IT8319304A1 - Highly sensitive analysis method - Google Patents

Description

DESCRIPTION

The present invention relates to a highly sensitive assay method. The fluorescence assay method, the luminescence assay method and other assay methods, such as highly sensitive assay methods, have been known up to now. However, in these methods samples such as body fluids cause errors, such as control value and abatement, in the assay due to contaminants in the body fluid.

The assay methods that cycle coeri zime are well known methods. However, in the above method, the detection reaction is required after the cycling reaction, which causes complicated operations and said detection reaction? itself influenced by the substance, as well as by what must be tested

Furthermore, it requires a particular reaction vessel for the fatty material and a quantity? extremely small of the reaction volume

Furthermore, in the known process, the unreacted coenzyme to be decomposed is decomposed by adding acid or an alkaline agent, heating and neutralizing the reaction mixture and in these operations it is important to add a quantity? of acid or alkaline agent in order to neutralize the reaction mixture and if the pH of the neutralized reaction mixture is varied, it determines the cycling ratio. These phenomena produce error in the assay.

Yup ? now found that, in the cycling reaction, the starting reaction itself is applied to the detection reaction. that allows you to avoid the need? of special operations for the reaction and the detection reagents. a particular reaction vessel required in the case of said improvement.

Yup ? also found that pH adjustment is easily achieved by adding a quantity in excess of lower alkyl ester of formic acid in order to avoid affecting the speed. of cycling following pH dispersions in neutralization,

It is an object of the present invention to provide an assay method in which the same cycling reaction? a revelation reaction without necessity? of the operation and reagent for the indication reaction and without affecting the substance other than the substrate to be assayed.

Another object of the present invention is to provide an assay method consisting not only of a term assay method but also of a time assay method.

A further object of the present invention is to provide a simple assay method which does not require a particular reaction vessel, without requiring quantity. extremely low reaction volume and that can? be conducted in a conventional vessel at constant temperature.

A further object of the present invention is to provide a method of testing in which the pH of the reaction mixture can be used. be easily adjusted by adding a quantity? in excess of lower alkyl ester of formic acid, in order to avoid the measurement error deriving from the cycling ratio influenced by the dispersion of the pH in the event that the exact addition of quantities? of acid or alkaline agent is not carried out until the reaction is neutralized after decomposition of the unreacted coenzyme. A further object of the present invention is to provide an assay method free of purification and concentration of the substance to be tested.

Implementation of the principles of this invention

are as follows:

According to the present invention, the oxide-reduced

is used in the main reaction not? limit

and has an action on the substance to be tested

as substrate together with NAD {P) + or NAD (P) H which

coenzyme. These enzymes can be new enzymes

or known enzymes. Examples of previously known enzymes and substrates are found in Enzyme Handbook, (Shiro Akabori Ed. Asakura pubi. Co. Tokyo).

The quantity of the reaction mixture is varied from 10 ml to 3 ml. The quantity of the coenzyme? equal to 0.1 moles up to 10 minutes. Quantity minimum measurable of the substance to be tested vary from 10 to 10-15 moles and the quantity? of coenzyme and in excess of the ratio of the action of the substance to be tested.

The preceding reaction takes place at an optimal pH of oxide-reductase and within the optimal temperature. Furthermore, the unreacted coenzyme is decomposed simultaneously with the termination of the main reaction. In this decomposition reaction system, in the case of the oxidized form coenzyme ???? or NADP +, alkali is added and heated to obtain the reduced form coenzyme, NADH or NADPH, which is formed from the main reaction. Furthermore, NAD ^ or NADP is obtained by adding acid and heating, in case the unreacted coenzyme is NADF or NADPH. Examples of alkalis are usually alkali hydroxides such as aqueous sodium hydroxide or aqueous potassium hydroxide. In addition, lithium hydroxide, sodium carbonate or carbonate can be used

_

aqueous potassium.

The alkali concentration must be such that said solution pH reveals an alkaline pH greater than 12. The preferred concentration ranges from 0.001M to 0.5M.

