Determination method of ammonia (ammonia ions) and diagnosis/determination kit for ammonia (ammonia ions)

Abstract

The invention relates to a determination method of ammonia (ammonia ions) content by means of technologies of enzymatic colorimetry and enzyme-linked assay, and composition and components of a reagent. The technical principle of determination is based on a series of catalytic reactions of ammonia kinase, propionyl coenzyme A carboxylase, and hydrogen cyanide synthetase. The invention further relates to a diagnosis / determination kit for ammonia (ammonia ions). The determination method provided by the invention has high sensitivity and small error. Therefore, the determination method and the kit provided by the invention can be widely applied to clinical medical / food inspection.

Description

technical field

[0001] The present invention relates to the technical field of medical / food inspection and determination, more specifically, the present invention relates to ammonia (ammonia ion) diagnosis / measurement method and kit thereof. Background technique

[0002] Ammonia determination methods include micro-diffusion method, ion exchange method, enzymatic method and ammonia electrode method. The most widely used methods are enzymatic method and blood ammonia analyzer analysis method based on ion selective electrode.

[0003] The diffusion method is that after the sample is alkalized, NH is released 3 , Titrate the released ammonia with acid, or use the Nessler reaction to form brown-yellow mercuric ammonium iodide for colorimetry. These methods need to be alkalized, and the endogenous ammonia formation will affect the accuracy and precision, so they are rarely used at present; the ion exchange method is more accurate than the diffusion method, and the CV is 8% to 13...

Examples

Embodiment 1

[0129] Embodiment 1: the mensuration of ammonia (ammonia ion) content in blood plasma

[0130] 1) Preparation of standard samples

[0131] Dissolve a certain amount of ammonia salt in water or buffer solution, and then adjust the ammonia concentration to 100 micromole / liter;

[0132] 2) Pretreatment of samples to be tested

[0133] Plasma samples are used as samples to be tested without pretreatment;

[0134] 3) Blank sample

[0135] Described water or buffer solution are used as blank sample, and its ammonia concentration is 0 micromole / liter;

[0136] B. Preparation of reagent solution:

[0137] The ammonia diagnosis / measurement reagent of the present embodiment is a single-dose reagent, which contains:

[0138] Tris(carboxymethyl)aminomethane-hydrochloric acid buffer 100mmol / L

[0139] Glycerin 1mol / L

[0140] NADPH0.25mmol / L

[0141] Ammonia kinase 16000U / L

[0142] Propionyl-CoA carboxylase 18000U / L

[0143] Hydrogen cyanide synthase 12000U / L

[0144] Adenosine...

Embodiment 2

[0160] Embodiment 2: the mensuration of ammonia (ammonia ion) content in blood plasma

[0161] 1) Preparation of standard samples

[0162] A certain amount of ammonia salt is dissolved in water, and then the concentration of ammonia (ammonia ion) is adjusted to 100 micromole / liter, as a standard sample;

[0163] 2) Preparation of samples to be tested

[0164] Plasma samples are used as samples to be tested without pretreatment;

[0165] 3) Blank sample

[0166] Described water is used as blank sample, and its ammonia (ammonia ion) concentration is 0 micromole / liter;

[0167] B. Preparation of reagent solution:

[0168] Ammonia (ammonia ion) diagnostic / determination reagent is a two-dose reagent, which contains:

[0169] Reagent 1 with the following composition:

[0170] Phosphate buffer 100mmol / L

[0171] NADH0.25mmol / L

[0172] Adenosine triphosphate 5mmol / L

[0173] Methylmalicyl CoA 5mmol / L

[0174] Hydrogen cyanide 5mmol / L

[0175] Monovalent phosphate 5mmol / L; ...

Embodiment 3

[0198] Embodiment 3: the mensuration of ammonia (ammonia ion) content in plasma sample

[0199] The mode of operation of this embodiment is the same as embodiment 2, but this embodiment:

[0200] B. Preparation of reagent solution:

[0201] Ammonia (ammonia ion) diagnostic / assay reagent is a three-dose reagent that contains:

[0202] Reagent 1 with the following composition:

[0203] Tris(carboxymethyl)aminomethane-hydrochloric acid buffer 100mmol / L

[0204] NADH0.25mmol / L

[0205] Adenosine triphosphate 5mmol / L

[0206] Methylmalicyl CoA 5mmol / L

[0207] Hydrogen cyanide 5mmol / L

[0208] Monovalent phosphate 5mmol / L;

[0209] Reagent 2 with the following composition:

