Immunoassay on a preblocked solid surface

Abstract: A method for immunoassay of a viral antigen is performed on a membrane precoated with an inert protein. Nonimmunological capture of antigen takes place by absorption onto the coated membrane. Captured antigen binds to a tracer which includes a label conjugated to a specific antibody, the inert protein concomitantly inhibiting nonspecific binding of tracer. The label may be an enzyme which converts a substrate to a detectable product or converts a blocked inhibitor to an inhibitor whereby a second enzyme is inhibited from converting a substrate to a product. The invention includes a kit of materials for performing an assay in accordance with the method of the invention.

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention . This invention relates to assay for a viral antigen. 2. Background of the Invention . A variety of assay systems which are both rapid and sensitive has been developed to detect or determine the concentration of a ligand in a liquid. Conventional immunoassays depend on the binding of the ligand to a specific antiligand, and have been particularly useful because they give high levels of specificity and sensitivity. These assays generally employ one of the above reagents in labeled form, the labeled reagent often being referred to as the tracer. Various means for labeling have been developed. Radioimmunoassay (RIA) procedures use radioisotopes as labels, provide high levels of sensitivity and reproducibility, and are amenable to automation for rapid processing of large numbers of samples. Fluoroimmunoassay (FIA) uses fluorochromes as labels, and provides direct detection of the- label by exciting the dye with excitation radiation of appropriate wavelength and detecting fluorescence therefrom. Enzymes have also been used as labels in immunoassay. In enzyme immunoassay (EIA), the enzyme labeled reagents are cheap to prepare and are highly stable thus giving a long shelf life, yet yield assays which approach the sensitivity of radioimmunoassay and which give objective results that can be determined either visually or with rather simple equipment, such as a spectrophotometer. In conventional EIA, an enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a color which is measured. Often, an unconjugated component, such as an antibody is immobilized on a solid support and serves to capture antigen in a specific binding reaction. Representative of such conventional EIA is U.S. Patent No. 3,654,090 to Schuurs et al. Bucher et al. in U.S. Patent No. 4,588,680 discloses assay for the M protein of various viruses, including Influenza A,B and C viruses. The assay includes disruption of the virus to release the M protein which is absorbed directly onto a polymeric support. PCT published application WO 86/02733 discloses assay for Herpes simplex virus (HSV) in which antigenic glycoproteins gA/B, gC and gD are absorbed directly onto a polymeric support or captured by specific monoclonal antibodies on the support. The direct addition of an antigen to a solid support in a column is known from WO 84/02193 as well. Further, an assay on a filter pretreated with an inert protein is known from GB-A-1 420 916. Sankolli et al. in Journal of Immunological Methods 104, 191 (1987) discloses an immunoassay for estradiol in which a solid support is pretreated with a specific anti IgG antibody. This antibody captures specific antiestradiol antibody which in turn captures estradiol and provides an assay of improved reproducibility. Armstrong et al. in U.S. Patent No. 4,497,899 discloses an assay for Chlamydia antigen in which the antigen is absorbed directly onto a solid support such as a bead, tube, strip, disk or microtiter plate. The absorbed antigen is then assayed by any conventional EIA, RIA or FIA technique. Further, the use of a membrane in an agglutination assay for detection of an antigen is disclosed in EP-A-0 280 560. In solid phase immunoassay, particularly for large molecular weight molecules, there is often a tendency for materials in the sample being assayed to attach in a nonspecific manner to the solid support. A particular cause of -loss of assay sensitivity or irreproducibility is nonspecific absorption of labeled antiligand conjugate (tracer) directly onto binding sites of the solid support which are not filled by antiligand. This problem has conventionally been addressed by blocking unoccupied binding sites, subsequent to application of antiligand, with a protein which does not react with the labeled conjugate. Cole et al., in U.S. Patent No. 4,407,943, discloses coating a porous membrane with a water insoluble protein, such a zein, affixing an antigen or antibody to the zein layer, and immobilizing an immunochemically neutral protein on the antigen to prevent nonspecific binding of extraneous protein. While this conventional post blocking has improved assay sensitivity, the sequential application of antiligand and blocking protein to a solid phase prior to ligand binding is cumbersome, time consuming and wasteful of expensive antiligand. Accordingly, there is a need for further improvement in solid phase immunoassay technology, particularly with