FR2576107A1 - METHOD OF SEARCHING FOR ANTIBODIES AND EQUIPMENT OR KIT FOR THE IMPLEMENTATION OF THIS METHOD - Google Patents

Abstract

THE SEARCH FOR RETROVIRUS ANTIBODIES, FOR EXAMPLE FROM AIDS VIRUS, IS CARRIED OUT ON BLOOD SAMPLES USING AN INSOLUBILIZED ANTIGEN COMPRISING A RETROVIRAL ANTIGENS BOUND TO GLOBULIN AND A CONTAINING A SOLID SUPPORT. ANTIBODY SPECIFIC TO RETROVIRAL ANTIGENS AND WHICH IS MARKED BY A NON-RADIOACTIVE DEVELOPER, THE LIQUID PHASE IS THEN SEPARATED FROM THE SOLID PHASE AND THE DETERMINANTS OF THE DEVELOPER TRACE ASSOCIATED WITH THE LIQUID PHASE AND THE SOLID PHASE ARE DETERMINED.

Description

Antibody search method and equipment or kit

  for the implementation of this process.

  The invention relates to an analytical method for the search for antibodies to retroviruses, as well as to new ones.

  viral isolates usable in this process, and more specifically

  the isolation of a virus linked to the T-lympho- virus

  human trope and its uses as well as its use

  other retroviruses in the analysis of sera for

  determine the presence of retrovirus antibodies, in particular

  especially those associated with immunodeficiency syndrome

acquired (AIDS).

  It has probably appeared in recent years that acquired immunodeficiency syndrome (AIDS) is caused by an infectious agent. Symptoms of this disease are limited to certain well-defined risk groups

  according to a scheme which strongly suggests a transmitted agent-

  susceptible to sexual or blood contact. Although the disease was first detected in the United States of America,

  it is increasingly manifested in other re-

regions, including in Europe.

  One of the means by which the disease is spread is believed to be the transfusion of donated blood from donors who are themselves infected with the AIDS virus. Pooling of donated blood means that if blood supplied by a donor carrying the AIDS virus is pooled with blood samples from other donors, all of the blood and other products derived from that pool can be contaminated, regardless of the sample size. Many blood donors with the AIDS virus are unaware of their contamination, especially in the initial stages, and other infected donors, even if they are aware of this fact, may not to be

  willing to reveal either that they are affected by the mala-

  die, or that they belong to a high risk group.

  A growing demand therefore exists for relative tests.

  Simple but fully reliable, making it possible

  kill on blood samples donated for trans-

  merge a routine search for the presence of the AIDS virus or AIDS virus antibodies. Blood donation

  who give a positive test result can be rejected.

  for transfusion and contamination of other samples

  tillings of blood by pooling with the sample

contaminated can be avoided.

  Research has been actively carried out for some

  two years to identify and isolate the responsible agent

  sand of AIDS, but a disagreement remains between various researchers in this field as for the identity and the precise nomenclature of this one. What was called

  an AIDS virus isolate was first reported

  port in 1983 by Montagnier and his collaborators in France who called it "Lymphadenopathy Associated Virus One" (LAV-1) (Virus Associated with Lymphadenopathy One). Almost a year later, Gallo and his collaborators in the United States published details on the isolation of another virus

  called the AIDS virus which they called "Human T-

  Lymphotropic Virus Type III ", (HTLV-III), (T-Lympho- Virus

Human Type III trope).

  We have now been able to isolate, from a tumor of the lymph node, a human T-lymphotropic retrovirus related to HTLV-III and LAV which we have called CBL-1. Our CBL-l is a stable isolate that can be

  grown in a host cell line and can be used

  read in an analysis for the detection of antibodies to

  AIDS virus in serum samples.

  Consequently, the invention provides a T-lym- retrovirus.

  human photrope CBL-l etiologically linked to AIDS.

  We have found that CBL-1 can be maintained for

  for extended periods of time in a leukemic T cell line called CCRF-CEM and described by Foley et al, Cancer 18, 522529 (1965) and another aspect of the invention provides CCRFCEM cells harboring

our CBL-1 virus.

  For confirmation, we have submitted samples

  tillings of CCRF-CEM cells harboring our CBL-1 virus at the National (now European) Collection of Animal Cell Cultures (NCACC) at the Public Health Laboratory Service Center for Applied Microbiology and Research,

  Porton Down, Salisbury, Wiltshire, SP4 OJG England.

  NCACC (ECACC) has been designated as the International Authority

  National Deposit under the Budapest Treaty of 1977.

  Our deposit was made on January 11, 1985 and received

deposit number 85 01 1101.

  Our CBL-l product was isolated from lymphocytes

  cultured from a biopsy of a tumor of the gan-

  lymphatic glion of a British patient undergoing treatment for an immunoblastic lymphoma associated with AIDS. Fragments of the biopsy were kept in a culture medium to which T cells were added and, after approximately two days, the CCRF-CEM cells were added to the culture which was kept in

  conditions such as primary lymphocytes dis-

  appear over a period of about a month as CCRFCEM cells proliferate and become infected with

  chronically by the CBL-1 virus.