[0210] Tris(carboxymethyl)aminomethane-hydrochloric acid buffer 100mmol / L

[0211] Sodium chloride 2mol / L

[0212] Propionyl-CoA carboxylase 28000U / L

[0213] Hydrogen cyanide synthase 50000U / L;

[0214] Reagent 3 with the following composition:

[0215] Tris(carboxymethyl)aminomethane-hydrochloric a...

technical field

[0001] The present invention relates to the technical field of medical / food inspection and determination, more specifically, the present invention relates to ammonia (ammonia ion) diagnosis / measurement method and kit thereof. Background technique

[0002] Ammonia determination methods include micro-diffusion method, ion exchange method, enzymatic method and ammonia electrode method. The most widely used methods are enzymatic method and blood ammonia analyzer analysis method based on ion selective electrode.

[0003] The diffusion method is that after the sample is alkalized, NH is released 3 , Titrate the released ammonia with acid, or use the Nessler reaction to form brown-yellow mercuric ammonium iodide for colorimetry. These methods need to be alkalized, and the endogenous ammonia formation will affect the accuracy and precision, so they are rarely used at present; the ion exchange method is more accurate than the diffusion method, and the CV is 8% to 13...

Examples

Embodiment 1

[0129] Embodiment 1: the mensuration of ammonia (ammonia ion) content in blood plasma

[0130] 1) Preparation of standard samples

[0131] Dissolve a certain amount of ammonia salt in water or buffer solution, and then adjust the ammonia concentration to 100 micromole / liter;

[0132] 2) Pretreatment of samples to be tested

[0133] Plasma samples are used as samples to be tested without pretreatment;

[0134] 3) Blank sample

[0135] Described water or buffer solution are used as blank sample, and its ammonia concentration is 0 micromole / liter;

[0136] B. Preparation of reagent solution:

[0137] The ammonia diagnosis / measurement reagent of the present embodiment is a single-dose reagent, which contains:

[0138] Tris(carboxymethyl)aminomethane-hydrochloric acid buffer 100mmol / L

[0139] Glycerin 1mol / L

[0140] NADPH0.25mmol / L

[0141] Ammonia kinase 16000U / L

[0142] Propionyl-CoA carboxylase 18000U / L

[0143] Hydrogen cyanide synthase 12000U / L

[0144] Adenosine...

Embodiment 2

[0160] Embodiment 2: the mensuration of ammonia (ammonia ion) content in blood plasma

[0161] 1) Preparation of standard samples

[0162] A certain amount of ammonia salt is dissolved in water, and then the concentration of ammonia (ammonia ion) is adjusted to 100 micromole / liter, as a standard sample;

[0163] 2) Preparation of samples to be tested

[0164] Plasma samples are used as samples to be tested without pretreatment;

[0165] 3) Blank sample

[0166] Described water is used as blank sample, and its ammonia (ammonia ion) concentration is 0 micromole / liter;

[0167] B. Preparation of reagent solution:

[0168] Ammonia (ammonia ion) diagnostic / determination reagent is a two-dose reagent, which contains:

[0169] Reagent 1 with the following composition:

[0170] Phosphate buffer 100mmol / L

[0171] NADH0.25mmol / L

[0172] Adenosine triphosphate 5mmol / L

[0173] Methylmalicyl CoA 5mmol / L

[0174] Hydrogen cyanide 5mmol / L

[0175] Monovalent phosphate 5mmol / L; ...

Embodiment 3

[0198] Embodiment 3: the mensuration of ammonia (ammonia ion) content in plasma sample

[0199] The mode of operation of this embodiment is the same as embodiment 2, but this embodiment:

[0200] B. Preparation of reagent solution:

[0201] Ammonia (ammonia ion) diagnostic / assay reagent is a three-dose reagent that contains:

[0202] Reagent 1 with the following composition:

[0203] Tris(carboxymethyl)aminomethane-hydrochloric acid buffer 100mmol / L

[0204] NADH0.25mmol / L

[0205] Adenosine triphosphate 5mmol / L

[0206] Methylmalicyl CoA 5mmol / L

[0207] Hydrogen cyanide 5mmol / L

[0208] Monovalent phosphate 5mmol / L;

[0209] Reagent 2 with the following composition:

[0210] Tris(carboxymethyl)aminomethane-hydrochloric acid buffer 100mmol / L

[0211] Sodium chloride 2mol / L

[0212] Propionyl-CoA carboxylase 28000U / L

[0213] Hydrogen cyanide synthase 50000U / L;

[0214] Reagent 3 with the following composition:

[0215] Tris(carboxymethyl)aminomethane-hydrochloric a...

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