Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54306—Solid-phase reaction mechanisms
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
  • G01N33/548—Carbohydrates, e.g. dextran
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
  • G01N33/56988—HIV or HTLV

Definitions

• the present invention relates to a sensitive assay method for detecting the presence or absence of antigens of a human retrovirus indicative of Acquired Immune Deficiency Syndrome (AIDS) , and AIDS-related complexes (ARC) in serum, plasma and other body fluids and in cell lysates, particularly, lysates of leukocytes or a subpopulation thereof.
• AIDS Acquired Immune Deficiency Syndrome
• ARC AIDS-related complexes
• the present invention also relates to test kits for carrying out the assay method.
• HIV-1 HTLV-III/LAV
• HIV-2 another human retrovirus
• the human retroviruses which are associated with AIDS are believed to be transmitted through intimate sexual contact as well as through blood. Infections caused by such human retroviruses result in the appearance of antibodies in the serum, semen and other body fluids of infected victims.
• the antibodies are generally directed against certain viral core and envelope proteins including the HIV-1 core proteins designated p 24 (24,000 MW) , and envelope proteins designated gp 41 (41,000 MW) , gp 120 (120,000 MW) and gp 160 (160,00 MW) .
• the core and envelope proteins have been identified by, among others, Western Blot techniques.
• the AIDS viruses cripple the body's immune defense system.
• the viruses target T 4 lymphocytes, in particular a T-lymphocyte subset known as helper lymphocytes. Infection with any one of the AIDS viruses results in reduction in the number and a change in the function of T 4 cells and/or an increase in Tg subsets; other T-lymphocytes as well as B-lymphocytes are eventually also affected by the virus, resulting in collapse of the immune system.
• the established screening process which involves detecting the presence of antibodies to the AIDS viruses, is not entirely satisfactory because detectable amounts of such antibodies are not produced until at least two to four months after primary infection. Therefore, early diagnosis of AIDS by the detection of antibodies during this so-called "gray area" is not possible.
• a technique for detecting retroviral antigens, rather than antibodies, in patients suspected of infection is a useful tool for early detection of AIDS. Early diagnosis facilitates treatment, and minimizes the risk of spreading the infection.
• HTLV-III Capture- Assay is impractical for mass screening because it is both time consuming, expensive and does not detect antigens directly from a test sample obtained from a patient.
• the present assay technique provides a method for detecting picogram amounts of retroviral antigens directly from serum, body fluids and lysed cell materials.
• the present invention provides a rapid and sensitive test system for detecting the presence of antigens of a human retrovirus indicative of AIDS or
• ARC by forming a triple sandwich immune complex comprising antibody-antigen-antibody anti-antibody.
• the method of the invention comprises incubating antibodies to a human retrovirus indicative of AIDS with a biological fluid for a time sufficient for the retroviral antibodies and antigens in the biological fluid to form a first immune complex comprising antibody-antigen; the first immune complex is then incubated with antibodies to the retrovirus for a time sufficient for the retroviral antibodies and first immune complex to form a second immune complex comprising antibody-antigen-antibody; the second immune complex is then incubated with retroviral anti-antibodies for a time sufficient to allow formation of a third immune complex comprising antibody-antigen-antibody-anti-antibody and detecting the formation of the third immune complex.
• the assay method of the invention may be carried out by either a modified ELISA (enzyme linked immunosorbance assay) or semi-solid phase enzyme-linked dot spot test.
• the dot spot test preferably employs nitrocellulose paper, typically cut into strips.
• picogram amounts of AIDS retroviral antigens are detectable directly from body fluids or lysed cell material. Results are obtainable in about 19 hours, less than 1/15 the time required for the conventional HTLV-III Capture Assay.
• a diagnostic test kit which permits on site testing for retroviral antigens.
• the kit comprises positive and negative reference controls, washing buffers, antibody reagents, nitrocellulose strips and enzyme conjugates necessary to carry out the method of the invention on test samples.
• the diagnostic kit also preferably includes predeveloped positive and negative reference strips and reagent control strips for evaluating the test results by visual comparison with the test strips.
• predeveloped strips facilitates reading the test results and virtually eliminates the need for trained personnel. This is especially important for on site testing where the facility may not have specialized equipment required for other testing systems.
