DETECION OF HUMAN T-CELL LEUKEMIA VIRUS TYPE III

Abstract: This invention discloses a method for detecting anti­ bodies to human T-cell leukemia virus Type III (HTLV-III), the virus associated with Acquired Immune Deficiency Syn­ drome (AIDS). The method involves use of a peroxidase labeled immunoglobulin which specifically binds to a com­ patible anti-human immunoglobulin previously bound to NTLV-III antibodies in test sera.

Description: The present invention is an enzyme linked immunoassay for the detection and measurement of small quantities of HTLV-III (AIDS). The assay process involves incubating human test sera suspected of containing AIDS human virus antibodies (HTLV-III antibodies) with HTLV-III virus protein bound to an insoluble immunosorbent. The amount of human immunoglobulin bound to the HTLV-III proteins is determined by first reacting said human antibodies with antibody against human immunoglobulins followed by reaction with a complex consisting of purified human immunoglobulin (1g) conjugated to peroxidase. The human 1g specifically binds to the HTLV-III antigens. The presence and measurement of test sera is determined by the addition of a hydrogen peroxidase solution which reacts with the peroxidase enzyme to form a peroxidase color reaction. In this invention it is noted that the imnunoglobulin bonded to peroxidase is human immunoglobulin. Evidence suggests that the retrovirus(es) of the HTLV family is an etiological agent of acquired immune deficiency syndrome (AIDS) based on the following: (1) there is precedence for an animal retrovirus cause of immune deficiency (feline leukemia virus in cats); (2) retroviruses of the HTLV family are T-cell tropic; (3) they preferentially infect "helper" T-cells (OKT4⁺); (4) they have cytopathic effects on various human and mammalian cells as demonstrated by their induction of cell syncytia formation; (5) they can alter some T-cell functions; (6) in some cases infection may result in selective T-cell killing; and (7) they are transmitted by intimate contact or through blood products. The presence of antibodies directed to cell membrane antigens of HTLV infected cells has been shown in sera of more than 40% of patients with AIDS [Essex et al., Science , 220:859 (1983)]. This antigen has since been defined as part of the envelope of HTLV. The original detection and isolation of the various HTLV isolates were made possible by two earlier developments: the discovery of T-cell growth factor (TCGF), also called Interleukin 2 (I1-2), which enabled the routine selective growth of different subsets of normal and neoplastic mature T-cells [Ruscetti, et al., J. Immunol ., 119:131 (1977); and Poiesz, et al., Proc. Nat. Acad. Sci. USA , 77:6134 (1980)] and the development of sensitive assays for detection of retroviruses based on reverse transcriptase assays. The methods of HTLV isolation and transmission involved a cocultivation procedure using permissive T-cells for the virus. The use of normal human T-cells in cocultivation experiments preferentially yielded HTLV of both subgroups with immortalizing (transforming) capability for some of the target T-cells. Disclosure of these discoveries is found in Gallo, et al., US-A-4,520,113 (publ. 28.05.85) and Gallo, et al., WO 85/04897 (publ. 07.11.85). Parallel references are Popovic, et al., "Detection, Isolation, and Continuous Production of Cytopathic Retroviruses (HTLV-III) from Patients with AIDS and Pre-AIDS," Science , 224:497, May 4, 1984; Schupbach, et al., "Serological Analysis of a Subgroup of Human T-Lymphotropic Retroviruses (HTLV-III) Associated with AIDS," Science , 224:503, May 4, 1984; and Sarngadharan, et al., "Antibodies Reactive with Human T-Lymphotropic Retroviruses (HTLV-III) in the Serum of Patients with AIDS," Science , 224:506, May 4, 1984. Other methods of detecting, isolating, and purifying HTLV-III virus or HTLV-III antibodies are disclosed in Gallo, et al., WO 85/04903 (publ. 07.11.85). "The Lancet" 30th June 1984, page 1438-1440 discloses a method for the determination of AIDS antigens by an indirect ELISA method in which bound AIDS antibodies are reacted with peroxidase-labelled goat anti-human immunoglobulin. The use of unlabelled anti-human immunoglobulin is not discussed. US-A-4,235,960 relates to a competitive enzyme-linked immunoassay by an ELISA method using two antibodies, wherein a first antibody is linked to a labelled antibody of the same species via a second bridging antibody against the first antibody and the labelled antibody. GB-A-2,114,289 relates to indirect methods for measuring bound antibodies. However this immunoassay is for class specific immunoglobulin antibodies, and is not directed to improved AIDS assays of high sensitivity. Summary of the Invention The present invention is another step in the discoveries leading to a therapeutic reagent