CN104459158B

CN104459158B - Aspartate aminotransferase Mitochondria Isoenzyme detection kit

Info

Publication number: CN104459158B
Application number: CN201410805113.9A
Authority: CN
Inventors: 邹炳德; 邹继华; 陆慧贤; 黄辛雷
Original assignee: NINGBO MEIKANG BIOTECHNOLOGY Co Ltd
Current assignee: Meikang biological Polytron Technologies Inc
Priority date: 2014-12-22
Filing date: 2014-12-22
Publication date: 2016-06-22
Anticipated expiration: 2034-12-22
Also published as: CN104459158A

Abstract

Disclosure one aspartate aminotransferase Mitochondria Isoenzyme detection kit, consist of: reagent 1: pH of buffer 6.5～8.00.02～0.5mol/L, L-Aspartic acid potassium 0.15～0.3mol/L, malic dehydrogenase 0.3～5KU/L, lactic acid dehydrogenase 0.3～5KU/L, people source cAST antibody 5～20mL/L, antibody stabilization agent 0.01～3%, enzyme stabilizers 0.01～10%, preservative 0.01～1%；Reagent 2: pH of buffer 7.0～9.00.02～0.5mol/L, α-ketoglutaric acid 0.005～0.02mol/L, reduced coenzyme Ⅰ 0.1～0.5mmol/L, preservative 0.01～1%, reduced coenzyme Ⅰ stabilizer 0.01～10%。The advantage with good stability。

Description

Aspartate aminotransferase Mitochondria Isoenzyme detection kit

Technical field

The present invention relates to technical field of medical examination, be specifically related to a kind of aspartate aminotransferase Mitochondria Isoenzyme (m-AST or mAST Mitochondrial form aspartate amino transferase) detection kit。

Background technology

Aspartate aminotransferase (Aspartateaminotransferase, AST) is widely present in the various histiocyte of human body, especially abundant with liver, heart tissue's cell content, and in normal human serum, AST activity is very low。In human body there are two kinds of isozymes in AST, and one is cAST, is mainly derived from cytoplasm, and another kind is mAST, is present in mitochondrion。Both isozymes are variant in genomic constitution, all different in aminoacid composition, immunological characteristic and half-life in vivo etc.。

When the slight pathological changes of body, histiocytic permeability of cell membrane increases, and cAST enters in blood system through cell membrane, but mAST has the protection of two-layer mitochondrial membrane, is not easily released into blood；And when body serious disease, necrocytosis, mitochondrion disintegrate occur, being attached to the mAST on mitochondrion can discharge into rapidly in blood。Therefore, serum mAST activity can reflect histiocytic degree of necrosis, if detecting high level mAST in serum, then shows that cardiac muscle, liver are badly damaged；Additionally the mAST half-life in blood is shorter than cAST, and when cell no longer destroys or repairs, in serum, mAST is quickly down to normal level, and therefore mAST is again the evaluation index of cardiac muscle, liver injury prognosis, and the diagnosis of disease, treatment are had important value。

At present, in serum, mAST measures mainly electrophoresis method, chromatography and immunodepression etc., especially possesses special, accurate, fast and convenient, to can be used for Aulomatizeted Detect advantage with immunodepression, is suitable for the application of clinical extensive specimen。Its Cleaning Principle is: people source cAST antibody forms immune complex with cAST effect in serum sample, make cAST activity inhibited, and mAST activity is unaffected, mAST with the reaction coupling of malic dehydrogenase (MDH) catalysis, can make reduced coenzyme Ⅰ (NADH) be oxidized to NAD⁺, by detecting the speed that certain wave strong point NADH absorbance declines, can be calculated the activity of mAST。Reaction equation is as follows:

