A method for enzymatic analysis and reagent therefor

Abstract: A method of enzymatic analysis utilizing color-development signal amplification system associated with enzymatic cycling of NAD-NADH interconversion in the presence of dehydrogenase and its substrate, wherein the dehydrogenase is selected from the group consisting of alcohol dehydrogenase derived from Zymomonas and amino acid dehydrogenase derived from thermophilic microorganisms is disclosed. The use of alcohol dehydrogenase derived from Zymomonas provides an extremely higher detection sensitivity than that in the conventional method. The use of amino acid dehydrogenase derived from thermophilic microorganisms improves reliability of the method for longer period of time than that in the conventional method.

Description: The present invention relates to a method of enzymatic analysis for detection of substances or components included in the body, foods and the like. More particularly, the present invention relates to enzymatic analysis utilizing a signal amplification system associated with oxidation-reduction cycling between nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) and it reduced form (hereinafter abbreviated as NADH). NAD and NADH are the most popular coenzymes included in the body, and which participate in oxidation-reduction reactions of various dehydrogenases by means of reversible changes between NAD and NADH which give and receive hydrogen atoms. NAD and NADH have different absorption peaks about at 260nm and 340nm respectively, and changes in their absorbancy is utilized for measurement of activities of various enzymes. A signal amplification system associated with the cyclic interconversion or cyclic oxidation-reduction reaction of NAD/NADH is known as a method for detection of NAD, or NADH in a high sensitivity (C. Bernofsky et al., Analytical Biochemistry, Vol. 52, pp. 452-458, 1973: A. Johannsson et al., Clinica Chimica Acta, Vol. 148, pp. 119-124, 1985: F. J. Dhahir et al., Clinical Chemistry, Vol. 38, pp. 227-232, 1992 and U.S. Patent No. 4,769,321). In this method, a color-development signal is amplified by repetition of the oxidation-reduction reactions between NAD and NADH (enzymatic cycling) to thereby detect the presence of NAD or NADH with a high sensitivity. Therefore, by correlating the presence or non-presence of NAD or NADH with a substance to be detected, the substance can be detected indirectly via the detection of NAD/NADH by this method. As will be seen from the reaction diagram shown hereunder, NAD or NADH act as a coenzyme, in the presence of dehydrogenase (e.g. alcohol dehydrogenase which is abbreviated hereinafter as ADH) and its substrate (e.g. ethanol) for the oxidation reaction of the substrate, during which NAD is reduced to NADH. Furthermore, NADH is oxidated into NAD in the presence of a tetrazolium dye and its reductase (diaphorase), at the same time the tetrazolium dye is reduced to formazan as the color-development signal. Under the conditions mentioned above, the oxidation-reduction reaction between NAD and NADH is repeated to thereby gradually increase the signal intensity. The detection technique of NAD or NADH with such a signal amplification system is applied to measurement of phosphatase activity (A. Johannsson et al., supra: and F. J. Dhahir et al., supra), and to measurement of NAD-synthetase activity (Hideo Misaki, Bio Industry, Vol. 7, pp. 775-787, 1990) and the like. In spite of that the method is colorimetric analysis, the method is now placed as an extremely excellent method in the field of immunoassay, especially in the measurement of alkaline phosphatase, since it can provide a potent detection sensitivity which is comparable to that in fluorescence technique (or fluorescent antibody technique) or color-development technique [Edited by Ishikawa et al., Kouso Men-eki Sokuteiho (in Japanese), the third edition, pp. 58-60, published by Igakushoin]. Heretofore, ADH derived from baker's yeast is utilized in general in an analytical system utilizing such an enzymatic cycling (repetitive interconversion) of NAD/NADH. The ADH of baker's yeast, however, has a very short activity-duration so that a large quantity of ADH must be added in a reagent for the analytical system to enable the repetitive oxidation-reduction reaction between NAD and NADH which in turn amplify a detectable color-development signal. The present inventors have exerted their efforts to increase the sensitivity of the enzymatic analysis and stability of dehydrogenase activity acting as a catalyst to provide hydrogen atoms to the system of enzymatic cycling between NAD and NADH, and found that ADH derived from microorganisms belonging to Zymomonas has an extremely potent activity compared with that of ADH derived from baker's yeast. The