DE3690028C2 - Method and test kit for analysing a biological sample for antibodies against retroviruses - Google Patents

Abstract

Published without abstract.

Description

The invention relates to a new assay for antibodies for retroviruses, in particular those with the acquired immunodeficiency syndrome (AIDS, from the Anglo-Saxon expression acquired immune deficiency syndromes) are associated.

The likelihood that the acquired immune deficiency syn drom (AIDS) is caused by an infectious agent has been evident for several years. The symptoms of this Disease were targeted to specific, well-defined risk groups confined in a pattern which is very likely makes the pathogen through sexual or blood contact will wear. Although this disease was initially reported in Ver purified states of America occurs increased incidence of the disease elsewhere, including in Europe, on.  

A method which is believed to make the sick is transmitted, is the transfusion of blood, wel donated by donors who themselves have AIDS virus are infected. Collecting donated blood means that if blood is donated by a donor which harbors an AIDS virus, with samples of Blood from other donors is pooled, then the whole Blood and the other products that come from this pool are contaminated, regardless of how small or large is the sample. Many of the blood donors who have AIDS Viruses are host to their infection, in particular in the early stages of infection, not consciously and others re infected donors, even if they are from their Knowing infection either refuse to admit that they have the Have illness or that they belong to a high risk group belong.

The invention has for its object, relatively simple but completely reliable tests according to which Samples from blood intended for transfusion purposes is due to the presence of AIDS virus or antibodies for AIDS virus can be routinely tested to provide.

There was also a need for an isolate (virus) that was found in such an assay can be used, e.g. H. one Isolate that are maintained for extended periods of time can and can be grown in a host cell line.

Donating blood, those who respond positively in the test can use transfusions purposes are rejected, and the contamination rer blood samples by combining with the contaminated th sample can be avoided.

Research on identification and isolation the pathogen responsible for AIDS has been actively operated for about 2 years. But there are two between the various researchers in this field  with regard to the exact identity and nomenclature none Accordance. About a so-called AIDS virus extract (Isolate) was first developed in 1983 by Montagnier and his colleagues gen in France who reported the material "Lymphadeno pathy Associated Virus One "(LAV-1). Approximately  a year later Gallo and his colleagues in the Ver United States details regarding isolation published another so-called AIDS virus, which then as "Human T-lymphotropic Virus Type III" (HTLV- III) designated.

GB 2 016 687 describes a stable reagent which is used for Conducting immunoassays is useful, which is a sugar coated immunoadsorbent bound to a solid support. The immunoadsorbent can be used in some Cases include pre-coating with an antibody. HTLV assays using HTLV antigens directly are absorbed into the cavities of a microtiter plate, are described in EP-A 136 798. U.S. 4,520,113 describes the detection of antibodies in sera from AIDS and pre-AIDS patients and describes the biochemical and immunological analysis of antigens in humans MTLV-III T cell leukemia virus are associated. In Science 225 (1984) 321-323 antibodies against the core Protein of the lymphadenopathy-associated virus (LAV) AIDS patients described.

The applicant was now able to get out of a lymph node tumor a human T-lymphotropic retrovirus, which is associated with HTLV-III and LAV is related to isolate and which of the applicant was designated as CBL-1. The CBL-1 mate rial by the applicant is a stable extract, which in a host cell line can be grown and in an assay for the detection of antibodies against the AIDS virus in Serum samples can be used.

The invention relates to a method for analysis a biological sample for antibodies against retroviruses, where you have the biological sample

• (1) with an immunoglobulin that has a specific contains antibodies for the retrovirus antigens, and which is marked with a dog tag, mixes
• (2) with an insolubilized antigen, which An genes of human retroviruses that are detected with the Retroviruses are etiologically related, bound to globulin or to monoclonal antibodies, the globulin or the antibody itself to an inert solid support is bound, in contact, and with which one
• (3) separates the liquid phase from the solid phase and the amount of identifier, either with the liquid or the solid phase associated.

The invention thus relates to the use of antigens human T-lymphotropic retrovirus CBL-1, which is etiologically too AIDS is related to the detection of antibodies against retro virus.

It has been found that CBL-1 has been in use for a long time leukemic T cell line, which is referred to as CCRF-CEM and by Foley et al, Cancer 18, 522-529 (1965) is held, and the invention relates to white further CCRF-CEM cells, which the CBL-1 according to the invention Host virus.

The applicant has samples from the CCRF-CEM cells, which the CBL-1 virus according to the application host at the National (now European) Collection of Animal Cell Cultures (NCACC) in Public Health Laboratory Service Center for Applied Microbiology and Re search, Porton Down, Salisbury, Wiltshire, SP4 OJG England deposited. The depository NCACC was named Inter national depositary under the Budapest Treaty recognized from 1977. The deposit took place on January 11 1985 and received the deposit number 85 01 1101.  