The reaction conditions are varied depending on the acali concentration, the heating temperature or the heating time. NAD + or NADP + is completely decomposed at 100 ° C in 10 minutes in 0.1N NaOH solution.

Examples of acid are those whose acidity? in aqueous solution it is lower than pH 2, as hydrochloric acid or sulfuric acid. Preferred concentrations of the acid are in the range of 0.001M-0.5M. The reaction conditions are varied depending on the acid concentration, the heating temperature and the heating time. For example NADH or NADPH pu? be decomposed at 50 ° C in 3 minutes in 0.1 N "HCl.

In the step of the neutralization reaction after decomposition of the unreacted coenzyme, in the event that NAD + or NADP + is decomposed by means of alkali, the reaction mixture is neutralized by the addition of lower alkyl ester of the formic acid. Examples of lower alkyl esters of formic acid are methyl formate, ethyl formate, propyl formate or butyl formate. According to the conventional method, the pH of the reaction mixture is dispersed if the addition of the acid and the mixing ratio are not exactly carried out. is varied causing an error in the assay value. On the other hand, in the neutralization reaction indicated above, even if a quantity is used in the neutralization. in excess of lower alkyl ester of formic acid, for example if you add a quantity? diecivolte in excess of the quantity? necessary for neutralization, pu? only react the quantity? of lower alkyl ester of formic acid corresponding to the quantity? of alkali and you can? then realizing the exact neutralization of the reaction mixture and yes. easily achieve pH adjustment.

In the event that NADH or NADPH is decomposed by acid, a slight amount is added. in excess of alkali for neutralization, by following the addition of the lower alkyl ester of the formic acid as indicated above for neutralization, then the reaction mixture can? be neutralized exactly.

Examples of alkalis are usually aqueous solutions of alkali hydroxides. The next stage of the reaction according to the present invention, i.e. the amplification reaction by means of the cycling of the coenzyme pu? be carried out by combining the oxido-reductase system, which operates on the remaining converted coenzyme corresponding to the substance to be tested and a quantity of in excess of the substrate and the conversion system in which the tetrazolium salt is converted to formazan by diaphorase or electron transfer room. The oxide-reductase of the above system is not? limited by its origin and? consisting of a dehydrogenase which requires the coenzyme rNAD (P) +. and forms NAD (P) H operating on a quantity? in excess of the specific substance.

Implementations of such enzymes and substrates are found in the "Enzyme Handbook" mentioned above.

The quantity of enzyme to use depends on the activity? of the enzyme, the type of substrate and the cycling ratio of the coenzyme.

The quantity molar of the substrate would be in a higher excess in comparison to that of the coenzi but of putting in the cycle and pu? be a concentration higher than the entity? maximum reaction of det? ta oxido-reductase and preferably? in the range of 0.1-50 U / ml in the reaction mixture. Examples of electron transfer substances are substances with activity. oxidizing on NAD (P) H to NAD (P) + and without adverse action on the cycling reaction of the coenzyme, for example phenazine metasulfate, Meldola blue and pyrocyanine. Their concentration can? be established in accordance with the reason for putting into the cycle and? preferably 1? g -

d l

salt concentration of | tetrazolium? somewhat? ^! - mitigated on the basis of solubility? of the tetrazolium salt and of the formazan finally formed and d? equal to 3--100 mg / ml of reagent. The looping reaction can? be conducted at room temperature up to 37 ° C, preferably 30-37 ° C. The reaction can? be blocked at the estimated time by adding acid such as hydrochloric acid or phosphoric acid. A surfactant is preferably added in order to avoid the precipitation of formazan.

Examples of surfactants are non-ionic surfactants such as Triton X-100 or Adekarol SO-14 (trade names). The concentration of the surfactant agent? 0.01-3% in the reagent. The addition of the surfactant agent allows an increase in sensitivity? of the assay and a stabilization of the formazan pigment. The colorimetric assay of the formazan pigment is so? format pu? to be with the density? optical (OD) at the specific absorption wavelength of the formazan, for example at 500-550 nm.