By above-mentioned reaction principle it can be seen that people source cAST antibody is the critical materials of mAST determination of activity system, can people source cAST antibody becomes accurately measure the key of mAST activity in human serum sample。But, according to this area general knowledge, when antibody is with solution state long-term storage, the phenomenons such as the generation that the generation of chemolysis thing, the generation of insoluble agglutinator, solubility associated complex can occur, thus causing that the biological activity of antibody is substantially reduced, therefore antibody is most to save as the best under-20 DEG C of environment, in order to keep the activity of antibody, can only provisional configuration test kit in use, and this is simply not proposed to commercial liquid reagent box。

Summary of the invention

The present invention is directed to the above-mentioned deficiency of prior art, it is provided that the aspartate aminotransferase Mitochondria Isoenzyme detection kit of a kind of excellent in stability。

In order to solve above-mentioned technical problem, the technical solution used in the present invention is: a kind of aspartate aminotransferase Mitochondria Isoenzyme detection kit, and this test kit is made up of following two liquid reagent, wherein:

Reagent 1:

Reagent 2:

Wherein, the described buffer in reagent 1 or 2 can be one or more in TRIS buffer, piperazine-N, N-double; two (2-hydroxyethanesulfonic acid) buffer, N-2-hydroxyethyl piperazine-N '-ethanesulfonic acid buffer, N-tri-(methylol) methylamino-2-hydroxy-propanesulfonic acid buffer, 4-(2-ethoxy)-1-piperazine propane sulfonic acid buffer, 3-morpholine-2-hydroxypropionate sodium buffer；

Described antibody stabilization agent can be the one in glycine and citric acid, glycine and heparin, citric acid and histidine, heparin and histidine combination, the combination of preferred histidine and citric acid, the weight proportion between said sequence combination is: histidine: citric acid=10～100:1；

Described enzyme stabilizers is one or several in trehalose, sucrose, bovine serum albumin, glycerol, Tween 80；

Described preservative is NaN₃Or the one in proclin300；

Described reduced coenzyme Ⅰ stabilizer is one or more in trehalose, bovine serum albumin, glycerol, Tween 80。

Advantages of the present invention and beneficial effect: the mAST detection kit of the present invention is by adding antibody stabilization agent, improve in test kit people source cAST antibody in the stability of liquid condition, additionally by combining interpolation enzyme stabilizers and reduced coenzyme Ⅰ stabilizer, therefore more effectively ensure that the stability that test kit is overall, greatly meet the demand of Clinical detection and diagnosis。

Specific embodiment

Below in conjunction with embodiment, the present invention is further described in detail, but is not limited to this。

Embodiment 1

A kind of mAST detection kit, including reagent 1 and reagent 2, wherein:

Reagent 1:

Reagent 2:

Embodiment 2

A kind of mAST detection kit, including reagent 1 and reagent 2, wherein:

Reagent 1:

Reagent 2:

Below the stability of the embodiment of the present invention 1 gained reagent is illustrated。

1, Antibody stability:

Following three group reagents of configuration by the following method:

Organize 1.: recipe configuration reagent described in embodiment 1, and store 18 months in 2～8 DEG C；

Organize 2.: except without antibody stabilization agent, all the other are recipe configuration reagent described in embodiment 1, and store 18 months in 2～8 DEG C；

Group is 3.: after 18 months, the newly configured reagent of formula (antibody of addition be-20 DEG C frozen same a collection of antibody) described in embodiment 1。

Above-mentioned three group reagents are placed on automatic clinical chemistry analyzer, location parameter is set in accordance with the following methods and measures the activity of 10 parts of human serum mAST simultaneously: reaction temperature 37 DEG C, test dominant wavelength 340nm, test commplementary wave length 405nm, tested serum sample/blank with reagent ratio is: sample/compare: reagent 1: reagent 2=18uL:240uL:60uL, reacts for dropping reaction。Method of testing is: sample/comparison first mixes with reagent 1 hatches 5min, add reagent 2 afterwards, reading is started after in 1.5min, and under measuring wavelength, monitor the change of each sample absorbance in 3min continuously, calculate the absorbance changing value △ A/min of average minute clock, and calculate mAST activity value: mAST activity (U/L)=(△ A according to below equation_Measure/min-△A_Blank/ min) × F (2840.3)。Measurement result is as shown in table 1。