present invention provides a new or improved method capable to detect NAD or NADH, and thus capable to detect various substances (e.g. cartino embryonic antigen, hereinafter abbreviated as CEA) which can be correlated with the presence or non-presence of NAD or NADH with a higher sensitivity and a higher stability than conventional method utilizing ADH derived from baker's yeast. In accordance with the first embodiment of the present invention, ADH derived from microorganisms belonging to the genus of Zymomonas is utilized as a catalyst to provide hydrogen atoms to the system of the enzymatic cycling between NAD or NADH which can amplify the color-development signal to thereby improve the detection sensitivity of the known method. Figs. 1 and 2 show successive or periodical changes in cycling rate and absorbancy (A490) in relation to CEA concentration respectively, wherein (--) denote the method of the 1st embodiment of the present invention wherein ADH derived from Zymomonas is utilized and (-○-) denote a conventional method wherein ADH derived from baker's yeast is utilized: In accordance with the 1st embodiment of the present invention, an analytical reagent which contains a diaphorase, a tetrazolium dye, a surfactant, a buffer solution, an ADH, and an alcohol is mixed with a sample which may or may not include NAD or NADH, then possible change in absorbancy of formazan which is produced by reduction of the tetrazolium dye when either one of NAD or NADH is present in the sample. The ADH to be included in the analytical reagent in this method is the one derived from the genus Zymomonas, and preferably, the ADH derived from Zymomonas mobilis. A sample of viable microorganisms belonging to Zymomonas can be obtained from competent organisms such as National Institute of Bioscience and Human Technology (the former name is Fermentation Research Instutute), American Type Culture Collection (ATCC) and the like. ADH can be isolated and purified from a culture fluid of Zymomonas. Cultivation of Zymomonas can be made by a conventional method in the same way for culture of ordinary bacteria. ADH can be purified in accordance with any conventional method for purification of ordinary proteins, however, it is preferable to utilize the method stated in an article, European Journal of Biochemistry. Vol. 154, pp. 119-124 (1986). ADH is added in the analytical reagent in a concentration between 0.01 - 1000 unit/ml, preferably 0.1 - 100 unit/ml. One unit of ADH activity is defined as the quantity of the enzyme which can oxidate 1 µmol of ethanol per minute at pH 8 and 30°C. Those components in the analytical reagent other than ADH and alcohol (in the 1st embodiment) can be the same with those in a conventional method. More specifically, any buffer solution having buffer action within neutral pH range, for example, phosphoric acid buffer, triethanol buffer and the like can be used. A typical pH range of the buffer is 5.0 - 10.0, preferably 7.0 - 9.0. A typical concentration range of the buffer in the analytical reagent is 10 - 1000 mM, preferably 50 - 500 mM. Examples of tetrazolium dye include 2-(p-nitrophenyl)-3-(p-iodophenyl)-5-phenyltetrazolium chloride (hereinafter abbreviated as INT), 3,3'-(3,3'-dimethoxy-4,4'-diphenylene) bis (2-(P-nitrophenyl)-5-phenyltetrazolium chloride), 2-(4',5'-dimethyl-2'-thyazolyl-3,5-diphenyltetrazolium bromide and the like. A typical concentration range of tetrazolium dye is 0.1 - 10 mM, preferably 0.5 - 2 mM. As to diaphorase, there is no specific limitation, however, it is preferable to use stable one such as diaphorase derived from Bacillus stearothermophilus. A typical concentration range in the analytical reagent is 0.01 - 1000 unit/ml, preferably 0.1 - 100 unit/ml. One unit of diaphorase activity is defined as the quantity of the enzyme which can reduce 1 µmol of INT per minute at pH 8.0 and 30°C. Any kind of surfactant such as non-ionic and ionic surfactant can be used, however, it is preferable to use Triton (trade mark of Rohm & Haas Co.) series surfactants, especially Triton X-100. A typical concentration range of the surfactant in the analytical reagent is greater than 0.001 w/v%, preferably 0.02 - 1 w/v%. In order to carry out the present invention, it is sufficient that all the aforementioned components of the analytical reagent are present together with a sample which may or may not include NAD or NADH at the beginning of the analysis, and there is no limitation to the order for mixing of these components and a sample. In other words, the analytical reagent can be prepared in situ or can be prepared preliminarily. NAD or NADH can be correlated with various substances to be detected in a conventional method. The merit of the 1st embodiment of the present invention is that NAD or NADH can be detected