DE3302743A1 - Highly sensitive assay - Google Patents

Abstract

A highly sensitive assay is described and is characterised by the following reaction stages: 1) a main reaction system in which the coenzyme is transformed according to the amount of the substance to be determined by the action of the oxidoreductase for the said substance in the presence of coenzyme NAD<+> or NADH, or NADP<+> or NADPH; 2) a decomposing reaction system in which the unreacted coenzyme is decomposed and neutralised at the end of the main reaction system; 3) a cycling reaction system in which the reaction is enhanced by means of coenzyme cycling, and which consists of a combination of the oxidoreduction reaction which transforms the excess substrate and coenzyme according to the amount of the substance to be determined, and of a converting reaction of tetrazolium salt to formazan in the presence of diaphorase or of an electron transfer substance and 4) a colorimetric measurement of the formazan formed in this way.

Description

DESCRIPTION

The invention relates to a highly sensitive assay. as highly sensitive assays are already the fluorescence assay / the luminescence assay and other methods are known. In these assays, however, samples / such as body fluids, errors, for example with regard to the control value and the erasure, which indicate contamination in the body fluids.

Cyclic coenzyme assays are known. In the known assays, however, a display reaction is required after the cycling reaction, which leads to a complication of the operability. This display reaction itself is also impaired by substances other than the substance to be determined.
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Furthermore, special reaction vessels are used for fatty materials and extremely small amounts of the reaction volume necessary.

In addition, in the known assays, unreacted coenzyme, which is to be decomposed, by adding acid or alkali, heating and neutralizing the reaction mixture is decomposed. In these processes it is important add the exact amount of acid or alkali to neutralize the reaction gas. If the pH of the neutralized reaction mixture fluctuates, then a fluctuation or pendulum ratio is brought about. These appearances lead to assay errors.

It has now been found that in the cycling reaction, the cycling reaction can even be used as an indication reaction, so that the special implementation of the indication reaction and the reagents required for this are no longer necessary

can come. Furthermore, endpoint assays and speed assays can be carried out / with advantageously no special reaction vessels being required.
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It has also been found that the pH adjustment can be easily accomplished by having an excess Amount of formic acid lower alkyl ester added to reduce cycling speed due to fluctuations in pH due to neutralization can be avoided.

The object of the invention is therefore to provide an assay in which the cycling reaction itself the Display or indication reaction is so that the execution of the display reaction and the necessary reagents can be omitted without the assay being interfered with by substances other than the substance to be determined

The invention is also intended to provide an assay that is not only an endpoint assay but can also be carried out as a rate assay. These assays are said to be feasible without special reaction vessels be and without extremely trace amounts of the reaction volume are required. The assays are also intended to be used in one conventional constant temperature box be feasible.

In the intended assay, the pH of the reaction mixture should also be easily adjustable by using a Excess amount of formic acid lower alkyl ester added in order to avoid measurement errors caused by the cycling ratio which is affected by fluctuations in pH when in the neutralization reaction no exact addition of the necessary amounts of acid or alkali after the decomposition of the unreacted coenzyme is carried out.

«••• ft ·· ··« · · ··

Finally, the desired assay should be feasible without purification and concentration processes for the substance to be determined be.

The essential principles of the present invention are as follows.

(1) Main reaction system:

AH ₂ + NAD (P) ⁺ A + NAD (P) H + H ⁺

Oxidoreductase

where AH ₂ or A: the substance to be determined

NAD (P) ⁺ : NAD ⁺ or NADP ⁺
. NAD (P) H: reduced form of NAD. or NADP

(2) Decomposition reaction:

NAD (P) ⁺ 7 / ^{only NAD} < ^P > ⁺ remains behind (

NAD (P) H J \ ^{only NAD} ( ^p ) ^H remains

Neutralization:

Neutralization of the alkali

MOH + HCOR ^ HCOOM + RAW

where M: alkali metal and R: lower alkyl

(3) Cycling response:

Substrate -n • -NAD (P) ⁺ -v , -> formazan

Dehydrogenase Y Y. Diaphorase. or elec-

'Yy J \ tronentransfersubstanz reaction product * r N NAD (P) H ^ ^ - tetrazolium salt

In the assay according to the invention, the oxidoreductase used in the main reaction is not subject to any restrictions. '

fen, and it has an effect on the substance to be determined as a substrate together with NAD (P) or NAD (P) H as a coenzyme. These enzymes can be either new enzymes or known enzymes. Examples of known enzymes and substrates can be found in "Enzyme Handbook" (Shiro Akabori Ed. Asakura publ. Co., Tokyo).