• the semi-solid assay method of the invention comprises forming and detecting triple sandwich immune complexes comprising antibody - antigen - antibody - anti-antibody; the triple sandwich immune complexes are formed by (1) incubating antibodies to a retrovirus indicative of AIDS with a biological fluid or lysed cell material to obtain a first immune complex comprising antibody - antigen; (2) incubating the first immune complex with antibodies to a retrovirus indicative of AIDS to obtain a second immune complex comprising antibody - antigen - antibody and (3) incubating the second immune complex with an anti-antibody against the antibody of step (2) to obtain a triple sandwich immune complex comprising antibody - antigen - antibody - anti-antibody.
• antibodies to a retrovirus indicative of AIDS are coated onto a solid phase, such as microtiter plates, round plastic or glass beads, approximately 0.5-2 mm. diameter or nitrocellulose paper.
• a solid phase such as microtiter plates, round plastic or glass beads, approximately 0.5-2 mm. diameter or nitrocellulose paper.
• Microtiter plates such as NUNC immunoplates, available from NUNC,
• nitrocellulose paper available from Schleicher and Schuell, Inc. , as Item No. -BA -83-, are suitable examples.
• Other types of membranes as are known to those skilled in the art may be substituted for nitrocellulose paper, including a inobenzyl-oxymethyl (ABM) blotting paper, aminoethylthio-ether (APT) blotting paper, diethylaminoethyl (DEAE) blotting paper and zeta-probe charged modified nylon.
• ABSM inobenzyl-oxymethyl
• APT aminoethylthio-ether
• DEAE diethylaminoethyl
• each test vessel i.e., microtiter well or nitrocellulose strip, preferably, 0.1 ug is used.
• the microtiter wells and beads are coated by known techniques, such as those described in General ELISA Procedure, Dako, Corp., order no. 23/8710.
• nitrocellulose strips When nitrocellulose strips are used, approximately 100 ul of a 0.06-0.12 ug/ml antibody solution is applied -to one side of each strip. Prior to applying the antibody to the strip, it is preferred to cover approximately 3/4 of the strip with a plastic material. The antibody solution is then applied to the exposed portion. The covered portion of the strip may be used as a handle without destroying the activity of the antibodies applied to the strip. It should be understood that the amount of antibody used as a coating on the solid phase may be varied depending on the totality of test conditions.
• Monoclonal antibodies may be substituted for the immune polyclonal antibodies previously mentioned. Monoclonal antibodies are useful in a test designed to detect a single core or envelope protein or a specific combination of selected proteins.
• Human antibodies to the AIDS retrovirus are obtained from the serum of infected individuals, either individual donors or pooled sera from multiple donors.
• Pig, goat, donkey, horse and/or cow immune antibodies are obtained by immunizing an animal in accordance with known techniques.
• an animal is first vaccinated with from 5-500 ug, preferably 100 ug of a retroviral lysate mixed with Freund's complete adjuvant (1:1).
• Booster injections begin 14-30 days after the first vaccination, and are administered approximately every 1-8 weeks, preferably every 2-4 weeks, and contain from 50-100 ug of viral lysate, with or without Freund's adjuvant depending on the antibody titer measured semi-quantitatively by ELISA.
• the serum is partially purified, preferably according to the conventional saturated ammonium sulfate method, although other purification techniques such as polyethyleneglycol precipitation (R.J. Carter et al, J. Immunol. Methods, 26:213 (1979)) may be used. Briefly, saturated ammonium sulfate (up to 50% of the starting volume of serum) is used to precipitate the serum. After discarding the supernatant.,..the.- precipitate is redissolved to the original starting volume of the serum with 25% PBS-ethyleneglycol
• the IgG rich fractions should optimally contain lmg/ l IgG, although fractions containing at least 0.6 mg/ l IgG are useful. If desired, total protein may be determined by the Lowry method.
• the IgG rich fractions which contain the desired antibodies to the retrovirus are stored in a refrigerator or freezer until used.
• 0.01% thimerosal is added as a preservative, although other preservatives known to those skilled in the art are also suitable.
• Human sera which as discussed hereinabove, may also be used as a source of the retroviral antibodies, are purified by the aforementioned saturated ammonium sulfate method.
• the partially purified antibodies are tested by ELISA and Western Blot. It is believed that for purposes of the present method an ELISA OD of 0.300-2.000 OD and Western Blot banding pattern showing activity against both core and envelope proteins are required.