  The CBL-1 virus has been shown to be related to the HTLV-III isolates previously described (a) by reverse transcriptase activity with a preference for Mg ++ cations; (b) by the morphology of the virus particles visualized by electron microscopy; (c) by indirect immunofluorescence specific for viral antigens in infected CCRF-CEM cells

  by CBL-l with sera prepared from pa-

  patients with AIDS; (d) by comparison with HTLV-III and LAV-l in analysis

  radioimmunological (radioimmunoassay - RIA) in solid phase.

  and is thus included in the taxonomic group of Retro-

  viridae containing lymphotropic viruses of human T cells. Preliminary DNA sequence analysis indicates that the sequence of the CBL-1 virus, although closely related to that of the other human Olympophotropic retroviruses described so far, is not identical to that of the products described above, but has some variation of

sequence in various places.

  It has been shown that the infected cells that we have deposited with ECACC continue to synthesize the virus and viral antigens over a period of at least

minus six months.

  Although we have found that CCRF-CEM cells are an ideal host for our CBL-1 virus, we have found that cells can also be used to harbor a similar virus that we isolated from the peripheral blood of the same patient on who

  the lymph node tumor biopsy was pre-

  levee and similar viruses isolated from other patients infected with AIDS-related viruses. We have thus shown that this cell line can provide a useful host for the in vitro culture not only of

  our product CBL-l but other related products.

  According to another aspect of the present invention, we pro-

  let's set up a method for analyzing a biological sample

  for the detection of a retrovirus antibody, characteristic

  in that the biological sample is brought into contact with (1) an insolubilized antigen comprising retroviral antigens linked to a globulin, the globulin itself being linked to an inert solid support; and

  (2) an immunoglobulin which contains a specific antibody

  of retroviral antigens and which is marked with a non-radioactive revealing tracer, and which is separated

  the liquid phase of the solid phase and determines the amount

  developer tracer associated with one or the other

of these phases.

  According to yet another aspect of the invention, there is provided a test equipment or test kit comprising (1) a first component which is an insolubilized antigen comprising retroviral antigens linked to a globulin, the globulin itself being linked to an inert solid support; and

  (2) a second component which is an immunoglobulin containing

  an antibody specific for retroviral antigens

  and marked with a non-radioactive developer.

  As noted above, CBL-1 finds primary use as a component of a test system for the routine monitoring of blood intended for transfusion to determine whether it is from a subject infected with the AIDS virus. We have developed a system based on simultaneous competitive analysis using immobilized CBL-1 antigens or other retroviral antigens such as

  solid phase, and the serum to be tested as a liquid phase.

  More specifically, our simultaneous competitive analysis is based on the immobilization of the CBL-1 antigen or other retroviral antigen by its binding to a solid phase, by means of a ligand, for example a globulin or another antibody, and allowing to the binding sites of

  this immobilized antigen to be the subject of a competi-

  tion from a mixture of a serum antibody to be tested and a known antibody to the AIDS virus which can be identified by the revealing agent. We have found that the most satisfactory results are

  obtained when the revealing agent is an enzyme tracer.

  The use of an enzyme tracer rather than a radioactive tracer has the advantage of eliminating the biological risk of radiation and an unexpected consequence

  is that it reduces the immune incubation of the es

sai an hour or less.

  Another possibility is to use immunofluorescence

  to reveal the presence of the known antibody linked to the anti

immobilized gene.

  As noted above, we found that the best

  their results are obtained when the antigen is attached to the solid phase via a ligand which is an antibody. One technique is to use a serum

  high titer human taken from someone who is conta-

  undermined asymptomatically by the AIDS virus. The ideal serum has a high titer of antibodies to the virus

  from AIDS but comes from a patient whose immune levels

  noglobulins are in the normal range.-Another approach is to use a monoclonal antibody against the retrovirus. If this is grown conventionally using mice, some benefits can be obtained as will be described in more detail below. The solid phase material is suitably polystyrene which can be used in the form of tubes,

  pearls or preferably plates presenting a-multipli-

  city-surface wells or other surface cavities.

  The gamma globulin prepared from the chosen serum, or other antibody, is then spread over the polystyrene,

  then the antigen is linked to the polystyrene coated with globu-

  line to form the first component of the test. This compound

  health can be kept wet or dry for

several months.

  The second component of the test comprises the iso- IgG fraction

  linked from the test serum and, when it is planned to reveal by means of an enzyme tracer, this IgG fraction can be conjugated with, for example, peroxidase

  horseradish (HRPO) according to known techniques.

  The test equipment according to the invention can be used by mixing the labeled IgG component with the serum to be tested and then putting the mixture of IgG and serum

  to be tested in contact with the insolubilized antigen, separately

  rant from one another the solid and liquid phases and

  by determining to what degree the IgG has bound to the antigen.

  According to concurrent competitive analyzes, the more the IgG component of the liquid phase is bound to the phase

  solid, the lower the amount of antibody or anti

  genes in the test serum. By choosing appropriate control values, it is thus possible to have a routine test in which, if HRPO is used

  as a revealing agent, the serum to be tested can be considered

  dere as free from AIDS antibodies or antigens as soon as the optical density (OD) of the test sample reaches a predetermined minimum level. Any sample of serum 8 to be tested which gives rise to the recording of an OD value below the predetermined level can therefore be rejected, thus avoiding possible contamination of a blood donation to be transfused with a virus or

an AIDS antibody.