The CBL-1 material according to the application was made from lymphocytes isolated in a biopsy of a lymph node tumor of a British patient who was on Immuno blast lymphoma associated with AIDS delt was. Fragments of the biopsy were taken in a culture medium, to which a T cell growth factor was added, and after about 2 days the CCRF-CEM cells became the Given culture, which kept in such conditions was that the primary lymphocytes over the course of about a month ago disappeared while the CCRF-CEM cells increased and was chronically infected with the CBL-1 virus the.

The CBL-1 virus has been shown to describe that previously extracts of HTLV-III is related since it

• (a) the activity of reverse transcriptase with has a preference for Mg⁺⁺ cations, and also
• (b) the morphology of the virus particles which identified by electron microscopy
• (c) by indirect immunofluorescence, which is viral for genes in CBL-1 infected CCRF-CEM cells with sera posed by AIDS patients, specific is
• (d) by comparison in the solid phase radioimmuno assay with HTLV-III and LAV-1,

and thus from the taxonomic group of the retro viridae, which includes human T cell lymphotropes Viruses included.

Preliminary DNA sequence analysis shows that the sequence of the CBL-1 virus, although closely related to that of the others human T-lymphotropic retroviruses that have so far been  were related, not with that of the previously described written materials is identical, but different where a sequence variation shows.

It has been shown that the infected cells involved in ECACC have been deposited, continuously viruses and virals Antigens synthesized for at least 6 months ren.

Although CCRF-CEM cells were found to be an ideal one Host for the CBL-1 virus according to the application was ge find that the cells can also be used to host a similar virus, which is from the Applicant from the peripheral blood of the same patient, from which the lymph node tumor biopsy was taken and similar viruses from other patients with AIDS related viruses are infected, have been isolated. The An Melderin has shown that this cell line is a valuable Host for in vitro breeding not only from the application according to CBL-1 material, but also from other ver applied materials.  

The invention further relates to a test kit that

• (1) a first component that insolubilizes antigen is which antigens from human retroviruses, those etiologically related to the retroviruses to be detected are bound to globulin or to monoclonal antibodies, contains, with the globulin or the antibody itself an inert solid carrier is bound, and
• (2) a second component, which is an immunoglobulin which is a specific antibody for the retrovirus Contains antigens, and which with an identifier is marked

includes.

As indicated above, the main use of CBL-1 is that there is a component in a test system for routine Transfusion blood testing is to determine if it is from a subject infected with AIDS virus. The applicant has a system based on the same early competitive assays using immobilized th CBL-1 antigens or other retrovirus antigens than solid phase and the test serum developed as a liquid phase.

More specifically, the simultaneous application is based on the application Competitive assay on the immobilization of the CBL-1 antigen or another retrovirus antigen by binding to a solid phase via a ligand, for example Globu  lin or another antibody, and then you allow that the binding sites on this immobilized antigen for the competing action of a mixture Test serum antibody and known antibody for AIDS virus, which is identified by the detection means can be available. The applicant has ge found that achieved the most satisfactory results can be if the detection agent is an enzyme marker is. The use of an enzyme marker instead of one Radioactive labeling has advantages because the radiation bio danger does not occur, and an unexpected consequence is that the assay immune incubation is at 1 hour or less can be shortened.

Another alternative is to use the immune system fluorescence to indicate the presence of a be Known antibody to an immobilized antigen is bound.

As stated above, it has been found that the best results nisse are obtained when the antigen adheres to the solid phase via a ligand that is an antibody. One technique is to use a high serum human serum Titer, which is from anyone who is asymptomatic with AIDS is infected. The ideal serum is contain a high titer of antibodies for AIDS viruses, will, however, come from a patient whose immune cell bulin values are in the normal range. Another possibility is the use of a monoclonal antibody, the ge compared to the retrovirus. Will this in bred in a known manner using mice, then certain benefits can be ensured, such as it is explained in more detail below.  

The solid phase material is conveniently poly styrene, which is in the form of tubes, beads or preferred as a plate with a variety of surface waves or other surface impressions or depressions becomes. Gamma globulin, which is from the selected serum or another antibody has been made applied to the polystyrene and the antigen turns on the globulin coated polystyrene to form the first component of the test. This component can stored wet or dry for several months.

The second component of the test involves the IgG fraction, which has been isolated from the test serum and if the Er recognition by means of an enzyme label, this can IgG fraction, for example with horseradish peroxidase (HRPO) be conjugated by methods known per se.

