METHOD FOR ASSAYING A BIOCOMPONENT

Abstract: Improved results are obtained in colorimetric bioassays using peroxidase as an enzyme label with 4-methoxy-l-­ naphthol as the reactive dye in combination with hydrogen peroxidase as the enzyme substrate.

Description: This invention relates to an improved method of conducting an immunoelectrophoresis assay, and especially for use in the diagnosis of cancer. The following general methods are known for the detection of a biocomponent, for example, serum protein in a sample: colour-staining, silver staining, autoradiography and immunological methods using a flurorimmunoassay (FIA), radioimmunoassay (RIA), all of which have been used from time to time in the diagnosis of cancer. The colour-staining method gives high backgrounds, unclear results and poor detection sensitivity. Silver staining and autoradiography are high in sensitivity and accuracy, but are operationally complicated and cannot be carried out promptly and simply. There are also waste disposal problems and their applicability is limited. The immunological methods can specifically detect single substances and have high sensitivity, give good quantitative determination and are of wide applicability. However, radioimmunoassays have a complicated operational procedure and are not applicable to double staining techniques and are thus of limited application. Enzyme immunoassays, in general, have high sensitivity and clarity and are comparatively simple and rapid to carry out. However, conventional methods for colouring enzyme-labelled gel immunoelectrophoresis assays, using an enzyme-labelled antibody, have problems of high background, lack of clarity and low sensitivity. Also, when carrying out gel immuno-electrophoresis assays a control marker or standard is generally made to migrate in the gel in a parallel but separate track. This method causes problems in the measurement of migration distances with any accuracy. In Electrophoresis 1983, 4 , 371-373 Takeda et al disclose the results of a study carried out on the separation and identification of different molecular species of human α-fetoprotein (AFP) in body fluid samples by double affinity electrophoresis with concavalin A and Lens cullinaris haemagglutinin A (LcH-A). It is not specifically stated, but it is known that the method of Takeda et al employs 4-chloro-1-naphthol as the chromogen in the subsequent development of the gel. In contrast to the method of Takeda et al, the present invention employs 4-methoxy-1-naphthol as the chromogen, it having been found by the present inventors that 4-methoxy-1-naphthol provides a much shorter colouring and migration time, a clearer migration diagram, and a markedly high sensitivity and resolving power as compared with 4-chloro-1-naphthol. 4-methoxy-1-naphthol is a known reagent for assaying hydrogen peroxide (EP-A-0060518) but it is not suggested that that chromogen is of any particular benefit in any particular application when compared either with 4-chloro-1-naphthol or any other known chromogen for the assay of hydrogen peroxide. Thus, in accordance with the present invention an assay method is provided in accordance with the preamble to claim 1, and wherein there is used in the development of gel after electrophoresis, a chromogenic composition comprising the combination of hydrogen peroxide and 4-methoxy-1-naphthol. In a more particular and preferred aspect of the present invention, there is added to the sample prior to electrophoresis, as an internal standard, a coloured protein-dye conjugate, the protein component of which has a different antigenic specificity from that of the biocomponent or target substance to be assayed, and there is incorporated into the gel-like carrier a receptor substance capable of undergoing a specific binding reaction with the standard protein. By adding a control marker combined with a colouring matter, eg. Bromophenol Blue, to the sample to be assayed as an internal standard, the reproducibility of the migration distance and the accuracy of measurement of the migration distance are considerably enhanced. The biocomponents assayable by the method of the present invention include: antigens, antibodies, enzymes, enzyme substrates, coenzymes, proteins glycoproteins, glucides, glycolipids, lipoproteins, nucleic acids, hormones, etc. and which are to be found in various body fluids, pathological tissue sections etc. The method of the invention is particularly applicable to the assay of body fluids such as blood, urine, saliva, gastric juice, intestinal juice, pancreatic juice, bile, tissue fluid, edema fluid, ascitic fluid, hydrothorax fluid, hydropericardia fluid, synovial fluid, cerebrospinal fluid, amniotic fluid, genital secretion fluid, mother's milk, nasal mucus, phlegm, tear, sweat, etc., and especially blood serum or plasma. Also included as target substances are labelled biocomponents comprising as the label an antigen, antibody, enzyme, enzyme substrate, coenzyme, protein, glycoprotein, glucide, glycolipid, lipoprotein, nucleic