phase from the solid phase and determining the quantity of revealing label associated with either the liquid or the solid phase.
2. A method according to claim 1 wherein the retrovirus is human T-lymphotropic retrovirus CBL-1.
3. A method according to claim 1 wherein the retrovirus is human T-lymphotrophic retrovirus HTLV-III, human T-leukaemia retrovirus HTLV-II or human T-leukaemia retrovirus HTLV-I.
4. A method according to any one of the preceding claims wherein the biological sample is serum or plasma from human blood.
5. A method according to claim 4 wherein the retrovirus is human T-lymphotropic retrovirus CBL-1 or human T-lymphotropic retrovirus HTLV-III and wherein the immunoglobulin labelled with the revealing agent is immunoglobulin from a human patient asymptomatically infected with the AIDS retrovirus but having a normal immunoglobulin level.
6. A method according to any one of the preceding claims wherein the revealing label is an enzyme label.
7. A method according to any one of the preceding claims wherein the globulin bound to the support is monoclonal antibody that will bind retrovirus antigen.
8. A test kit comprising (1) a first component which is an insolubilised antigen comprising retrovirus antigens bound to globulin, the globulin itself being bound to an inert solid support and,
(2) a second component which is an immunoglobulin which contains specific antibody to the retrovirus antigens and which is labelled with a non-radioactive revealing label.
9. A kit according to claim 8 wherein the retrovirus is human T-lymphotropic virus CBL-1.
10. A kit according to claim 8 wherein the retrovirus is human T-lymphotropic virus HTLV-III, human T-leukaemia virus HTLV-II or human T-leukaemia virus HTLV-I.
11. A kit according to claim 8 wherein the retrovirus is human T-lymphotropic retrovirus CBL-1 or human T-lymphotropic retrovirus HTLV-III and wherein the immunoglobulin is from a human patient asymptomatically infected with the AIDS retrovirus but having a normal immunoglobulin level.
12. A kit according to any one of claims 8 to 11 wherein the revealing label is an enzyme label.
13. A kit according to any one of claims 8 to 12 wherein the globulin bound to the support is monoclonal antibody that will bind retrovirus antigen.
14. Human T-lymphotropic retrovirus CBL-1 etiologically related to AIDS.
15. Leukaemic T-cells CCRF-CEM harbouring human T-lymphotropic retrovirus CBL-1.
16. Insolubilised human T-lymphotrophic retrovirus CBL-1 antigens bound to globulin, the globulin itself being bound to an inert solid support.
17. Insolubilised antigens according to claim 16 wherein the globulin is from a human patient asymption already infected with AIDS retrovirus but having a normal immunoglobulin level.
18. Insolubilised antigens according to claim 16 wherein the globulin is monoclonal antibody that will bind retrovirus antigen.
19. A method of assay according to claim 1 substantially as hereinbefore described with reference to any one of the Examples.
20. A test kit according to claim 8 substantially as hereinbefore described with reference to any one of the Examples.
21. Insolubilised antigens according to claim 16 substantially as hereinbefore described with reference to any one of the Examples.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB858500918A GB8500918D0 (en) | 1985-01-15 | 1985-01-15 | Viral isolates |
US75660485A | 1985-07-19 | 1985-07-19 | |
US756604 | 1985-07-19 | ||
GB8500918 | 1985-07-19 |
Publications (2)
Publication Number | Publication Date |
---|---|
AU5238586A true AU5238586A (en) | 1986-08-13 |
AU591647B2 AU591647B2 (en) | 1989-12-14 |
Family
ID=26288664
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU52385/86A Ceased AU591647B2 (en) | 1985-01-15 | 1986-01-13 | Improvements relating to viral isolates and their use |
Country Status (15)
Country | Link |
---|---|
AT (1) | ATA900186A (en) |
AU (1) | AU591647B2 (en) |
BE (1) | BE904030A (en) |
BR (1) | BR8604533A (en) |
CH (1) | CH675636A5 (en) |
DE (2) | DE3690028T (en) |
DK (1) | DK437386A (en) |
FI (1) | FI863704A0 (en) |
FR (1) | FR2576107B1 (en) |
GB (1) | GB2179449B (en) |
HU (1) | HU204929B (en) |
IT (1) | IT1191841B (en) |
NL (1) | NL8620004A (en) |
SE (1) | SE8603803L (en) |