The substance can? be tested using the above method. According to the method of the present invention, one can? realize not only the essay. at term but also the kinetic assay. The following examples illustrate the present invention, but are not intended to be limiting.

Example 1

Quantitative assay of NAD +:

(Reagent)

(1) 0.02M phosphate buffer solution (pH 8.0)

(2) Adekatol-INT solution (INT (100 mg) dissolved in 2% Adekatol-S0-145 solution (100ml))? (3) Diaphorase or electron transfer substance.

1) diaphorase solution (NADH) 100 U / ml.

2) solution of phenazine metasulfate (PMS) 100mg / 20ml.

3) 10 mg / 20 ml pyrocyanine solution, i

4) Meldola's blue solution 10mg / 20ml.

(4) Ethanol

(5) Alcohol dehydrogenase 250 U / ml.

(6) NAD + 10mM solution (NAD + is formed in the reaction and unreacted NADH is decomposed by acid and heating, then neutralized with alkali and then completely neutralized with ethyl formate).

(7) HC1 0, IN

(Operation)

In each of the separate test tubes were added respectively: phosphate buffer (0.5ml), Adekatol-INT (0.2ml), ethanol (20> ul), water (30 ^ and alcohol dehydrogenase (0.1ml) .

Diaphorase (NADH) or electron transfer substance (50) was added and incubated at 37 ° C for 3 minutes. Then the solution of NAD + 10 M (0, 10, 20, 40, 60, 80 or 100 ^ 1) was added respectively and each volume adjusted to a total of 1.0 ml and incubated at 37? C for exactly 10 minutes. 0.1N HCl was added to each of the tubes to stop the reaction and the OD value at 50 nm was measured. Water (0.1ml) was added in place of NAD as a control. (Result)

The result is shown in Figure 1 in which the quantity? of NAD, together with the diaphorase or electron transfer substance and the density? of optical absorption have a good linearity. The result shows an exact cycling reaction and sensitivity. high.

Example 2

Effect of the oxido-reductase used in the cycling reaction:

(Reagents)

(1) 0.2 M phosphate buffer, pH 8.0

(2) Adekatol-INT solution equal to that of example 1

(3) diaphorase solution (NADH)

(4) substrate solution

l) 100 mM ethyl alcohol

2) 100 mM glycerol-3-phosphate solution

3) 100 mM lactic acid

4) 100 mM malic acid

(5) oxido-reductase

'

1) solution of alcohol dehydrogenase (ADH) 25000 J / ml 2) solution of glycerol-3-phosphate dehydrogenase (G-3-PDH) 600 U / ml

3) lactate dehydrogenase solution (LDH) 1500 J / ml 4) malate dehydrogenase solution (MDH) 4600 J / ml 5) NAD + 10 solution

6) HC10, IN

(Operation)

In each of the separate test tubes, phosphate buffer (0.5ml), Adekatol-INT (0.2ml), diaphoresis (NADH) (50.4.1), substrate solution (50yu? L), were added separately. oxide-reductase which operates on said substrate (5 / * 1) and water (95 y, 1) and was subjected to incubation at 37 ° C for 3 minutes.

Was a solution of NAD + 10 ^ ?? added? (0, 10, 20, 40, 60, 80 or .100 \ respectively) to each enzyme, he adjusted. the total volume to 10 ml and is subjected to incubation at 37 ° C for exactly 10 minutes. 0.1N HCl (2ml) was added to each tube to stop the reaction and measured. the OD value at 500 nm. Water (100yul) was used in place of the NAD + solution as a control.

(Result)

The result is shown in Figure 2.

In each oxido-reductase, a good linearity is found, ci? which indicates the exact cycling reaction and high sensitivity.

Example 3

Quantitative assay of NADP +:

(Reagent)

(1) 0.2 M phosphate buffer, pH 8.0

(2) di-iAdek? T? L-INT solution (INT (100 mg) dissolved in 2% Adekatol -S0-145 solution (100ml)) (3) diaphorase-NADPH solution (NADPH) 100 U / ml (4) solution d? I, substrate j

1) glucose-6-phosphate (, G-6 a-P) 100 mM

2) glutamic acid (GA) 100 mM

(5) oxide reductase solution

1) solution of glucose-6-phosphate dehydrogenase? J (G? 6? PDH) 1000 U / ml.