acid, hormone, avidin or biotin. As the receptor there may be used a peroxidase conjugate e.g. a peroxidase labelled antibody, protein A, enzyme, enzyme substrate, coenzyme, nucleic acid, hormone-receptor, specifically bound protein (B-2 protein-bound protein, C-reactive protein, etc.), avidin, biotin, or lectin etc. Preferably the receptor is peroxidase (POD)-labelled protein A. Since the various assay conditions such as the amount of assay sample, the amount and the ratio of enzyme-labelled receptor, the amount of substrate, the reaction time and temperature, etc. depend on the substance to be assayed, the titer of the receptor to be used etc., the most suitable conditions for each assay sample must be selected. For example, when POD-labelled protein A is used in staining the gel after electrophoresis, unbound substances are first eliminated from the gel after electrophoresis, and then a predetermined amount of POD-labelled protein A is added dropwise to the gel surface, followed by allowing the assay sample to stand at approximately room temperature, preferably at 15 to 25°C, for 30 to 45 minutes. Then, the assay sample is washed with an appropriate buffer solution for 30 to 40 minutes, and then hydrogen peroxide and 4-methoxy-1-naphthol are added thereto to initiate the enzyme reaction. After the precipitate line is thoroughly coloured (usually after 2 to 3 minutes), the assay sample is washed to remove unreacted substances, decolorised and dried. In the washing and decolorisation steps any appropriate reagent may be used, but it is preferable in view of simplicity and rapidity to use running water. After washing and decolorisation, the assay sample is dried slowly since too rapid drying causes unevenness, making the surface of the gel rough and interfering with the assay. Thus, it is preferable to carry out drying at a temperature from room temperature to 60°C. Preferably the concentration of POD-labelled protein A is adjusted to 0.01 to 0.5% (W/V) by appropriate dilution, the preferred diluting solution being a barbital buffer solution at a pH of about 8.6. The preferred NaCl concentration of the barbital buffer solution is 0.05 to 0.5M. The colouring reagent solution is a solution of about 1% 4-methoxy-1-naphthol in an appropriate solvent, which is further diluted to 0.001 to 0.1% when used. The preferred diluting solution is a Tris-hydrochloric acid buffer solution at a pH of about 7.0 to 8.5. The method of preparing the gel for the electrophoresis is not particularly critical, and any conventional method can be used. For example, a predetermined amount of agar, agarose or the like is added to distilled water or a suitable buffer such as a barbital buffer solution at a pH of about 8.6 or a Tris-hydrochloric acid buffer solution, and heated at a temperature from 60 to 80°C with gentle stirring to make a solution. The solution is then spread onto a flat plate and left to cool, thereby to coat the plate with the gel. The gel concentration can be adjusted to the particular characteristics (molecular weight, configuration etc.) of the biocomponent or target substance and the labelled receptor combination and the reaction product (colouring matter). Suitable gel concentrations will usually be 0.5 to 2.0% (W/V), preferably 0.8 to 1.0% (W/V). An antiseptic may be added to the gel as appropriate. When assaying a biocomponent of body fluid by gel electrophoresis, it is usual to use a control marker for the migration which is migrated in parallel with the sample. In the conventional method, the migration conditions may well differ between the control marker and the biocomponent due for example to local structural deformations of the gel, unevenness in the gel structure, electrical load conditions, temperature difference, etc., resulting in poor reproducibility. There is also a problem in the accuracy of measurement values. In accordance with the present invention it has also now been found that measurement values having better accuracy and reproducibility than those of the conventional method can be obtained by using a protein having a different antigen specificity from that of the bio-component, as an internal standard, and incorporating into the carrier gel a receptor capable of being specifically bound to the internal standard protein. In particular, when a mixture of the internal standard substance coloured by a colouring matter (for example, Bromophenol Blue, which will be hereinafter referred to as BPB) with a body fluid is subjected to electrophoresis on a gel-like carrier containing antibodies against the biocomponent and against the internal standard substance, measurement values having a better accuracy and reproducibility can be obtained. As the internal standard substance usable in the present invention, a protein having no cross reactivity with the biocomponent is employed. Suitable proteins include human serum albumin, bovine serum albumin, egg albumin, chick serum albumin, etc. Particularly preferred are egg albumin