The test kit according to the invention can be used to mix the mar IgG component with the test serum and contact brine gene of the mixture of IgG and test serum with the insolubi lized antigen, separation of the solid and liquid phases from each other and determining the extent to which the IgG bound to the antigen can be used. Correspond The larger the concurrent competitor assays is the extent to which the IgG component of the liquid Phase is bound to the solid phase, the lower the Amount of antibody or antigen in the test serum. By It is therefore possible to select a suitable control value Routine test to have where if HRPO as a marker the test serum is used as free of AIDS antibodies or antigen can be viewed provided that the optical density (OD) of the test sample a predetermined Minimum value reached. Any sample of the test serum, wel  che an OD count below the predetermined value that is on was created, can therefore be rejected be a transfusion with a blood donation that possibly contaminated with AIDS virus or antibodies is avoided.

As an alternative to CBL-1 as a retrovirus antigen in the Assay procedure and the diagnostic test kit according to the above other retroviruses can be used. These can be other human retroviruses that are etiological with the AIDS virus, such as the human T-lymphotropic retro virus HTLV-III or other human retroviruses like that human T-leukemia virus, HTLV-II or HTLV-I or tieri retroviruses, such as the Maedi-Visna or bovine leukosis virus are related.

As will be explained in more detail below, the indi direct binding of human retrovirus antigens to the solid Phase over the globulin a certain duplication of the Results, and the test format allows for easy, quick le and precise determination of the presence or absence contamination of blood samples by relatively little trained and inexperienced technicians.

Description of the drawings

Fig. 1 shows an electron micrograph with 81000 magnification, where CBL-1 particles germinate, and are shown localized at the border of the CCR-CEM host cells.

Figure 2 shows the solid phase ELISA for anti-HTLV-III. The figure shows the optical densities obtained in a series of tests of clinical sera, including those that are reactive and non-reactive to anti-HTLV-III, and results previously known from radioimmunoassay.

In FIG. 3, results similar to those in FIG. 2 are shown, but this time for anti-HTLV-I, determined according to a similar, single-stage, simultaneously competing ELISA.

The following examples illustrate the invention.

example 1 Isolation and characterization of CBL-1

The CBL-1 virus was isolated from lymphocytes derived from a British patient's lymph node tumor biopsy, who was treated for immunoblast lymphoma, which associated with acquired immune deficiency syndrome (AIDS), cul activated. The lymph node biopsy was broken up into small fragments cut and placed in a culture medium, which consists of RPMI-1640 basic medium, supplemented with 10% fetal calf serum and 10 µg / ml phytohaemagglutinin. After 24 hours which was conditioned medium, which T cell growth factor harvested from mixed cultures from tonsillar Lymphocytes contained, added, and the phytohaemaggluti nin was removed. 48 hours after the initiation of the Culture antiserum for interferon alpha was added. To CCRF-CEM cells (hereinafter referred to as CEM cells len referred) also added. The CEM cells are a permanently growing leukemic T cell line developed by Foley et al (1965) Cancer 18: 522-9.

The primary disappear under the breeding conditions Lymphocytes in the course of 4 weeks of culture, during the  CEM cells grow exponentially. The CBL-1 virus produces through the primary lymphocytes, infected the CEM cells, and after 8 weeks of culture, the CEM cells are chronic infected with the CBL-1 virus. Preserving the CBL-1-in infected CEM cells did not require T cell growth factor or anti-interferon alpha. This CBL-1 infected CEM cells were obtained from NCACC under the Filing number 85 01 1101.

It could be shown that the CBL-1 virus is related to the previously described isolates of HTLV-III / LAV, since it (a) has the activity of a reverse transcriptase with a preference for Mg²⁺ cations, and by the morphology the virus particles, which were made visible by electromicroscopy (see FIG. 1), and (b) by indirect immunofluorescence, which were used for the viral antigens in CBL-1-infected CEM cells with sera, produced by AIDS patients , specific, and by comparison with other AIDS virus extracts in the solid phase RIA. The CEM / CBL-1 cells have continued to synthesize the virus and viral antigens over the course of 6 months.

Example 2 1. Antigen production

A raw, frozen and thawed cell lysate is made in the following way se manufactured. HT-H9 cells infected with HTLV-IIIB can to a maximum density in a suspension culture to grow. The cultures are cooled to 4 ° C and 0.25% v / v β-propiolactone are added over 2 hours. The Cells are then centrifuged out of the culture fluid and Pelletized at low speed. The cell pel  lets are resuspended in distilled water again and subjected to three freeze / thaw cycles. Every cycle the rest of the cell pellets are carefully resuspended. After the third cycle, the extract becomes 0.25% vol / vol adjusted with β-propiolactone and at 4 ° C for 2 hours that held. It is then at 37 ° C for a half Hour warmed. During this time, the pH in the Be held high by 7.4. The raw lysate is clarified and at Stored at -20 ° C. Before use, it is placed in TRIS buffer, supplemented with 0.1% BSA and detergent, diluted. The choice of Detergent and its concentration is important and it has been de found that the use of Tween 20 in a con concentration of 0.1% is optimal, whereby the activity of the Antigen preparation amplified three times or sometimes more becomes. After dilution in TRIS-BSA / Tween 20, the anti incubated for 30 minutes at 37 ° C. It is during this Stage that antigen enhancement occurs.

