CA1291949C - Cloth enzyme immunoassay - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A novel device for carrying out immunoassay techniques on antigens or haptens is provided herein. The device is a com-bination of a macroporous hydrophobic synthetic polymer cloth selected from the group consisting of woven polypropylene, non-woven polypropylene, woven polyester, non-woven polyester, woven nylon, non-woven nylon, woven polyethylene, and non-woven poly-ethylene, the cloth having such a structure that it: can accom-modate a large volume of liquid per surface area thereof, has a large surface area, and has minimum flow resistance; and an antibody or an antigen directly adsorbed thereon and directly absorbed therein.

Description

This invention relates to the detection of antigens or haptens based on immunoassay techniques.
An antigen is an extraneous substance which, when introduced into the body of vertebrates, causes the production of an antibody which can specifically complex with that antigen. Any substance, for example a protein, which is not normally present in certain organisms, can cause the formation of antibodies when it infiltrates into or is applied to an organism under suitable conditions. An antibody once produced is also capable of binding a hapten, i.e., a relatively small and simple compound which may be the determinant group of a given antigen. The hapten is capable of binding with the specific antibody but is incapable itself of giving rise to the production of an antibody, unless it is bound to an antigenic carrier. These small molecular weight antigens (haptens) may require conjugation with large molecular weight carriers in order to elicit antibody production. This antigen-antibody complexing is the basis of immunoassays.
The binding interaction between an antigen or a hapten and its antibody is specific and sensitive. Other types of materials that participate in similar specific and sensitive binding interactions are: enzymes and their substrates; hormones;
vitamins; metabolites; and pharmacological agents; and their receptors and binding substances.
Since virtually any foreign compund can be made immunogenic, the domain of immunoassays is unlimited.

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Diagnostic tests claim a large share of the health care market. In both human and veterinary medicine, the definitive diagnosis of infectious diseases require the demonstration of the infectious agents or their components. Traditional cultural methods for the detection of pathogens are slow, expensive and of uncertain sensitivity, and require extensive laboratory facilities. To overcome some of these disadvantages, specific binding assay techniques have provided analytical methods for determining various organic substances of diagnostic, medical, environmental and industrial importance which appear in liquid mediums at very low concentrations. Specific binding assays are based on the specific interaction between the ligand, i.e. the bindable analyte under determination, and a binding partner therefor. When one of the ligand and its binding partner is an antibody and the other is a corresponding hapten or antigen, the assay is known as an immunoassay. In addition several immunological tests are now commercially available, namely:
agglutination test~; immunofluorescent tests; and enzyme immunoas~ays. Hohever, many of these tests require the use of microscopes, spectro-photometers, or other laboratory facilities, limiting their use under field conditions. Prompt and effective control of diseases depends on rapid and simple field tests.
Radioimmunoassay employs a radioactive isotope as the label.
Such an assay necessarily must follow the heterogeneous format since the monitoraole character of the label is qualitatively .,,, , ~

~19~9 unchanged in the free- and bound-species. ~ecause of the inconvenience and difficulty of handling radioactive materials and the necessity of a separation step, homogeneous assay systems have been devised using materials other than radioisotopes as the label component, including enzymes, bacteriophages, metals and organometallic complexes, coenzymes, enzyme substrates, enzyme activators and inhibitors, cycling reactants, organic and inorganic catalysts, prosthetic groups, chemiluminescent reactants, and fluorescent molecules. Such homogeneous specific binding assay systems provide a detectable response, e.g., an electromagnetic radiation signal, e.g. chemiluminescence, fluorescence emission, or color change, related to the presence of amount of the ligand under assay in the liquid sample.
Immunoassay diagnose infectious diseases by detecting either increased titers of antibodies against pathogen antigens or the presence of the pathogens or their antigens. Antigen assays offer more definitive diagnosis of infectious diseases as the eapaeity to produce antibodie~ remains in subjects which have reeovered from the disease.
Enzyme immunoassays use enzyme-labeled immunoreagents (antibodies or antigens) for the detection of captured antigens or antibodies.
Absorption is a simple and rapid means of immobilization of immunoreaetants. Since antibodies and many antigens contain hydrophobic regions in their structures, they bind readily to , ' ` . ,i. .

hydrophobic surfaces. Most commonly used enzyme immunoassays depend on the adsorption of immunoreactants onto either a solid surfRce or membrane. Solid phases, e.g. microtiter plates, tubes or beads, and plastics, e.g. polystyrene, polyvinyl chloride, nylon, and polymethacrylate have commonly been used. Although nitrocellulose membranes have been used to adsorb antigens as well as antibodies, these are thin and can only accommodate a small volume of test sample per given surface area. Furthermore, since their pore sizes are small, their effective washing requires a vacuum suction apparatus which holds them airtight.
It is also known that it is possible to prepare porous fibres which have enzymes embedded therein, the latter retaining, by their being so immobilized, their catalytic properties, while at the same time the enzyme is prevented from escaping and being dispersed in the raction mass and thus contaminating the reaction product.
The patent literature is replete with descriptions of techniques and means for effecting immunoassays. A
repre8entative selectlon of such patents include the following:
1) Canadian Patent 1,031,257 issued May 16, 1978 to R.Dietrich for "Carrier for Immunological Determinations"
(directed to a device comprising an immunologically-reactive material on an object carrier or a film, the immunologically-reactive material being in a lyophilised and self-adhering form);

2) Canadian Patent No. 1,060,342 issued August 14, 1979 to O. Lostia et al for "Fibres Incorporating Antibodies, Antigens and Antisera, Method For Their Preparation and Their Use"
(directed to a polymeric structure comprising a porous artificial fibre where the substance occluded in the fibre was antibodies, antigens or antisera, and where the pores of the fibre were of such nature as to prevent escape of the occluded substance but to allow for the penetration of the agent that is to be reacted with that substance);

3) Canadian Patent No. 1,083,036 issued August 5, 1980 to G. Bolz for "Indirect Solid Surface Test For Antigens or Antibodies", (directed to a specifically-described procedure for determining reacted labeled antibody);

4) Canadian Patent No. 1,107,195 issued August 18, 1981 to D. Wagner et al for "Specific Binding Assay Employing Polystyrene As Separating Agent" (which provided a specific binding assay method using nonion-exchange cross-linked polystyrene for determining a ligand in, or the ligand-binding capacity of, a liquid medium);

5) Canadian Patent No. 1,108,986 issued September 15, 1981 to D. Wagner et al for "Specific Binding Assay Employing Polyvinyl Alcohol As Separating Agent" (which provided a specific binding assay method using nonion-exchange cross-linked polyvinyl alcohol for determining a ligand in or the ligand binding capacity of a liquid medium);

~19 ~t 6) Canadian Patent No. 1,152,430 issued August 23, 1985 to J. Gordon et al for "Protein On Nitrocellulose Sheet Support"
(directed to a solid support for proteins consisting of a porous nitrocellulose sheet containing an electrophoretically-transferred replica of an electropherogram.of proteins in a gel);

7) Canadian Patent No . 1, 199, 269 issued January 14, 1986 to V.A. Marinkovitch for "Multiple Component Binding Assay System and Method of Making and Using it" (directed to a diagnostic kit which included a support having a plurality of cotton threads supported in a predetermined spaced relation for simultaneous contact with a liquid test sample);

8) U.S. Patent No. 3,951,741 patented April 20, 1976 by R.F. Devlin for "Sensitized Matrix For Detection of Disease"
(directed to a specific sensitized matrix for diagnosing both infectious and non-infectious diseases, including an insoluble, inert, pliable and wettable matrix having a network of pores, and a protein polymer network immobilized in that network of pores);

9) U.S. Pa~ent No . 4,168,146 patented September 18, 1979 by A.O. Grubb et al for "Immunoassay With Test Strip Having Antibodies ~ound Thereto" (directed to a diagnostic te~t device useful for immunochemical quantification, which was a carrier strip comprising a silica-modified micro-porous polymer having finely-divided silica substantially-uniformly embedded in a particularly-recited permeable, continuous polymeric matrix);