and chick serum albumin. As the receptor for the standard protein to be incorporated into the carrier gel a monoclonal or polyclonal antibody specific to the protein standard is preferably used. Suitable colouring agents for colouring the internal standard protein include Bromophenol Blue, Bromothymol Blue, etc. To avoid tailing by colouring matter unbound during the migration, it is preferable to adjust the concentrations of these colouring agents to 0.001 to 0.1% (W/V). In the method of the present invention, the colouring agent, internal standard protein, and body fluid are either mixed directly with one another, or a mixture of the colouring agent and the internal standard protein is first smeared onto a flat plate for reaction and dried, and then the body fluid is added dropwise to the flat plate and mixed with the colouring agent protein conjugate. The latter procedure is preferred because the concentration of the component in the body fluid does not influence the result, the stability of the internal standard substance is preserved and the technique is simple and rapid. The present invention is illustrated in, but not to be restricted by, the following Examples and by reference to the accompanying drawings, in which: Figure 1 shows a standard curve in AFP standard serum assay in Example 1. Figure 2 shows the result of AFP measurement in amniotic fluids in Example 2. EXAMPLE 1 Investigation of quantitative determinability of the target substance by rocket electrophoresis: a) Preparation of AFP standard serum: AFP standard serum (104l ng/ml, made by DAKO Co. Denmark) was diluted with normal human serum (AFP: 5ng/ml or less) to prepare AFP standard serums (100, 200, 400, 600 and 800 ng/ml). b) Preparation of anti-AFP-Containing agarose plate: 20 µl of anti-AFP serum (made by DAKO Co.) were added to 10ml of 1.0% agarose (dissolved in a barbital buffer solution having a pH of 8.6 and an ionic strength of 0.05, made by FMC Marine Colloids Division Co, USA) and mixed. The anti-AFP serum-containing agarose gel was then poured into a gel mould (110 x 125 mm, made by FMC Marine Colloids Division Co.) and coagulated to prepare an anti-AFP-containing agarose plate having a thickness of about 1mm. c) Preparation of POD-labelled protein A solution : 20 µl of POD-labelled protein A (made by E Y Laboratories, USA) were added to 10ml of a dilute saline solution (prepared by dissolving 14.0g of NaCl in 1l of barbital buffer solution having a pH of 8.6) to make a 0.2% POD-labelled protein A solution. d) Preparation of colouring reagent : Just before application, 200 µl of 4-methoxy-1-naphthol (made by Aldrich Co, USA), 10ml of 0.05M Tris-hydrochloric acid buffer solution (solution made by mixing aqueous trishydroxymethyl-aminomethane at 12.114 g/l and 0.1M HCl in a ratio of 100:77, pH 7.6) and a drop of 30% H₂O₂ were mixed together to make a colouring reagent. e) Measurement of AFP : 10 µl each of the AFP standard serums (100, 200, 400, 600, and 800 ng/ml) obtained in said section (a) were poured into holes (4.0mm in diameter) on the anti-AFP-containing agarose plate prepared in said section (b), and subjected to electrophoresis at 3mA/cm for 90 minutes at 10°C. After migration, the plate was treated with an absorbent pad (made by Nihon Shoji Co, Japan) for 2-3 minutes to eliminate unbound substances, and then washed with a phosphate buffer solution (pH 7.2) for 30 minutes. Then, the plate was dried again with an absorbent pad, and 10ml of the POD-labelled protein A solution prepared in said section (c) was poured onto the flat plate. After the reaction had proceeded for 30 minutes, the steps of drying with the absorbent pad, washing with the phosphate buffer solution (pH 7.2) for 30 minutes and drying with the absorbent pad were repeated in the same manner as above. 10ml of the colouring reagent prepared in said section (d) were then added to the plate and reacted for 2 - 3 minutes. Following that the plate was washed with water for 2 to 3 minutes, dried with an absorbent pad and the migration distances measured. By means of this procedure a standard curve is obtained as shown in Figure 1. EXAMPLE 2 MEASUREMENT OF AFP IN AMNIOTIC FLUID BY CONCANAVALIN-A CROSS-IMMUNOELECTROPHORESIS (CAIE) : a) Preparation of assay sample: Amniotic fluids collected from 16 women, 7 to 18 weeks pregnant were centrifuged (3,000 rpm for 20 minutes) and NaN₃ was added to the supernatants to a concentration of about 0.001% (W/V). The mixtures were freeze-dried at -20°C to make assay samples. b) Preparation of Con-A-containing agarose plate : To 10 ml of 1.0% agarose (dissolved in a barbital buffer solution having a pH of 8.6 and an ionic strength of 0.05) were added 600µg/ml of Con-A (Pharmacia Fine Chemicals Inc. Sweden). 