The antigen preparation obtained in this way de, can be used to express HTLV-III Antigen under different defined culture conditions follow. The antigenicity can be found in solid-state RIA Using solid phase anti-HTLV-III and a second Reagent containing ¹²⁵-J anti-HTLV-III, quantitative to be grouped. In the last analysis, the anti gene used in a dilution which has a P-N ratio nis of 10: 1 with 1 to 2% mark binding in property negative sera (see below under the assay procedure) allowed. There was a variable rejection of viral anti genes in the supernatant fluid of the cells, but in relati relationships became the main mass of the detectable Antigen left in the cell pellets. This was true for CBL-1 and for other isolates (extracts) of HTLV-III, which in  CEM cells or HT-H9 cells were present. There was none Limitation of antigen purity (see below), and it was right to increase antigen expression in the cells maximize. The antigen preparation used here was easy to manufacture, probably with a higher degree loot per volume of cell culture as an antigen, which from over liquid has been prepared and is stable at -20 ° C. It can easily be inactivated by β-propiolactone. One can also strongly expect that by using it Tween 20 the titer of any infectious Virus is decreased.

2. Antisera

The reagent for the assay is made from human serum high titer, which comes from people who are asymptomatically infected with the AIDS retrovirus. The out choice of sera for reagent preparation was important. The Serum had a high titer of anti-HTLV-III according to RIA and came from a patient whose immunoglobulin levels were in the normal range. In practice it is in general required reagents from a small number of sera deliver that meet the above criteria, and the work of the reagents in the assay. So this reagent cien are non-infectious, they are used as the starting material used Sera by heating at 56 ° C for 30 minutes treated.

(a) Preparation of anti-HTLV-IIIB globulin

This material is used for in-situ cleaning on a festival phase HTLV-IIIB antigen used. In this strategy lie considerable advantages. Globulin from heat treated  Serum is pre-twice with 40% saturated ammonium sulfate received. This is the easiest reagent for the Globu Lin coating that makes it possible to either total to use serum or purified IgG. The globulin was used with an optimal dilution, which by individual titration of the reagent must be determined. The solid phase material was polystyrene in the form of Cavities and was coated with globulin. The free binding sites were then filled with an inert one Protein - in this case 0.1% bovine serum albumin (BSA) - saturated. The coated and abge saturated solid phase became wet at 4 ° C for months stored. Drying gave an even more stable product. To coat the solid phase, HT-H9 / HTLV-IIIB- Lysate as described above in TRIS-BSA tween buffer to a dilution which, in subsequent tests, the Possibility revealed that 1 to 2% of the label was negative Serum was bound, diluted. The coating was on most effective during a 2 day incubation of the antigen at room temperature in the solid phase and then wet Store at 4 ° C until use. Preliminary tests showed that the solid phase in this form for several months is stable. However, it can also without signi fictional loss of potency can be dried and remains stable for several months.

(b) labeling of globulin

The IgG described in 2 (a) above is treated with HRPO under Ver use of heterobifunctional derivatives in the usual Conjugated way, using a molar substitution ver ratio of 1: 3 receives. This material was in association with the solid phase in one described in 2 (a) above  Enzyme immunoassay for the detection of antibodies for AIDS Retroviruses used in samples of body fluids.

Another IgG sample was used for comparison purposes ¹²⁵-J up to a specific activity of 15 µCi per µg protein marked.

Example 3 Single stage competition ELISA for Anti-CEM / CBL-1 Serumrea genzien

A human serum that has a high titer of anti-HTLV III was used for the production of coated Globulin, as previously described in Example 2.2, used. Polystyrene cavities with a round bottom are made with an opti paint dilution of globulin coated and then with an inert protein, in this case 0.1% BSA.

The same serum is used as the source for Immunoglobulin used, which by ion exchange Chromatography on DE 52, as previously described, cleaned has been. IgG was used with horseradish peroxidase in a young woman carry substitution ratio of 3 molecules horseradish Peroxidase coupled per 1 molecule of IgG.

The effect of the conjugate is increased by incubation with Kavi previously with an indirect antibody with viral Antigens derived from HTLV-III infected cells and coated with antigens on uninfected cells. The optimal dilution of the conjugate was found to be the genome where you get maximum color binding with the infected cell antigen and minimal color binding with the non infected cells.  

