IMPROVED ENZYME IMMUNOASSAY AND A KIT THEREFOR

Abstract: The use, in an enzyme immunoassay, of a detecting support carrying thereon enzyme detecting reagent fixed at a first set of localised predetermined positions in detecting enzyme labels immobilized at a corresponding and second set of fixed localised predetermined positions carried on an immunological reaction support, the immobilization of which labels is the result of an immunoassay reaction, said detecting support being superimposible over said reaction support with the respective sets of positions in contact to permit detection of enzyme labels. A kit for use of the assay method is provided.

Description: This invention relates to an improved enzyme immunoassay. In particular, it relates to a modification in enzyme immunoassays which is useful for the diagnosis and therapy of various diseases. It is useful in the preventive and therapeutical treatment of various diseases to clinically assay specific active substances including hormones, enzymes and antibodies in biologically derived samples such as blood, urine and cerebrospinal fluids and tissues. Such clinical assays are conducted as part of the routine work of medical centers and hospitals. However, conventional methods usually take much time, require a relatively large amount of sample and involve complicated procedures. These are inconvenient for both the patients and those who are involved in medical practice. Therefore, it has been desired to develop an assay which requires only a small amount of sample and which gives an accurate result in a short period of time. Immunoassays have recently widely been used as a clinical assay. These are based on the specific reaction between two immunologically active substances, (e.g. an antigen and an antibody), one of which is labelled with a detectable marker. Numerous markers are known for use in immunoassay, for example, enzymes, radioisotopes and fluorophores. Among them, enzymes are especially useful for accuracy because of their substrate specificity. In a typical enzyme immunoassay, a test sample is treated with an immunologically active substance (A) (e.g. an antigen or an antibody) which has previously been labeled with an appropriate enzyme. Any immunologically active substance (B) (e.g. an antigen or an antibody) contained in the test sample is specifically bound with the substance (A) and then the enzymatic activity in the bound substance is measured by a conventional method, usually by colorimetry, The reaction is usually carried out in the presence of an appropriate substrate (e.g. which can be colored by the catalytic effect of said enzyme). According to this method, the amount of the substance (B) in the test sample can quantitatively be determined. It is well known that enzyme immunoassay includes "heterogeneous immunoassay" (e,g, "ELISA") and "homogeneous immunoassay" (e.g, "EMIT®"). The former employs a phase separation procedure as an integral part of the method, while the latter is conducted in a single reaction mixture. ELISA is applicable to a wide range of substances in comparison with other methods. This invention provides an improvement in the ELISA technique. ELISA can be classified into two assays by the type of reaction, that is, a competitive reaction or a non-competitive reaction. In the competitive assay, a test sample containing an unknown amount of an antigen (B) is reacted with a known amount of an immobilized antibody (A) in the presence of an enzyme-labeled antigen (B'), wherein the antigen (B) and the enzyme-labeled antigen (B') competitively react with the immobilized antibody. After the reaction, the immobilized antibody (A)-bound to enzyme-labeled antigen (B') is separated from the reaction mixture (usually by decantation), and the catalytic (enzymatic] activity of the bound product is measured. In this competitive reaction, the amount of the enzyme-labeled antigen (B') bound to the antibody (A] is decreased by the amount of the antigen (B) contained in the test sample, in other words, the amount of the antigen (B) in the test sample is inversely proportional to the amount of enzyme-labeled antigen (B') which is bound to the immobilized antibody. Accordingly, by measuring the enzymatic activity of the bound enzyme-labeled antigen, there can be measured the amount of the antigen (B) contained in the test sample. On the other hand, in the non-competitive immunoassay (e.g. sandwich assay), an immobilized antibody (A) is firstly contacted with a test sample containing an unknown amount of an antigen (B), and the bound sample is