respect to avoidance of nonspecific binding. SUMMARY OF THE INVENTION The invention relates to a method for determining a viral antigen in a liquid comprising: a) combining a first liquid suspected of containing a viral antigen with a membrane coated with an inert protein and a tracer including an enzyme whereby said viral antigen is absorbed onto said coated membrane and binds to said tracer to give a bound fraction including said enzyme on said membrane; b) separating said membrane from said first liquid by causing said first liquid to pass through said membrane; c) passing a second liquid containing a substrate for said enzyme through said membrane, said substrate being converted by said enzyme on said membrane to a colored product; and d) detecting said viral antigen by a signal associated with the color of said product. Another aspect of the invention is a kit of materials useful in performing an assay in accordance with the method of the invention. Thus, the invention provides an assay for a viral antigen in which substantially all binding sites of a solid support are filled with an inert protein. Antigen capture onto the support containing inert protein is accomplished without a specific capture antibody and thereby avoids the time consuming and labor intensive step of producing specific capture antibody. The inert protein inhibits substantially all nonspecific binding of other protein, including tracer, which would otherwise reduce assay sensitivity. Since the inert protein is readily available and inexpensive, the invention provides a simplified assay of significant cost savings. BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1-3 illustrate the results of assays performed by the method of the invention for RSV, Influenza A and HSV respectively. DETAILED DESCRIPTION While this invention is satisfied by embodiments in many different forms, there will herein be described in detail preferred embodiments of the invention, with the understanding that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the embodiments illustrated and described. The scope of the invention will be measured by the appended claims. The immunoassay of the invention may be configured to detect the presence of a viral antigen in the sample or to determine its concentration. The viral antigen may be from any source, and may be a viral antigen present in a body fluid, or it may be isolated from a body fluid and subsequently introduced into a different liquid, such as buffer. In other cases, the viral antigen may be from a source other than a body fluid, as, for example, a culture of microorganisms or a cellular extract thereof. Preferred are viral antigens present in a body fluid, such as Adenovirus, Parainfluenza 3 virus and, most preferably, Herpes simplex virus, HSV, Respiratory syncytial virus, RSV, and Influenza A, Flu A. The invention will hereinafter be described generically in terms of: a viral antigen. Turning now to a detailed description of the assay components, the membranes may be of nylon or nitrocellulose. A particularly preferred membrane is a modified nylon membrane commercially available from Pall Corp., Glen Cove, New York, under the trade names Immunodyne® and Biodyne® A,B and C. Any inert protein may be coated onto the membrane which does not interfere with the subsequent binding reaction between antigen and tracer and which does not substantially bind nonspecifically to other proteins in the assay medium. Representative nonlimiting examples of suitable inert proteins are casein and albumin, although others will be evident to those skilled in the art. Coating of the inert protein to the membrane may be carried out by any suitable method, preferably by incubating the membrane with a solution of the protein whereby the protein is physically absorbed into the polymeric matrix of the surface of the membrane. The membrane having a coating of inert protein is exposed to the sample suspected of containing the viral antigen. Preferably, the coated membrane is incubated with the sample in a transient, flow-through format for 1 to 15, preferably 5 minutes at a temperature of 0 to 50°C, preferably ambient temperature. By this procedure, antigen in the sample is absorbed onto the coated membrane in proportion to its concentration in the sample. In addition, it has been found that viral antigen is absorbed preferentially even when the sample contains a large excess of extraneous protein, such as is the case when the sample is a body fluid. The tracer of the invention is an antibody having an enzyme conjugated thereto. Any enzyme may be used which may be conjugated to the antibody and for which a substrate convertible to a colored product exists. Suitable enzymes are, for example, cyclases, isomerases and peroxidases. A preferred peroxidase is horseradish peroxidase. Preferred enzymes are hydrolases such as peptidases, esterases, phosphatases and glycosidases. The most preferred tracers include alkaline phosphatase or carboxyesterase conjugated to the antibody. Conjugation of enzymes to antibodies is well-known and fully understood by those skilled in the art. Alternatively, the enzyme may be encapsulated in a liposome. Encapsulation