with a higher sensitivity than the conventional one, since the ADH derived from Zymomonas has a potent activity which can enhance the enzymatic cycling of NAD/NADH which in turn amplify the color-development signal. In general, a quantity of a mixture containing dehydrogenase (1st embodiment: ADH derived from Zymomonas ,), its substrate (1st embodiment: alcohol,), a tetrazolium dye, a diaphorase, a surfactant, and a buffer solution in a predetermined ratio is added to a quantity of a sample which may or may not include NAD or NADH at a temperature at which the enzymes are operable (e.g. 20-40°C), then the resultant mixture is subjected to colorimetric analysis. The colorimetric analysis can be made by rate assay method in which changes in absorbancy is periodically measured, or by end point assay method in which the enzymatic cycling is performed for a predetermined period, then the enzymatic reaction is terminated by adding acid thereto, and finally absorbancy is measured. In either methods, commercially available spectrophotometers can be utilized. Now the present invention will be further described in detail hereunder for better understanding of the present invention by way of Examples. However, it should be noted that the present invention is not limited thereto. Firstly, preparation of ADH derived from Zymomonas and alkaline phosphatase labelled anti-CEA antibody which is utilized in some of the following Examples will be explained. PREPARATION OF ADH DERIVED FROM ZYMOMONAS Utilizing the medium specified hereunder, Zymomonas mobilis ATCC29191 was cultured under the conditions at 37°C, pH 5.0. composition of the medium glucose 15% yeast extract 0.5% potassium dihydrogenphosphate 0.05% magnesium phosphate 7-hydrate 0.05% calcium pantothenate 0.001% biotin 0.00002% After cultivation, the microbial cell mass was collected by centrifugation. To 70 g of the collected cell mass, 400 ml of 30 mM dipotassium hydrogenphosphate solution containing 0.5 mM ammonium iron (II) sulfate, 10 mM ascorbic acid, 0.1% Nonidet P-40, 0.0002% DNase I, and 0.02% lysozyme was added, and the resultant suspension was incubated at 30°C for 2 hours. The resultant suspension was then subjected to centrifugation to obtain a supernatant. The pH of the supernatant was adjusted to 6.5 by adding thereto 2-morpholinoethane solfonic acid containing 30 mM sodium chloride, 2 mM magnesium chloride, 0.5 mM ammonium iron (II) sulfate, and 10 mM ascorbic acid. The resultant liquid was passed into a BLUE SEPHALOSE CL-6B (trade mark of PHARMACIA Co.) column (3.0 cm diameter x 15 cm long), then the column was washed with 400 ml of the same buffer, followed by elution with 1 mM NAD, ADH active fraction was collected, concentrated, and dialyzed, to thereby ADH was prepared. PREPARATION OF ALKALINE PHOSPHATASE LABELLED ANTI-CEA ANTIBODY To 2 mg of alkaline phosphatase (derived from calf intestine, purchased from Boehringer-Mannheim Yamanouch K.K.) 3 mg of succinimidil-(n-maleimid-methyl)-cyclohexane-1-carboxylate (purchased from Zieben Chemical Co.) was added, and the resultant mixture is subjected to reaction at pH 7.0, 30°C for 10 minutes, thereby a maleimide groups was introduced into a molecule of alkaline phosphatase. To the resultant solution, 1 mg of anti-cartino embyonic antigen (hereinafter abbreviated as anti-CEA antibody) Fab' was added at pH 5, thereby alkaline phosphatase labelled anti-CEA antibody (having cross-linking between maleimide groups and thiol groups) was prepared. Then, objects and outlines of the following Examples will be explained. Example 1 demonstrates NAD detection sensitivity of the analytical reagent prepared in accordance with the 1st embodiment of the present invention in comparison with that of a conventional analytical reagent utilyzing ADH originated from baker's yeast. Example 2 demonstrates practical applications of the present invention for detection of CEA. The principle of the CEA enzyme immunoassay in Example 2 is as follows: Anti-CEA antibody is preliminarily fixed to a microtiter plate. A sample which may or may not include CEA is pipetted in the plate. When CEA is included in the sample, CEA is captured. Then alkaline phosphatase labeled anti-CEA antibody is pipetted into the plate. If CEA is captured to the plate, the alkaline phosphatase labeled anti-CEA antibody is captured (sandwich method). After washing of the plate, nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADP) pipetted into the plate, whereat NADP is converted to NAD by the catalytic function of the alkaline phosphatase in quantity-depending manner, and the cyclic interconversion of the resultant NAD can amplify the color-development signal by the analytical reagent of the present invention. It is well known in the art that various substances can be