15ml of the Con-A-containing agarose gel were then poured into a gel mould (110 x 125 mm, FMC Marine Colloids Division Co.) and coagulated to make a Con-A-containing agarose plate having a thickness of about 1mm. c) Measurement of AFP : 10µl aliquots of the samples prepared in section (a) were poured into holes (4mm in diameter) in the Con-A-containing agarose plate prepared in said section (b), and subjected to one-dimensional electrophoresis. Migration was carried out with a barbital buffer solution (pH 8.6) at 3 mA/cm for 90 minutes at 10°C. As the control, 5 µl of 3% (W/V) human serum albumin [fraction V, made by Sigma Co. USA, containing 0.01% (W/V) BPB] were used and the 6 cm-migration position was marked. After the one-dimensional electrophoresis, the agarose plate was cut into strips about 1 cm wide parallel to the direction of migration, which were then placed on top of the anti-AFP-containing agarose plate prepared in Example 1, section (b). The assembly was then subjected to further electrophoresis in a direction perpendicular to the first direction at 2.5 mA/cm for 2 hours at 10°C. After the migration, the plate was treated with an absorbent pad (made by Nichon Shoji Co. Japan) for 2 to 3 minutes, for elimination of unbound substances, and then washed with a phosphate buffer solution (pH 7.2) for 30 minutes. The plate was again dried with an absorbent pad, and reacted with 10 ml of the POD-labelled protein A solution prepared in Example 1, section (c). After the reaction had proceeded for 30 minutes, the steps of drying with an absorbent pad, washing with a phosphate buffer solution (pH 7.2) for 30 minutes and drying with an absorbent pad were repeated in the same manner as above, and then 10 ml of the reagent solution prepared in Example 1, section (d) were added and reacted for 2 to 3 minutes. After washing with water for 2 to 3 minutes and drying with an absorbent pad, the distance from the albumin position obtained in the one-dimensional electrophoresis to a migration peak shown in the two-dimensional electrophoresis was measured. AFP in the amniotic fluid is divided into two fractions by means by Con-A-CAIE. The fraction having an immune precipitate peak at the AFP-migrated position on the lectin-free agarose flat plate by one-dimensional electrophoresis is Con-A unadsorbed AFP (peak b), whereas the peak shown on the cathode side therefrom is Con-A-adsorbed AFP (peak a). Figure 2 shows a graph on which proportions of peaks b are plotted with changes in pregnancy period, defining the total height of peaks a and b as 100%. That is, Figure 2 shows that the Con-A adsorbed AFP in the amniotic fluid tends to increase with the elapse of time. EXAMPLE 3 MEASUREMENT OF AFP IN THE SERUM OF PATIENTS SUFFERING FROM HEPATIC CARCINOMA BY CON-A-CAIE a) Preparation of assay sample : Sera from 47 cases of primary hepatic carcinoma and 8 cases of metastatic hepatic carcinoma were diluted with normal human serum (AFP: 5 ng/ml or less) to prepare assay samples having not more than 15,000 ng/ml of AFP. b) Measurement of AFP : Measurement was carried out in the same manner as in Example 2. As a result, typical migration patterns of sera of patients suffering from hepatic carcinoma by Con-A-CAIE are classified into the type in which a single peak a appears (Type 1), the type in which two fractions, peaks a and b appear (Type 2), and the type in which a single peak b appears (Type 3). As shown in Tables 1 and 2, the AFP in the Serum of primary hepatic carcinoma can be divided into Type 1 (single peak a) and Type 2 (peak a > peak b), whereas that in the metastatic hepatic carcinoma can be divided into Type 2 (peak a < peak b) and Type 3 (single peak b), and thus, this is useful for the differential diagnosis of cancer. AFP FRACTION APPEARANCE FREQUENCY BY CON-A-CAIE Fraction Pattern Fraction as appeared Primary hepatic carcinoma Metastatic hepatic carcinoma Type 1 a 29 (61.7%) 0 Type 2 a, b 18 (38.3%) 6 (75%) Type 3 b 0 2 (25%) 47 8 EXAMPLE 4 PROCEDURE FOR MEASURING AFP IN SERUM OF CANCER PATIENT USING CHICK SERUM ALBUMIN (FRACTION V, made by Sigma Co. USA) AS AN INTERNAL STANDARD a) Preparation of assay sample: A BPB (made by Sigma Co.) solution was prepared at 0.005% (W/V) with the addition of 0.0025% (W/V) chick serum albumin. 10µl of the BPB solution were smeared onto an agarose plate and dried. A drop (25µl) of serum of a patient suffering from hepatic carcinoma was added to the plate through a Pastem pipette to make an assay sample. b) Preparation of LCH containing agarose plate : A plate was prepared in the same manner as in Example 2, section (b) except that lentil lectin (LCH) (Pharmacia Fine Chemicals) was added to 1.0% agarose in place of Con-A to a final concentration of 200 µg/ml. c) Prepatration of agarose plate for two-dimensional migration : A plate was prepared according to Example 1, section (b), except that 20 µl of anti-AFP serum (made by DAKO Co.) and 10 µl of anti-chick serum albumin serum (made by Cappel Co. USA) were added to 10 ml of 1.0% agarose. d) Measurement of AFP : 10µl aliquots of the assay sample prepared in said section (a) were poured into holes in the LCH-containing agarose plate prepared in said section (b) and subjected to one-dimensional electrophoresis under the same