CEM / CBL-1 antigen

Cells that are constantly infected with the CEM / CBL-1 virus, were in the non-adhesive mixing bowl up to the maximum density doors bred. The stirring cultures are cooled to 4 ° C and then brought to 0.25% volume with β-propiolactone. After 2 hours of incubation at 4 ° C, the cell pellets harvested and subjected to the freeze and thaw cycles and then clarified by centrifugation as previously described. The tissue culture extract is then a further 2 hours treated the incubation at 4 ° C with 0.25% β-propiolactone and then hydrolyzed as before. In this case it is Diluent for cell disruption and freezing Thawing cycle phosphate buffered salt, which with 0.1% Tween 20 is added. The antigen preparations are on then titrated on a solid phase, which with high Titer anti-HTLV-III globulin is coated. The dilution, which have an OD 450 nm between 1.0 and 1.2 in the fol given defined conditions, was used for the subsequent test.

Optimization and format or size of the tests

Cavities coated with high titer globulin and then tied or scared off who with 100 µl of an optimal dilution of CEM / CBL-1-An incubated. After an overnight incubation in the room the antigen extracts from the dishes washed, which then dried under such conditions were found to have stability Activate immobilized CEM / CBL-1 virus antigens. To the 25 µl of antibody-positive and antibody-negative will be tested Active serum in separate bowls and 75 ul ELISA Be conjugate in detergent-supplemented distilled water  added to the cavities. The mixture of test serum and Kon jugat is served in the bowls during different times conditions under different conditions (immune incubation) incubated. The cavities are then three times with saline Tween (0.8% sodium chloride solution, supplemented with 0.1% Tween 20) washed. After three washes, the cavities filled with salt tween and can be used for 2 minutes Absorb the moisture at room temperature. Then the Washing process repeated with three washing cycles. After that who the 100 µl chromogen substrate, in this case 3.3 ′, 5.5′- Tetramethylbenzidine (TMB) added. After 20 minutes of inku bation at room temperature is the chromogen release by Addition of 50 µl of 4 normal sulfuric acid terminated. The Dye release was measured spectrophotometrically at 450 nm measure up. The working dilution of some particular CEM / CBL-1 antigen is assumed to be the dilution at which reliably has an optical density between 1 and 1.2 with negative serum, if it is in ELISA under opti paint conditions, defined as 1 hour of immune incubation at 45 ° C was used.

Results

When comparing the results you get at that known RIA using a solid phase and ¹²⁵-J-labeled IgG, described as in Example 2, obtained and the ELISA results obtained in this example holds, two unexpected benefits become apparent, too Speak in favor of ELISA. First, you get from subopti paint antigen preparations, which result in poor results in RIA give good results with the same titer with ELISA. Second, as explained in the table, it is with RIA not possible, incubation times are less than 3 hours to reduce. But there is a need for a quick one  Assay, d. H. 1 to 2 hours in total, with the transfusion implementation, and this is only possible with ELISA.

TABLE

Time / temperature experiments in RIA with ¹²⁵-J-labeled IgG

FIG. 2 shows the results of the ELISA test for 56 clinical sera, which in this case originate from haemophilic patients and were treated with commercially available and unavailable factor VIII concentration and originated from homosexuals. It is clearly evident from the figure that there is a clear separation between sera which are reactive for anti-CEM / CBL-1 and non-reactive sera.

In this particular example, the middle one is optical Density of 16 sera that are reactive for CEM / CBL-1 antibodies are, 0.085 (0.037 to 0.327), and the mean optical Density for 40 sera that did not reak for this antibody tiv is 1.16 (0.738 to 1.352). The serum a comes from a terminal AIDS patient and is weakly reactive in IF and direct binding assays. Sera b and c are not reactive when tested again.

It was exactly the same scheme in an overview of the Blood transfusion sera used in the UK. Until To date, over 12,000 sera have been donated checked. 3 sera gave screen test responses, however, found when testing again, that only one donor was reactive. The serum was repeatedly reactive, was titrated bar and demonstrated strong reactivity at the Immunofluorescence against infected, but not against over uninfected cells. The random reactivity of Sera unrelated to CEM / CBL-1 infections was extremely low. Follow on en Attempts to titrate sera which are reactive for anti-HTLV III, showed that the titers were largely those are the same if they are both according to the CEM / CBL-1 ELISA as well as tested according to the direct assays. This was verified by subsequent testing of the sera by Patients were removed using an acute seroconver sion after the primary HTLV-III infection. Again there was roughly no difference in salary or to Time of initial reactivity in both the direct Binding assay as well as the competitor ELISA. Under use Using the same assay were sera from patients with oligo- and pan-reactive anti-lymphocyte antibodies and Sera from patients with autoimmune diseases at the same  competitor ELISA not active. In summary, show these values indicate that specificity and sensitivity this competitive ELISA for antibodies to AIDS ver applied retrovirus are high.