of enzymes into liposomes and conjugation of liposomes to antibodies for use as tracers in immunoassay is likewise wholly conventional. The choice of substrate of course depends on the enzyme component of the tracer and a wide variety of substrates are well-known for each class of enzymes. Preferred substrates are those which form insoluble precipitates on membranes. When the enzyme is a peroxidase, a preferred substrate is diaminobenzidine. Preferred substrates for hydrolases are indolyl derivatives. For example, when the enzyme is alkaline phosphatase, a preferred substrate is 3-indolyl phosphate. When the enzyme is an esterase, preferred substrates are 3-indolyl acetate and butyrate. These substrates are all well-known in the art. In accordance with the assay method of the invention, the membrane coated with inert protein and having viral antigen captured thereon, as described above, is incubated with a solution of the tracer in a liquid to induce immunological binding of the antigen and antibody component of the tracer. The membrane having a bound antigen-antibody fraction thereon may then be separated from the liquid phase of the assay medium by any suitable method, preferably by causing the liquid to pass through the membrane. Liquid flow through the membrane may be by gravity or preferably may be enhanced by capillary action induced by absorbent material positioned under the membrane. The membrane may then be suspended in a second liquid such as water, saline or buffer having the enzyme substrate dissolved therein. Enzyme in the bound fraction converts the substrate to a product detectable by a signal associated with color. Thus, the signal detected may be the development or disappearance of a color, or a change from one color to another, or a change in the rate at which the substrate is converted to the product, for example, the color of a substrate may be observed to remain unchanged for a specified length of time. It is preferred that the substrate be colorless until cleaved by the enzyme label to give a colored product. The extent of color formation is proportional to antigen concentration, which may be determined by assaying liquid samples having predetermined quantities of antigen therein and comparing color intensities. Measurements may be made either instrumentally or, preferably with the naked eye. Another aspect of the invention is a reagent kit or package of materials for performing an assay for a viral antigen in accordance with the method of the invention. The kit includes a membrane, coated with an inert protein, an antibody having an enzyme conjugated thereto, a substrate for said enzyme, and a housing containing an absorbent material positioned adjacent said membrane. The kit may also include a second enzyme, standards for the ligand, as, for example, one or more ligand samples of known concentration, or it may include other reagents, enzyme substrates, or other labeled or unlabeled specific ligands, antiligands or complexes thereof useful in carrying out the assay. It may include solutions, such as saline or buffers. The components of the kit may be assembled in a housing, preferably plastic, containing a material positioned under the membrane, such as absorbent paper, to facilitate flow of assay liquids through the membranes by capillary action. EXPERIMENTAL Inhibition constants were measured in 50 mM Tris pH = 8.0. Enzyme and inhibitor were incubated at ambient temperature for 20 minutes. Substrate for the enzyme was then added and the rate of hydrolysis was followed spectrophotometrically. The substrate for PLE and RLE was o-nitro-phenylbutrate and for AChE was acetyl thiocholine and Ellman's reagent. The following examples are provided to further describe the invention but are not to be considered in any way as limitative of the invention. EXAMPLE I Assay for Respiratory Syncyctial Virus (RSV ) A membrane filter stack was assembled with the following configuration: Top layer - Three micron Immunodyne Immunoaffinity Membrane, (Pall, East Hills, New York, #BIA0030HC5). Precoated by immersion in phosphate buffered saline containing 0.3% casein for 30 minutes at ambient temperature. Next layer - Non-woven rayon sheet (Schleicher and Schuell, Keene, New Hampshire; #5-S). Bottom layer - Cellulose absorbent pads (2) (Filtration Sciences, Mount Holly Springs, Pennsylvania; #ED 320-200) The membrane layers were encased in a plastic holder which includes a receiving well formed above the top layer. Within this well was fitted a flow restriction insert which has an aperture more narrow than the receiving well and sits flush against the top membrane. An antigen stock was prepared with respiratory syncytial virus (RSV) (Long strain) infected HEp-2 cells diluted in a buffer containing: 250 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris HCl), 150 mM sodium chloride (NaCl), 10 mM ethylenediaminetetraacetate (EDTA), 4% (v/v) polyoxyethylene sorbitan monolaurate (Tween 20), 1% n-acetyl cysteine, 0.2% sodium azide (NaN₃), pH 8.5. Control antigen was prepared in a similar manner from uninfected HEp-2 cells. A 150 µl aliquot of this antigen (or control) was applied to the device and allowed to