conditions as in Example 2, section (c), and the position of BPB marker was marked after the migration. The agarose plate was cut into strips parallel to the direction of migration and the strips placed on the agarose plate prepared as in said section (c). The assembly was then subjected to electrophoresis under the same conditions as in Example 2, section (c) but in the perpendicular direction and then coloured. Distances to the respective migration peaks from the position of chick serum albumin precipitate peak as the point of origin were measured. The precipitate peak of chick serum albumin was not crossed with the precipitate peak of serum of a patient suffering from hepatic carcinoma, and formed at the substantially same position as that of BPB marker after the one-dimensional migration. According to the present invention, biocomponent assays can be carried out with lower background, higher clearness, sensitivity, rapidity, simplicity, reproducibility and accuracy as compared with conventional methods using 4-chloro-1-naphthol as the chromogen. In particular, the measurement sensitivity is about 20-times higher, for example, from 2,000 ng/ml to about 100 ng/ml. Furthermore, higher reproducibility and accuracy can be attained by introduction of an internal standard system and measurement times can be considerably shortened, with a considerable increase in measurement sensitivity and accuracy. For example, in the case of the Con-A-CAIE of example 2, the two-dimensional migration time, which usually takes 16 hours can be reduced to 2 hours. Also, the colouring time, which takes 30 minutes to one hour according to the conventional method, can be reduced to only 2 to 3 minutes. Furthermore, the antibody concentration during the two-dimensional migration can be enough at 0.15 to 0.3% owing to the increased sensitivity. Moreover the stained gels show no fading and can be permanently preserved. This is a further advantage.

Claims: An immunoelectrophoresis assay method, which comprises subjecting a sample containing a biocomponent or a target substance to be assayed to electrophoresis on a gel like carrier and visibly developing the separated band or bands of the biocomponent or target substance to be assayed by treating the gel containing the electrophoresed sample with a peroxidase-labelled receptor conjugate capable of undergoing specific binding to the biocomponent or target substance, washing the treated gel to remove unbound conjugate, and chromogenically developing the bound peroxidase label by reacting the bound label with an enzyme substrate comprising hydrogen peroxide and a chromogen, characterised in that the chromogen used is 4-methoxy-1-naphthol. A method according to Claim 1, characterised in that the biocomponent to be assayed is an antigen, antibody, enzyme, enzyme substrate, coenzyme, protein, glycoprotein, glucide, glycolipid, lipoprotein, nucleic acid or hormone. A method according to Claim 2, characterised in that the biocomponent to be assayed is a α-fetoprotein (AFP), human chorionic gonadotropin (HCG), Γ-glutamyltransferase (Γ-GTP) or carcinoembryonic antigen (CEA). A method according to Claim 1, wherein the target substance to be assayed is a labelled biocomponent containing as the label an antigen, antibody, enzyme, enzyme substrate, coenzyme, protein, glycoprotein, glucide, glycolipid, lipoprotein, nucleic acid, hormone, avidin or biotin. A method according to any one of Claims 1-4, characterised in that the peroxidase (POD)-labelled conjugate is a POD-labelled antibody, protein A, enzyme, enzyme substrate, coenzyme, nucleic acid, a hormone-receptor, specifically bound protein, avidin, biotin or lectin. A method according to any one of Claims 1-5, characterised in that there is added to the sample prior to electrophoresis, as an internal standard, a coloured protein-dye conjugate, the protein component of which has a different antigenic specificity from that of the biocomponent or target substance to be assayed, and wherein there is incorporated into the gel-like carrier a receptor substance capable of undergoing a specific binding reaction with the standard protein. A method according to Claim 6, wherein the protein used as the internal standard is bovine serum albumin, human serum albumin, chick serum albumin or egg albumin and the receptor is the corresponding anti-albumin antibody. A method according to Claim 6 or 7, wherein the internal standard protein comprises 0.001 to 0.1% (W/V) colouring agent. A method according to any one of Claims 6-8, wherein the internal standard protein is coloured with Bromophenol Blue. A diagnostic method which comprises subjecting a clinical sample from a patient having, or suspected of having carcinoma, to an immunoelectrophoresis assay as claimed in any one of Claims 1-7, and using the results of the assay to make or confirm the diagnosis.

Inventors: ISHIGURO, TATSUYA
Applicants: KYOWA MEDEX CO. LTD.
Application year: 1985
Main classification: G01N33
IPC codes: G01N33, G01N27, C12Q1