WO (1) | WO1986004423A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU577937B2 (en) * | 1985-02-05 | 1988-10-06 | United States of America, as represented by the Secretary, U.S. Department of Commerce, The | A method for detecting htlv-iii neutralizing antibodies in sera |
AU579715B2 (en) * | 1985-02-26 | 1988-12-08 | United States of America, as represented by the Secretary, U.S. Department of Commerce, The | Detection of human t-cell leukemia virus type iii |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB8500918D0 (en) * | 1985-01-15 | 1985-02-20 | Cancer Res Inst | Viral isolates |
US4748110A (en) * | 1985-09-25 | 1988-05-31 | Abbott Laboratories | Immunoassay for HTLV-III antigens |
US5364933A (en) * | 1986-03-03 | 1994-11-15 | Institut Pasteur | Methods of immunopurification of antigens of human immunodeficiency virus type 2 (HIV-2) |
US6514691B1 (en) | 1986-01-22 | 2003-02-04 | Institut Pasteur | Peptides of human immunodeficiency virus type 2 (HIV-2), antibodies against peptides of HIV-2, and methods and kits for detecting HIV-2 |
US4839288A (en) * | 1986-01-22 | 1989-06-13 | Institut Pasteur | Retrovirus capable of causing AIDS, antigens obtained from this retrovirus and corresponding antibodies and their application for diagnostic purposes |
CA1278259C (en) * | 1986-02-26 | 1990-12-27 | William C. Saxinger | Competitive elisa for the detection of antibodies |
US4904581A (en) * | 1986-05-06 | 1990-02-27 | Epitope, Inc. | Method of detecting AIDS virus infection |
SE8701765L (en) * | 1987-04-28 | 1988-10-29 | Statens Bakteriologiska Lab | METHOD OF ANALYSIS AND AGENTS FOR THIS |
AU615670B2 (en) * | 1987-09-04 | 1991-10-10 | Murex Diagnostics Corporation | Immunoassay and biological constructs for use therein |
US5115098A (en) * | 1990-02-28 | 1992-05-19 | President And Fellows Of Harvard College | End-blocked peptides inhibiting binding capacity of gp120 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2016687B (en) * | 1978-03-20 | 1982-09-08 | Abbott Lab | Sugar coated reagents for solid phase immunoassay |
EP0136798A3 (en) * | 1983-08-25 | 1986-02-19 | Biotech Research Laboratories Inc. | Titre plate and assay kit for detection of antibodies in human serum and their production and use |
IE58321B1 (en) * | 1984-04-23 | 1993-09-08 | Us Health | Isolation of proteins of HTLV-III, serological detection of antibodies to HTLV-III in sera of patients with aids and pre-aids conditions, and detection of HTLV-III infection bi/immuno-assays using HTLV-III infection by immuno-assays using HTLV-III and its proteins |
US4520113A (en) * | 1984-04-23 | 1985-05-28 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Serological detection of antibodies to HTLV-III in sera of patients with AIDS and pre-AIDS conditions |
US4652599A (en) * | 1984-04-23 | 1987-03-24 | The United States Of America As Represented By The Department Of Health And Human Services | Method of continuous production of retroviruses (HTLV-III) from patients with AIDS and pre-AIDS using permissive cells |
AU580425B2 (en) * | 1985-05-24 | 1989-01-12 | United States of America, as represented by the Secretary, U.S. Department of Commerce, The | Competitive elisa for the detection of antibodies |
-
1986
- 1986-01-13 BE BE0/216129A patent/BE904030A/en not_active IP Right Cessation
- 1986-01-13 GB GB08620371A patent/GB2179449B/en not_active Expired
- 1986-01-13 DE DE19863690028 patent/DE3690028T/de active Pending
- 1986-01-13 HU HU86767A patent/HU204929B/en unknown
- 1986-01-13 AT AT0900186A patent/ATA900186A/en not_active Application Discontinuation
- 1986-01-13 IT IT19072/86A patent/IT1191841B/en active
- 1986-01-13 BR BR8604533A patent/BR8604533A/en unknown
- 1986-01-13 CH CH3710/86A patent/CH675636A5/de not_active IP Right Cessation
- 1986-01-13 DE DE3690028A patent/DE3690028C2/en not_active Expired - Lifetime
- 1986-01-13 FR FR868600376A patent/FR2576107B1/en not_active Expired - Lifetime
- 1986-01-13 NL NL8620004A patent/NL8620004A/en unknown
- 1986-01-13 AU AU52385/86A patent/AU591647B2/en not_active Ceased
- 1986-01-13 WO PCT/GB1986/000023 patent/WO1986004423A1/en active Application Filing
- 1986-09-11 SE SE8603803A patent/SE8603803L/en not_active Application Discontinuation
- 1986-09-12 FI FI863704A patent/FI863704A0/en not_active Application Discontinuation
- 1986-09-12 DK DK437386A patent/DK437386A/en not_active Application Discontinuation
