WO1986004423A1

WO1986004423A1 - Improvements relating to viral isolates and their use

Info

Publication number: WO1986004423A1
Application number: PCT/GB1986/000023
Authority: WO
Inventors: Robin Weiss; Richard Tedder; Rachanee Cheingsong-Popov; Bridget Ferns
Original assignee: Institute Of Cancer Research
Priority date: 1985-01-15
Filing date: 1986-01-13
Publication date: 1986-07-31
Also published as: FR2576107A1; GB8620371D0; AU5238586A; DK437386D0; NL8620004A; DE3690028T; HUT41129A; DE3690028C2; DK437386A; BE904030A; SE8603803L; BR8604533A; FI863704A; GB2179449B; GB2179449A; CH675636A5; AU591647B2; SE8603803D0; HU204929B; ATA900186A

Abstract

Samples e.g. transfusion blood, are assayed for antibodies to retrovirus, e.g. AIDS virus, using an insolubilised antigen comprising retrovirus antigens bound to globulin, the globulin itself being bound to an inert solid support; and an immunoglobulin which contains specific antibody to the retrovirus antigens and which is labelled with a non radio active revealing label, and the liquid phase is then separated from the solid phase and the quantity of revealing label associated with either the liquid or the solid phase determined. The use of enzyme labelled antibody in competition with test sera for binding on the insolubilised antigen permits better identification of antibody containing specimens. The retroviruses may be a HTLV-I, II or III or a new retrovirus isolate CBL-1 etiologically related to AIDS.

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

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.