  Other retroviruses can be used as an alternative to CBL-1 as retroviral antigens in

  the method of analysis and in the diag test equipment

  nostic of the invention. It can be other retro-

  human viruses etiologically linked to AIDS such as

  Human T-lymphotropic retrovirus HTLV-III or other retro-

  human viruses such as leukemic virus HTLV-II or HTLV-I, or animal retroviruses such as

  maedi-visna or that of bovine leukosis.

  As will be described in more detail below, the indirect binding of human retroviral antigens to the solid phase via globulin provides a certain amplification of the results and the test formula allows a simple, rapid and precise determination of the presence or absence of contamination in blood samples by relatively unskilled technicians

and inexperienced.

  The invention will be better understood thanks to the exemplary embodiments described below by way of illustration, and to the appended drawings, in which: FIG. 1 represents an electron micrograph at magnification of 81,000 showing particles of

  CBL-1 that detach from CCRF-CEM host cells and if-

  killed at the limit of these; - Figure 2 shows the effectiveness of ELISA (test known as "Enzyme Linked Immuno-Sorbent Assay", Analysis by immunoabsorption with enzyme binding) in solid phase for anti-HTLV III. The figure presents the optical densities obtained by testing a certain number of clinical sera, some reactive and others not reactive for anti-HTLV III, results known in advance by radioimmunological analysis; - Figure 3 shows data similar to that of

  Figure 2, but this time for anti-HTLV I, deter-

  undermined by a competitive one step ELISA

similar to the previous one.

EXAMPLE 1.

  ISOLATION AND CHARACTERIZATION OF CBL-1.

  The CBL-1 virus was isolated from cultured lymphocytes

  ves from a lymph node tumor biopsy

  of a British patient undergoing treatment

  are for immunoblastic lymphoma associated with acquired immunodeficiency syndrome (AIDS). The lymph node biopsy was cut into small fragments and placed in a culture medium consisting of RPMI-1640 base medium supplemented with 10% fetal calf serum and 10 µg / ml phytohemagglutinin. After 24 hours, conditioned medium containing T cell growth factor harvested from cultures was added.

  mixed tonsil lymphocytes, and the phyto-

  hemagglutinin. 48 hours after the start of the

  culture, interferon alpha antiserum was added.

  CCRF-CEM cells (called -

  see below CEM cells). CEM cells are a constantly growing leukemic T cell line

  described by Foley et al (1965) Cancer 18; 522-9.

  Under these conditions of common culture the primary lymphocytes disappeared in four weeks of culture while the CEM cells proliferated exponentially. The CBL-1 virus produced by primary lymphocytes infected CEM cells and after eight weeks in culture the CEM cells were chronically infected with the CBL-1 virus. Maintaining CEM cells infected with CBL-1 did not require T cell growth factor or anti-alpha interferon. These CEM cells infected with CBL-1 were deposited as indicated more

  top with the NCACC under the deposit number 85 01 1101.

  CBL-1 virus has been shown to be related to isolates

  HTLV-III / LAV previously described (a) by an acti-

  speed of reverse transcriptase with a preference for the cations Mg ++, by the morphology of the particles of

  viruses visualized by electron microscopy (see fi-

  gure 1) and (b) by specific indirect immunofluorescence

  than for viral antigens in infected CEM cells

  CBL-1 with sera prepared from AIDS patients and by comparison with a

  another isolate of AIDS virus in RIA in solid phase.

  CEM / CBL-1 cells continued to synthesize the

  viruses and viral antigens over a period of six months.

EXAMPLE 2

1. Preparation of the antigen.

  A raw lysate of frozen-thawed cells was prepared

  as follows. HT-H9 cells were left infected

  ted by HTLV-IIIB grow at maximum density in suspension culture. The cultures were cooled to 4 ° C and 0.25% v / v f-propiolactone was added for two hours. We then separated the culture fluid and

  agglomerated the cells by centrifugation at low speed.

  The cell pellet was resuspended in distilled water and subjected to three freeze / thaw cycles. At each cycle, the residue from the cell pellet was carefully resuspended. After the third cycle,

  the extract was brought to 0.25% vol / vol with -propiolac-

  tone and kept at 4 C for two hours; we

  then warmed it to 37 C for half an hour. Pen-

  During this time, the pH was maintained in the region of

  7.4. The crude lysate was clarified and stored at -20 C.

  Before use, it was diluted in TRIS buffer (2-

  amino-2-hydroxymethyl-1,3 propanediol) containing 0.1% bovine serum albumin (BSA) and detergent. The choice of detergent and its concentration were important and an optimum was observed by the use of Tween 20

  at 0.1% concentration, amplifying the activity of the pre-

  paration of antigens by a factor of three or sometimes even more. After dilution in TRIS BSA / Tween 20, the antigen was incubated for 30 minutes at 37 C. It is at this stage

  that there is an increase in the activity of antigens.

  In this way, the antigen preparation can be used.

  to monitor the expression of the HTLV-III antigen

  under various defined culture conditions. The anti

  genicity can be quantified by RIA in solid phase using anti-HTLV-III in solid phase and a second reagent, comprising anti-HTLV-III marked with I. In the final analysis, the antigen has been used at a dilution that allows a P: N ratio of 10: 1 with

  1 to 2% binding to the tracer in the presence of negative sera

  tifs (see analysis method below). Variable loss of viral antigens in the supernatant fluid was observed on the cells but in relative terms the mass of detectable antigen remained in the cell pellet. This

  is true for CBL-1 and other HTLV- isolates

  III carried in CEM cells or HT / H9 cells.

  There are no constraints on the purity of the antigen

  (see below) and the ex-

  pressure of the antigen in cells. The preparation

  of antigen used here is easy to manufacture, likely-

  higher yield per volume of cell culture

  laire that the antigen prepared from the supernatant fluid and appears stable at -200C. It can easily be

  inactivated by la'n-propiolactOne. We can also expect

  tend to reduce the use of Tween 20

  the title of any infectious virus.

2. Antisera.

  The reagent for the analysis was prepared from sera

  high titer humans taken from infected people

  asymptomatically undermined by the AIDS retrovirus.

  The selection of sera for the preparation of the reagent is important. Serum had high anti-HTLV-III titer

  by RIA but came from a patient whose immunity levels

  noglobulins were in the normal range. In practice, it is usually necessary to prepare reagents from a small number of sera meeting the above criteria and to test the effectiveness of the reagents for analysis. In order to make these reagents non-infectious, the starting sera were treated by heating at 56 ° C.

during 30 minutes.

  (a) Preparation of anti-HTLV-IIIB globulin. This product was used to purify the HTLV-IIIB antigen in situ

  on a solid phase. This strategy leads to

  considerable tages. The globulin from the treated serum was precipitated twice by ammonium sulfate at% of saturation. It is the simplest reagent for recovery with globulin although it is possible to use either whole serum or IgG

  purified. Globulin was used at an optimal dilution.

  male that had to be determined by individual titration

  of the reagent. The solid phase material was polysty-

  reins as a sink; it was coated with globulin and then the excess binding sites were neutralized with an inert protein - in this case 0.1% bovine serum albumin (BSA). The solid phase covered

  and neutralized was kept wet at 4 C for

  for months; drying gives a more stable product.

  For recovery of the solid phase, the HT-H9 / HTLVIIIB lysate described above was diluted in the buffer.

  TRIS / BSA / Tween to a dilution which, in subsequent tests

  quents, allowed the binding of 1 to 2% of the tracer with a negative serum. The recovery was carried out in the most efficient way by a two-day incubation of antigen at room temperature in the solid phase followed by storage in a wet state at 4 ° C. until use. Preliminary experiments have shown that the solid phase was stable in this form for several months. However, it can also be dried without significant loss of activity and remain stable for

several months.

(b) Labeling of globulin.

  The IgG described above in 2 (a) was conjugated with HRPO using heterobifunctional derivatives in the usual manner to provide a molar substitution ratio of 1: 3. This product was used in combination with the solid phase described in 2 (a) above in a

  enzyme immunoassay for the detection of

  AIDS retrovirus body in body fluid samples. For comparison, another IgG sample was labeled with 125I at a specific activity of 15

i / Ci per pg of protein.

EXAMPLE 3.

  Competitive one-step ELISA for anti-CEM / CBL 1.

Serum reagents.

  We used a human serum containing a high titer

  anti-HTLV-III for the preparation of coated globulin

  strictly as described previously in Example 2.2.

  Polystyrene wells having a round bottom configuration were covered with an optimal dilution of globulin, then they were neutralized with an inert protein, in this case 0.1% BSA. The same serum was used as the source of immunoglobulin, which was purified by ion exchange chromatography on DE 52 as previously described. IgG has been coupled with

  horseradish peroxidase to an approximate degree of substitution

  matif of three molecules of horseradish peroxidase for

an IgG molecule.

  The efficiency of the conjugate was determined by incubation with respect to previously covered wells, by

  through an indirect antibody, through anti

  viral genes from cells infected with HTLV-III and by cells not infected with antigens. We considered as optimal dilution of the conjugate that which gave the maximum colored bond with the antigen

  infected cells and maximum color production

  male with uninfected cells.

CEM / CBL antigen 1.

  Cells persistently infected with CEM / CBL 1 virus were grown to maximum density

  in non-adherent shaking cultures. The cul-

  tures with stirring were cooled to 4 ° C. and then brought to 0.25% by volume with P-propiolactone. After two hours of incubation at 4 ° C., the cell pellet was harvested and subjected to freeze / thaw cycles, then clarified as above by centrifugation. Culture extract

  tissue was then treated for a new incubation

  tion for two hours at 4 ° C. with 0.25% of '-propiolactone and then hydrolyzed as above. In this case, the diluent for cell bursting and cycling

  freeze / thaw was physiological buffered phos-

  phate, supplemented with 0.1% Tween 20. We then titrated

  antigen preparations on a solid phase coated

  anti-HTLV-III high titer globulin green, and

  used as working dilution for subsequent tests

  quents the dilution giving an OD at 450 nm between

* 1.0 and 1.2 under the conditions defined below.

Optimization and test formula.

  Wells covered with high titer globulin, and then neutralized, were incubated with 100 µl of the dilution

  optimal CEM / CBL 1 antigen.

  tion at room temperature, the anti-extract was removed

  gene by washing the wells and drying them under conditions

  which have been found to promote the stability of immobilized CEM / CBL 1 viral antigens. For the assay, 25 µl of antibody positive and antibody negative sera were placed in separate wells, and 75 µl of ELISA conjugate was added to the wells in distilled water supplemented with detergent. The mixture of test serum and conjugate (immune incubation) was incubated in the wells

  for varying 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 two minutes at room temperature, after which the procedure

  was repeated with three wash cycles. In-

  subsequently, 100 ivl of chromogenic substrate was added to

  in this case 3.3 ', 5.5'tetramethylbenzidine (TMB).

  After twenty minutes of incubation at room temperature, the chromogenic reaction was stopped by adding four times normal sulfuric acid. We measured

  color formation by spectrophotometry at 450 nm.

  We considered as working dilution for each anti

  particular CEM / CBL 1 gene that which reliably gave an optical density between 1 and 1.2 with a negative serum when used in ELISA in

  optimal conditions, defined as an incubation

one hour immune response at 450C.

  Results. The comparison between the results obtained in a conventional RIA using the solid phase and an IgG labeled with 1251 described in Example 2 and the ELISA results obtained in the present example demonstrated two unexpected advantages in favor of ELISA. First, suboptimal antigen preparations which gave

  poor RIA results have been able to pro-

  in the same way, give good results in ELISA. Second, as illustrated in Table 1, it has

  not been possible with the RIA to reduce incubation times

  tion within three hours. There is a need for rapid research, one to two hours in total, in transfusion preparations, and this was only possible with ELISA.

TABLE 1

  Variable time and temperature experiments in RIA with

IgG labeled with 125I.

  Control sera Time in hours Temperature_ S r m _ _on Negative Cutoff Positive

18) 1.13 0.28 0.08

3) TA 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) 450C 0.40 0.14 0.07

1) 0.27 0.13 0.05

*% of entry level binding.

  Figure 2 shows the ELISA test results of

  56 clinical sera, in this case from hemo-

  philes treated with commercial concentrates and not

  Factor VIII and homosexuals. It can be seen in the figure that a clear separation has been made between the sera reactive for anti-CEM / CBL 1 and the non-reactive sera. In this particular example, the average optical density of 16 sera reactive for the CEM / CBL 1 antibody was 0.085 (0.037 to 0.327) and the density

  average optics of 40 sera not reactive for this anti

  body was 1.16 (0.736 to 1.352). The serum a was obtained from an AIDS patient who was convicted and was poorly reactive to immunofluorescence and direct binding tests. Sera b and c were non-reactive during a

new test.

  The exact same formula was used in one study

  on Blood Transfusion sera in the United Kingdom.

  To date, more than 12,000 donor sera have been tested. Three sera gave reactions to the tests

  screening. However, when retried, a don-

  only a single neur was found to be reactive. Serum was repeatedly reactive, titratable, and exhibited strong immunofluorescence reactivity towards

  infected cells but not uninfected cells.

  Random reactivity of sera not linked to infection

  by CEM / CBL 1 was therefore extremely low.

  Subsequent titration experiments for antiHTLV-III reactive sera demonstrated that the titers were roughly the same in the ELISA CEM / CBL 1 assays

  and direct analyzes. This was confirmed by the exa-

  men later of sera taken from patients undergoing

  an acute seroconversion following a primary infection with HTVL-III. Again there was no

  big no difference in level or reaction time

  initial activity between direct link analysis and competitive ELISA. Using the same test method,

  patient sera containing anti-lympho- antibodies

  oligo and pan-reactive cytes, and patient sera

  with autoimmune disease, have not been reacted

  tifs in competitive ELISA. Considered together these

  data indicate specificity and high sensitivity

  of this competitive ELISA for the antibody of the retro-

AIDS-related virus.

EXAMPLE 4.

  Competitive one-step ELISA for anti-HTLV-I Sera and reagents were selected and prepared in the same way as for the immunological analysis of CEM / CBL i in Example 3, except that used

  cells infected with HTLV-I for the preparation of anti

  gene and sera from patients contaminated with HTLV-I as

source of reagents.

  Figure 3 shows the optical density provided by 32 sera

  clinical trials, including three known low-dose controls

  tifs. A clear differentiation was manifested between reactive and non-reactive sera, the average optical density of five reactive sera for this antibody being 0.099 (0.060 to 0.129) and the average optical density of 24 non-reactive sera for this antibody being 1.64 (1.48 to 1.83). The test is robust and the conditions can be varied, but in practice we have decided to adopt as an optimum one hour immune incubation at 45 C. This corresponds to the conditions adopted for the detection of

CEM / CBL antibody 1.

  The same test formula was used to screen more than 1,000 non-standard British blood donors and none

  reactive serum has not been identified. However, a

  selective selection of some 75 donors of African origin

  caine et caribbean helped identify a single donor

  HIV positive giving for the first time.

  The procedure described above was repeated but in use

  reading sera from patients infected with HTLV-II as

  source of recovery globulin and as source of IgG.

  Cells infected with HTLV-II were used as

  source of antigen. These reagents were chosen and pre-

  dressed in the same way as previously described in the example

  ple 2 for the anti-HTLV-III RIA.

EXAMPLE 5

  Production and use of test equipment.

  1. Reagents At least the internal surface of the wells was covered with a multi-well polystyrene plate containing 96 wells of a purified human antibody to CBL-1. The antibody came from a heat treated serum from a human infected with the AIDS retrovirus. After covering the polystyrene with the antibody, the residual binding sites were neutralized with an inert protein, in this case bovine serum albumin. The antigen used for insolubilization on the plates of polystyrene was CBL-1. This was first treated with F propiolactone viricide and a suspension of inactivated CBL-1

  in TRIS / BSA / Tween buffer was incubated with poly-

  styrene treated for about two days at room temperature. In this way, the CBL-1 was dissolved by

  indirect bond to the polystyrene support via the

  medium of the antibody. This component is the first component

health testing equipment.

  The second component of the test equipment was prepared by labeling the same human antibody which was used to coat the polystyrene as described above. The antibody was labeled by conjugation with peroxidase

  horseradish (HRPO) using conjugation techniques

  its classics. This HRPO-labeled antibody was kept in a lyophilized state and reconstituted immediately before use using a suspension in a

aqueous thinner.

  In addition to the two components mentioned above, it is

  desirable to include in the test equipment

  auxiliary devices so as to make all available

  the necessary development and control reagents.

  For equipment using HRPO as a tracer, it is convenient to use 3,3 ', 5,5' -tetramethylbenzidine

257610?

  (TMB) as a substrate for the enzyme; the TMB has been

  used in tablet form, each tablet provides

  sufficient substrate for 96 wells. Immediately before use, the TMB tablets were dissolved in an aqueous solution containing trisodium citrate and hydrogen peroxide. It is also desirable to provide positive and negative control sera. The negative control serum was normal human serum not reactive for the antibody to CLB-1 in this test. The control serum

  positive was a heat treated human serum, reacted

  tif for the CBL-1 antibody in the test procedure.

  In addition, it is desirable to provide, as cut-off control serum, a human serum treated with reactive heat (at cut-off level in the test) for the antibody to CBL-1 and also a washing fluid comprising serum.

physiological and Tween 20.

  2. Use of test equipment.

  The HRPO-labeled antibody was reconstituted immediately-

  before use. The test was carried out using

  25 microliters of serum sample per well.

  Two wells received a negative control serum and one a positive control serum, three a cut-off control serum and the test samples were introduced into the remaining wells. 75 microliters of the reconstituted HRPO-labeled antibody were introduced into each

  well, then we covered the well plate and incubated it

  open on a heating block at 45 for one hour, or alternatively at 20 to 25 ° C overnight for an incubation of at least 16 hours. At the end of the incubation period, the plate was washed and added to each well

  portions of 100 microliters of substrate solution.

  The plate was then incubated for 20 minutes at 18-25 C, after which a blue color developed in the wells containing the negative samples. Portions of 50 microliters of 2M sulfuric acid were then added to each well, the blue color becoming

2576T107

  then yellow. The absorbance of each well was then read at 450 nm, within 30 minutes of adding the acid.

  sulfuric, using a micro plate reader

  well. 3. Results The results were evaluated as follows: the mean absorbance of the three control sera was calculated

  cutoff. We then considered negative for the anti

  body of the AIDS retrovirus, within the limits of the test system, any sample with equal absorbance

  or higher than the average value of the cut-off indicator.

  Any sample with an absorbance below the average of the cut-off indicator or up to 10% above

  of it has been tested again.

  Any serum that appears repeatedly positive in this test has been retested for confirmation

  using immunofluorescence or the separation method

  ration by electrophoresis and transfer known as

"Western Blot" designation.

  The procedure described above has been tested with 1600 routine samples from blood bank donors with negative results. However, when we tested

  other clinical specimens from patients with

  both a clinical diagnosis of AIDS, of para-AIDS (Aids-

  relatex complex) or other diseases, positive results have been detected which in almost all cases was confirmed by the immunofluorescence test and another enzyme immunoassay. We got

  the results shown in Table 2.

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TABLE 2

  Reactivity of sera from blood donors and patients with AIDS, AIDS-related conditions and other diseases. Positive for Positive for Number Antibody Antibody Clinical sample group with test confirmation

Blood donors 1600 0 -

AIDS 4 4 4

  para-AIDS 39 39 39 High-risk groups 7 7 7 Other diseasesb Mononucleosis

infectious 48 0 -

  Hepatitis B 25 3c 2 Syphilis 117 2 2

Rheumatoid arthritis

milk 9 O Other infectionsd 37 O

  aConfirmation tests include immunofluores-

  cence and an alternative enzyme immunoassay.

  b Only positive samples confirmed.

  A positive sample to two achievements of the test on

  four, negative upon confirmation.

  dIncludes rubella (18 cases), parvovirus (9), cytomegalovirus

(5), toxoplasmosis (5).

  The principle of using a human antiserum to bind a viral antigen to the solid phase gives a solid phase which itself reflects the pattern of viral antigens

  best recognized by humans. This may be before-

  gous because there will then be a good adaptation between the mixture of antigens captured and the profile of human antibodies. In addition, the mild conditions used for the preparation of antigens may allow the preservation of delicate conformational epitopes not shown in

  gradient purified and exploded preparations used

  read in the other solid phase tests. It is now possible to adjust the mixture of solid phase antigens by covering the solid phase with a murine monoclonal antibody of defined specificity so that the

  immobilized antigens have a specific predeter-

  undermined. The data provided here demonstrates the superiority

  of the test when a selected monoclonal antibody reacts

  Health with p25 (a 25 K Dalton protein) is used on the solid phase. The advantages relate to the quantity

  lower antigen required for proper research

  table (Table A) and on the increased sensitivity of the resulting assay, see Table B, which shows that endpoint dilutions of individual sera give greater inhibition (i.e., lower DO reading )

  in the monoclonal test than in the polyclonal test. August

  Mention of sensitivity made the assay more effective in detecting early production of antibodies compared to many other assays. In addition, the use of a murine antibody on the solid phase avoids the potential problem of cross-linking by rheumatoid-type factors between human IgG of the solid phase and the human IgG conjugate in homologous trials. In practice, a small number of sera have been identified which regularly exhibit a higher degree of inhibition in the monoclonal test compared to the polyclonal test. This phenomenon is apparently due to the reactivity of low level and of low titer which binds the conjugate to the solid phase in a non-specific way in the homologous tests only. This or a similar phenomenon may also explain another unexpected finding, namely that the distribution of reactivity in optical density of normal sera is much narrower in the monoclonal test than in a polyclonal test carried out simultaneously. Given the simplicity found in the use of a monoclonal antibody for a retroviral gag gene product, essentially providing a competitive assay for the human anti-HTLV-III response against the nucleus, it is likely that the use of antibodies for defined env products will allow the development of tests

  analogs for antibodies against antigens of

  selected loppes. This will allow a much more precise study of the human response to different antigens.

  which may be of clinical importance

  ques, prognostics and infection control.

TABLE A

  Titration of the CEM / CBL 1 antigen on solid phases

polyclonal and monoclonal.

  Dilution Coating of the solid phase of the antigen of the Polyclonal Monoclonal antigen * Neg.Cou- Positive Neg C for pure pure

1.34 ** 0.69 0.05 NT NT NT

1.14 0.45 0.03 1.85 0.86 0.11

0.82 0.45 0.03 NT NT NT

NT NT NT 1.50 0.37 0.09

NT NT NT 1.36 0.38 0.08

NT NT NT 1.23 0.28 0.07

100 NT NT NT 1.18 *** 0.27 0.07

  * Cut - weak positive control ** OD at 450 nm under standard test conditions for anti-CEM-CBL-1 *** "Working" title of the antigen preparation NT = Not tested

TABLE B

  OD at 450 nm of dilutions of 19 sera titrated at 50% of the final point of inhibition, tested on polyclo- solid phases

nales and monoclonals.

  Coating of the solid phase Polyclonal Monoclonal Serum Positive control 0.08 0.09 Cutoff 0.76 0.55 Negative 1.39 1.42 Hemophiliac -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

  (lymphadenopathy 2 0.84 0.58 general 3 0.73 0.56 persistent) 4 0.60 0. 48 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 test is carried out by a

  determination of tracer associated with the solid phase. However

  dant, the level of tracer associated with the starting serum being

  or can be determined, it is also possible to monitor

  The reduction in radioactive count or level of enzyme associated with the liquid phase after the known labeled sera and the tested sera have been brought into contact with the solid phase antigen, and the reduction in tracer level.

  the liquid phase used as a measure of the anti-

body of sera tested.

  The advantages of concurrent competitive testing can be

summarized under the following headings.

Antigen preparation.

  Although Example 2 describes the use of purified HTLV-III viruses, the increased binding resulting from the use of an immobilized antibody makes it possible to use for recovery a preparation of crude cell lysate when such a

  antigen is not by itself capable of direct binding

  usefully in a solid phase. This measure sup-

  takes precedence over the constraint constituted by the need for an anti

  highly purified gene for the production of diag-

  nostic and makes their manufacture easier. This advantage is not applicable to direct research in which a coating of whole human globulin would give a signal

  inevitable with the ultimate anti-human globulin tracer.

Test formula.

  Using an undiluted volume of serum offers an advantage

  considerable tage at blood centers where the need for an initial dilution would increase the workload. The ratio between the volumes of serum and enzyme tracer can vary from 1: 1 to 1:10 with only a slight deterioration in the efficiency of the test. The optimum is 1: 3 with 25 / 1l of serum and 75/1 of enzyme tracer, which

  provides good differentiation between positive sera

  tives and negatives. Test times can be reduced

  an hour or less using an ELISA technique.

  Sensitivity A measure of the sensitivity of any test for the detection of AIDS antibodies is given by the proportion of sera collected from condemned AIDS patients which remain positive. With direct testing, it seems that a

  variable number of patients lose responsiveness for anti

  body. This does not happen with competitive testing, which suggests that the sensitivity of competitive testing is

  as good or better than other popular tests

ment used.

  Specificity There is no reason to expect a competitive test

  presents the major problem of falsely positive reactivity

  Fri The inclusion of lymphocyte membrane antigens in the envelope of HTLV virions certainly leads to a false

  reactivity, as well as the presence of IgG aggregates adhered

  rant non-specifically to the solid phase. Such a pheno-

  leads does not give rise to a reactivity in the comprehensive test

  touchy. Non-specific effects of a variable protein concentration can be minimized by choosing an appropriate serum / tracer ratio (1: 3, see above) and by using a cutoff value greater than 50%. inhibition. Sera which produce an inhibition of tracer binding equal to or greater than that provided by an internal control serum are considered positive for the screening test but the test must be repeated. Given the low level of false reactivity, expected unless

  of l per 5,000, it is unlikely that false reactions

  positive in competitive ELISA will create a problem numerically as big as they seem to do in direct testing. This is particularly so when direct tests are performed without a control antigen. We

  expects this to be the case in transit centers

  fusion of the fact that the use of a double-

  the amounts of reagent required in the direct tests.

  In summary, the combination of a simple formula, a sensi-

  high bility and high specificity makes the test com-

  ideal petitive for screening blood donations and diagnostic use. In addition, when the centers use a direct test for anti-HTLV-III, it may be preferable to confirm the reactivity of the serum by a new test in the competitive test rather than by submitting the sera to the " Western Blot ", more laborious and less sensitive, which can be recommended by Food

  and Drug Administration in the United States.

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Claims (18)

Claims.   1. Method for analyzing a biological sample for the search for a retrovirus antibody, characterized in that the biological sample is brought into contact with (1) an insolubilized antigen comprising retroviral antigens linked to a globulin , the globulin itself being linked to an inert solid support; and   (2) an immunoglobulin which contains a specific antibody   of retroviral antigens and which is marked with a non-radioactive tracer, and which is separated   the liquid phase of the solid phase and determines the amount   developer tracer associated with one or the other of these phases.   2. Method according to claim 1, characterized in that e   that the retrovirus is the human T-lymphotropic retrovirus hand CBL-l.   3. Method according to claim 1, characterized in that   that the retrovirus is the human T-lymphotropic retrovirus   hand HTLV-III, the human T-leukemia retrovirus HTLV-II   or the human T-leukemic retrovirus HTLV-I.   4. Method according to one of the preceding claims,   characterized in that the biological sample is a human blood plasma or serum.   5. Method according to claim 4, characterized in that   that the retrovirus is the human T-lymphotropic retrovirus   hand CBL-1 or the human T-lymphotropic retrovirus HTLV-III,   and in that the revealed agent immunoglobulin   is an immunoglobulin from a human patient asymptomatically infected with the AIDS virus but   having a normal immunoglobulin level.   6. Method according to one of the preceding claims, characterized in that the revealing tracer is an enzyme tracer.   7; Method according to one of the preceding claims,   characterized in that the globulin bound to the support is   a monoclonal antibody - suitable for fixing - an antigen -retro-   viral. 8. Equipment or test kit comprising (1) a first component which is an insolubilized antigen comprising retroviral antigens linked to a globulin, the globulin itself being linked to an inert solid support; and   (2) a second component which is an immunoglobulin containing   an antibody specific for retroviral antigens   and marked with a non-radioactive developer.   9. Equipment according to claim 8, characterized in that the retrovirus is the human T-lymphotropic retrovirus CBL-1. 10. Equipment according to claim 8, characterized in that the retrovirus is the human T-lymphotropic retrovirus HTLV-III, the human T-leukemic retrovirus HTLV-II or   the human T-leukemic retrovirus HTLV-I.   11. Equipment according to claim 8, characterized in that the retrovirus is the human T-lymphotropic retrovirus CBL-l or the human T-lymphotropic retrovirus HTLV-III and in that the immunoglobulin comes from a human patient asymptomatically contaminated with the AIDS retrovirus   but having a normal immunoglobulin level.   12. Equipment according to one of claims 8 to 11,   characterized in that the revealing tracer is an enzyme tracer.   13. Equipment according to one of claims 8 to 12,   characterized in that the globulin bound to the support is   a monoclonal antibody capable of fixing a retro- viral.   14. Human T-lymphotropic CBL-1 retrovirus logically to AIDS.   15. CCRF-CEM T-leukemia cells harboring retro- CBL-1 human T-lymphotropic virus.   16. Insolubilized human T-lymphotropic CBL-1 retroviral antigens linked to a globulin, the globulin itself   being bound to an inert solid support.   17. insolubilized antigens according to claim 16, characterized in that the globulin comes from a human patient asymptomatically contaminated with the retrovirus   of AIDS but having a normal level of immunoglobulin.   18. insolubilized antigens according to claim 16,   characterized in that globulin is a mono- antibody   clonal proper to fix a retroviral antigen.