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1~'31~'~9 10) U.S. Patent 4,277,561 patented July 7, 1981 by D.
Monget et al for "Support For The Determination of Enzyme Activities and Process" (directed to a support for the determination of enzyme activity in a biological extract wherein the support comprised a fibrous material impregnated with a substrate and a particularly-recited water-soluble pH
stabilizer);

11) U.S. Patent No. 4,347,311 patented August 31, 1982 by H.H. Schmitz for "Enzyme Immunoassay For Determining Antigen lC Specific Antibodies and Test Kit For Carrying Out This Assay"
(directed to a highly sensitive enzyme immunoassay procedure for determining antibodies which are specific to antigens by coating a particularly-recited solid support with an antibody);
and 12) U.S. Patent No. 4,442,204 patented April 10, 1984 by A.C. Greenquist for "Homogeneous Specific Binding Assay Device and Preformed Complex Method" (directed to a test device comprising a solid carrier member, e.g., a fibrous web matrix, e.g. paper, or A polymeric film or gel, incorporated with specifically-reci~ed reagents for a homogeneous specific binding as8aY system).
Accordingly, those concerned with the development and use of immunoassay techniques and related devices have recognized the desirability for further improvements and it is an object of one broad aspect of the invention to provide a rapid, accurate method for the quantitation of antigen or antibody on a solid surface.

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It is an object of a further aspect of the invention to provide a method to provide rapid and sensitive immunoassays.
An object of a further aspect of the present invention is the provision of a relatively simple yet high effective and sensitive diagnostic test for the detection of specific disease states, both infectious and non-infectious.
An object of yet another aspect of this invention is the use of the hydrophobic cloths to make immunoassays rapid and simple.
An object of yet another aspect of this invention is to enable the use of hydrophobic cloths for immunoassays for antigens and haptens as well as for antibodies.
By a broad aspect of this invention, an immunoassay device is provided comprising the combination of a macroporous hydro-phobic cloth selected from the group consisting of woven poly-propylene, non-woven polypropylene, woven polyester, non-woven polyester, woven nylon, non-woven nylon, woven polyethylene, and non-woven polyethylene, the cloth having such structure that it:
can accommodate a large volume of liquid per surface area thereof, has a large surface area, and has a minimum flow resis-tance, and an antibody or an antigen directly adsorbed thereon and directly absorbed therein.
The immunoassay device may be treated with an antiserum containing an antibody specific for the antigen being tested, or it may be treated with a purified antibody bearing the appro-priate specificity. The antibodies present in the antiserum may be partially denatured prior to being applied to the hydro-~gl9 ~

phobic macroporous cloth, e.g. by exposure to a low pH environ-ment, e.g, a pH of 2.5, or by heating. Alternatively, the antibodies may be affinity-purified prior to being applied to the hydrophobic macroporous cloth. The antibody may be provided by diluted antiserum.
It is preferred that the immunoassay device be in the form of the above-described macroporous hydrophobic synthetic polymer cloth bonded to a different material, thereby to provide an antibody-coated test strip that may be handled throughout an assay procedure. Thus, the present invention embraces the bonding, in any suitable manner, of the so-treated above-described macroporous hydrophobic synthetic polymer cloth to a dipstick.
By another aspect of this invention an immunoassay method is provided for detecting an antigen, the method comprising: a) treating a surface of macroporous hydrophobic synthetic polymer cloth selected from the group consisting of woven polypropylene, non-woven polypropylene, woven polyester, non-woven polyester, woven nylon, non-woven nylon, woven polyethylene, and non-woven polyethylene, the cloth having such structure that it: can accommodate a large volume of liquid per surface area thereof, has a large surface area, and has a minimum flow resistance, with an antibody, thereby to have an antibody directly adsorbed thereon and directly absorbed therein: b) incubating the treated cloth with a sample to be tested for the antigen; c) washing the l~i9L'~9 incubated cloth with a buffer to remove unadsorbed sample; d) incubating the washed cloth with an enzyme-antibody conjugate prepared by coupling a purified antibody specific for the antigen to an indicator enzyme; e) washing the incubated cloth with a buffer to remove unreacted conjugate; and f) detecting remaining enzyme-antibody conjugate by incubation in a chromogenic Qubstrate indicator solution to produce a visible colour upon product formation.
By still another aspect of this invention, an immunoassay method for detecting an antigen is provided comprising: a) treating a surface of a macroporous hydrophobic synthetic polymer cloth selected from the group consiQting of woven polypropylene, non-woven polypropylene, woven polyester, non-woven polyester, woven nylon, non-woven nylon, woven polyethylene, and non-woven polyethylene, the cloth having such structure that it: can accommodate a large volume of liquid per surface area thereof, has a large surface area, and has a minimum flow resistance, with an antigen, thereby to have an antigen to be assayed directly adsorbed thereon and directly absorbed therein; b) applying to the 4urface of the treated cloth, a mixture of the antigen being assayed and an enzyme-antibody conjugate specific for the antigen being aQsayed; c) treating a control identical macroporous hydro-phobic synthetic polymer cloth with a mixture of the antigen being assayed and an enzyme specific for the antigen being assayed to have the mixture of the antigen being assayed and an ,~1 ~`9~949 enzyme specific for the antigen being assayed adsorbed thereon and absorbed therein; d) incubating both the treated cloths ~ubstantially simultaneously; e) washing each of the incubated cloths with an identical buffer solution; and f) detecting the antigen by incubation of both the cloths in an identical chromogenic substrate indicator solution to produce a visible colour upon product formation, the amount of antigen being determined by the difference in intensity of the colour between the cloth containing the free antigen and the cloth containing the antigen-enzyme-Qpecific mixture.
Consequently, the immunoassay procedure in one embodiment of thi# invention may consiqt of the following: a hydrophobic macroporous synthetic polymer cloth having such structure that it: can accommodate a large volume of liquid per surface area thereof, has a large surface area, and has a minimum flow resistance, is treated to have adsorbed thereon and absorbed therein, either an antiserum containing an antibody . ,p~"~.

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-specific for the antigen being tested, or a purified antibody bearing the appropriate specificity, and is subsequently incu-bated with the test ~ample purported to contain the antigen.
That treated cloth is then washed with an appropriate buffer to remove any unadsorbed and unabsorbed material, and is then incu-bated with an enzyme-antibody conjugate prepared by coupling purified antibody specific for the antigen to a suitable indi-cator enzyme. That treated cloth is then washed with buffer to remove unreacted conjugate, and the remaining conjugate is detected by incubation in a chromogenic substrate-indicator solu-tion which produces a visible colour upon product formation.
The immUnoaQSay procedure of another a~pect of the invention may consist of the following: a test sample containing the anti-gen to be aQ~ayed i~ mixed with a Quitable enzyme-antibody con-jugate, e.g. diluted horseradish peroxidase (HRP)-antibody con-jugate specific for the antigen of interest, and an aliquot of this mixture is incubated with an antigen-treated hydrophobic synthetic polymer cloth. A control hydrophobic ~ynthetic polymer cloth is treated with a mixture of the same enzyme-antibody con-~ugate but without the free antigen.
After washing with a suitable buffer solution, e.g. PBST, tho~e cloths incubated with an antigen-conjugate mixture fail to produce the same intensity of colours ~upon incubation in ABTS-indicator) as cloths which were incubated with a control mixture consisting of the conjugate in the absence of free antigen.