for acquired immune deficiency syndrome (AIDS). Furthermore, the present invention represents an additional refinement in AIDS detection techniques. In a first aspect of the invention there is provided a method for assaying to determine the amount of antibodies to human T-cell leukemia virus type III present, characterised in that: (a) AIDS virus antibodies present in the sample are bonded to HTLV-III antigens linked to an insoluble immunosorbent and are incubated with anti-human immunoglobulin; (b) peroxidase-labelled human Ig is added; (c) a colour former is added which reacts with the peroxidase; and (d) the amount of HTLV-III antibodies is indirectly measured by measuring activity of said peroxidase. In a second aspect of the invention there is provided a method for assaying to determine the amount of antibodies to human T-cell leukemia virus type III present in a sample solution characterised in that (a) HTLV-III- antibodies present in the sample are linked to an insoluble solid immunosorbent (b) anti-human immunoglobulin is reacted with said antibodies; (c) said anti-human immunoglobulin which is bound to said antibodies is further reacted with peroxidase-labelled human immunoglobulin, said human immunoglobulin specifically binding to said anti-human immunoglobulin; (d) colour former is added which specifically reacts with said peroxidase; and (e) activity of the peroxidase is measured in order to indirectly measure the amount of HTLV-III antibodies present in said sample solution. In the process of the present invention it is of interest that the peroxidase enzyme is utilized in amounts which are termed greater enzymatic amounts and a preferred amount is to use a moderate excess of peroxidase which can be added to the immune material. The Ig component of the process is selected from any available Ig antibody such as IgA, IgD, IgE, IgG, and IgM. A preferred temperature for the process is in the cold and specifically at or about 1°C. A range of 1-50°C may be tolerated. A preferred peroxidase is horseradish peroxidase. A preferred ratio of peroxidase to Ig or gamma globulin is 5:1. Material Information Disclosure Sternberger, Immunocytochemistry , "The unlabelled Antibody Enzyme Method," Ludwig Prentice Hall, 1974, pp. 129-171, discloses an unlabelled antibody of PAP (peroxidase-antiperoxidase) procedure. The present invention is a modification of the Sternberger procedure, which is suitable for use with animals and uses peroxidase bound to antiperoxidase. This procedure only applies to animal reagents because a human PAP reagent is unavailable since it requires immunizing a human with peroxidase. Furthermore, the human PAP step of Sternberger is substituted in the present invention for human IgG labelled with peroxidase. See particularly Figure 2. Maggio (ed.) Enzyme-Immunoassay , CRC Press, Inc., P. 44 et seq. (1980) and Nakane, et al., J. Histochem., Cytochem., Vol. 22, p. 1084 (1974) disclose methods of periodate coupling (peroxidase-labelled immunoglobulins) but do not disclose a method for detection of antibodies to HTLV-III. Kurstak (ed.), Viral Immunodiagnosis , Academic Press, Inc., pp. 3-30 (1974) discloses a variety of immunoperoxidase techniques and represents the state of the art. Sasse, et al., U.S. Patent No. 4,235,960, discloses a competitive, double-antibody enzyme-linked immunoassay. Specific Disclosure In the HTLV-III assay method of the present invention, anti-human immunoglobulin, which specifically binds to HTLV-III antibodies, is added to human test sera suspected of containing the AIDS virus antibodies previously coupled to solid immunosorbent test strips. The amount of inmunoglobulin bound to the test sera is measured by an enzyme-inmunoassay wherein human immunoglobulin labeled with peroxidase is incubated with the test strips. The amount of HTLV-III is measured by measuring the amount of inmunoglobulin/peroxidase complex bound to the anti-human inmunoglobulin previously bound to HTLV-III antigens. A preferred methodology illustrating the above procedure is disclosed in Example 1 and illustrated in Figure 2. The first immunoglobulin, anti-human IgG, IgA, IgD, IgE, or IgM is preferably prepared from goat. Other animal anti-human immunoglobulin may be used, such as rabbit, hamster, chicken, rat, guinea pig, sheep, horse, or mouse. These immunoglobulins are commercially available. The human immunoglobulin/peroxidase complex is the label by which HTLV-III antigens can be detected and measured. This complex may be formed, in the preferred embodiment, by chemically coupling purified human immunoglobulin and peroxidase. One method of preparation of this complex is described by Nakane, et al., J. Histochem. Cytochem. , Vol. 22, p. 1084 (1974). Horseradish peroxidase, a colorless protein (apoenzyme) combined with a dark brown iron-porphyrin (and composed of 