contacted with a known amount of an enzyme-labeled antibody (A'). After the reaction, the bound product of the antigen (B)-the enzyme-labeled antibody (A') is separated from the reaction mixture, and the amount of the bound enzyme-labeled antibody is measured on the basis of the catalytic (enzymatic) activity by a conventional method. The amount of the bound enzyme-labeled antibody is proportional to that of antigen (B) in the test sample. Thus, the amount of the antigen in the test sample can quantitatively be determined. As mentioned above, these enzyme immunoassays employ an antigen (or antibody) immobilized on the surface of a suitable solid carrier. The solid carriers include various kinds of natural or artificial products, such as polyacrylamides, nylons, and glass. In general, porous beads carriers are used in ELISA assay. When the bead carrier is used as a solid phase in ELISA, an immobilized substance (e.g. antigen) which is obtained by fixing the protein on the surface of the beads is reacted with an enzyme-labeled substance (e.g. antibody) competitively or non-competitively. To this reaction mixture, any suitable color-producing reagents such as pigments which can be colored by the catalytic effect of the enzyme is added, and colored reagents are extracted from the reaction mixture. Then, the extract is subjected to spectrophotometric measurement. However, conventional ELISA assay using a solid beads carrier has to be conducted in liquid phase and requires troublesome laboratory procedures such as separation and extraction of colored reagents and spectrophotometrical measurements of the extract. Because of such laboratory procedures, this method cannot give the desired data rapidly. It is very important to get rapid and correct information from these assays in order to apply the most suitable diagnoses and treatments to each patient. This is especially significant in emergency cases. Thus, it has been desirable to find an improved immunoassay suitable for clinical assay of diseases, particularly for diagnosis of diseases in the early stage, for instance, in diseases involving antigen-antibody reaction or various infectious diseases. EP-A-0111042 discloses a diagnostic testing slide which is treated with a flame or corona discharge to provide a single hydrophilic region on which mixing of immunoreactive substances can take place but does not clearly disclose immobilisation of such substances. US-A-3936357 discloses a diagnostic test device which allows a sample to be brought into contact sequentially with different reagents, using layers of absorbent material connected by hinges. The final step in such a sequence bringing the sample into contact with a colour indicator. This device is also a single site reaction device. US-A-3933954 similarly discloses a hinged arrangement whereby an analyte is brought into contact with reagents, again at a single site. EP-A-0165288 (this document belongs to the prior art according to Article 54(3)EPC) discloses a device allowing chemical analysis to be carried out. The device is comprised of segments which can be brought into contact with each other, wherein the segments contain sites which reagents and samples are present. Folding allows a sample to be contacted with reagents thus carrying out the analysis. The applicants have aimed to develop an improved enzyme immunoassay which can measure an immunologically active substance in the living body using a rapid and simple procedure with high accuracy. They have now found that the desired assay can be effected by using a membrane carrier for immobilizing proteins whereon hydrophilic spots are formed in combination with a coloring-membrane on which a pigment is fixed in spots corresponding to the hydrophilic spots of the membrane carrier. The present invention aims to provide an improved enzyme immunoassay which can be carried out by a simple procedure to give an accurate result in a short period of time. A further aim of this invention is to provide a kit for use in carrying out the assay of this invention. The present invention will be described with reference to the accompanying drawing, in which; Fig. 1 shows a schematic plain view of one embodiment of an immuno-active membrane with hydrophilic spots used in this invention. Fig. 2 shows a schematic sectional view of one embodiment of a kit for the enzyme immunoassay of this invention. The invention relates to a method for assaying an immunologically active substance (B) in a test sample which comprises; (a) preparing an immuno-active membrane of a hydrophobic polymer sheet having