drain through the flow restriction insert and onto the top membrane layer. (Liquid is drawn through the top membrane by the capillary action of the supporting absorbent layers.) The flow restriction insert was then removed, and to the device was added 150 µl of a wash solution consisting of 50 mM Tris HCl, 150 mM NaCl, 0.2% NaN₃, pH 7.2 (Tris buffered saline (TBS)), additionally containing 1 mg/ml of rabbit IgG. A solution containing 27 µg/ml of anti-RSV antibody conjugated to alkaline phosphatase was prepared in a buffer containing 50 mM Tris HCl, 100 mM NaCl, 200 mM sodium phosphate, 1% casein, 1 mM magnesium chloride, 0.1 mM zinc chloride, and 1 mM 2-mercaptoethanol, pH 7.5. A 150µl aliquot of this mixture was added to the device and allowed to absorb into the membrane stack. Following a brief (two minute) incubation, the device was washed with 300 µl of TBS (without IgG). A 150 µl solution containing 0.33 mg/ml nitroblue tetrazolium, 1% methanol, and 0.2% NaN₃ was added to the device. This was followed by the addition of 150 µl of a solution containing 12 mM levamisole in 50 mM 2-amino-2-methyl-1-propanol acetate (AMP HOAc), 0.2% NaN₃, 19 mM magnesium chloride, at pH 9.8. Following a five minute incubation at ambient temperature, the color forming reaction was stopped by the addition of 150 µl of a solution containing 200 mM potassium phosphate, 10 mM EDTA, 0.2% NaN₃, pH 7.2. The color density of the resulting membrane was measured with a reflectance densitometer (Gretag, Seattle, Washington, model 183). The results of an experiment performed with a series of antigen dilutions are presented in the Figure 1. EXAMPLE II Assay for Influenza Virus, Type A An assay for influenza virus was performed in a manner similar to Example I with the following exceptions: 1) The antigen stock was prepared from Madin-Darby canine kidney (MDCK) cells infected with Influenza A (WSN strain). 2) Top layer membrane was 3 micron Biodyne C (Pall, East Hills, New York; #BNPCH5) instead of Immunodyne. 3) Antigen stock buffer was prepared with Tris acetate instead of Tris hydrochloride, contained no sodium chloride, and additionally contained 1 mM ethylene bis(oxyethylenenitrilo)tetraacetic acid (EGTA). 4) The conjugate diluent contained 100 mM instead of 50 mM-Tris, and 150 mM instead of 100 mM NaCl. 5) Levamisole concentration was 16 mM instead of 12 mM. The results of an experiment performed with a series of antigen dilutions are presented in Figure 2. EXAMPLE III Assay for Herpes Simplex Virus (HSV) An assay for herpes simplex virus (type I and II) was performed in a manner similar to the Example I with the following exceptions: 1) The antigen stock was prepared from Vero cells infected with HSV type II. The results of an experiment performed with a series of antigen dilutions are presented in Figure 3. Thus, the invention provides an assay for a viral antigen which does not include a specific capture antibody for the antigen. Instead, a solid support is precoated with an inert protein and antigen is captured directly on the coated support. Further, the inert protein, even though applied as a precoat prior to antigen capture, prevents substantially all nonspecific binding of other proteins, such as tracer, which would otherwise reduce assay sensitivity.

Claims: A method for determining a viral antigen in a liquid comprising: a) combining a first liquid suspected of containing a viral antigen with a membrane coated with an inert protein and a tracer including an enzyme whereby said viral antigen is absorbed onto said coated membrane and binds to said tracer to give a bound fraction including said enzyme on said membrane; b) separating said membrane from said first liquid by causing said first liquid to pass through said membrane; c) passing a second liquid containing a substrate for said enzyme through said membrane, said substrate being converted by said enzyme on said membrane to a colored product; and d) detecting said viral antigen by a signal associated with the color of said product. The method of Claim 1 wherein said inert protein is selected from the group consisting of casein and albumin. The method of Claim 1 wherein said tracer further comprises an antibody specific for said antigen having said enzyme conjugated thereto. The method of Claim 1 wherein said tracer further comprises an antibody specific for said antigen and a liposome having said enzyme encapsulated therein, said liposome being conjugated to said antibody. A kit of materials for performing an assay for a viral antigen comprising a membrane coated with an inert protein, an antibody having an enzyme conjugated thereto, a substrate for said enzyme, and a housing containing an absorbent material positioned adjacent said membrane. The kit of Claim 5 further comprising at least one other reagent selected from the group consisting of an enzyme, an enzyme substrate, buffer and saline.

Inventors: HENDERSON GLENN L, HOKE RANDAL A, HOPKINS ANNE C, MCLAURIN DANIEL A, HENDERSON, GLENN L., HOKE, RANDAL A., HOPKINS, ANNE C., MCLAURIN, DANIEL A.
Applicants: BECTON DICKINSON CO, BECTON DICKINSON AND COMPANY
Application year: 1989
Main classification: G01N33
IPC codes: G01N33