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
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  • 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
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  • 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
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  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/81—Packaged device or kit
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  • 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
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  • Y10S436/804—Radioisotope, e.g. radioimmunoassay
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  • Y10S436/00—Chemistry: analytical and immunological testing
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  • Y10S436/808—Automated or kit
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  • Y10S436/823—Immunogenic carrier or carrier per se

Definitions

• 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) .
• AIDS acquired immune deficiency syndrome
• AIDS acquired immune deficiency syndrome
• 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.
• 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) .
• CBL-1 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.
• 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.
• NCACC National (now European) Collection of Animal Cell Cultures
• 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
• 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.
• 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.
• 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,
• CBL-1 a second component which is an i ⁇ tmunoglobulin which contains specific antibody to the retrovirus antigens and which is labelled with a non-radioactive revealing label.
• 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.
• 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.
• 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.
• 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.
• HRPO horseradish peroxidase
• 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.
• 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.
• retroviruses 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.
• 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.
• 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.
• 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.
• 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.
• culture medium composed of RPMI-1640 basal medium supplemented with 10% foetal calf serum and 10 ug/ml phytohaemagglutinin.
• conditioned medium containing T-cell growth factor harvested from mixed cultures of tonsillar lymphocytes was added, and phytohaemagglutinin removed.
• CCRF-CEM cells hereafter called CEM cells
• CEM cells are a permanently growing leukaemic T-cell line described by Foley e_t al (1965) Cancer 18: 522-9.
• 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
• 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 ⁇ -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 ⁇ -propiolactone and kept at 4 C for 2 hours; it was then warmed to 37 C for half an hour.
• 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.
• 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) .
• 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 ⁇ -propiolactone. The use of Tween 20 would also be expected greatly to reduce the titre of any infectious virus.
• 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 _ir ⁇ 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) .
• BSA bovine serum albumin
• 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.
• 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.
• 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 ⁇ -propiolactone.
• 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% ⁇ -propiolactone and then hydrolised as previously.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• HRPO horseradish peroxidase
• 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.
• HRPO horseradish peroxidase
• ancillary reagents so that all the necessary revealing reagents and control reagents were available.
• HRPO horseradish peroxidase
• TMB 3,3' ,5,5'-tetramethylbenzidine
• 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.
• 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.
• 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.
• Drug Addict 1 0.50 0.44 2 0.66 0.50 3 0.95 0.73 4 0.85 0.63
• the assay is conducted by a determination of label associated with the solid phase.
• 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.
• 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.
• 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.

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• 1986
  • 1986-01-13 WO PCT/GB1986/000023 patent/WO1986004423A1/en active Application Filing
  • 1986-01-13 AU AU52385/86A patent/AU591647B2/en not_active Ceased
  • 1986-01-13 GB GB08620371A patent/GB2179449B/en not_active Expired
  • 1986-01-13 FR FR868600376A patent/FR2576107B1/fr not_active Expired - Lifetime
  • 1986-01-13 BR BR8604533A patent/BR8604533A/pt unknown
  • 1986-01-13 BE BE0/216129A patent/BE904030A/fr not_active IP Right Cessation
  • 1986-01-13 HU HU86767A patent/HU204929B/hu unknown
  • 1986-01-13 NL NL8620004A patent/NL8620004A/nl unknown
  • 1986-01-13 DE DE19863690028 patent/DE3690028T/de active Pending
  • 1986-01-13 IT IT19072/86A patent/IT1191841B/it active
  • 1986-01-13 DE DE3690028A patent/DE3690028C2/de not_active Expired - Lifetime
  • 1986-01-13 AT AT0900186A patent/ATA900186A/de not_active Application Discontinuation
  • 1986-01-13 CH CH3710/86A patent/CH675636A5/de not_active IP Right Cessation
  • 1986-09-11 SE SE8603803A patent/SE8603803D0/sv not_active Application Discontinuation
  • 1986-09-12 DK DK437386A patent/DK437386D0/da not_active Application Discontinuation
  • 1986-09-12 FI FI863704A patent/FI863704A0/fi not_active Application Discontinuation

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