47.0% C, 13.2% N, 7.35% H, 0.43% S and 0.12% Fe) is available commercially in a highly purified crystalline form. In the first step, reactive amino groups on HRP are blocked by alkylation with dinitrofluorobenzene (DNFB). Blocked HRP is then activated for coupling by periodate cleavage by the sequential addition of NaIO₄ and H₂O (pH 8) followed by addition of immunoglobulin-NH₂ (pH 8) followed by addition of immunoglobulin-NH₂ and NaBH₄ (borohydride). The result is the IgG-peroxidase complex. Even though this coupling procedure is well known and commonly used, this invention may use other coupling methods. Although IgG is preferred, IgA, IgD, IgE, or IgM may be used. The immunosorbent test strips used in the present invention are insoluble and capable of attaching HTLV-III antigens. This immunoadsorbent is present as an insoluble surface and may be agarose in bead form, carbohydrates such as dextran, cellulose, or nitrocellulose, plastics such as polystyrene, polycarbonate, polypropylene, or polyamide, or inorganic materials such as glass or silica gel. HTLV-III antigens may be chemically coupled to the immunosorbent or coated onto the surface of the immunosorbent. The present invention employs the immunoglobulin specific combining property for two different purposes. The first purpose is the same as in labeled antibody methods; that is, immunoglobulin is used directly or indirectly as a reagent for selective localization of the tissue antigen under investigation. However, instead of visualizing this reaction by labeling the immunoglobulin, visualization is done by using specific immunoglobulin binding a second time, this time to bind onto the immunoglobulin an enzyme that is detectable by a suitable histochemical substrate. The double use of immunoglobulin specifically in sequence depends on the bivalence of the immunoglobulin. The color former may be any chromogenic reagent which reacts with the peroxidase labeled human immunoglobulin in order to produce color. The preferred reagent contains hydrogen peroxide and dianisidine. Other chromogenic reagents conventionally used are o-phenylene diamine, p-phenylenediamine, 5-amino-salicylic acid, pyrogallol and similar solutions, such as diaminobenzidine. The method for assaying HTLV-III antibodies may be incorporated into a test kit characterized by: a) an insoluble immunosorbent which may be in bead, strip, plate, or test cavity form; b) anti-human immunoglobulin capable of binding to HTLV-III antibodies; c) human immunoglobulin labeled with peroxidase, said human immunoglobulin being capable of bonding to the anti-human immunoglobulin noted above; d) a color former or chromogenic reagent suitable for reaction with peroxidase; and e) a means for measuring the activity of the peroxidase after contact with the chromogenic reagent. Description of the Figures Figure 1 shows identification of HTLV-III in different test sera taken from suspected AIDS patients. Figure 2 is a schematic diagram illustrating the invention. Example 1 Identification of HTLV-III antigens recognized by Ugandan sera collected in 1972-1973. Absorbance values of samples tested for HTLV-III antibody were expressed in ratio to a standard normal control serum and samples with ratio values greater than the [(mean + 2sd) = 2.0] for normal donors were subjected to a confirmatory test requiring positive reactivity with the major HTLV-III viral bands separated by SDS-PAGE (200 µg of virus per slab, 12% acrylamide) and transferred to nitrocellulose sheets by electroblotting overnight at 15°C and 30V. The sheets were cut into strips and incubated with human test sera diluted 1:400 in 5% non-fat dried milk containing 0.02% merthiolate for 16 hours at 4°C. The strips were washed with wash Buffer, PBS containing 0.5% deoxycholate, 0.5% Triton® X-100 and 1 mM phenyl-, methyl-, sulfonylfluoride (PMSF). Next the strips were incubated at room temperature for 1 hour with goat antihuman IgG Fc fragment (Cappel labs) diluted 1:400 in Buffer 1, 20 mM Tris-HCl pH 7.5 containing 1mM EDTA, 0.2 M NaCl, 0.3% Triton® X-100, 2mg/ml bovine serum albumin, and 4% normal goat serum. The strips were washed as above and incubated for 1 hour at room temperature with human IgG conjugated with horseradish peroxidase (2 µg/ml) in PBS containing 0.5% Tween® 20, and 5% normal goat serum. After washing, the peroxidase color reaction was developed by incubation in 25 mM tris pH 7.5 containing 0.02% hydrogen peroxide and 0.025% o-dianisidine. Panels (A) and (B) of Figure 1 show 39 different sera tested in consecutive experiments. Strips are arranged in order of ascending screening ratios from left to right; lane numbers refer to the original position of the strip within the nitrocellulose blot. Lanes A1 and B1 contain normal control human serum; A2 and B2 contain positive control human serum from an AIDS patient.