a number of hydrophilic spots thereon, on to which spots a prefixed amount of an immuno-active substance (A) or (B) has been immobilized, (b) preparing a detecting-membrane having a number of spots which contain a detectable reagent which can be detected by the catalytic effect of an enzyme at the corresponding positions to those of the immuno-active membrane, (c) contacting the immuno-active membrane with the test sample and standard samples containing predetermined amounts of substance (B), together with, simultaneously or successively, substance (A') or substance (B') where the immobilized substance is substance (A) or substance (A') where the immobilized is substance (B), substances (A') and (B') being substances (A) and (B), respectively, but each carrying an enzyme label, whereby immunoreactions are permitted to occur. (d) adding a substrate to each spot of the immuno-active membrane, (e) superimposing the detecting membrane on the immuno-active membrane so that the spots in both membranes correspond to each other, (f) detecting the detectable reagent and thereby determining the amount of substance (B) in the test sample. The preparation of the immuno-active membrane and the coloring-membrane and the use of the present invention will now be more fully explained. (1) Preparation of the immuno-active membrane The immuno-active membrane used in this invention can be prepared using various kinds of hydrophobic polymers. The desired number of hydrophilic spots can be formed on the surfaces of the hydrophobic sheets by known procedures in the art. For the purpose of this invention, it is convenient to use a membrane-carrier which has been disclosed in Japanese Patent Application No. 277656/1984 (hereinafter, it is referred to as "spot-membrane"). Thus, for example, a hydrophobic porous polymer sheet such as porous polyolefin sheet (e.g. polypropylene or polyethylene sheet) is put between two shielding boards (e.g. aluminum boards) perforated with numerous small holes (a) of a suitable size. These are exposed to low-temperature plasma from both sides of the shielding boards. The resulting membrane has numerous hydrophilic spots corresponding to the holes of the shielding boards. This membrane may then be treated with 0.02 - 10 % aqueous octamethylenediamine in order to promote the immobilization (fixing) of an immuno-active substance (protein) thereon (the amount of protein adsorbed on a spot depends on the concentration of the octamethylene-diamine). The membrane may be washed with phosphate buffer (PB) or phosphate buffered saline (PBS), then treated with glutaraldehyde to introduce aldehyde groups. This membrane is washed again with PB or PBS. An immuno-active substance may then be immobilized on to the spot-membrane mentioned above by usual methods. This substance is capable of reacting with the substance to be assayed in the test sample, or is its own analogue. For example, when the substance to be assayed (B) is an antigen, the substance to be immobilized may be a known amount of an antibody (A), specific for the substance (B) (including its analogs) (vice versa, when the substance (B) is an antibody, the substance to be immobilized (A) is an antigen). The spot-membrane may be any shape and size, but is conveniently in the shape of a rectangle as shown in the accompany Fig. 1. The shape may also be a square, round or any other shape. The size is also not vital but may be 2 to 12 cm x 6 to 20 cm,preferably 5 cm x 10 cm for easy handling. The thickness is usually 25 to 100 µm. The number and size of the spots (a) are also not specific, but the spots are usually formed at 4 to 32 spots per membrane at a size of 0.5 to 15 mm in diameter. This procedure is illustrated by using albumin as the test substance (B) (antigen) in the following description. Firstly, an anti-albumin antibody (A) is prepared as follows. Animals such as rabbits, goats or guinea-pigs are immunized against albumin by a multiple injection course of a highly purified globulin-free albumin. Then, antiserum is harvested from bloods samples collected from the immunised animals. An anti-septic agent is added to this antiserum. The anti-albumin antibody (A) may also be a commercially available one. The anti-albumin antibody (A) is diluted with a buffer such as PBS to 1/1500 - 1/3000 fold. The anti-albumin antibody (A) is then immobilized onto the spot-membrane as follows. The diluted antibody (A) is added on to each spot of the membrane at an amount of 5-100 µl], preferably 10-20µl]. Alternatively, the spot-membrane may be immersed in 100-1000µl [preferably 