The amount of the reaction mixture varies between 10 ml and 3 ml. The amount of the coenzyme is 0.1 mol to 10 mmol. The minimum measurable amount of the substance to be determined is 10 to 10 mol. The amount of coenzyme is above the Action ratio of the substance to be determined.

The aforementioned reaction takes place at the optimal pH of the oxidoreductase and within an optimal temperature range away. Thereafter, unreacted coenzyme is decomposed, with the main reaction being terminated at the same time. at In the case of the oxidized form of the coenzyme NAD or NADP, alkali is added to this decomposition reaction system, and it is heated to maintain the reduced form of the coenzyme NADH or NADPH, which is formed in the main reaction. Furthermore, NAD or NADP is maintained by adding acid and heating if the unreacted Coenzyme is NADH or NADPH. Examples of suitable alkalis are customary alkali hydroxides, such as aqueous sodium hydroxide or potassium hydroxide. Aqueous lithium hydroxide, sodium carbonate or potassium carbonate can also be used be used.

The alkali concentration should be such that the pH of the solution is above 12. A preferred concentration is 0.001M to 0.5M.

The heating conditions are varied depending on the alkali concentration, the heating temperature or the heating time. NAD oder.NADP is completely decomposed NaOH solution for 10 minutes by heating at 100 ⁰ C in 0.1N.

An example of an acid which lowers the acidity of the aqueous solution to a pH value below 2 is hydrochloric acid or sulfuric acid. The preferred concentration of the acid is 0.001M to 0.5M. The heating conditions can be varied depending on the acid concentration, the heating temperature and the heating time. For example, NADH or NADPH can be decomposed by heating for 3 minutes at 50 ⁰ C in 0.1N HCl.

at the stage of the neutralization reaction after the decomposition of the unreacted coenzyme is in the case that NAD or NADP has been decomposed by alkali, the reaction mixture is neutralized by adding lower alkyl formate. Examples of suitable formic acid lower alkyl esters are methyl formate, ethyl formate, propyl formate and Butyl formate. In the conventional assay, the pH of the reaction mixture and the cycling ratio will vary if the addition of the acid is not made accurately. can vary, which can lead to determination errors.

In the neutralization reaction described above, even if an excessive amount of formic acid lower alkyl ester in the neutralization, for example the 10-fold excess over that required for neutralization Amount, is added, only the amount of formic acid lower alkyl ester corresponding to the amount of alkali is used, so that an exact neutralization of the reaction mixture can be obtained and the adjustment of pH can be easily performed.

in the event that NADH or NADPH are decomposed by acid, a slight excess of alkali is added for neutralization. Then, as in the previous case, formic acid lower alkyl ester added for neutralization, whereupon the reaction mixture can be exactly neutralized.

Examples of alkali are customary aqueous solutions of alkali hydroxides. The next stage of the reaction in the invention

according to assay, namely the amplification reaction by coenzyme cycling can be carried out in such a way that the oxidoreductase system, which converts the remaining coenzyme according to the substrate to be determined or an excess amount of the substrate, and the conversion system, in the tetrazolium salt by diaphorase or a Electron transfer substance is converted, combined. The oxidoreductase of the system described above is not subject to any restrictions with regard to its origin. It is a dehydrogenase that ^{needs coenzyme NAD (P) +} and forms NAD (P) H by acting on an excess amount of the specific substance.

■ Examples of such enzymes and substrates can be found in the aforementioned "Enzyme Handbook".

The amount of enzyme used depends on the enzyme activity, the type of substrate and the coenzyme cycling ratio away.

The molar amount of the substrate should be present in a larger excess compared to the amount of cycling coenzyra, and the substrate can be present in a concentration which is greater than the maximum reaction rate of said oxidoreductase. Preferably it is 0.1 to 50 U / ml in the reaction mixture. An example of an electron transfer substance is a substance having an oxidation activity on NAD (P) H to NAD (P) ⁺ without adversely affecting the coenzyme cycling reaction. Individual examples are phenazine methosulfate, Meldola blue and pyrocyanine. The concentration of this substance can be selected according to the cycling ratio, and it is preferably 1 pg to 1 mg / ml of the reaction mixture. Examples of suitable tetrazolium salts are 3.3 · - (3.3 · -0 ^ βϋιοχγ-4,4′-diphenylene) -bis- [2- (p ~ nitrophenyl) -5-phenyl-tetrazolium chloride] (NTB) and 2 - (p-

Nitrophenyl) -3- (p-iodophenyl) -5-phenyltetrazolium chloride (INT) or 2- (4 ·, 5'-dimethyl - ^ '- thiazolyl) -3,5-diphenyltetrazolium bromide (4,5-MTT) .. The concentration of the tetrazolium salt is rather limited by the solubilities of the tetrazolium salt and the formazan formed in the end and is 3 to 100 mg / ml of the reagent.

The cycling reaction can be carried out at 30 to 37 ⁹ C at room temperature to 37 ° C, preferably. The reaction can be stopped at the estimated time by adding an acid such as hydrochloric acid or phosphoric acid. A surfactant may preferably be added to prevent the formazan from precipitating.

Examples of suitable surfactants are nonionic surfactants such as Triton X-100 or Adekarol SO-14 (Trade Mark). The concentration of the surface active Mean is 0.01 to 3% for the reagent. The addition of the surfactant leads to an increase the sensitivity of the assay and stabilization of the formazan pigment. The colorimetric assay of the formazan pigment thus formed can be measured by measuring the optical Density (OD) at the specific absorption wavelength of the formazan, for example 500 to 550 nm will.

The substance can be determined by the method described above. In the assay according to the invention cannot only an end point determination but also a speed determination be guided.

The invention is illustrated in the examples.

Example 1

Quantitative assay of NAD:
(Reagents)
(1) 0.2M phosphate buffer solution (pH 8.0)

(2) INT-Adekatol solution (INT (100 mg), dissolved in 2% Adekatol-SO-145 solution (100 ml)

(3) Diaphorase or electron transfer substance 1) Diaphorase (NADH) solution 100 U / ml

2) Phenazine methosulfate (PMS) solution 100 mg / 20 ml

3) Pyrrocyanine solution 10 mg / 20 ml

4) Meldola blue solution 10 mg / 20 ml

(4) ethanol

(5) alcohol dehydrogenase 250 U / ml

(6) 10 mM NAD ⁺ solution (In the main reaction, NAD ^{+ is} formed, and unreacted NADH is decomposed by acid and heating, followed by neutralization with alkali and completely further neutralized with ethyl formate.)

(7) 0.1 N HCl

(Execution)

Phosphate buffer (0.5 ml), INT-Adekatol (0.2 ml), ethanol 20 μl), water (30 μl) and alcohol dehydrogenase (0.1 ml) are placed in separate test tubes. Diaphorase (NADH) or electron transfer substance (50 μΐ) was added and incubated for 3 minutes at 37 ⁰ C. Then 10 μl of NAD ⁺ solution (0, 10, 20, 40, 60, 80 or 100 μl) was added and each volume was adjusted to a total of 1.0 ml. ^{It was incubated at 37 °} C. for exactly 10 min. 0.1N HCl (2 ml) was added to each test tube to stop the reaction and the OD was measured at 50 nm. Kasser (0.1 ml) was added in place of NAD as a control.

(Results)

The results are compiled in FIG. The amount at NAD together with the diaphorase or electron transfer substance and the optical absorption density are in good linearity. The result shows an exact cycling response and the high sensitivity of the assay.

Example2

Effect of the oxidoreductase used in the cycling reaction:

(Reagents)

(1) 0.2M phosphate buffer pH 8.0

(2) INT-Adekatoi solution, as in Example 1

(3) Diaphorase (NADH) solution
(4) substrate solution

1) 100 mM ethyl alcohol

2) 100 mM glycerol-3-phosphate solution

3) 100 mM lactic acid

4) 100 mM malic acid
(5) oxidoreductase

1) Alcohol dehydrogenase (ADH) solution 25 OCG u / ml

2) Glycerol-3-phosphate dehydrogenase (G-3-PDH) solution

600 u / ml

3) Lactate dehydrogenase (LDH) solution 1500 U / ml 4) Malate dehydrogenase (MDH) solution 4600 U / ml

5) 10 μΜ NAD ⁺ solution

6) 0.1 N HCl

(Execution)

Phosphate buffer (0.5 ml), INT-Adekatol (0.2 ml), Diaphorase (NADH) (50 μΐ), substrate

solution (50 μΐ), oxidoreductase, which acts on the above substrate (5 μΐ), and water (95 μΐ) given. It was ^{incubated at 37 °} C. for 3 min.

10 μΜ NAD ⁺ solution (0, 10, 20, 40, 60, 80 or 100 μΐ) was added to the individual enzymes. It was adjusted to a total volume of 10 ml and ^{incubated at 37 °} C. for exactly 10 min. 0.1N HCl (2 ml) was added to each test tube to stop the reaction. The OD was measured at 500 hm. Water (100 μΐ) was used as a control instead of NAD solution.

(Results)

The results are shown in FIG. With every oxidoreductase a good linearity is shown, which indicates the exact cycling response and the high sensitivity.

Example 3

Quantitative assay of NADP:

(Reagents)
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(1) 0.2 M phosphate buffer pH 8.0

(2) INT-Adekatol solution (INT (100 mg), dissolved in 2% Adeka-

tol-SO-145 solution (100 ml))

(3) NADPH diaphorase (NADPH) solution 100 U / ml (4) substrate solution

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

2) 100 mM glutamic acid (GA)
(5) Oxidoreductase solution

1) Glucose-e-phosphate dehydrogenase (G-6-PDH) solution 1000 U / ml

2) Glutamate dehydrogenase (GlDH) solution 1000 U / ml

(6) 10 μΜ NADP ⁺ solution

COPY

(7) 0.1 N HCl

(Execution) :

Phosphate buffer (0/5 ml) / INT-Adekatol (0.1 ml), diaphorase (NADPH) (100 μΐ), substrate (50 μΐ) / enzyme (50 μΐ) and water (30 μί) were placed in separate test tubes, and it was incubated at 37 ° C for 3 min. Then 10 μΜ NADP ⁺ (0, 10, 20, 40, 60, 80 and 100 μΐ) were added in each case. The total volume was adjusted to 1.0 ml by adding water. ^{It was incubated at 37 °} C. for exactly 10 minutes. 0.1 N HCl (2 ml) was added to stop the reaction and the OD was measured at 500 nm. Water (100 μΐ) was added as a control instead of the NADP solution.

. '

(Results)

The results are shown in FIG. Good linearity was obtained. The NADP can be more sensitive together with glucose-6-phosphate dehydrogenase and glutamate dehydrogenase.

Example 4
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Quantitative Assay of Malic Acid:

(Sample)

Malic acid solution (malic acid; 0, 1, 2, 4, 6, 8 and 10 μΜ) 20 μΐ

(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 (120 U / ml) solution (100 μΐ)

(2) 0.2N NaOH

(3) ethyl formate

(4) Reagent II: mixed solution of 0.5 H phosphate buffer pH 8.0

(0.1 ml), ethyl alcohol (10 μΐ), alcohol dehydrogenase (250 U / ml) (50 μΐ), Diaphora-

se (NADH) (100 u / ml) (100 μΐ), 1% NTB solution (20 μΐ) and Triton-X-100 solution (20 μΐ).

(5) 0.1 N HCl

(Execution)

. The samples are placed in separate test tubes and reagent I (0.2 ml) was added. It was then ^{incubated at 37 °} C. for 15 min. NaOH 0.2 N (0.1 ml) was added to the reaction mixture and ^{heated to 100 °} C. for 10 min. It was then immediately cooled and then neutralized with ethyl formate (20 μΐ). After a 5 minute incubation at 37 ° C II (0.3 ml) was added to reagent, and incubated for exactly 10 minutes at 37 ⁰ C. 0.1 N HCl (2.5 ml) was added to stop the reaction and the OD at 550 nm was measured. Water (100 μΐ) was used as a control instead of the NAD solution.

(Results)
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The results are shown in FIG. The amount of malic acid is related to the OD with less error. The malic acid was determined with high sensitivity.

Example 5

Quantitative assay of γ-aminobutyric acid:

(Sample)

Human serum (50 μΐ) containing γ-aminobutyric acid (GABA) (0, 10, 20, 30, 40 and 50 μΜ) was added.

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(Reagents)

(1) Reagent I: mixed solution of 0.1 M pyrophosphate buffer /

pH 8.3 (1.0 ml), 10 mM NADP ⁺ solution (0.2 ml), 0.1 M a-ketoglutarate solution (1.0 ml) and

GABASE (2 U / ml, 0.4 ml, commercially available enzyme from Pseudomonas fluorescens)

(2) 0.5 N NaOH solution

(3) ethyl formate

(4) Reagent II: mixed solution of INT adekatol solution (0.1 ml,

INT (100 mg), dissolved in 2% Adekatol-SO-145 solution (100 ml)), Diaphorase (NADPH) solution (100 u / ml, 1.0 ml), 0.1 M glucose-6 ~ phosphate solution (0.2 ml) and glucose-6-phosphate-de-. hydrogenase solution (100 u / ml, 2.0 μΐ).

(Execution)

Reagent I (0.35 ml) was added to the sample and ^{incubated at 37 °} C. for 30 minutes. 0.5N NaOH (0.1 ml) was added and it was heated for 10 min to 100 ⁰ C. After cooling, ethyl formate (20 μΐ) was added for neutralization, and it was incubated for 5 min at 37 ° C. Reagent II (0.2 ml) was added and it was incubated for exactly 10 min at 37 ° C. 0, 1 N HCl (2 mL) was added to stop the reaction and the OD at 500 nm was measured, and a control without GABA was used.

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(Results)

The results are shown in FIG. The amount of GABA in human serum can be plotted on a straight line in relation to the OD value. GABA can be highly sensitive and can be determined without separating the protein.

COPV

Explanation of the drawings:

Figure 1: Standard curve of NAD using Diaphorase and other electron transfer substances as an indication reaction

Figure 2: Standard curve of NAD, which shows the effect of the oxidoreductase used in the cycling reaction »

10. 'Figure 3: Standard curve of NADP, which shows the effect of the shows oxidoreductase used in the cycling reaction

Figure 4: Malic acid standard curve
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Figure 5: Standard curve of GABA added to the human serum became

COPY

Claims (18)

KRAUS & WEISERT PATENT LAWYERS AND APPROVED REPRESENTATIVES IN FRONT OF THE EUROPEAN PATENT OFFICE WALTER KRAUS DIPLOMAL CHEMIST DR.-IN G. ANNEKÄTE WEISERT DIPL.-INQ. SPECIALIST DIRECTION CHEMIE GARDSTRASSE 16 - O-BOOO MUNICH 71 ■ TELEPHONE 089 / 797O77-707O78 · TELEX O5-2121β θ kpat <S TELEGRAM KRAUSPATENT 3590 WK / to TOYO JOZO KABUSHIKI PATENT KAISPRÜCHE Tagata, Japan Highly sensitive assay 1. Highly sensitive assay as to what is to be determined Substance characterized by the following Response levels: (1) a main reaction system in which the coenzyme corresponding to the amount of the substance to be determined by the action of the oxidoreductase for said substance in the presence of coenzyme Ni
NADPH is converted; Presence of coenzyme NAD ⁺ or NADH, or NADP ⁺ or ^ (2) a decomposition reaction system in which the unreacted Coenzyme is decomposed and neutralized upon termination of the main reaction system; (3) a cycling reaction system in which the reaction means a coenzyme cycling is strengthened, and that from a combination the oxidoreduction reaction, the excess substrate and coenzyme according to the amount to be determined Substance converts, and a conversion reaction from tetrazolium salt to formazan in the presence of diaphorase or an electron transfer substance · and (4) a colorimetric measurement of the formazan so formed. 2. Assay according to claim 1, characterized in that g e k e η η - indicates that the molar ratio of the coenzyme in the main reaction system is above the molar ratio of the substance to be determined lies. 3. Assay according to claim 1, characterized in that g e k e η η - draws that unreacted coenzyme in the case of NAD and NADP by adding aqueous alkali hydroxide, Heating and neutralizing decomposed. 4. Assay according to claim 3, characterized in that g e k e η η - shows that the concentration of the aqueous alkali hydroxide results in a pH of above 12. 5. Assay according to claim 4, characterized in that sodium hydroxide is used as the alkali hydroxide xide or potassium hydroxide is used. 6. Assay according to claim 3, characterized in that the heating is complete Carries out decomposition of NAD or NADP. 7. Assay according to claim 3, characterized in that one neutralization by addition caused by formic acid lower alkyl ester. 8. Assay according to claim 1, characterized in that unreacted coenzyme in the case ■ - 3 - decomposed of NADH or NADPH by adding aqueous acid and heating, and that one neutralized. 9. Assay according to claim 8, characterized in that g e k e η η - shows that the concentration of the aqueous acid gives an acidity below pH 2. 10. Assay according to claim 9, characterized in that the acid used is a substance det / which results in a pH value of less than in aqueous solution. 11. Assay according to claim 10, characterized in that the acid used is hydrochloric acid or sulfur acid used .. 12. Assay according to claim 8, characterized in that the heating is complete Carries out decomposition of NADH or NADPH. . 13. Assay according to claim 8, characterized in that the neutralization in the way carries out that a small excess of an alkali metal hydroxide solution is added and formic acid lower alkyl ester immediately admits. 14. Assay according to claim 13, characterized in that the alkali metal hydroxide is sodium hydroxide or potassium hydroxide is used. 15. Assay according to claim 7 or 13, characterized in that the formic acid lower alkyl ester Methyl formate, ethyl formate, propyl formate or butyl formate used. 16. Assay according to claim 1, characterized in that the electron transfer substance used is g e k e η η - COPY in the cycling reaction phenazine methosulphate, Meldola blue or pyrocyanine is used. 17. Assay according to claim λ, characterized geke η η - draws / that one adds a surface-active agent. 18. Assay according to claim 17 / characterized / that one is used as a surface-active agent a nonionic surfactant is used.