Example 4 Single stage competitor ELISA for Anti-HTLV-I

Sera and reagents are selected and prepared in the same manner as in the CEM / CBL-1 immunoassay of Example 3, except that cells infected with HTLV-I are for the antigen preparation, and Sera from patients infected with HTLV-I can be used as the source for the reagents. In Fig. 3 the optical density is shown, which is obtained with 32 clinical sera including three known weak positive controls. There is a clear difference between reactive and non-reactive sera, with the mean optical density of 5 reactive sera for this antibody 0.099 (0.060 to 0.129) and the mean optical density for 24 sera that are not reactive for this antibody 1.64 (1.48 to 1.83). The test was stable and the conditions can be varied in practice. However, it was decided that an immuno-incubation of 1 hour at 45 ° C was considered optimal. This is in parallel with the conditions for the detection of CEM / CBL-1 antibodies.

The test was used in the same scheme to over 1000 to examine unselected British blood donors, and no reactive sera were identified. In the opposite A selective test of about 75 donors was also given  a single donor from Africa and the Caribbean that was seropositive for the first time.

The above procedure was repeated using Sera from patients infected with HTLV-II as Source for coating with globulin and as a source for IgG. The cells infected with HTLV-II were identified as Antigen source used. These reagents are on same way as before in Example 2 for Anti-HTLV-III RIA described, selected and manufactured.

Example 5 Manufacture and use of a test kit 1. Reagents

A polystyrene plate with many cavities (a multi corrugated plate), which contained 96 cavities, min at least on the inner surface of the cavities with ge Purified, heat treated antibody for CBL-1 coated. The antibody comes from a heat treated serum of a person who is diagnosed with the AIDS retro virus was infected. After coating the polystyrene the free binding sites become with the antibody saturated with an inert protein, in this case with Bovine serum albumin. That for the insolubilisie antigen used on the polystyrene plates CBL-1. This was first done with the viricidal β-propiolactone treated, and then a suspension of inactivated tem CBL-1 in TRIS-BSA-Tween buffer with the treated  Incubate polystyrene for about 2 days at room temperature beer. In this way, the CBL-1 was created by indirect Binding to the polystyrene support via the antibody in solubilized. This component is the first component of the test kit.

The second component of the test kit is manufactured in which is the same human antibody that is used for loading layering of the polystyrene as described above, ver was marked. The antibody was obtained by Kon Jugation with horseradish peroxidase (HRPO) using marked conjugation methods known per se. The one with HRPO labeled antibody is freeze-dried stored and immediately before use with a suspension in an aqueous diluent reconstituted.

In addition to the two components mentioned above it is preferred to include auxiliary reagents in the test kit, so all the necessary detection reagents and control reagents are available. For this kit at the HRPO is used as a marker, it is useful 3,3 ', 5,5'-tetramethylbenzidine (TMB) as a substrate for the Enzyme to use, and the TMB was used in tablet form Provided, each tablet is sufficient Substrate for the 96 wells resulted. Just before The TMB tablets are used in an aqueous Solution containing tri-sodium citrate and hydrogen peroxide holds, solved. It is also preferred to be positive and ne to provide negative control sera. The negative control serum is normal human serum used for the antibody for CBL-1 is not reactive in this test. The positive con troll serum is heat-treated human serum, which  for antibodies to CBL-1 is reactive in the test procedure. In addition, it is preferred to use a shortened control serum, a heat-treated human serum (based on the shortened Ge stop in the test) for antibodies CBL-1 is reactive, and just if a wash solution containing salt and Tween 20 to provide.

2. Use of the test kit

Immediately before use, the HRPO-labeled Anti body reconstituted. The trial is using of 25 microliter samples of serum per well guided. Two wells are with negative control serum and one loaded with positive control serum, three with shortened control serum (the expression shortened refers refer to the Anglo-Saxon expression "cut-off") and the Test samples are filled into the remaining cavities. 75 microliter portions of the reconstituted HRPO-labeled Antibodies are placed in each well, and the The plate with the cavities is then covered and placed on one Heating block at 45 ° C for 1 hour or alternatively at 20 to 25 ° C overnight for at least 16 hours incubated. Towards the end of the incubation period, the Plate washed, and 100 microliter portions of the substrate solution Solution are added to each cavity. The plate will then incubated at 18 to 25 ° C for 20 minutes. Then ent a blue color develops in the cavities, the ne negative samples included. 50 microliter parts from 2M Schwe Rock acid is then added to each cavity, and the blue color changes to yellow. The absorption of everyone Cavity at 450 nm is removed within 30 minutes of the Add sulfuric acid using a reader read for a microtiter plate.  

3. Results

The results are obtained in the following way. The mitt Lower absorption of the three truncated control sera will be calculates. Any sample that shows absorption, wel che is equal to or greater than that of the middle shortened control value, is used as a negative antibody for AIDS Retrovirus viewed within the confines of the test system. Any samples with an absorption below that medium shortened comparison sample or up to 10% above that of the shortened mean comparison sample checked again.