• the retroviral antibodies obtained in the manner previously described are useful as both the antibody coated onto the solid phase, and for the formation of the aforementioned second immune complex. While it is preferred to use antibodies obtained from different sources for each purpose, the same antibodies may be used for both. That is, for example, when porcine immune antibody is used to coat the test vessel, it is preferred to use non-porcine antibodies for the formation of the second immune complex. However, when the same antibody is used, prior to formation of the second immune complex, it is necessary to neutralize any excess binding sites remaining on the coating antibody. It is believed that this procedure avoids undesirable binding of the anti-antibody required for formation of the third immune complex to the coating antibody, and therefore, preserves the integrity of the test system. The excess binding sites are generally neutralized by adding anti-antibodies against the coating antibody. Thus, for example, if the coating antibody is porcine IgG, the neutralizing antibodies are anti-porcine IgG. Forminq A Triple Sandwich Immune Complex
• the retroviral antibodies are coated onto microtiter wells in accordance with known techniques. 0.06-0.12 ug, preferably, 0.1 ug (100 ul of a 1 ug/ml solution) of antibody is coated onto each well. Typically, 100 ul of a 0.1% BSA solution is then added to the wells to block non-specific binding, although other reagents as are known may be used, such as gelatine. Approximately
• test sample 100 ul of a test sample, either body fluid or lysed cell material, is added to each well, and preferably incubated overnight (approximately 16 hours) at 4°C to thereby obtain a first immune complex comprising antibody-antigen.
• the liquid is discarded and the plates are washed 5 times, preferably with PBS-Tween, although other buffers may be used, such as Tris. No soaking time is required for the washes. Then, 100 ul of an approximately 1 ug/ml solution of retroviral antibodies are added to each well and incubated preferably for 1 hour at 37°C to thereby obtain a second immune complex comprising antibody-antigen-antibody. If desired, this incubation may be overnight at 4°C. Upon completion of the incubation, the liquid is discarded and the plates washed 5 times with buffer, as previously described.
• anti-antibody preferably, 100 ul of a 1 ug/ml solution of an anti-antibody, i.e., an antibody to the retroviral antibodies used to obtain the second immune complex, is added to each well and incubated for 1 hour at 37°C to obtain a third immune complex comprising antibody-antigen-antibody-anti-body.
• the anti-antibodies are against the retroviral antibodies used to obtain the second immune complex.
• porcine IgG is used for this purpose, the anti-antibody is anti-porcine IgG. Rabbit or goat antiserum is preferred for this purpose.
• Such antisera are commercially available from Calbiochem and
• the commercially available anti-antibody preparations have an IgG concentration of from 0.6-1.2 mg/ml.
• the stock material is diluted accordingly, as previously described.
• the anti-antibody is labeled with a detectable atom such as a radioisotope or fluorescein, so that the triple sandwich immune complexes may be detected by radioimmunoa ' ssay or immunofluorescent assay techniques. If a radionuclide is used, i 125 is preferred.
• the triple sandwich immune complex is detected by immunoenzymatic assay.
• the anti-antibody is biotinylated, preferably with either biotin or biotin
• the plates are washed 5 times as previously described.
• 1 mg/ml avidin of strepavidin enzyme conjugate solution is diluted from 1:1000 - 1:5000, preferably, 1:1000.
• the avidin or strepavidin is enzyme conjugated, in accordance with known methods, to horseradish peroxidase, alkaline phosphatase, or
• Beta-D-galactosidase It should be understood that other enzymes as are known to those skilled in the art are also useful. It is presently preferred to use avidin or strepavidin conjugated with horseradish peroxidase. Upon completion of the incubation of the enzyme and biotinylated third immune complex, the liquid is discarded and the plates washed 5 times as before. Then 100 ul of an appropriate enzyme substrate specific for the enzyme conjugate used, diluted accordingly, is added and incubated for 10-20 minutes at room temperature in the dark. Typically, the microtiter plate or other vessel, such as tube is covered with ' aluminum foil. The reaction of enzyme with an appropriate substrate is a color producing reaction. The substrate orthophenyldia ine (OPD) is preferred when horseradish peroxidase is used, although
• OPD is commercially available as tablets from Electro Nucleonics Inc. and is prepared in accordance with the manufacturer's instructions prior to use (i.e., one tablet is dissolved in 5 ml of the substrate buffer supplied) and 100 ul of the prepared solution is added. After completion of the incubation period, the color producing reaction is stopped by addition of 50 ul of 2N H2S0 4 . Other reagents may be used to stop the reaction such as H 2 0.