correlated with NAD or NADH, thus the method of the present invention can be applied for detection of the substances in a higher sensitivity and a higher reliability than the conventional method. Example 1 (1) PREPARATION OF ANALYTICAL REAGENT An aqueous solution containing following components was prepared. sodium phosphate (pH 8.0) 250 mM INT 1 mM ethanol 4 % Triton X-100 0.1 % diaphorase (derived from Bacillus stearothermophilus , purchased from Seikagaku Kogyo) 3 unit/ml ADH (derived from Zymomonas mobilis ) 1.6 unit/ml As a control (Control 1), an aqueous solution was prepared with the same components, except that the ADH was substituted with the one derived from baker's yeast (purchased from Sigma Co.). (2) METHOD To each well of 96 well microtiter plate (purchased from Corning Co.), 100µl aliquot of the previously prepared analytical reagent was pipetted, then 10 µl aliquot of 1µM NAD was added to each well. The titer plate was set into an automatic analyzer Biomek-1000 [by Beckman Instrument Inc. (USA)] to measure increase in absorbancy at 490 nm per minute (A490). At the same time cycling rate (%) based on the values at 0 minute was calculated from the measured values. (3) THE RESULTS The results are shown in Table 1 and Fig. 1 respectively. As will be seen from Table 1 and Fig. 1, absorbancy and cycling rate were rapidly decreased in Control 1 in which ADH derived from baker's yeast was utilized, whereas absorbancy and cycling rate were almost constant in the reagent in accordance with the present invention (Example 1) in which ADH derived from Zymomonas mobilis was utilized. It is exemplified that the sensitivity for detection of NAD in Example 1 is 2.5 times of that in a conventional method (Control 1) after 5 minutes, 8 times after 25 minutes. Time (min.) Absorbancy (A490)/Cycling rate (%) Example 1 Control 1 1 0.0109/100 0.0109/100 5 0.0109/100 0.0043/39.4 15 0.0105/96.3 0.0019/17.4 25 0.0100/91.7 0.0012/11.0 Example 2 To each well of a microtiter plate [purchased from Oy Medix Biochemica (Finland)] fixed with anti-CEA antibody, in different concentrations, 0, 1.25, 2.5, 5.0, 10.0, 20.0 ng/ml, 50 µl aliquot of cartino embryonic antigen was pipetted, then 50 µl aliquot of 1 µg/µl alkaline phosphatase labeled anti-CEA antibody was further pipetted. The resultant microtiter plate was left undisturbed for 2 hours at a room temperature for antigen-antibody reaction. After the reaction, the plate was washed with 20 mM tris-hydrochloric acid buffer (pH 7.2) containing 0.2 % of * Tween 20, 0.2 % bovine serum albumin, and 0.15 M sodium chloride, 50 µl aliquot of 0.5 M diethanolamine (pH 9.8) containing 1 mM NADP and 1 mM magnesium chloride was added to each well, and reaction was allowed for exactly 10 minutes at a room temperature, then 100 µl aliquot of the same analytical reagent as used in Example 1 was pipetted in each well and reaction was allowed for 10 minutes at a room temperature. The reaction was terminated by adding 50 µl aliquot of 1 N hydrochloric acid to each well, absorbancy (A490) was measured by automatic microtiter plate analyzer, Biomek-1000. As a control, the same analysis was carried out, except that the analytical reagent was substituted with an analytical reagent (Control 2) which is the same with Control 1 in Example 1. The results are shown in Table 2 and Fig. 2 respectively. As will be seen from Table 2 and Fig. 2, the method of the 1st embodiment of the present invention (Example 2) provides excellent detection sensitivity that amount to about 4 times of that in a conventional method (Control 2) in the CEA detection system. Concentration of CEA (ng/ml) Absorbancy(A490) Example 2 Control 2 0 0.040 0.030 1.25 0.149 0.055 2.5 0.239 0.082 5.0 0.463 0.144 10.0 0.751 0.198 20.0 1.303 0.345

Claims: A method of analysis utilizing a color-development signal amplification system based on enzymatic cycling of NAD-NADH interconversion in the presence of an alcohol dehydrogenase derived from microorganisms belonging to Zymomonas and its substrate. The method of claim 1, wherein said alcohol dehydrogenase is derived from Zymomonas mobilis. A reagent for enzymatic analysis utilizing a color-development signal amplification system based on enzymatic cycling of NAD-NADH interconversion in the presence of a dehydrogenase and its substrate, which consists essentially of a diaphorase, a tetrazolium dye, a surfactant, a buffer solution, an alcohol dehydrogenase derived from Zymomonas, and a substrate therefor.

Inventors: SHINZAKI AKIHIRO, SUZUKI TADAO, WATANABE MIWA, SHINZAKI, AKIHIRO, SUZUKI, TADAO, WATANABE, MIWA, SHINZAKI, AKIHIRO, C/O UNITIKA LTD., SUZUKI, TADAO, C/O UNITIKA LTD., Watanabe, Miwa, c/o Unitika Ltd.
Applicants: UNITIKA LTD, UNITIKA LTD.
Application year: 1993
Main classification: C12Q1
IPC codes: C12Q1