2x) '? Solution of glutamato-dehydrogenase (G1DH)

1000 U / ml

(6) Solution of NADP + IO

(7) HC10, IN

(Operation)

Phosphate buffer (0.5ml), Adekatol-INT (0.1ml), diaphorase (NADPH) (100yul), substrate (50: / <tl), enzyme (50yu1) and water were added to separate test tubes, respectively. (30yul) and subjected to: incubation at 37 ° C for 3 minutes, then NADP + 10 / U M (0.10, 20, 40, 60, 80 and 100 / <1) was added. The total volume was adjusted to 1.0 ml by addition of water and incubated at 37 ° C for.

10 minutes exactly. HC10.1N (2ml) was added to stop the reaction and measured. the OD value at 500 nm.

Water (100 / -? 1) was added in place of the NADP solution as a control.

(Result)

The result is illustrated in Figure 3, in which a good linearity was obtained. and NADP + pu? be tested with high sensitivity? together with glucose-6-phosphate dehydrogenase and glutamate dehydrogenase.

Example 4

Quantitative assay for malic acid:

(Sample)

Malic acid solution (malic acid: 0, 1, 2, 4, 6, 8 and 10y * M) 20 pX.

(Reagents)

(1) reagent I: mixed solution of 0.2 M phosphate buffer pH 8.0 (0.2 ml), 10 mM NAD + solution (0.1 ml) and malate dehydrogenase solution

(120 U / ml) (100yul).

(2) 0.2 N NaOH

(3) ethyl formate

(4) Reagent II: mixed solution of 0.5 M phosphate buffer pH 8.0 (0.1 ml), ethyl alcohol (lOyul)? alcohol dehydrogenase (250 U / ml) (50 ^ 1), diaphorase (NADH) (100 U / ml) (100 ^, 1), 1% NTB solution (20 ^ * 1) and Triton X- solution 100 (20 '^ 1). (5) HC10, IN

(Operation)

Samples were added in separate test tubes and reagent I (0.2 ml) was added, then incubated at 37 ° C for 15 minutes.

0.2 N NaOH (0.1 ml) was added to the reaction mixture and heated. -at 100? C for 10 minutes, it cooled -,! i d? : immediately and neutralized? with ethyl formate (20 yu * 1). After incubating at 37 ° C for 5 minutes, reagent II (0.3 ml) was added and incubated at 37 ° C for exactly 10 minutes. HC10.1N (2.5ml) was added to stop the reaction and measured. the OD value at 550 nm. Water (100yul) was added in place of the NAD + solution as a control.

(Result)

The result is shown in figure 4. Ls quantity? of malic acid? in relation to the OD value with small error. Was malic acid tested with sensitivity? high.

Example 5

Quantitative assay for gamma-aminobutyric acid:

(Sample)

Human serum (50 li1), to which gamma-aminobutyric acid (GABA) (0, 10, 20, 30, 40 and 50yUM) was added.

(Reagents)

(1) Reagent I: 0.1 M pyrophosphate buffer mixed solution, pH 8.3 (1.0 ml), 10 mM NADP + solution (0.2 ml), 0.1M alpha-ketoglutarate solution (l , C mi) and? GABASE (2 U / mL, 0.4 mL, commercially available enzyme from Pseudomonas fluorescens)

(2) 0.5N NaOH solution

(3) ethyl formate- (4) Reagent II: mixed induction of? Solution of Adekatol-INT (0.1ml, INT (100mg) dissolved in 2% Adekatol SO-145 solution (100ml)) , diaphorase solution (NADPH) (100 U / ml 1.0 ml), 0.1 M glucose-6-phosphate solution (: 0.2 ml) and glucose-6-phosphate dehydrogenase solution (100 U /ml,.2,0yw]). (Operation)

Reagent I (0.35 mL) was added to the samples and incubated at 37 ° C for 30 minutes.

0.5N NaOH (0.1 ml) was added and heated? at 100? C Der 10 minutes. After cooling, ethyl formate (20 µl) was added for neutralization and incubated at 37 ° C for 5 minutes. Reagent II (0.2 ml) was added and incubated at 37 ° C for exactly 10 minutes. 0.1 N HCl (2.mi) was added to stop the reaction and the OD value at 500 nm was measured. A control without GA? BA was used .;

(Results)

The result is shown in figure 5. The quantity? of GABA in human serum pu? be represented with a linear trend in relation to the value of OD. GABA pu? be tested with high sensitivity? and without protein separation.

In the attached figures:

Figure 1: Standard NAD + curve using diaphorase i and other electron transfer substances as the detection reaction.

Figure 2: NAD + standard curve showing the effect of the oxide-reductase used in the cycling reaction.

Figure 3: NADP + standard curve showing the effect of the oxide-reductase used

Claims (18)

R I V E N D I C A Z I O N I 1. - In the highly sensitive test method for the test substance, the improvement comprising the following reaction steps: 1) main reaction system that converts the coenzyme corresponding to a quantity? of substance to be tested by the action of the oxido-reductase for said substance in the presence of coenzyme NAD + or NADH, or NADP + or NADPH; 2) ssis.t? But? Of? Decomposition reaction to decompose and neutralize the unreacted coenzyme at the end of the main reaction system; 3) cycling reaction system which amplifies the reaction by means of the cycling of the coenzyme consisting of a combination of redox reaction which operates on the substrate in excess and on the converted coenzyme corresponding to the quan tit? of the test substance and the conversion reaction from tetrazolium salt to formazane in the presence of diaphorase or electron transfer substance; And 4) colorimetric measurement of formazan cos? format. 2. - Method of essays? according to claim 1, wherein the molar ratio of the coenzyme in the main reaction system? in excess compared to the molar ratio of the substance to be tested. 3. A test method according to Claim 1, wherein the unreacted coenzyme in the case of NAD and NADP + is decomposed by adding aqueous alkaline hydroxide and by heating and neutralizing. 4.?; Test method according to claim 3, wherein the concentration of aqueous alkaline hydroxide? a concentration that produces a pH greater than 12. 5.? Test method according to claim 4, wherein the alkaline hydroxide is sodium hydroxide or potassium hydroxide. 6. A test method according to Claim 3, in which the heating is carried out until complete decomposition of NAD + or NADP + .. 7. A test method according to Claim 3, wherein neutralization is achieved by adding lower alkyl ester of formic acid. 8. A test method according to Claim 1, wherein the unreacted coenzyme, in the case of NADH or NADPH, is decomposed by adding aqueous acid and by heating and neutralizing. 9. - Test method according to claim 8. wherein the concentration of the aqueous acid? a concentration that produces an acidity? pH below 2. 10. A test method according to claim 9, wherein said acid? a substance that produces an acidity? of pH lower than 2 in its aqueous solution. 11. Test method according to claim 10, wherein the acid? hydrochloric acid or sulfuric acid. 12. A test method according to Claim 8, wherein heating is carried out until complete decomposition of NADH or NADPH. 13.? The assay method of claim 8 wherein neutralization is carried out by adding a small excess of alkali hydroxide solution and immediately adding lower alkyl ester of formic acid. 14. A test method according to claim 13, wherein said alkaline hydroxide? sodium hydroxide or potassium hydroxide. 15. Test method according to claims 7 or 13, wherein the lower alkyl ester of the formic acid? methyl formate, ethyl formate, propyl forinate or butyl formate. 16. A test method according to claim 1, wherein the electron transfer substance in the cycling reaction? phenazine methosulfate, Meldola blue or pyrocyanine. 17. A test method according to Claim 1, wherein a surfactant is added. 18. - Test method according to claim 17, wherein the surfactant? a non-ionic tensing agent.