300-400µl], of the diluted antibody at 4°C - 30°C for 10 minutes to 48 hours, preferably 5 to 24 hours. After washing with PB or PBS, normal serum from goat, rabbit or guinea-pig, or bovine serum albumin is poured into each spot in order to block unbound groups on the membrane. The resultant membrane is washed again with PB or PBS. The immuno-active membrane may be used per se , but is preferably used in the form of a kit as shown in accompanying Fig. 2. That is, the immuno-active membrane (1) is placed on a suitable holding board (2) such as a glass or plastic board (thickness: about 1 mm) and covered with a flame board (3) with holes corresponding to the spots (a) of the immuno-active membrane (1). The holding board (2) is preferably colorless. It may be more convenient to put all of the components (1), (2) and (3) in an appropriate container (5). In Fig. 2, it is shown as attached with a coloring-membrane (4). The thickness of this kit can be 0.5 to 5 mm. (2) Preparation of coloring membrane The coloring-membrane is prepared by using the same polymer sheet as used for the spot-membrane as above or a filter paper, wherein a color-producing reagent such as pigment is immobilized at the regions corresponding to those of the spots of the immuno-active membrane. This procedure is illustrated in the following description. (i) Where the same polymer sheet as that for the spot-membrane is used: The spot-membrane as prepared above(without immobilization of an immuno-active substance) is adhered to a glass or plastic film such as polyphenylene film with the aid of agar or double-coated adhesive tape. A solution of a pigment (concentration: 0.01 - 0.1 %) in water or 1 % agar solution is added to spot in a proportion of 10-20µl/spot. The coloring-membrane should be stored in the dark (under deaeration). (ii) Where filter paper is used: Alternatively, the coloring-membrane can be prepared using filter papers such as TOYO FILTER, No.2, No.131, No.520, or No.514A. The filter paper is dipped in 1 % agar containing 0.01 = 0.1 % pigment. It is preferred to keep the resulted membrane in dark and dry condition at a temperature of 25 - 37°C. This filter paper is cut into pieces of the same size and shape as the spot on the immuno-active membrane (1). Then, these pieces are adhesively aligned on a suitable support such as glass or polyethylene sheet, preferably a colorless one. (iii) The coloring-membrane can also be prepared from a plate having wells on its surface. This plate may be a polyethylene or polypropylene sheet of about 1 mm thickness. Wells are formed corresponding to spots on the immuno-active membrane. The previously determined amounts of agar containing a pigment may be added into the wells, then the plate dried. This process is suitable for many materials other than agar, for example, gelatin, dextrin and polyethyleneglycol. Color producting reagents used in this invention include many pigments such as 5-aminosalicylic acid, o-phenylenediamine, 2,2-azyno-di-(3-ethylbenzyl-thiazoline)-6'-sulphonic acid (ABTS), o-nitrophenyl-β-D-galactoside and phenyl phosphate disodium. The pigment is selected depending on the type of enzyme. For example, when the enzyme is peroxidase (HRP), the pigment may be ABTS, and when the enzyme is β-D-galactosidase, the pigment may be disodium phenylphosphate. Immunoassays can be conducted as follows. (3) Immunological reaction (a) Sandwich assay (non-competitive assay) An aqueous solution of albumin (B) to be tested (each 50 - 500µl) is poured onto each spot of an immuno-active membrane immobilized with anti-albumin antibody (A) and allowed to react for 10 minutes to 48 hours, preferably, 30 minutes to 3 hours at 4 - 30°C, preferably, at 25°C. After or during the reaction, an enzyme-labeled anti-albumin antibody (A') which has been diluted to 1/10 - 1/3000 with a buffer such as PBS containing surfactants (e.g. Tween®) is added to each spot. The membrane is maintained at 4 - 37°C, preferably at 30 -35°C for 10 minutes to 2 hours, preferably 30 to 60 minutes. (b) Competitive assay Albumin (B) to be tested and an enzyme-labeled albumin (B') are dissolved in PBS containing surfactants such as Tween, separately. These solutions are combined together in a certain proportion, and the resulting mixture in added to each spot of an anti-albumin antibody (A)-immobilized immuno-active membrane. They are immunologically reacted for 10 minutes to 48 hours at 4 - 35°C, preferably, for 10 to 60 minutes at 20 - 30°C. (c) Competitive hindrance assay An aqueous solution of albumin (B) to be tested is poured onto