While it is not desired to be bound by any theory, it is believed that antigen present in the test sample combines with the conjugate, thus preventing its interaction with the antigen-treated cloth. In this manner, the presence of antigen in a test sample will diminish the amount of colour produced in the test, while the control sample (minus free antigen) gives proof of the functional integrity of the conjugate.
The detection of B. abortus antigen LPS, using the immuno-assay with LPS-coated polyester cloth has thus been provided as another embodiment of this invention. The immunoassay, which requires only one incubation with the immunological reagent, pro-vides an internal control for the quality of the reagent used.
Before describing various aspects of thig invention, a description of the reagents used will be given. Chemicals used were of the analytical reagent grade. All biochemicals were purchased from Sigma Chemical Co. Distilled water ~H~O) was employed as a universal solvent. Antigens and bovine antisera were provided by the Animal Diseases Research Institute ~ADRI) in Nepean, Ontario. Some materials employed as solid phases (i.e., cellulose cotton and nylon cloths) were acquired locally, whereas non-woven polypropylene filter cloth was purchased from Aldrich Chemical Co. and a variety of polyester cloths were obtained from DuPont.

lZ919 ~9 - 13a -EnzYme ImmunoassaY Reaqents (1) 0.06 M carbonate buffer ~pH 9.6) NaHCO3 3.8g Na2CO3 1.93g Add H2O and NaOH (if necessary) to 1,000 ml.
~2) 0.01 M phosphate-buffered saline (pH 7.2) (PBS) NaH2PO4.2H20 0.31g Na2HPO4 l.lg NaCl 8.5g . . .~. ~.

(3) P~S_with _W EN 20 (PBST) PBS 1000m TWEEN 20 [TWEEN 20 is the registered Trade Mark of an emulsifier comprising polyoxyethylene derivatives of fatty acid partial esters of sorbitol anhydrides of Atlas Chemical Industries] 0.5ml (4) Indicator svstem for horseradish Deroxidase (ABTS-indicator) 0.05 M citrate buffer (pH 4.5) 20ml 40 nM 2,2'-Azino-di-(3-ethylbenzthiazoli~e sulfonic acid) ABTS) 0.5ml 0.5 M H202 0.02ml (5) Indicator sYStem for alkaline phos~hatase diethanolamine 2.62g P-nitrophenyl phosphate .025g Add H2O and HCl to 25 ml to obtain a final pH 9.8.
Prep_rat _n of AffinitY Purified Anti-Brucella Antibodies from Bovine Antiserum Anti-Brucella antibodies were purified from bovine antiserum by the affinity p~rification method. It is based on the adsorption of anti-Brucella antibodies onto the antigenic surface of whole killed P. abortus cells mixed with antiserum. Cells with adsorbed antibodies can then be separated from the serum by centrifugation, and the antibodies can be recovered by exposure to a low pH environment with subsequent removal of the cells by centrifugation. The method is simple to perform, inexpensive, and usually results in high yields of specific antibodies.

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Ten milliliters of standard plate test antigen, consisting of whole heat-killed B. _bortus cells (strain 413, biotype 1) .~uspended in phenol-saline (i.e., 0.85% NaCl and 0.5% phenol in H70) at a concentration of 4 x 10" cells/ml, were dispensed in a 50 ml-capacity round bottom polycarbonate centrifuge tube. The cells were pelleted by centrifugation at 10,000 x g, for 10 minutes. The pellet was then washed twice in 0.1 M glycine-HCl (pH 2.24) to remove any acid-soluble material present on the cell surface, followed by two washings in 0.1 M Tris-HCl buffer (pH
7.0). Care was taken to disperse the cells as gently as possible during resuspension (a glass stirring rod is convenient for this purpose). To the final washed pellet was added 25 ml of bovine antiserum, in which the cells were dispersed. The suspension was allowed to stand at room temperature for 30-40 minutes, with gentle stirring every 5-10 minutes. The suspension was then centrifuged as above and the supernatant discarded. The resulting pellet was w~shed three times in 25 ml of 0.1 M Tris-HCl buffer (pH 7.0) to remove any loosely adsorbed material.

3rucella-specific'antibody was recovered by resuspending the final washed pellet in 25 ml of 0.1 M glycine-HCl (pH 2.24). the cells were immediately removed by centrifugation and antibody-rich supernatant was transferred to a vessel containing 10 ml of 1.0 M Tris-HCl buffer (pH 8.0) in order to abrogate the harsh low pH environment. The remaining cell pellet was processed in this manner a second time to improve antibody recovery, and the final ,, ~,; . ''' ' ~91~49 supernatants were pooled. The antibody solution was then dialyzed against PBS for 24 hours at 4C, with at least three changes of buffer. Precipitate material arising in the dialysate was stored at -80C until use. Whenever necessary, the protein in the dialysate was concentrated using an Amicon protein concentrator.
Unless otherwise specified, antiserum used in the Examples was serum obtained from chronically infected cattle which have high titers of anti-Bru_ella antibody. B. abortus cells used were heat-killed standard plate test antigen (whole cells) which is strain 413, biootype 1, suspended in phenol-saline.
Preparation of an Enzyme-antibodv Coniuqate bv the Periodate Oxidation Method A modified version of the method developed by Nakane and Kawaoi was employed for conjugating horseradish peroxidase with anti-Brucella antibody. For this, highly purified horseradish peroxidase (RZ~3) was used, where RZ is the light absorbance ratio of A4~3/A7,~ for a given enzyme solution. Five milligrams of horseradish peroxidase (Sigma~ type VI, RZ-3.0) were dissolved in a mixture of 1.0 ml of 0.3 M sodium carbonate (pH 8.1) and 1.0 ml of 0.08 M NaIO4. The solution was mixed gently for 30 minutes at room temperature, and 1.0 ml of 0.16 M ethylene glycol was then added. This was further mixed at room temperature for 1 hour. The latter solution was then dialyzed against 5 l of 0.01 M sodium carbonate buffer (pH9.5) at 4C for 16-24 hours. To ,~' ' ' ,' , 19 ~

this dialysate was added 0.5 ml of PBS containing ca. 5 mg of affinity purified anti-Brucella antibody and 0.5 ml of 1.01 M
carbonAte buffer (pH 9.5). The solution was incubated for 3 hours at room temperature, with gentle mixing. At the end of the incubation, 5 mg of NaBH4 were added to the solution which was then left to stand at 4~C for 3 hours. The solution was then dialyzed extensively against PsS at 4C. Any precipitable material formed in the dialysate was removed by centrifuging for 10 minutes in an eppendorf microfuge. The resulting supernatant constituted the stock conjugate, which was designated conjugate, and was stored at -20C until use.
Collection of Antiserum An adult cow suffering from chronic brucellosis was bled at one-week intervals over a period of approximately three weeks.
At each bleeding, ca. 1 1 of blood was collected in glass bottles and immediately incubated at 37 for 2 hours, followed by overnight storage at 4~C to allow for complete clot formation.
The serou~ liquid was decanted into separate vessel and centrifuged at 8,'000 X g to remove any remaining blood cells.
The supernatant thus obtained was stored at -20C.
A large stock of antiserum, which was used as a source of antibody for all subsequent experiments, was prepared by pooling all serum samples obtained after the prescribed succession of bleedings, and was stored at -80C until use.

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~9~9 ~9 In the accompanying drawings, Figure 1 is a graph showing the effect of varying the treating concentration on the signal generated in the polypropylene cloth-based cloth enzyme immunoassay;
Eigure 2 is a graph showing the partial denaturation of bovine antiserum antibodies by audification;
Eigure 3 is a graph showing the partial denaturation of bovine antiserum and affinity purified antibodies by heat; and Eigure 4 is a graph showing diluted antiserum as a source of coating antibody.
The following are Examples of this invention:
I(a) Determination of the Optimum Affinity Purified Coating Antibody Concentration for the Poly~roDvlene-based Cloth EnzYme ImmunoassaY
Polypropylene filter cloth pieces (6 x 6 mm) were coated with 50 ml of various concentrations of affinity purified anti-Brucella antibody per piece (a concentration range of 0.2 to 1.0 mg of protein/ml was chosen). After the required overnight incubation perio~, the cloths were washed with PBST and ' subsequently incubated with 30 ~l of B. abortus cells (strain 413, biotype 1) diluted to 4 x 10~ cells/ml in PBS, for 30 minutes at room temperature. A series of negative control cloths (incubated without antigen) was also included. The cloths were then washed nith PBST and probed with 25 ul of conjugate diluted ,.` ' ' .

g 1:1,000 in PBST, and incubataed for 30 minutes at room temperature. After washing with PsST, the cloths were assayed for retained horseradish peroxidase activity by immersion in 0.5 ml of ABTS-indicator solution for 30 minutes, at room temperature, and the reaction was stopped by addition of 0.5 ml of 0.1 M NaF. Absorbance was read at 650 nm.
As shown in Figure 1, the assay response peaks at a concentration of 0.2 mg/ml.

I(b) Pretreatment (Partial Denaturation) of the Coating Antibodv The use of antiserum for the direct coating of the cloth was investigated as an effective and economical alternative to purified antibody.
In order to achieve maximum immobilization of the antibodies present in the antiserum, a simple procedure was developed for the pretreatment of the coating antiserum to incur partial denaturation of the antibodies, thus rendering them more capable of interacting with the hydrophobic cloth surface, to the exclusion of other serum proteins which might compete for binding sites on the cloth. In order to improve the detectability of the 2() cloth enzyme immunoassay employing bovine antiserum as the source of coating antibody, partial denaturation procedure~ were developed using an acidic pH and heat. The following describes an investigation undertaken to determine the optimum time of exposure to a pH 2.5 environment for the improved immobilization of antibodies from antiserum on polypropylene.

, !~ . !,, 1 Z9~949 Three separate 0.85 ml aliquots of antiserum were mixed with 0.3 ml each of 0.4 M glycine-HCl buffer (pH 1.5) to produce a final pH 2.5. A zero-time exposure sample consisting of 0.85 ml of antiserum plus 0.6 ml of 1.0 M Tris-HCl buffer (pH 7.0) was also prepared as a control. Each acidified sample was allowed to stand at room temperature for either 5, 10, or 20 minutes, after which they were immediately neutralized by addition of 0.3 ml of 1 . O M Tris-HCl (pH 8.0). These samples were then used to coat polypropylene filter cloth pieces. The effect of time of exposure to pH 2.5 on the cloth enzyme immunoassay signal generated is presented in Figure 2.
Figure 2 shows that an approximate five-fold improvement in the assay's sensitivity was achieved by exposure of the antiserum to pH 2.5 for 10 minutes.
In a second experiment, the effect of exposing bovine antiserum and affinity purified antibodies to heat on their abilities to serve as sources of coating antibodies was examined.
One millilitet samples of anti~erum and the affinity purified antibody dialysate (containing 0.27 mg of protein/ml) were 20 incubated for 10 minutes at either 25, 65, 70, 75, or 80C.

These were then allowed to cool to room temperature and used to coat polypropylene cloths as previously. The assay protocol employed was the same as in the previous experiment, with the exception that the antibody-coated cloths were incubated with 30 ' ."' '` ~ , ~`';' ' ''' 1~91~ 9 ml of B. abortus plate test antigen diluted to 4 x 107 cells/ml in PBS. The results of this experiment are presented in Figure 3, where the signal generated in the immunoassay is plotted against temperature for both antiserum and affinity-purified antibody.
~ igure 3 shows that an even greater improvement in sensitivity was obtained by heating the serum at 75C for 10 minutes. The sensitivity of the assay employing affinity purified antibody remained essentially unchanged over the range of temperatures tested.
These experiments have demonstrated the usefulness of either exposure to a low pH or to heat in improving the sensitivity of the a~say using antiserum as the source of coating antibody.
Without wishing to be bound by theory, it is believed that the improvement in the sensitivity of the antiserum-based immuno-assay i~ a consequence of an increase in the hydrophobicity of the denatured Fc region of the antibodies, which in turn cause~
these to be adsorbed more ~trongly to the hydrophobic cloth sur-face and in greater numbers. The partially denatured ~tate may also ensure that the anbitodies adhere to the solid phase in a more ideal orientation, with the Fc region affixed to the cloth ~urface and the Fab segment~ free to interact with the antigen.
Treating the affinity-purified antibodies with heat did not seem to confer any particular advantage. Thus, these affinity-puxi-fied antibodies cannot be beneficially altered further by heat treatment.

I(c) Diluted Bovine Antiserum as a source of Coating Antibody The feasibility of applying diluted antiserum in the cloth enzyme immunoassay was investigated in the following experiment.
Aliquots of bovine antiserum were diluted 2, 4, 6, 8, and 10 times in PBS. The diluted samples were partially denatured by heating at 75nc for 10 minutes. These were then cooled to room temperature and applied to 6x6 mm polypropylene filter cloth pieces, which were subsequently employed in the cloth enzyme immunoassay according to the procedure used in the previous example. The results are presented in Figure 4, where the cloth enzyme immunoassay signal generated is plotted against the serum dilution factor.
Figure 4 shows that there was no appreciable decline in the sensitivity of the assay throughout the range of coating serum dilutions examined. Therefore, it was concluded that bovine antiserum diluted 1:10 in PBS, with subsequent heating at 75C
for 10 minutes, can serve as a suitable source of coating antibody in the cloth enzyme immunoassay. The precise dilution factor used for'a given batch of antiserum will, of course, depend on the specific antibody titer of that serum.

(II) DetectabilitY of the Cloth Enzvme Immunoassav For Brucella Antigens The detectability of Brucella cells and lipopolysaccharide antigens (LPS) by the polypropylene cloth enzyme immunoassay was examined in this Example. Unless otherwise stated, all antibody-,,,., '.,1~
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coated cloths used in the following experiments were prepared with bovine antiserum diluted 1:10 in PBS and heated at 75C for 10 minutes.
II(a) Detectability of the Whole Cell Assay Employinq Antibody-Coated PolYpro~ylene Eilter cloth Antibody-coated polypropylene filter cloth pieces were incubated for 30 minutes at room temperature with 30 ~1 of B.
bortus cell suspensions containing either 1.2 x 105, 1.2 x 10~, or 1.2 x 103, cells in PBS. Each cloth in this series of l experiments was prepared in quadruplicate, as were cloths to which 30~ul of PBS containing no antigen was added. These were then washed with PBST and probed with 25~ul of conjugate diluted 1:1000 in PBST, for 30 minutes at room temperature. At the end of this period, the cloths were washed with PBST and assayed for retained horseradish peroxidase by immersion in ABTS-indicator solution for 3 hours, in order to optimize the final enzyme signal. The enzyme reaction was stopped by addition of 0.5 ml of 0.1 M NaF. Table 1 shows the relation~hip between the quantity of antigen added 'and the corresponding cloth enzyme immunoassay signal obtained.

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Table 1. Detectability of the Whole Cell Assay Employing Antibody-Coated Polypropylene Filter Cloth ~. af Gel~s ~ A 650 Per Cloth 1 2 3 4 -1.2 x lC 5 0.1~6 0.213 0.234 0.245 1.2 x 10 4 0.107 0.080 0.10$ 0.075 1.2 x lO 3 0.054 0.050 O.G60 0.055 O 0.060 O.C51 0.058 0.055 Table 1 ~how~ that the polypropylene cloth immunoassay can detect 104 cells using a 36 mm2 polypropylene cloth.
II(b) Detectabilitv of Brucella LPS bY the PolyproPylene Cloth ImmunoassaY
The assay procedure employed for the detection of whole cells using antibody-coated polypropylene filter cloth was applied to the detection of B. abortus LPS. In this Example, antibody-coated cloth~ were incubated for 30 minutes at room temperature with 30 ~l of PBS containing either 3, 0.3, or 0.03 ng of LPS, or PBS alone. Each cloth was prepared in quadruplicate. These were then proce#sed in the immunoassay as previously described. The results are presented in Table 2.

Table 2. Detectability of LPS by Polypropylene Cloth Immunoassay . __ _ A ~n_ ~C~*h ~ 1 2 3 4 ~.00 1.~0 1.3~ 1.440 1.1~
0.~ 0.~1 0.~ 0.216 0.2 0.~ 0.~3 0.~ 0.~ 0.
_ 0.~ 0.~ 0.~3 0.~

Table 2 shows that the detectability of this assay occurred at approximately 0.3 ng ~or 300 picograms) of LPS applied per cloth piece.
II(c) Performance of the ImmunoassaY Under Simulated Clinical Conditions In the routine diagnosis of brucel 108is, ~rucella organisms are often recovered from infected animals in milk; vaginal secretions; supramammary, retropharyngeal, internal iliac, and lumbar lymph nodes; spleen tissue; uterine tissue; and in some instances, blood. As these materials constitute complex environ-ments for the detection of antigens, a determination was made to whether or not undefined sample components might be prohibitive to antigen detection by the immunoassay. Another aspect of clinical ~pecimens examined was the interference of anti-Brucella antibodies present in the samples to the antigen assay by the immunoassay. The ability of the polypropylene filter cloth-based as#ay to detect B. abortus antigens in body fluids and tissue homogenates of bovine origin was examined in Example II.

`~`

19'~`S' Antibody-coated cloth was prepared as in the previous Example.
II~d) Detection of B. Abortus in Bovine BodY Fluids and Tissue Homoqenates In order to ascertain the ability of the immunoassay to detect B. abortus antigens in simulated clinical specimens, tissues obtained from a cow which was a serological reactor for B. abortus, but culture negative, were artificially innoculated with whole cells and assayed for the presence of antigen as described below. The presence of endogenous circulating antibody specific for B. abortus offered an opportunity to assess the performance of the assay under conditions which might theoreti-cally interfere with the capture of antigen in such samples. The possible inhibitory effect of endogenous antibody was alleviated by subjecting the test samples to extreme heat (3 hours, 70C) prior to performing the assay.
Ti~sue homogenates were prepared by homogenizing in a stomacher and adding sufficient PBS to produce a fluid consis-tency ~approximately 0.5 ml per gram of tissue). Homogenates were made from the following bovine tissues; inguinal lymph nodes, spleen, and uterine horn.
A sample from each homogenate was inoculated with sufficient B. abortu~ cells to give a final concentration of 8 x 10 cells/ml. Milk and serum samples from a healthy animal were . ~

1~91~

likewise inoculated. These samples, along with their uninocu-lated counterparts, were heated at 70C for 3 hours, after which they were cooled to room temperature. Antibody-coated poly-propylene filter cloths were then added to triplicate test tubes containing 0.5 ml of heated sample ~one cloth/tube) and incubated for 30 minuteq at room temperature with constant gentle shaking.
The cloths were then removed and processed in the immunoassay as previously described. Cloths were assayed for retained conjugate by immer~ion in 0.5 ml of ABTS-indicator solution for 30 minutes.
The re~ults are shown below in Table 3.
Table 3. Detection of Antigens Suspended in Body Fluids and Tissue Homogenates.

S~nqi~e a ~x~ic Ctrt~nolC

A 0.15 ¦ 0.240 ¦ 0. Z 4 1 2 o~o33 B 0.164 0.177 0.170 0.051 0.060 0.061 C 0.1~? 0.170 0.162 O.oQ2 O.C27 o.o20 D 0.159 0.165 0.162 O.C20 0.030 O.OeD
E 0.175 0.192 0.195 O.C45 0.042 0.0 0 a) Tissue homogenates from spleen ~A), inguinal lymph nodes ~B), unterine hore ~C), plu~, normal bovine serum ~D) and normal bovine milk ~E).
b) Cloths incubated with inoculated samples.
c) Cloths incubated with uninoculated samples.
Table 3 demon~trates the as~ay re~pon~e for each sample tested. In most cases, the control signals ~i.e., those obtained ~;.

1~31~ ~

from the uninoculated samples) remained low, whereas those arising from the corresponding inoculated samples were distinct.
The experiment demonstrates the ability of the immunoassay to detect B. abortus antigens suspended in various biological samples.
III Detection of Bovine Viral Diarrhea (BVD) Antiqen The applicability of the ability of the immunoassay to detect antigens of bacterial origin to the detection of viral antigens will now be described in this Example.
Bovine viral diarrhea is an enteric disease posing a serious threat to exposed livestock. The etiological agent is a virus which i8 referred to as bovine viral diarrhea (BVD) antigen. At present, an immunoasQay method is not used for the detection of BVD antigen in clinical specimens.
The strategy employed for the BVD assay was similar to that used previously for the detection of Brucella antigens: poly-propylene filter cloth was coated with anti-BVD antibodies and ub~oquontly incubated with BVD antigen, and the captured material was detected by probing with an enzyme-antibody con-jugate ~prepared by coupling antibodies from anti-BVD antiserum to horseradi~h peroxidase by the periodate oxidation method~.
Whole antiserum was employed in order to make the required con-jugate. The feasibility of using whole antiserum for this pur-pose wa~ first te~ted by conjugating whole anti-Brucella antiserum with horseradish peroxidase and applying the resulting .~

conjugate in the B. abortus immunoassay, thus affording an opportunity to compare the performance of such a conjugate with an established system.
III~a) Svnthesis and Testinq of a Brucella-specific Enzvme-Antibody Coniuqate Made With Whole Bovine Antiserum Whole bovine anti-Brucella antiserum was conjugated to horseradish peroxidase according to the periodate oxidation method: 0.5 ml of whole serum was dialyzed overnight at 4C
against 0.01 M sodium carbonate buffer (pH 9.5). The resulting dialysate was mixed with 5 mg of periodate-activated horseradish peroxidase and incubated for 2.5 hours at room temperature.
Sodium borohydride (NaBH4) was then added as prescribed, and incubated for 3 hours at 4C against PBS, and the final dialysate wa# microfuged (10 minutes, at 4C). The cleared dialysate constituted the conjugate stock.
The performance of this conjugate in the immunoassay was compared to that of the conjugate prepared with purified anti-body. Two series of Brucella-specific antibody-coated poly-propylene filter cloth pieces were incubated with 30 ~1 of B.
abortus cells diluted to 4 x lOn, 4 x 107, or 4 x 106 cells/ml in PBS, for 30 minutes at room temperature. The cloths were then waO-hed in PBST, and one series was probed with 25 ~1 of the conjugate prepared with affinity purified antibody diluted 1:100 in PBST while the other series was probed with 25 ~1 of the conjugate prepared with whole antiserum diluted 1:100 in PBST.
These were incubated for 30 minutes, at room temperature, and subsequently washed with PBST. The cloths were assayed by immersion in 0.5 ml of ABTS-indicator solution for 30 min. The results are shown below in Table 4.
Table 4. Relative Performance of Conjugate D in the Immunoassay.

A 650~
~. ~ C~ ~ ~ ~
~rC~*h 1 2 1 2 1.2x106 0.~2 0.932 0.631 0.5~3 1.2x10 0.3~5 0.~0 0.153 0.180 1.2x10~ 0.081 0.~ 0.081 0.080 O 0.010 0.012 0.~0 o.o~

* prepared by coupling affinity purified antibody to HRP
** prepared by coupling whole antiserum to HRP
According to Table 4, the response of the immunoassay using conjugate~ was somewhat dampened throughout the series of antigen dilutions tested, as compared to the performance of conjugate~ in the assay. However, the Example demonstrates the feasibility of employing such a conjugate.
III~b) AdaPted ImmunoassaY for the Detection of BVD Antiqen A BVD-4pecific conjugate wa~ prepared by coupling whole anti-BVD antiserum to hor~eradish peroxidase, in order to adapt the immunoassay for the direct detection of BVD antigen.

,~

A sample of anti-BVD antiserum (precise titer unknown) was obtained from a cow vaccinated wtih killed BVD virus. An anti-BVD serum protein-horseradish peroxidase conjugate was prepared by coupling 0.5 ml of anti-BVD antiserum dialyzed against sodium carbonate buffer to 5 mg of horseradish peroxidase as in the previous Example. The resulting product was designated conjugate3.
The standard antigen employed in the immunoassay originated from a commercial BVD vaccine, consisting of killed whole virus Quspended in saline containing an unknown quantity of bovine serum albumin (added as a stabilizer) and a variety of anti-microbial agents. The quantity of viral antigen per unit volume of the vaccine was not disclo~ed by the manufacturer. The vaccine was dialyzed overnight at 4C again~t PBS prior to u~e.
Polypropylene filter cloth pieces were coated with 60 ~1 of partially denatured anti-BVD antiserum ~i.e., heated at 75C for 10 minutes~ and incubated at room temperature as previously.
Antibody-coated cloths were incubated for 30 minutes at room temperature with 30 ~1 of dialyzed BVD vaccine diluted either 1:10, 1:100 or 1:1,000 in PBS. Undiluted vaccine and PBS alone were also included in the series, and each cloth was prepared in quadruplicate. After the incubation period, the cloths were washed with PBST and probed with 25 ~1 of conjugate3 diluted 1:100 in PBST, as previously. These were then assayed by immersion in 0.5 ml of ABTS-indicator solution for 30 minutes.
The results are ~hown below in Table 5.

.~

~g~.~'?~

Table 5. Detection of BVD Antigen by the Immunoassay r ~
~y~nn~L~n 1 Z ~ 4 u~u~ 1.2~ 1.1S0 1.~0 1.2C0 1:10 0.2~ 0.280 0.2~ 0.210 1:10~ 0.091 0.110 0.115 0.095 1 :10CO o.~o o.a71 o.css o.cso ~ a~y~n O.C55 O.C53 O.C51 0.0~

Table 5 shows that the detectability of the BVD assay, in the form tested, was fixed somewhere in the range of 1:10 to 1:100 dilution of the dialyzed vaccine. These results clearly demonstrate the ability of the immunoassay to detect BVD antigen.
III(c) Alternative Solid Phases for ~se in the Immunoassav A series of Examples was carried out to determine the usefulness of other materials as solid phases for capture and detection of B. abortus cells.
Polypropylene filter cloth was compared to a variety of other materials employed as solid phases in the immunoassay. The material~ tested were 100% nylon cloth (acquired locally), woven polyester cloth ~acquired locally), nonwoven polyester cloth ~Dupont), cellulose acetate membrane ~elman), cellulose nitrate membrane ~Schleicher and Schuell), analytical paper ~Schleicher and Schuell), and a polyethylene filter (1.5 mm thickness, `~`

1~915~

supplier unknown). These were all cut into 6 x 6 mm square pieces, and coated with 60 ~l of partially denatured bovine anti-Brucella antiserum diluted 1:10 in PBS, as previously. The materials were then incubated with 30 ~l of B.
abortus cells diluted to 4 x 107 cells/ml in PBS for 30 minutes at room temperature. A parallel series to which PBS containing no antigen was added was also included, and each solid phase was prepared in duplicate. After the incubation period, the materials were washed with PBST and probed with conjugate (prepared with affinity purified antibody~ as usual. These were then assayed by immer~ion in 0.5 ml of ABTS-indicator solution for 30 minutes. The performance of each solid phase in the assay i~ shown below in Table 6.
Table 6. Performances of Various Solid Phases Employed in the Immunoassay 6s0 ¦ b ~1~ ~EEe 1 2 1 1 2 ~n~l~.e ~ clo~ 0.428 0.4~0 0.012 0.010 ~lonclo~ 0.~ 0.3~ 0.011 o.o~
W~en ~ clo~ 0.840 O.~oO 0.035 0.
~w~n ~ clo~ C 0.810 0.792 0.033 O.OE~
G~l~oæ æxt*e m~n# 0.2~ 0.1o 0.1C0 0.1 G~l~o~ ni~ mE~ne 0.2~2 0.23 0.1~ 0.1~
yl~ 0.450 0.4~ 0.061 0.~2 ic~ p~xr 0.416 0.4~ 0.400 0.421 a) Cloths incubated with antigen b) ClothQ incubated without antigen c) A variety of DuPont polyester cloths were examined and the results with these cloths were similar to data ~hown here Table 6 shows that of the solid phases tested, the highest signals were obtained with the woven polyester and nonwoven poly-ester cloths. Other materials that are useful in the immunoassay are nylon cloth and the polyethylene filter, which produce Qimi-lar results to those obtained using polypropylene filter cloth.
The main advantage of hydrophobic clothg as supportQ in an immunoa8gay iQ that they provide a large volume for abgorption per ~urface area for antigen-antibody interaction. Polyethylene, nylon, and polyester cloths, by virtue of their hydrophobic characteristics, have been found both to ad~orb and to abQorb antibodies and thus provided a large 4urface area for antigen capture. Other materialg amenable to the immunoassay concept ~hould have included cellulose acetate and cellulose nitrate membranes and filter paper. However as the regults above indicated, they are not ugeful according to a~pects of the present invention.
IV ComParison of the ImmunoassaY Usina PolYPropylene Filter _oth, a Flat PolYProPvlene Sheet and a PolYgtvrene Microtiter Plate Surface The immunoassay respon~e arising from the use of an antibody-coated pla~tic oolypropylene gheet and a polystyrene microtiter qurface, which have very limited surface areag ~ Q ~

available for antibody adsorption, and hence antigen capture, to the assay response obtained using polypropylene filter cloth when various quantitie~ of antigen are applied was compared.
A flat polypropylene sheet, cut into 6 x 6 mm pieces, was coated with anti-Brucella antibodies as were 6 x 6 mm pieces of polypropylene filter cloth. Several wells in a polystyrene microtiter plate were also coated. These were then incubated with 30 ~l of PBS containing either 1.2 x 10', 1.2 x 106, 1.2 x 105, 1.2 x 10,4, B. abortus cells (plate test antigen), or PBS
alone, for 30 minute~ at room temperature. The materials were subsequently proce~sed in the immunoassay as in the previous Example, with the exception that enzyme activity was as~ayed by immersion in 0.5 ml of ABTS-indicator solution for 3 hours. Each determination was done in triplicate, and the results are shown below in Table 7.
Table 7. Detectability of the Immunoassay Employing Three Different Solid Phases.

~.ofG~lsb~ P~e A 650 12 X 1012 X 10612 ~ U12 X 1O O
Pclyprcpylere 1 5. oe 1. oe 0.29 0.09 0.04 F~lter 2 5.12 1.24 0.29 O.C9 o.o3 Cloth 3 5.30 1.15 0.24 0.08 o.o3 Plastic 1 l. oe 0.43 O.OS 0.03 0.04 Polyprcpylene 2 1.01 0.32 0.07 o.oe 0.04 Sheet 3 0.~6 0.87 0.07 0.04 0.03 Poly~n~ne 1 1.08 o.~8 0.08 0.02 o.o3 Mi.~titer Plabe 2 1.22 0.35 0.09 o.o3 0.04 9urf3De 3 1.01 0.30 0.08 O.C4 0.02 Table 7 shows that the successful detection of very small quantities of antigen must require a sufficiently large capturing surface in order to increa~e the probability of interaction between the solid phase and the antigen during the limited incu-bation period involved. This expectation is confirmed by the results obtained using an antibody-coated plastic polypropylene sheet and a polystyrene microtiter plate surface as solid phases, which failed to detect small quantities of antigen to which the polypropylene cloth responded, and which showed a greatly diminished sensitivity throughout the range of antigen concen-tration tested.
V Commercial Adaptation of the Immunoassa~: Dipstick of Hvdrophobic Cloth A commercial form of the immunoassay was developed for application of the assay in any number of circumstances (e.g., diagnostic laboratory and field testing, etc.). One practical form consists of affixing a small rectangular piece of poly-propylene filter cloth to a ~trip, e.g. of cellulose acetate, which allows for the easy retrieval of the antibody-coated cloth from test samples and provides a convenient means of handling the cloth throughout the assay procedure. It is necessary to ensure that the bond created between the polypropylene cloth and the cellulose acetate does not alter the properties of the former or reæult in any structural features at the cloth/strip junction which might cause non-specific retention of the conjugate.

A

~9~

A bond was created by first dissolving one edge of a cellulose acetate strip having the dimensions 2 1/2" x 1/4"
polypropylene cloth piece of the same thickness, making sure not to allow any overlapping of one edge over the other. Upon evaporation of the acetone, a strong bond was formed between the cellulose acetate strip and the polypropylene filter cloth piece.
The cloth portion of the resulting test strip was coated with antibody by applying 100 ~1 of partially denatured bovine anti-Brucella antiserum diluted 1:10 in PBS and incubating overnight at room temperature, followed by washing with PBST as previously.
The antibody-coated test strip was tested in the immunoassay in the manner described below.
Test strips were incubated with either 30 ~1 of PBS
containing 1.2 x 106 B. abortus cells (plate test antigen) or 30 ~l of PBS alone, for 30 minutes at room temperature. The~e were then washed with PBST and incubated for 30 minutes at room temperature with 25 ~l of conjugate' diluted 1:1,000 in PBST.
The cloth portions of the test strips were then washed with PBST, and were subsequently assayed for retained enzyme activity by immersion in 1 ml ABTS-indicator solution for 30 minutes with gentle shaking. The reaction was stopped by addition of 0.5 ml of 0.1 M NaF and absorbance was read at 650 nm. Each determina-tion was performed in quadruplicate, and the results of the assay are shown below in Table 8.

Table 8. Application of Antibody-Coated Test Strips in the Immunoassay _ . A 650 ~, ~ ~ A~ l ~rTe~ ~ 1 2 ¦ 3 4 1.2xl0~0.4ll0.4~ 0.415 0.435 _ o.oe3 0.016 o.oeo o.oe1 The results of the assay demonstrate the ability of the antibody-coated test strips to detect B. abortus antigens at the concentration tested. The background level of enzyme activity was negligible, thus satisfying one of the important requirements of the immunoassay. These results were reproducible.
V APPlication of a Hvdrophobic Cloth as an Adsorbent and Absorbent of Antiqen in Immunoassav for B. Abortus LPS.
The following experiment shows the application of hydrophobic cloth as an ad~orbent and absorbent of antigen for the immunoassay.
Polyester non-woven cloths (those known by the Trade-mark SONTARA 8100 of Du Pont) were incubated with 50 ~l per 6 x 6 mm piece of 2 ~g/ml solution of B. abortus LPS in PBS, overnight at room temperature. The cloths were then washed with PBST.
~SONTARA 8100 is a macroporous non-woven 100% polyester cloth B

- 38 a -having a Frazier air permeability (in M3/m2.5 at 124 Pal of 1.1 of thickness 1.02 mm so that the pores exceed 20 ~m in diameter).
Each cloth was incubated for 30 min, at room temperature, with 25 ~1 of either of the following preparations: 50 ~1 of PBS
containing 90 ng of B. abortus lipopolysaccharide plus 25 ~l of antibody-horseradish peroxidase conjugate diluted 10,000 X in PBST, 50 ~1 of PBS containing a ng of lPS plus 25 ~l of diluted conjugate, 50 ~l of PBS containing 0.9 ng of lipopolysaccharide plus 25 ~l of diluted conjugate, or 50 ~l of PBS alone plus 25 ~l of diluted conjugate.

~' ~3 The cloths were then washed with PBST and assayed for HRP by immersion in 0.5 ml of ~BTS-indicator for 90 min, at room tem-perature. The reaction was stopped by addition of 0.5 ml of 0.1M
NaF, and absorbances were read at 650 nm. The results of this experiment: are recorded in Table 9 below.
Table 9 ~ Immunoassay for the Detection of B. Abortus LPS
-LPS (ng) A Visual observation appl ied per cloth 1 650 2 0.02 0.01 yes 3 0.127 0.115 yes 0.3 0.22 0.20 no 0 0.10 0.23 --The re~ults show clearly that at least 30 ng and 3 ng of LPS
were detectable by simple visual examination, where the corre-sponding samples produced less colour than the control. However, it i~ equally important to note that the assay might succe~sfully detect even lower quantitie~ of LPS by colorimetric measurement.
This assay offers the di~tinct advantages of providing an inter-nal control for conjugate quality and requiring only one incuba-tion step for the decisive immunological reaction. Such advan-tage~ make this as~ay more easily adaptable for field testing.

It is believed that the detectability limit of this assay may be improved by coating the polyester cloth with a limited quantity of antigen, making the free antigen present in the test sample more competitive for the conjugate.
By another aspect of this invention, an improved enzyme-antibody conjugate having a higher specific activity has been provided which should allow for the use of highly diluted conjugate in the a~say, thu~ deccribing the quantity of free antigen required to prevent attachment of the conjugate to the cloth (thus increa~ing the sensitivity of antigen detection).
The immunoassay device of aspects of thi~ invention thus employs hydrophobic cloths as ~urface~. Hydrophobic fabric~
~cloths) of plastics, e.g. polypropylene and polyester, are moderately priced because of their large commercial demand as textiles and filters. These cloth~ offer the fcllowing advan-tages over the previous adasorption supports: they can accomodate a larger volume of sample per area; have a larger surface area for binding immunoreactants and for immunoreactions; are easily washed because of minimum flow resistance; and have both strength and durability.
The immunoassay procedure for detecting antigens in test samples using antibody-coated cloths was found to be fast and simple, requiring only the most basic instruments found in most research and clinical laboratories. The assay i~ al~o designed 80 that a qualitative result can be obtained in field test situa J 1~ L111 ~

tions, where access to instrumentation is limited or non-existant.
The immunoassay procedure is a rapid and simple procedure which can be applied for the direct detection of antigens in test samples. The assay can be easily and economically adapted for field testing, where a positive result could easily be distin-gui~hed from a negative one by visual assessment of the sub-strate-indicator, (e.g. AsTS), which produces a blue-green colour in the presence of horseradish peroxidase. The "dipstick" format of the field kit makeQ the manipulation of antibody-coated cloth throughout the procedure simple and convenient, so that the test can be performed by untrained hand~. Furthermore, thi~ format obviates the need for even common laboratory equipment, and all necessary reagents can be easily supplied in the form of a kit.
Although horseradish peroxidase was used as an indicator, any other suitable enzyme, e.g. alkaline pho~phatase and galactosidase, can be employed in the enzyme-antibody conjugate for the detection of antigen~. Also, monovalent antigens e.g.
toxins ~i.e., hapten~ can be detected by a competitive assay form of the immunoassay method. In this form of the aQsay, a ~tandard preparation of the monovalent antigen could be immo-bilized on the cloth surface by adsorption or via a hydrophobic carrier. A teQt sample suspected of harboring the antigen would then be mixed with an enzyme-antibody conjugate specific for that antigen and incubated with the antigen-coated cloth. A negative control in which a representative sample devoid of antigen is mixed with the conjugate would be incubated with a separate antigen-coated cloth. Since the presence of free antigen in the test sample should prevent binding of the conjugate to the cloth surface, the assay result would be obtained by comparing the amount of enzyme immobilized on the test cloth with that obtained on the negative control cloth. Thus, the immunoassay is amenable to a variety of assay forms, the exact form being determined by the nature of the specific antigen being detected.
The results obtained indicate that several types of hydrophobic cloth~ can be used as #olid phases for the adsorption of antibodies. The#e include polypropylene, polye_ter, nylon, and polyethylene cloth#, all of which were found to be suitable ad#orbent# for antibody, e.g. anti-Brucella antibody. All those cloths have proven successful for the detection of antigens, e.g., B. abortu# antigens.
It has been found that whole bovine antiserum containing antibody with the appropriate antigen-specificity can be u#ed to coat hydrophobic cloth when heated at 75C for 10 minute#. Thi#
obviates the need for purified antibody preparations, which are time-con~uming to produce and may entail some expense. However, in order to minimize the potential for cross-reactions it is preferred that enzyme-antibody conjugate# be prepared using purified antibodies. Since the conjugate can be diluted up to 1,000 time#, only a small amount of conjugate ~tock need be pre ` ;. ~

pared in this manner, thus maintaining the ease and economy of each individual test.
The detection of Brucella abortus ~the causative agent of bovine brucellosi~) was used to test the utility of the method of an aspect of this invention. Using the hydrophobic cloth car-riers of an a~pect of this invention coated with whole antiserum preheated at 75C for 10 minutes, the immunoassay was able to detect 0.3 nanograms of B. abortus lipopoly~accharide and 104 B.
abortus whole cells. The polypropylene cloth-based immunoassay was also ~ucces~fully adapted for the detection of bovine viral diarrhea (BVD) antigen.
As noted above Brucella abortus was used to examine the performance of hydrophobic cloth as adsorbents of~immunore-actants. B. abortus causes brucellosis, a serious disease of humans and cattle. Confirmation of the brucellosis by the cultural diagnosis is a slow, complicated process of uncertain sensitivity. Rapid, simple and sensitive detection of Brucella antigen~ will facilitate confirmation and thus surveillance of brucellosis and it~ control.
Polypropylene cloth has been found to have excellent proper-tie# as a solid phase in the immunoa~say. The fact that poly-propylene filter cloth is available in a nonwoven filter cloth form gives it the added advantage of retaining a stable fabric structure ~i.e., no loose edges) even under agitated conditions.
Furthermore, polypropylene filter cloth i~ easily adapted for the preparation of commercial test kits.

y~

1~91Si ~9 The detection of B. abortus antigens by antibody-coated cloths i8 only one example of the method of aspects of this invention for the study of microbial antigen detection by cloth enzyme immunoassay. The immunoassay method is amenable to the detection of any given number of microbial antigens, provided that these are sufficiently small to be retained on the antibody-coated cloths throughout the assay procedure. In cases where antigens, e.g. whole cells, are too large for effective retention on the cloths, important antigenic components thereof might be dissociated from the surface by simple chemical or mechanical means 80 as to facilitate detection.
For example, the present invention in other aspects i~
applicable to many immunologically reactive materials, e.g.
proteins, peptides, polysaccharides, etc. which are of decisive ~ignificance for an immunological determination, i.e. the presence of the~e materials is the determining factor in the immunological test procedure. These materials can be detected in the body fluids of humans and animals using immunological prin-ciple~ or can ~erve for their detection. Especially suitable immunologically-reactive materials are pathogenic and vaculta-tively pathogenic organisms, e.g., parasites, protozoa, bacteria or viruses or their immunologically-active components, isolated antibodies from humans and animal~, serum constituents, toxins, hormones, enzymes, alkaloids, cell and tissue extracts, sub-stances with a small molecular weight, e.g., insulin, anngio-tensin and urokinaYe, biogenic amines, blood cells, particles chemically or physically covered with antigens or antibodies, e.g., erythrocytes or latex particles.
The following Table provides a selection of typical diseases or conditions which can be determined with the aid of the immuno-assay device in accordance with aspects of the present invention according to the immunologically reactive materials lyophilised thereon.
TABLE
Antiqen Disease Toxoplasma gondii Toxoplasmosis Entamoeba histolytica Amoebiasis Trypano~oma cruzi Chagas Trypano~oma gambiense/rhodesiense Sleeping sickness Leishmania donovani Leishmaniasi~
Schistosoma mansoni Schistosomiasis Echinococcus granulosu~ Echinococcosi~
Filariae Filariasis Fasciola hepatica Fascioliasi~
Pla~modia Malaria Candida species Candidiasis Aspergilli A~perigillosi~

Mycropolyspora faeni/Micromonospora vulgaris Farmer s lung 4~

Treponema pallidum Syphilis Neisseria gonorrhoeae Gonorrhea Neissseria meningitis Meningitis Brucella abortus Brucellosis Mycoplasma pneumoniae Pneumonia Australia antigen Acute hepatitis Herpes simplex virus HerpeQ simplex Influenza virus Flu Cell nuclei Systemic lupus erythrematosi~ or Scleroderma Cryptococci CryptococcosiQ
Torulopsis species Systemic mycosis H-antigen Salmonella ~flagellar)

Claims (11)

1. A cloth enzyme immunoassay device comprising the combination of a microporous hydrophobic cloth selected from the group consisting of woven polypropylene, non-woven polypropylene, woven polyester, non-woven polyester, woven nylon, non-woven nylon, woven polyethylene, and non-woven polyethylene, said cloth having such structure that it: can accommodate a large volume of liquid per surface area thereof, has a large surface area, and has a minimum flow resistance, and an antibody or an antigen directly adsorbed thereon and directly absorbed therein.

2. The immunoassay device of claim 1 wherein said antibody is provided by an antiserum containing an antibody specific for the antigen being tested.

3. The immunoassay device of claim 2 wherein said anti-bodies present in said antiserum have been partially denatured.

4. The immunoassay device of claim 3 wherein said partial denaturization is achieved by exposure to a low pH environment.

5. The immunoassay device of claim 3 wherein said partial denaturization is achieved by heating.

6. The immunoassay device of claim 1 wherein said antibodies present in said antiserum have been affinity-purified.

7. The immunoassay device of claim 1 wherein said antibody is provided by a purified antibody bearing the appropriate specificity.

8. The immunoassay device of claim 1 wherein said antibody is provided by diluted antiserum.

9. The immunoassay device of claim 1 in the form of said hydrophobic synthetic polymer cloth bonded to a different material, thereby to provide an antibody-coated test strip that may be handled throughout an assay procedure.

10. An immunoassay method for detecting an antigen, said method comprising:
a) treating a surface of a macroporous hydrophobic synthetic polymer cloth selected from the group consisting of woven polypropylene, non-woven polypropylene, woven polyester, non-woven polyester, woven nylon, non-woven nylon, woven polyethylene, and non-woven polyethylene, said cloth having such structure that it: can accommodate a large volume of liquid per surface area thereof, has a large surface area and has minimum flow resistance, with an antibody, thereby to have an antibody directly adsorbed thereon and directly absorbed therein;
b) incubating said treated cloth with a sample to be tested for the antigen;
c) washing said incubated cloth with a buffer to remove unabsorbed sample;
d) incubating said washed cloth with an enzyme-antibody conjugate prepared by coupling a purified antibody specific for said antigen to an indicator enzyme;

e) washing said incubated cloth with a buffer to remove unreacted conjugate; and f) detecting remaining enzyme-antibody conjugate by incubation in a chromogenic substrate indicator solution to produce a visible colour upon product formation.

11. A immunoassay method for detecting an antigen, said method comprising:
a) treating a surface of a macroporous hydrophobic synthetic, polymer cloth selected from the group consisting of woven polypropylene, non-woven polypropylene, woven polyester, non-woven polyester, woven nylon, non-woven nylon, woven polyethylene, and non-woven polyethylene, said cloth having such porosity that it: can accommodate a large volume of liquid per surface area thereof, has a large surface area, and has minimum flow resistance, with an antigen, thereby to have an antigen to be assayed directly adsorbed thereon and directly absorbed therein;
b) applying to the surface of said treated cloth, a mixture of the antigen being assayed and an enzyme-antibody conjugate specific for said antigen being assayed;
c) treating a control said identical macroporous hydrophobic synthetic polymer cloth with a mixture of said antigen being assayed and an enzyme specific for said antigen being assayed to have said mixture of said antigen being assayed and an enzyme specific for said antigen being assayed directly adsorbed thereon and directly absorbed therein;
d) incubating both said treated cloths substantially simultaneously;
e) washing each of said incubated cloths with an identical buffer solution; and f) detecting said antigen by incubation of both said cloths in an identical chromogenic substrate indicator solution to produce a visible colour upon product formation, the amount of antigen being determined by the difference in intensity of the colour between said cloth containing said free antigen and said cloth containing said antigen-enzyme-specific mixture.