Claims: An assay method for the determination of the amount of antibodies to human T-cell leukemia virus type III present, characterised in that: (a) AIDS virus antibodies present in the sample are bonded to HTLV-III antigens linked to an insoluble immunosorbent and are incubated with anti-human immunoglobulin; (b) peroxidase-labelled human Ig is added; (c) a colour former is added which reacts with the peroxidase; and (d) the amount of HTLV-III antibodies are indirectly measured by measuring activity of said peroxidase. An assay method for the determination of the amount of antibodies to human T-cell leukemia virus type III present in a sample solution characterised in that (a) HTLV-III- antibodies present in the sample are linked to an insoluble solid immunosorbent (b) anti-human immunoglobulin; are reacted with said antibodies; (c) said anti-human immunoglobulin which are bound to said antibodies are further reacted with peroxidase-labelled human immunoglobulin, said human immunoglobulin specifically binding to said anti-human immunoglobulin; (d) colour former is added which specifically reacts with said peroxidase; and (e) activity of the peroxidase is measured in order to indirectly measure the amount of HTLV-III antibodies present in said sample solution. The method of claim 2, characterised in that antihuman immunoglobulin is one member selected from the group of immunoglobulins consisting of IgA, IgD, IgE, IgG and IgM. The method of claim 3, characterised in that said immunoglobulin is animal immunoglobulin. The method of claim 2, characterised in that said insoluble solid immunosorbent is selected from one member of the group consisting of agarose in bead form, carbohydrates such as dextran, cellulose, or nitrocellulose, plastics such as polystyrene, polycarbonate, polypropylene, or polyamide, and inorganic material such as glass or silica gel. A method according to any of claims 2 to 5 characterised in that a) a sample solution suspected of containing HTLV-III antibodies or antibodies exhibiting known HTLV-III specificity is contacted with an insoluble immunosorbent under conditions in which said antibodies will link to said immunosorbent; b) anti-human immunoglobulin are added to said antibodies linked to said immunosorbent under conditions in which said immunoglobulin will bond to said antibodies; and c) peroxidase-labelled human immunoglobulin is added to said immunosorbent under conditions in which said human immunoglobulin will bond to said anti-human immunoglobulin; A test kit for assaying HTLV-III antibodies characterised by a) an insoluble immunosorbent in bead, strip, plate, or test cavity form; b) anti-human immunoglobulin capable of bonding to HTLV-III antibodies; c) human immunoglobulin labelled with peroxidase, said human immunoglobulin being capable of bonding to the anti-human immunoglobulin noted above; d) a colour former or chromogenic reagent suitable for reaction with peroxidase; and e) a means for measuring the activity of the peroxidase after contact with the chromogenic reagent.

Inventors: GALLO, ROBERT CHARLES, SAXINGER, CARL W.
Applicants: US COMMERCE, THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY, UNITED STATES DEPARTMENT OF COMMERCE
Application year: 1986
Main classification: G01N33
IPC codes: G01N33, C12Q1