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU577937B2 (en) * | 1985-02-05 | 1988-10-06 | United States of America, as represented by the Secretary, U.S. Department of Commerce, The | A method for detecting htlv-iii neutralizing antibodies in sera |
AU579715B2 (en) * | 1985-02-26 | 1988-12-08 | United States of America, as represented by the Secretary, U.S. Department of Commerce, The | Detection of human t-cell leukemia virus type iii |
Also Published As
Publication number | Publication date |
---|---|
DE3690028C2 (en) | 1995-05-24 |
GB8620371D0 (en) | 1986-10-01 |
NL8620004A (en) | 1986-12-01 |
DK437386D0 (en) | 1986-09-12 |
GB2179449A (en) | 1987-03-04 |
IT1191841B (en) | 1988-03-23 |
FI863704A (en) | 1986-09-12 |
CH675636A5 (en) | 1990-10-15 |
DK437386A (en) | 1986-09-12 |
BR8604533A (en) | 1987-07-14 |
SE8603803D0 (en) | 1986-09-11 |
WO1986004423A1 (en) | 1986-07-31 |
HU204929B (en) | 1992-02-28 |
HUT41129A (en) | 1987-03-30 |
IT8619072A0 (en) | 1986-01-13 |
ATA900186A (en) | 1993-12-15 |
FR2576107A1 (en) | 1986-07-18 |
BE904030A (en) | 1986-07-14 |
FI863704A0 (en) | 1986-09-12 |
SE8603803L (en) | 1986-09-11 |
AU591647B2 (en) | 1989-12-14 |
GB2179449B (en) | 1988-12-21 |
FR2576107B1 (en) | 1990-08-31 |
DE3690028T (en) | 1987-03-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Gorny et al. | Generation of human monoclonal antibodies to human immunodeficiency virus. | |
US4716102A (en) | Purified AIDS-associated virus ARV-2 | |
Kelen et al. | Rapid detection of Australia/SH antigen and antibody by a simple and sensitive technique of immunoelectronmicroscopy | |
AU591647B2 (en) | Improvements relating to viral isolates and their use | |
US6492104B1 (en) | EIA test using nondenatured HIV antigen for early detection of HIV infection | |
WO1993021346A1 (en) | Assay for detection of hiv antigen and hiv antibody | |
US4912030A (en) | Viral isolates and their use in diagnosis | |
RU2082417C1 (en) | Antiviral agent, vaccine based on thereof, method of its preparing, therapeutic agent and method of detection of rna-viruses | |
US4806467A (en) | Method for the detection of equine infectious anemia and other retrovirus infections using a competitive enzyme-linked immunoabsorbent assay and reagents useful in the same | |
WO1995032311A1 (en) | Non-hiv antibodies as disease markers | |
Hashida et al. | Shortening of the window period in diagnosis of HIV-1 infection by simultaneous detection of p24 antigen and antibody IgG to p17 and reverse transcriptase in serum with ultrasensitive enzyme immunoassay | |
Dawson et al. | Reliable detection of individuals seropositive for the human immunodeficiency virus (HIV) by competitive immunoassays using Escherichia coli-expressed HIV structural proteins | |
EP0386713B1 (en) | Immunoassay for antibodies to HIV | |
US4877725A (en) | Immunoassays for antibodies which bind to the acquired immunodeficiency virus | |
JP3421336B2 (en) | Differentiation between HTLV-I and HTLV-II using synthetic peptides | |
JPS63500401A (en) | Competitive ELISA for detection of antibodies | |
EP0951646B1 (en) | THE IMMUNOENZIMATIC ASSAY FOR THE DIAGNOSIS OF EQUINE INFECTIOUS ANEMIA VIRUS DISEASE BY USING RECOMBINANT PROTEIN (rGP90) DERIVED FROM EQUINE INFECTIOUS ANEMIA VIRUS | |
US7026133B2 (en) | Method and composition for the diagnosis of equine infectious anemia virus disease by using the recombinant capsid protein virus (p26) | |
CA2037466A1 (en) | Detection of anti-hiv antibodies | |
SU1687611A1 (en) | Method for diagnosticum preparation for viruses express- indication | |
AU625720B2 (en) | Monoclonal antibodies to specific antigenic regions of the human immunodeficiency virus and methods for use | |
Nielsen et al. | Antigen-antibody reaction in solution in capture competition immunoassay for human immunodeficiency virus antibodies | |
WO1998027428A1 (en) | THE IMMUNOENZIMATIC ASSAY FOR THE DIAGNOSIS OF EQUINE INFECTIOUS ANEMIA VIRUS DISEASE BY USING RECOMBINANT PROTEIN (rGP90) DERIVED FROM EQUINE INFECTIOUS ANEMIA VIRUS | |
JPH029393A (en) | Production of monoclonal antibody to be specifically reacted with bovine infectious pathogenic virus | |
JPH09510009A (en) | Discrimination of HTLV-I and HTLV-II using synthetic peptides |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |