CN1760374A

CN1760374A - Method for testing activity of 5'-nucleotidase, diagnosis kit for 5'-nucleotidase

Info

Publication number: CN1760374A
Application number: CN 200410064905
Authority: CN
Inventors: 王尔中
Original assignee: Individual
Current assignee: Individual
Priority date: 2004-10-11
Filing date: 2004-10-11
Publication date: 2006-04-19

Abstract

A reagent kit for diagnosing 5'-nucleotidase is composed of buffer liquid, inosine monophosphate, monovalence phosphate, nucleotide phosphatase, xanthine oxidase, peroxidase, stabilizer and reduced chromogen group. A process for measuring the activity of 5'-nucleotidase includes such steps as proportionally mixing specimen with reagents, enzyme coupling reaction and biochemically analyzing the final resultant to determine the variation in light absorptivity of master wavelength and in turn the activity of 5'-nucleotidase. Its advantages are high sensitivity and precision, and no pollution.

Description

5 '-activity of 5 '-nucleotidase measuring method and 5 '-the phosphonuclease diagnostic kit

Technical field

The present invention relates to a kind ofly to measure 5 '-method of activity of 5 '-nucleotidase, the invention still further relates to simultaneously be used to realize 5 of this method '-the phosphonuclease diagnostic kit, belong to medical test determination techniques field.

Background technology

Medical research shows, 5 '-phosphonuclease (5NT) increases and is mainly seen in the obstructive jaundice, also is found in liver cancer and hepatitis.When the concurrent cholangitis of cholestasis, primary and Secondary cases cholehepatocirrhosis and chronic hepatitis, the 5NT rate of rise is higher than alkaline phosphatase; The susceptibility that 5NT raises when liver tumor and hepatic granuloma is higher than alkaline phosphatase.Because 5 '-activity of 5 '-nucleotidase do not have physiological and raise, and is not only responsive than alkaline phosphatase for diagnosis infant's hepatopathy and gestational liver function cholestasis, and specificity is arranged.Therefore measure 5 '-activity of phosphonuclease is significant for the diagnosis of disease.

5 '-activity determination method of phosphonuclease is a lot, mainly contains isotope substrate method, chemical phosphorus acid test method (nineteen twenty-five).Understand according to the applicant, generally adopt the isotropic substance Substrate test method at present in the world, method is: 5 '-phosphonuclease acts on H ³-dUMP, after the termination reaction,, try to achieve 5 by the ion exchange column analytical results '-activity of 5 '-nucleotidase.Perhaps utilize 5 '-phosphonuclease produces inorganic phosphorus after acting on single adenosine phosphate, analyze content of inorganic phosphorus with Chemical acid method again and calculated 5 '-activity of phosphonuclease.

Method of isotope substrate is complicated to also have isotopic contamination, and needs Isotope analyzer, therefore is difficult to apply conscientiously.Determination of inorganic phosphorus is the method for nineteen twenty-five invention, and accuracy is bad, and strong acid contaminate environment or the like also is not suitable for applying.

Summary of the invention

Propose a kind of can overcome 5 of above prior art shortcoming '-measuring method of activity of 5 '-nucleotidase, provide simultaneously in order to realize 5 of this method '-the phosphonuclease diagnostic kit.Adopt reagent in this test kit not only can on ultraviolet analyser or half, automatic clinical chemistry analyzer, carry out 5 '-activity of 5 '-nucleotidase is measured, and finding speed is fast, accuracy is high, thereby can obtain practical applying.

The present invention measures 5 '-step of activity of 5 '-nucleotidase is as follows:

1), with sample with mainly by the reagent mix of inosine list phosphoric acid, unit price phosphoric acid salt, Phosphatase, nucleotide, XOD, peroxidase, the combination of reduced form chromogen, make it to take place the reaction of following principle:

Inosine list phosphoric acid+water 5 phosphonucleasesInosine+phosphoric acid salt

Inosine+phosphoric acid salt Phosphatase, nucleotideXanthoglobulin+ribose 1-phosphate

Xanthoglobulin+water+oxygen XODUrate+hydrogen peroxide

Hydrogen peroxide+reduced form chromogen combination PeroxidaseThe indoleamine chromogen

Or quinone-imine chromogen+water

2), detect the end reaction thing in the speed that predominant wavelength 400--600nm absorbancy rises, calculate 5 '-the active size of phosphonuclease.

Usually step 2) the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer device, detect the speed that predominant wavelength 400--600nm absorbancy rises, calculate 5 '-the active size of phosphonuclease.

The blending ratio of above sample and reagent by volume is 1/10 to 1/250, and the mensuration temperature of above process is controlled at 20 ℃ to 50 ℃ conventional scopes, and the reaction times was controlled at conventional 2-30 minute.

The present invention uses 5 '-enzyme linked reaction such as phosphonuclease coupling Phosphatase, nucleotide, XOD, peroxidase, with inosine list phosphoric acid is substrate, and reduced form chromogen (Chromogen) combined system, colourless reduced form chromogen combination is oxidized to coloured quinone-imine chromogen (Quioneimine) or indoleamine chromogen (Indamine) dyestuff, therefore the height (different dyes absorbing wavelength difference is between 400--600nm) of measuring dyestuff content can directly reflect 5 '-size of activity of 5 '-nucleotidase.The advantage of this enzyme linked reaction system has: (1) its utilize will be colourless the reduced form chromogen make up be oxidized to coloured quinone-imine chromogen or indoleamine chromogen dyestuff and reflect 5 '-activity of 5 '-nucleotidase, the good advantage of tolerance range is arranged.(2) reacted constituent of this enzyme linked reaction system composition all adds, and is not subjected to the pollution of inside and outside source material, and test result is accurate.

In order to realize 5 of the inventive method '-the phosphonuclease diagnostic kit can be single agent, comprising:

Damping fluid 40--200mmol/l

Inosine list phosphatase 11--50mmol/l

Unit price phosphoric acid salt 0.2--10mmol/l

Phosphatase, nucleotide 500--50000U/l

XOD 500--50000U/l

Peroxidase 500--50000U/l

Stablizer 10--80% (cumulative volume)

Reduced form chromogen combination 0.1--20mmol/l

3-methyl-2-Proxel hydrazone (3-methyl-2-benzothiazolinone-hydrszone is called for short MBTH) that above reduced form chromogen combination can be 0.1-20mmol/l or the amino anti-arsenic of 4-(4-aminoantipyrine) of 0.1-20mmol/l combine with one of following 14 kinds of compositions of 0.1--20mmol/l:

PHENOL 99.8 MIN ((CARBOLIC ACID)) (phenol)

N-ethyl-N-(3-thiopropyl)-m-thialdine amine (N-ethyl-N-(3-sulfopropyl)

-m-anisidine is called for short ESPAS)

N, and the two ethyls of N--m-Tolylamine (N, N-Diethyl-m-toluidine)

2, and the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-(2,4-Dichloriphenol)

2,4, and 6-three bromo-3-hydroxyl-Phenylsulfonic acid (2,4,6-Tribromo-3-hydroxy-

Benzenesulfonic acid is called for short TBHB)

3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid (3,5-Dichlorophenolsulfonic acid) of 5-

3, and the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-(3,5-Dichloro-2-hydroxy-

Benzenesulfonic acid is called for short DHBS)

N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt (N-ethyl-N-

(2-hydroxy-3-sulfopropyl)-m-toluidine sodium is called for short TOOS)

Three bromine hydroxy-benzoic acid (Tribromohydroxybenzoic acid)

Two monomethylanilines (Dimethylaniline is called for short DMA)

N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine (N-Ethyl-N-(2-hydroxy-

3-sulfopropyl)-m-toluidine is called for short EHSPT)

2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid) (2,2 '-azino-bis (3-

Ethylbenzthiazoline-6-sulfonic acid) is called for short ABTS

Vanillic acid (Vanillic acid (4-hydroxy-3-

Methoxybenzoic acid) is called for short HMB)

(3-methyl-ethyl-hydroxyaniline is called for short 3-methyl-ethyl-hydroxyanilines

MEHA)

Also above single agent can be made into following pair of agent, more help eliminating inside and outside source inosine and xanthoglobulin and pollute:

Reagent I

Damping fluid 40--200mmol/l

Unit price phosphoric acid salt 0.2--10mmol/l

Phosphatase, nucleotide 500--50000U/l

XOD 500--50000U/l

Peroxidase 500--50000U/l

Stablizer 10--80% (cumulative volume)

Reduced form chromogen combination (one) 0.1--20mmol/l

Reduced form chromogen combination (one) can be that the amino anti-arsenic of 4-of the 3-methyl-2-Proxel hydrazone of 0.1-20mmol/l or 0.1-20mmol/l is one of in the two.

Reagent II

Damping fluid 40--200mmol/l

Inosine list phosphatase 11--50mmol/l

Stablizer 10--50mmol/l

Reduced form chromogen combination (two) 0.1--20mmol/l

Reduced form chromogen combination (two) can be one of following 14 kinds of compositions of 0.1--20mmol/l: PHENOL 99.8 MIN ((CARBOLIC ACID)), N-ethyl-N-(3-thiopropyl)-m-thialdine amine, N, the two ethyls of N--m-Tolylamine, 2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-, 2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid, 3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-, 3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt, three bromine hydroxy-benzoic acid, two monomethylanilines, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine, 2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid), vanillic acid, 3-methyl-ethyl-hydroxyanilines.

The prescription of two agent is not limited only to above-mentioned prescription, and reduced form chromogen combination () and (two) can transpositions.The composition of reagent I wherein, peroxidase can be placed on reagent II, so can form multiple formulations, does not describe in detail one by one.

Reagent can also be made into following three reagent, not only more help eliminating inside and outside source inosine and xanthoglobulin and pollute, also more help the stable of reagent:

Reagent I

Damping fluid 40--200mmol/l

Unit price phosphoric acid salt 0.2--10mmol/l

Stablizer 10--50mmol/l

Reduced form chromogen combination (one) 0.1--20mmol/l

Reagent II

Damping fluid 40--200mmol/l

Phosphatase, nucleotide 500--50000U/l

XOD 500--50000U/l

Peroxidase 500--50000U/l

Stablizer 10--80% (cumulative volume)

Reagent III

Damping fluid 40--200mmol/l

Inosine list phosphatase 11--50mmol/l

Stablizer 10--50mmol/l

Reduced form chromogen combination (two) 0.1--20mmol/l

Three doses prescription is not limited only to above-mentioned prescription, and reduced form chromogen combination () and (two) can divide any position that is opened in reagent I, II or III.The composition of reagent I wherein, unit price phosphoric acid salt can be placed among the reagent II, the composition of reagent II, Phosphatase, nucleotide, XOD, peroxidase etc. also can be placed among the reagent I, peroxidase can also be placed on reagent III, so can form multiple formulations, does not describe in detail one by one.

The pH scope of buffer reagent can be 6.0-11.0.Buffer reagent can be three (carboxymethyl) aminomethane-hydrochloric acid (Tris-HCl) damping fluid, phosphoric acid salt (Phosphate) damping fluid, trolamine (Triethanolamine) damping fluid, 2-amino-2-methyl-1-propanol (2-Amino-2-methyl-1-propanol) damping fluid, imidazoles (Imidazole) damping fluid or glycylglycine (Glycylglycine) damping fluid etc., but is not limited only to these damping fluids.

In addition, in order to reduce the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stablizer 10-80% or 10-50mmol/l among reagent I, the reagent II of the reagent I of above single agent, two agent, reagent II or three doses, the reagent III, stablizer can be one or more in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, sulfuric acid amine or the salt etc.

Experiment shows, takes all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, no matter is single agent, two agent or three doses, the present invention 5 of following system component relation '-the phosphonuclease diagnostic kit is comparatively desirable:

Damping fluid 80--120mmol/l

Inosine list phosphatase 11--5mmol/l

Unit price phosphoric acid salt 2--8mmol/l

Phosphatase, nucleotide 5000--12000U/l

XOD 5000--12000U/l

Peroxidase 5000--12000U/l

Stablizer 10--50% (cumulative volume)

10--50mmol/l

Reduced form chromogen combination 0.1--10mmol/l

Because the present invention utilizes Enzymology method, enzyme digestion reaction to have the high characteristics of specificity fully, is not vulnerable to the interference of other materials of inside and outside source.Easy, the easy handling of enzyme process method.The specificity of enzyme digestion reaction impels test result accurate.Enzyme digestion reaction all is to carry out under buffer conditions, does not have environmental pollution problem.The enzyme process method does not need special, extra instrument, and testing cost is cheap.Therefore can guarantee to have higher test accuracy, be more convenient for applying.

In addition, one of striking features of the present invention is to eliminate inside and outside source inosine and hypoxanthic pollution, eliminate the first half that inside and outside source inosine and hypoxanthic effect occur in the entire reaction time period, be consumed totally at time second half section contaminated inside and outside source inosine and xanthoglobulin, and second half section time test 5 '-the needed inosine of activity of 5 '-nucleotidase and xanthoglobulin all be result from 5 '-activity of phosphonuclease.

Embodiment

The present invention is further illustrated below in conjunction with examples of implementation.

Embodiment one (single agent)

5 of present embodiment '-the phosphonuclease diagnostic kit comprises:

Damping fluid 80mmol/l

Inosine list phosphatase 11 mmol/l

Unit price phosphoric acid salt 2mmol/l

Phosphatase, nucleotide 5000U/l

XOD 5000U/l

Peroxidase 5000U/l

Stablizer 50% (cumulative volume)

The amino anti-arsenic 2mmol/l of 4-

PHENOL 99.8 MIN ((CARBOLIC ACID)) 10mmol/l

On automatic clinical chemistry analyzer, set: temperature 37,10 minutes reaction times, test predominant wavelength 495-505nm, more than the test commplementary wave length 600nm, tested 5 '-volume ratio of phosphonuclease sample and reagent is 1/25, the Direction of Reaction is positive reaction, 1 minute time of lag, 2 minutes detection times.

After adding sample and reagent, make it to mix, following reaction take place:

Xanthoglobulin+water+oxygen XODUrate+hydrogen peroxide

Or quinone-imine chromogen+water

The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 495-505nm absorbancy rises, thereby calculate 5 '-the active size of phosphonuclease.

Present embodiment uses 5 '-enzyme linked reactions such as phosphonuclease coupling Phosphatase, nucleotide, XOD, peroxidase, with inosine list phosphoric acid is substrate, and reduced form chromogen (Chromogen) combined system, colourless reduced form chromogen combination is oxidized to coloured quinone-imine chromogen (Quioneimine) or indoleamine chromogen (Indamine) dyestuff, therefore the height (different dyes absorbing wavelength difference is between 400--600nm) of measuring dyestuff content can directly reflect 5 '-size of activity of 5 '-nucleotidase.

Embodiment two (two agent)

5 of present embodiment '-the phosphonuclease diagnostic reagent has:

Reagent I

Damping fluid 100mmol/l

Unit price phosphoric acid salt 5mmol/l

Phosphatase, nucleotide 8000U/l

XOD 8000U/l

Peroxidase 8000U/l

Stablizer 50% (cumulative volume)

The amino anti-arsenic 1mmol/l of 4-

Reagent II

Damping fluid 100mmol/l

Inosine list phosphoric acid 3mmol/l

Stablizer 30mmol/l

2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid 1mmol/l

Measure 5 '-during activity of 5 '-nucleotidase, temperature is controlled at 30 ℃, 15 minutes reaction times, test predominant wavelength 546nm, more than the test commplementary wave length 600nm, tested 5 '-volume ratio of phosphonuclease sample and reagent is 1/25, the Direction of Reaction is positive reaction, 1 minute time of lag, 2 minutes detection times.

Concrete determination step is:

Xanthoglobulin+water+oxygen XODUrate+hydrogen peroxide

Or quinone-imine chromogen+water

The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 546nm absorbancy rises, thereby calculate 5 '-the active size of phosphonuclease.

The reaction times of each reactions steps was controlled at 15 minutes.

Embodiment three (three doses)

5 of present embodiment '-the phosphonuclease diagnostic reagent is three doses, has:

Reagent I

Damping fluid 120mmol/l

Unit price phosphoric acid salt 8mmol/l

Stablizer 50mmol/l

3-methyl-2-Proxel hydrazone 2mmol/l

Reagent II

Damping fluid 120mmol/l

Phosphatase, nucleotide 12000U/l

XOD 12000U/l

Peroxidase 12000U/l

Stablizer 50% (cumulative volume)

Reagent III

Damping fluid 120mmol/l

Inosine list phosphoric acid 5mmol/l

Stablizer 20mmol/l

Two monomethylaniline 2mmol/l

On automatic clinical chemistry analyzer, set: 25 ℃ of temperature, 20 minutes reaction times, test predominant wavelength 578nm, more than the test commplementary wave length 700nm, tested 5 '-volume ratio of phosphonuclease sample and reagent is 1/25, the Direction of Reaction is positive reaction, 1 minute time of lag, 2 minutes detection times.

Concrete determination step is:

Xanthoglobulin+water+oxygen XODUrate+hydrogen peroxide

Or quinone-imine chromogen+water

The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 578nm absorbancy rises, thereby calculate 5 '-the active size of phosphonuclease.

Embodiment four

5 of present embodiment '-the phosphonuclease diagnostic reagent has:

Reagent I

Damping fluid 120mmol/l

Unit price phosphoric acid salt 6mmol/l

Phosphatase, nucleotide 12000U/l

XOD 12000U/l

Peroxidase 12000U/l

Stablizer 50% (cumulative volume)

The amino anti-arsenic 1mmol/l of 4-

Reagent II

Damping fluid 120mmol/l

Inosine list phosphoric acid 2mmol/l

Stablizer 20mmol/l

Two monomethylaniline 2mmol/l

Measure 5 '-during activity of 5 '-nucleotidase, temperature is controlled at 30 ℃, 10 minutes reaction times, test predominant wavelength 578nm, more than the test commplementary wave length 700nm, tested 5 '-volume ratio of phosphonuclease sample and reagent is 1/25, the Direction of Reaction is positive reaction, 1 minute time of lag, 2 minutes detection times.

After adding sample and reagent, make it to mix, following reaction take place:

Inosine list phosphoric acid+water 5 phosphonuclease enzymesInosine+phosphoric acid salt

Xanthoglobulin+water+oxygen XODUrate+hydrogen peroxide

Or quinone-imine chromogen+water

The reaction times of each reactions steps was controlled at 5 minutes.

In a word, experiment showed, and adopt measuring method of the present invention can draw required measurement result by ultraviolet analyser or half, automatic clinical chemistry analyzer device fully, and highly sensitive, tolerance range good, is not subjected to the pollution of inside and outside source material.

Claims

1. one kind 5 '-the activity of 5 '-nucleotidase measuring method, step is as follows:

Xanthoglobulin+water+oxygen XODUrate+hydrogen peroxide

Or quinone-imine chromogen+water

According to claim 1 described 5 '-the activity of 5 '-nucleotidase measuring method, it is characterized in that: described step 2) be, the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the speed that predominant wavelength 400--600nm absorbancy rises, calculate 5 '-the active size of phosphonuclease.

3, according to claim 1 or 2 described mensuration 5 '-method of activity of 5 '-nucleotidase, it is characterized in that: temperature is controlled at 20 ℃ to 50 ℃ scopes, and the reaction times was controlled at 2-30 minute.

4, according to claim 1 or 2 described mensuration 5 '-method of activity of 5 '-nucleotidase, it is characterized in that: tested 5 '-ratio control of phosphonuclease sample and reagent is 1/10 to 1/250.

5. one kind 5 '-the phosphonuclease diagnostic kit, be grouped into by following one-tenth:

Damping fluid 40--200mmol/l

Inosine list phosphatase 11--50mmol/l

Unit price phosphoric acid salt 0.2--10mmol/l

Phosphatase, nucleotide 500--50000U/l

XOD 500--50000U/l

Peroxidase 500--50000U/l

Stablizer 10--80% (cumulative volume)

Reduced form chromogen combination 0.1--20mmol/l

According to claim 5 described 5 '-the phosphonuclease diagnostic kit, it is characterized in that: described reagent is made into single agent, two agent or three doses.

According to claim 5 or 6 described 5 '-the phosphonuclease diagnostic kit, it is characterized in that: comprise that also stablizer is at least a in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, ammonium sulfate or the salt.

According to claim 5 or 6 described 5 '-the phosphonuclease diagnostic kit, it is characterized in that: the pH scope of described buffer reagent is 6.0～11.0, and described buffer reagent is a kind of in three (carboxymethyl) aminomethane-hydrochloride buffer, phosphate buffered saline buffer, trolamine damping fluid, 2-amino-2-methyl-1-propanol damping fluid, imidazole buffer or the glycylglycine damping fluid.

9, according to claim 5 or 6 described 5 '-the phosphonuclease diagnostic reagent, it is characterized in that: one of following 14 kinds of compositions of 3-methyl-2-Proxel hydrazone that described reduced form chromogen is 0.1-20mmol/l and 0.1-20mmol/l combine: PHENOL 99.8 MIN ((CARBOLIC ACID)), N-ethyl-N-(3-thiopropyl)-m-thialdine amine, N, the two ethyls of N--m-Tolylamine, 2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-, 2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid, 3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-, 3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt, three bromine hydroxy-benzoic acid, two monomethylanilines, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine, 2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid), vanillic acid, 3-methyl-ethyl-hydroxyanilines.

10, according to claim 5 or 6 described 5 '-the phosphonuclease diagnostic reagent, it is characterized in that: described reduced form chromogen is combined as by the amino anti-arsenic of the 4-of 0.1-20mmol/l and one of 14 kinds of compositions below the 0.1-20mmol/l and combines: PHENOL 99.8 MIN ((CARBOLIC ACID)), N-ethyl-N-(3-thiopropyl)-m-thialdine amine, N, the two ethyls of N--m-Tolylamine, 2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-, 2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid, 3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-, 3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt, three bromine hydroxy-benzoic acid, two monomethylanilines, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine, 2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid), vanillic acid, 3-methyl-ethyl-hydroxyanilines.