Any sera that are repeated positive in this test Results were given below for confirmation purposes Use the immunofluorescence test or Western blotting tests checked again.

The procedure described above was based on 1600 routine blood bank donor samples tested with negative results. Was which other clinical specimens from patients with kli African diagnosis of AIDS, an AIDS-related complex or other diseases tested, you got positive Results, and in practically every case there was a loading confirmation by the immunofluorescence test and by a other enzyme immunoassay. The following results were obtained nits.  

TABLE

Reactivity for sera from blood donors and patients with AIDS, AIDS-associated conditions and other diseases

The principle of using a human antiserum for the Binding of viral antigen to the solid phase gives one solid phase, which itself is the pattern of viral antigens reflected. This was best recognized in humans. This can be an advantage because it is a good "fit" between the mixture of the captured Antigens and the profile of human antibodies is obtained. Furthermore, the mild conditions that lead to antigen Manufacture used to preserve more delicate or complicated, misshapen epitopes that in the gradient of the cleaned and destroyed preparation that are used in other solid phase assays, are not present. It is now possible to mix the mixture Solid phase antigens adjust by looking at the solid Phase defined with monoclonal mouse antibody cificity coated so that the immobilized antigens have a predetermined specificity.

The values shown here show the superior nature of the Tests when a selected monoclonal antibody that reacted with p25 (a 25 K Dalton protein) on the solid Phase is used. The advantages are the low amount of antigen required for an appropriate assay is required (Table A) and sensitive in the increased of the resulting test, see Table B, where ge shows that endpoint dilutions (i.e. low OD readings) of the individual sera a higher inhibition monoclonal compared to polyclonal Tests result. Increasing the sensitivity causes  that the test is more effective in detecting antibody production compared to many other assays. In addition ver avoids the use of mouse antibodies on the solid Phase the potential problem of the rheumatoid-like fac toren cross-linking between solid phases human IgG and Human IgG conjugate in the homologous assay. Became practical few sera identified which consistently a size degree of inhibition in the monoclonal assay Show baseline compared to the polyclonal assay Basis. This phenomenon is probably due to the low content and low titer reactivity, which Conjugates to the solid phase in a non-specific way binds only in the homologous assay. This or something similar Phenomenon can also be another unexpected fact explain that the distribution of the optical density reactiv normal sera are significantly more narrow in the assay is more monoclonal than at the same time guided polyclonal assay.

Since it turned out to be easy, a monoclonal To use antibodies for a retroviral gag gene product you essentially get a competitive assay for the core human anti-HTLV III response, and it is likely Lich that the use of antibodies for defined env- Products developing analog assays for antibodies for selected envelope antigens allowed. This will be a we considerably closer examination of the humane response different virus antigens allow which of Wich activity in clinical, prognostic and infection con troll exams can be.  

TABLE A

Titration of CEM / CBL-1 antigen on polyclonal and monoclonal solid phases

TABLE B

OD 450 nm dilutions of 19 sera, titrated for 50% inhibition end point, tested with polyclonal and monoclonal solid phase

In the above examples, the assay is determined the label associated with the solid phase carried out. However, since the marking content with is associated with the starting serum, can be determined it is also possible to reduce the radioactive Count or the enzyme content associated with the liquid phase is associated with a known labeled serum and the test sera in contact with the solid phase antigen were brought, and the decrease in the salary of the Mar The liquid phase is used as a measure of the antibody content in the test serum is used to determine.

The advantages of the simultaneous competition assay can can be summarized under the following headings.

Antigen production

Although in Example 2 the use of purified HTLV-III Viruses are described as allowing increased binding comes from the use of the immobilized antibody, the use of a raw cell lysate preparation for loading stratification when it is alone, such a type do not directly engage in any useful way solid phase binds. This level removes the restriction that highly purified antigen for the production diagnosti shear tests must be used, and this will result in their Making lighter. This is an advantage which for the direct assay is not applicable where an entire human globulin coating an unavoidable signal with the finished anti-human globulin label de.  

Format or size

The use of a volume of undiluted serum bie a great advantage for blood transfusion centers where, if an initial dilution would be required, the working load would be greater. The relationship between serum and enzyme labeled volume can be within the range of 1: 1 to 1:10 with little change in the test result can be varied. The optimum is 1: 3, with 25 µl serum and 75 µl of enzyme label are used and obtained a good differentiation between positive and negative Sera. The assay time can be below 1 hour or less Using the ELISA technique.

sensitivity

A measure of the sensitivity of any assay to AIDS Antibody is determined by the ratio of the sera terminal AIDS patients must be removed which remains positive determines. For direct assays it seems that there are different numbers of Patients lose their antibody reactivity. This occurs not in the competition assay on what suggests that the Sensitivity of the competitive assay is as good or better like that of one of the assays currently used.

Specificity

A competitive assay is not expected to be large Problems with incorrect positive reactivity occur. The inclusion of lymphocyte membrane antigens in the envelope of HTLV virions certainly leads to false reactivity, like the presence of aggregates of IgG, which are not stick specifically to the solid phase. Such a phenomenon  does not result in an increase in the reactivity of the competition assay. Nonspecific affects of variable protein concentration ions can be selected by selecting appropriate serum / label ratios (1: 3, as above) are kept to a minimum and by using the shortening of over 50% inhibition. Sera, which is an inhibition of label binding or greater than that of the internal control serum, are considered positive as a screen test. The test should however, be repeated. Because of the low values for false reactivity assumed to be below 1 at 5000 it is unlikely to be false positive reak with the competitor ELISA numerically very difficult results, as is the case with other direct assays of the Case is. This is especially true where the direct Assays can be performed without control antigen. Man assumes that this is the case with transfusion centers as the use of control antigen meets the requirements doubles for reagents in direct assay the.

Summary

The combination of a simple format, highly sensitive speed and high specificity causes the competition assay ideal for screening blood donations and for diag nostic use is suitable. In addition, he can where centers use a direct assay for anti-HTLV-III the, be better to determine the serum reactivity for testing in the competitive assay instead of taking the sera the more labor-intensive and less sensitive Western Blotting tests by the FDA in the association recommended countries.

Claims (17)

1. Method for the analysis of a biological sample for antibodies against retroviruses, in which the biological sample

• (1) mixed with an immunoglobulin which contains a specific antibody for the retrovirus antigens and which is labeled with a recognition marker,
• (2) with an insolubilized antigen containing antigens from human retroviruses, which are aetiologically related to the retroviruses to be detected, bound to globulin or to monoclonal antibodies, the globulin or the antibody itself being bound to an inert solid support Brings in contact, and where you
• (3) separates the liquid phase from the solid phase and determines the amount of identifier that is associated with either the liquid or the solid phase.

2. The method according to claim 1, characterized records that the insolubilized anti gene antigens of the human T-lymphotropic retrovirus CBL-1 sums up. 3. The method according to claim 1, characterized records that the insolubilized anti gene antigens of the retroviruses human T-lymphotropic retrovi rus HTLV-III, human T-leukemic retrovirus MTLV-TI or human T-leukemic retrovirus HTLV-I.   4. The method according to any one of the preceding claims, characterized in that the biological Sample is serum or plasma from human blood. 5. The method according to claim 4, characterized records that the antigen used antigens of Retroviruses human T-lymphotropic retrovirus CBL-1 or hu manes T-lymphotropic retrovirus MTLV-III, and that Immunoglobulin labeled with the recognition agent Is immunoglobulin from a human patient who is asymptomatic with the AIDS retrovirus, however has a normal immunoglobulin level. 6. The method according to any one of the preceding claims, characterized in that the recognition marker is an enzyme marker. 7. Test kit that

• (1) a first component, which is an insolubilized antigen, which contains antigens of human retroviruses, which are aetiologically related to the retroviruses to be detected, bound to globulin or to monoclonal antibodies, the globulin or the antibody itself being bound to an inert solid Carrier is bound, and
• (2) a second component, which is an immunoglobulin, which contains a specific antibody for the retrovirus antigens and which is labeled with a recognition marker,

includes. 8. Kit according to claim 7, characterized net that the antigen used antigens of human T- lymphotropic virus CBL-1.   9. Kit according to claim 7, characterized net that the insolubilized antigen used antigens of the human T-lymphotropic virus MTLV-III, human T-leuk Aemic virus MTLV-II or human T-leukemic virus MTLV-I includes. 10. Kit according to claim 7, characterized net that the insolubilized antigen used antigens of the human T-lymphotropic retrovirus CBL-1 or human T-lym includes photropenic retrovirus MTLV-III and that the immune globulin comes from a human patient, the asymptoma table is infected with the AIDS retrovirus, but a nor paint immunoglobulin levels. 11. Kit according to one of claims 7 to 10, characterized ge indicates that the identifier is an En is zymmarker. 12. Use of antigens of the human T-lymphotropic Retrovirus CBL-1, etiologically related to AIDS, for detection of antibodies against retroviruses. 13. Leukemic T-cells CCRF-CEM, which the human T- host lymphotropic retrovirus CBL-1. 14. Insolubilized human T-lymphotropic retrovirus CBL- 1 antigens bound to globulin or to monoclonal anti body, the globulin itself attached to an inert solid Carrier is bound. 15. Insolubilized antigens according to claim 14, characterized characterized in that the globulin from a hu manic patient who is asymptomatic with the AIDS retrovirus is infected, but a normal immunoglobulin has bulin levels.   16. Insolubilized antigens according to claim 14, characterized characterized in that the globulin is a monoclo is a natural antibody that binds retrovirus antigen.