each spot of albumin (B)-immobilized immuno-active membrane. Then, an enzyme-labeled anti-albumin antibody (A) is added thereto. The resulting membrane is treated in the same manner as described in the above (a). Preferable enzymes for the above enzyme-labeled active substance can be chosed from typically used enzymes in ELISA, for example, horseradish peroxidase (HRP) , β-galactosidase and alkaline phosphatase. The enzyme may be bound to the antigen or antibody to be labeled by a conventional method such as glutaraldehyde procedure, periodic acid procedure or maleimide procedure. (4) Determination The enzyme bound to the immuno-active membrane (1) noncompetitively (3-a) or competitively (3-b and -c) may be detected according to the following steps. (a] Wash the resultant immuno-active membrane with PB or PBS. (b) A mixture containing a substrate component other than those immobilized on the coloring-membrane is added dropwise into each spot (a) of the immuno-active membrane (1). The composition of the substrate varies depending on the enzyme. For example, when the enzyme is HRP and the pigment is ABTS, the substrate may be a mixture of 0.003 5 H₂O₂, 0.03 M citric acid and 0.06 M disodium phosphate (pH 4.4). (c) The coloring membrane (4) is superposed on the surface of the immuno-active membrane (1) so that the spots of both membranes correspond. In this step, the coloring membrane becomes wet and colored. (d) After standing for a while (e.g. 1 - 10 minutes), the coloring membrane is removed to stop the reaction. Alternatively, about 5 ml of 0.05 % sodium azide may be added into each spot (a) to stop the reaction, It is preferable to use an adhesive tape, such as Limbro/Titrek (Plate Seal) , (manufactured by Flow Laboratories Inc.) or Celotape (manufactured by Sekisui Chemicals Inc.) for protecting the surface of the coloring membrane (4) against oxidation. (e) The color of the spots corresponding to the test sample (test spots) is compared to that of the spots which have not been treated with the test sample (control spots) and their intensity evaluated by naked eye or with a densitometer. The kit of this invention may be washed by dipping all the components in washing solution such as PBS and draining throughly, and may then be used repeatedly. However, when the test sample is infectious, after sealing the container (5), the kit can be discarded. The assay of this invention is applicable to various ELISA assays, and does not require troublesome laboratory procedures as in conventional assays. This assay is useful to determine a lot of immunologically active substances in biological samples collected from many kinds of organisms, especially human. These substances include hormones, growth hormone, insulin, glucagon, human-placental gonadotropins, steroid hormones, and prostaglandin; and further d-fetoprotein, tumor fetal antigen, ferritin, IgA, IgM, IgG, IgE, d₁-microglobulin, β₂-microglobulin, basic fetoprotein, HBS antigen, bile acid, various drugs, as well as antibodies arising from bacterial infections or viral infections, and antibodies involved in autoimmune diseases. According to this invention, a sample can be subjected to an immunological reaction immediately after being collected from subjects such as patients, and accurate results can be obtained in a short period of time. This invention is particularly useful for diagnoses of various diseases without troublesome laboratory procedures. This means that anyone can conduct the enzyme immunoassay, if necessary. Since the kit of this invention is portable, immunoassays can be done anywhere and anytime as needed. The following Example is provided to further illustrate and examplify the present invention. Example 1) Preparation of spot-membrane A sheet of plastic membrane (150 mm x 150 mm) was cut off from a commercially available microporous polymer sheet (Juragard #2500, 25 µm in thickness, maximum pore diameter 0.04 - 0.4 µm, hydrophobic, manufactured by Polyplastics Inc.), and the sheet was put between two aluminum shielding boards perforated with 36 holes of 6 mm diameter. They were subjected to plasma-treatment (100 mA, 500 V) with water vapor for 3 minutes from both sides to give a spot-membrane. 5 - 10 µl of 0.3% aqueous octamethylenediamine was added to each spot and allowed to stand for about 5 minutes. Following the removal of excess liquid, the resulted membrane was immersed in 5 % glutaraldehyde solution and washed with distilled water. 