• optical density of the liquid contained in the wells is measured in a spectrophotometer at 492 nm (reference 650 nm) as an indication of the presence or absence of antigens to a retrovirus associated with
• the Behring ELISA Processor II is specifically designed for the purpose, but other instruments are also suitable.
• the results of the test are evaluated by visually comparing the color developed by the positive and negative controls to the color produced by the test sample.
• a diagnostic test kit for the detection of the presence or absence of antigens of a retrovirus indicative of AIDS, which bind with retroviral antibodies
• the test kit comprises retroviral antibodies, retroviral anti-antibodies, and means for detecting the formation of triple sandwich immune complexes between the antibodies, antigen and anti-antibodies.
• the means are present in an amount sufficient to perform said detection.
• the presence of the triple sandwich immune complexes may be detected by radioimmunoassay, immuno-enzymatic and im unofluorescent techniques, preferably, immuno-enzymatic means.
• a preferred test kit includes nitrocellulose test strips, a set of tubes containing prediluted positive and negative control references, a reagent control, and at least one sample tube containing a predetermined volume of buffer to which a predetermined amount of test sample is added.
• the reagent control is provided to assure that the reagents are functional.
• the nitrocelluose strips are coated on one side with from
• the preferred kit also includes a second antibody to the
• AIDS retrovirus from a source different from the coating antibody, a biotinylated anti-antibody against the second retroviral antibody, an enzyme substrate, color reaction stopping reagent, and washing buffer.
• predeveloped positive and negative reference strips and reagent control strips are provided in the kit.
• the predeveloped strips are used to evaluate the test results by a visual comparison with the sample test strips after completion of a color reaction.
• the test is considered positive if a test sample produces a color comparable to that of the positive control.
• the presence of absence of antigens of a human retrovirus indicative of AIDS may be detected directly from body fluids and lysed cell materials.
• Leukocytes are of particular interest.
• Leukocytes are collectable from the buffy coat resulting from centrifugation of heparinzed blood in accordance with known techniques.
• the cells so collected are typically lysed with 2.5% saponin or 0.1
• Nonidit NP40, available from Sigma
• Other known lysing techniques are also considered useful.
• the viral lysate is typically an inactivated, partially purified viral lysate which on testing in accordance with the method described herein gives an OD reading of between
• the viral lysate may be purified by known techniques, and inactivated by the known psoralen-UV irradiation method or heat (56°C for one hour) .
• a partially purified, inactivated HIV-1 lysate is commercially available from Protatek
• a NUNC flat bottomed 96 well immuno plate was coated with goat anti-HIV-1 IgG obtained partially purified from Protatek International, Inc.
• the titer of goat IgG was measured to be approximately 1.-0 mg/ml as determined by the Mancini technique against rabbit affinity purified anti-goat IgG (Mancini, G. et al, Immunochem. 2:235, 1965).
• the IgG was also tested by Quick Western Blot Assay and it was determined that antibodies to p24 and gp41 were present.
• the reference rabbit anti-goat IgG was obtained from Calbiochem, Catalog 401511.
• the goat antibody was diluted 1:1000 with PBS and 100 ul was added to each well of the immuno plate.
• the immuno plate was sealed with Parafilm and incubated overnight (16 to 18 hours) at 4°C, followed by washing 5 times with PBS on a Behring ELISA Processor II washing machine. After the final wash 0.1% (v/v) bovine serum albumin (BSA) diluted in PBS buffer was added to each well. The plate was then incubated for one hour at room temperature followed by washing 5 times as previously described.
• the assay was carried out in the following manner. An HIV-1 lysate, obtained from Protatek International, Inc. was diluted with PBS, 1:1,000, 1:10,000, 1:100,000 and
• the plate was washed five times with PBS - Tween, as previously described. Then, 100 ul of an approximately 1 mg/ml solution of human anti-HIV-1 IgG (in PBS) was added to each well. The human IgG was tested prior to use and demonstrated the presence of HIV core and envelope antibodies. Using NOR PARTIGEN plates it was determined that the Human IgG had an IgG concentration of about 1 mg/ml. (The PARTIGEN plates were obtained from Calbiochem Catalog No. 91001 and the IgG concentration was determined using the method described by the manufacturer.) The plates were incubated at

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• 1987
  • 1987-03-25 DK DK151087A patent/DK151087A/da not_active IP Right Cessation
• 1988
  • 1988-03-23 AU AU17801/88A patent/AU1780188A/en not_active Abandoned
  • 1988-03-23 WO PCT/US1988/001026 patent/WO1988007586A1/en unknown

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