Table 1mAST detection kit Antibody stability result

By table 1 result it can be seen that be not added with the reagent of antibody stabilization agent after storage 18 months, measured result exceeds about 10% than newly configured reagent, illustrates that the antibody in liquid reagent is deposited 18 months artifact activity and decreased, it is suppressed that ability declines to some extent；And with the addition of the reagent of antibody stabilization agent, the result of mensuration with newly join reagent and remain to keep consistent (± 3%), it was shown that antibody stabilization agent can protect the biological activity of antibody in liquid reagent effectively, improves the stability of antibody。

2, heat stability:

Recipe configuration reagent described in embodiment 1, is placed in heat damage 7d under 37 DEG C of water bath, measures and records the blank absorbency changing value of reagent in 7d, and measure the mAST value of 10 parts of human serum samples in 7d。

Each Blank absorbance values in 37 DEG C of water-bath 7d of table 2mAST detectable

Determination of serum result after 37 DEG C of water-bath 7d of table 3mAST detectable

By table 2 and table 3 result it can be seen that 7d placed by reagent 37 DEG C, blank absorbency is without significant change, and determination of serum result is close to comparison, and result is good。

The people source cAST antibody of the present invention is commercially available prod, such as the GOT1Antibodies that Thermoscientific company sells。

The above embodiment of the present invention is the description of the invention and cannot be used for the restriction present invention, and any change in the implication suitable with claims of the present invention and scope is all considered as being included in the scope of claims。

Claims

1. an aspartate aminotransferase Mitochondria Isoenzyme detection kit, it is characterised in that: this test kit is made up of reagent 1 and 2 two kinds of liquid reagents of reagent, wherein:

Reagent 1:

PH of buffer 6.5 ~ 8.0 0.02 ~ 0.5 mol/L, L-Aspartic acid potassium 0.15 ~ 0.3 mol/L, Malic dehydrogenase 0.3 ~ 5 KU/L, Lactic acid dehydrogenase 0.3 ~ 5 KU/L, People source cAST antibody 5 ~ 20 mL/L, Antibody stabilization agent 0.01 ~ 3 %, Enzyme stabilizers 0.01 ~ 10 %, Preservative 0.01 ~ 1 %,

Reagent 2:

PH of buffer 7.0 ~ 9.0 0.02 ~ 0.5 mol/L, α-ketoglutaric acid 0.005 ~ 0.02 mol/L, Reduced coenzyme Ⅰ 0.1 ~ 0.5 mmol/L, Preservative 0.01 ~ 1 %, Reduced coenzyme Ⅰ stabilizer 0.01 ~ 10 %；

Described antibody stabilization agent is the one in glycine and citric acid, glycine and heparin, citric acid and histidine, heparin and histidine combination；

2. aspartate aminotransferase Mitochondria Isoenzyme detection kit according to claim 1, it is characterized in that: the described buffer in reagent 1 or 2 is TRIS buffer, piperazine-N, N-double; two (2-hydroxyethanesulfonic acid) buffer, N-2-hydroxyethyl piperazine-N '-ethanesulfonic acid buffer, N-tri-(methylol) methylamino-2-hydroxy-propanesulfonic acid buffer, 4-(2-ethoxy)-1-piperazine propane sulfonic acid buffer, one or more in 3-morpholine-2-hydroxypropionate sodium buffer。

3. aspartate aminotransferase Mitochondria Isoenzyme detection kit according to claim 1, it is characterised in that: the described combination that antibody stabilization agent is histidine and citric acid。

4. aspartate aminotransferase Mitochondria Isoenzyme detection kit according to claim 1, it is characterised in that: described preservative is NaN₃Or the one in proclin300。