2) Preparation of immuno-active membrane Rabbit anti-HSA serum (R8411) was diluted with PBS to 1/200. The resultant solution was added to each spot of the spot-membrane obtained in 1) at an amount of 2O µl/spot. The spot-membrane was allowed to stand for 24 hours at 4°C The antiserum was removed with an aspirator, and the membrane washed. To these spots, HSA labeled with HRP(HRP-HSA) (test spot NO. B₀), 10 mg/l of HSA (ibid. B₁₀) and 100 mg/l of HSA (ibid. B₁₀₀) were added dropwise, respectively. The membrane was allowed to react at 25°C for 20 minutes and 40 minutes respectively. The excess liquid was removed by aspiration, followed by washing. 3) Preparation of coloring-membrane TOYO FILTER PAPER (No.151) was immersed in 1 % agar solution containing 0.01 % or 0.1 % ABTS(ABTS-agar), then the filter paper was air-dried. This filter paper was cut into circular pieces (6). These pieces were then aligned on double coated adhesive tape. Juragard membrane (2500) was attached around the edge of each piece of filter paper. On the piece of filter paper, 15 µl of substrate solution (0.0034 % H₂O₂ in 0.03 M citrate-phosphate buffer, pH 4.4 was poured. When the substrate solution was completely diffused, the resulting coloring-membrane was superposed on the immuno-active membrane. They were reacted for 2 to 15 minutes at 25°C. The coloring of the coloring-membrane was measured colorimetrically and evaluated as follows. The results are shown in Table 1. The symbols in the table indicate the observed degree of coloring on a scale of -(not colored) up to +++. The control assay was conducted using immuno-active membrane containing only HRP-HSA and substrate mixture which lacks H₂O₂. These results indicates that the coloring-membrane develops color corresponding to the concentration of the substances (HSA) in the test sample.

Claims: A method for assaying an immunoactive substance (B) in a test sample which comprises; (a) preparing an immunoactive membrane of a hydrophobic polymer sheet having a number of hydrophilic spots thereon, on to which spots a prefixed amount of an immunoactive substance (B), or substance (A) capable of immunoreacting with (B), has been immobilized, said hydrophilic spots being prepared by subjecting said sheet to a low temperature plasma-treatment using oxygen, oxygen-containing gas or water vapor, (b) preparing a colouring-membrane having a number of spots which contain a colour-producing reagent which can be detected by the catalytic effect of an enzyme, at positions corresponding to those of the immunoactive membrane, (c) contacting the immuno-active membrane with the test sample and standard samples containing predetermined amounts of substance (B), together with, simultaneously or successively, substance (A') or substance (B') where the immobilized substance is substance (A) or substance (A') where the immobilized is substance (B), substances (A') and (B') being substances (A) and (B), respectively, but each carrying an enzyme label, whereby immunoreactions are permitted to occur, (d) adding a substrate to each spot of the immunoactive membrane, (e) superimposing the colouring membrane on the immunoactive membrane so that the spots on both membranes correspond to each other, and (f) comparing the colour of the spots treated with the test sample with that of the remaining spots treated with the standard samples, and thereby determining the amount of substance (B) in the test sample. A method as claimed in claim 1 wherein the hydrophobic polymer sheet is porous. A method as claimed in claim 1 or claim 2 wherein the hydrophobic polymer sheet is a polyolefin sheet, e.g. polypropylene. A method as claimed in any one of claims 1 to 3 wherein the colouring-membrane is prepared by immobilizing a pigment at the regions corresponding to those of the hydrophilic spots. A method as claimed in any one of claims 1 to 4 wherein the assay is conducted as a sandwich assay, as a competitive assay or as a competitive hindrance assay. A method as claimed in any one of claims 1 to 5 wherein the hydrophilic spots are circular and of 0.5 to 15 mm diameter. An immunoassay kit for use in the method of any of claims 1 to 6 which comprises an immuno-active membrane, a supporting board and a flame board in a suitable container and a detecting membrane. The use of an immuno-assay kit as claimed in claim 7 in the determination of an immunologically active substance in a biological sample i.e. in diagnosis other than when practised on the human or animal body.

Inventors: HORIGUCHI, KAZUO, KAKU, SHIGEKI, NAKANISHI, SETSUKO, NISHIMURA, MASAJI, UO, JUNKO
Applicants: SHIONOGI & CO., LTD.
Application year: 1986
Main classification: G01N33
IPC codes: G01N33