CN102788880A

CN102788880A - Heart-type fatty acid binding protein detection reagent kit and preparation method thereof

Info

Publication number: CN102788880A
Application number: CN2012102911109A
Authority: CN
Inventors: 谢兵
Original assignee: BEIJING ENJIHE BIOTECHNOLOGY Co Ltd
Current assignee: BEIJING ENJIHE BIOTECHNOLOGY Co Ltd
Priority date: 2012-08-16
Filing date: 2012-08-16
Publication date: 2012-11-21

Abstract

A heart-type fatty acid binding protein detection reagent kit, the kit body of the reagent kit comprises two reagents inside: reagent R1: buffer solution, polyethylene glycol, sodium azide, sodium ethylene diamine tetracetate, and bovine serum albumin; reagent R2: buffer solution, latex particles bound with heart-type fatty acid binding protein monoclonal antibody, Tween-20, sodium azide, sodium ethylene diamine tetracetate, and bovine serum albumin. Mixing a sample with the reagents in a specific volumetric ratio to conduct a series of reactions, placing the reactant under a semi/full automatic biochemical analyzer, and detecting the change speed of light absorbance at the position of 500nm dominant wavelength, thus the concentration of heart-type fatty acid binding protein can be figured out. The reagent kit has the advantages of accuracy, stability and convenience.

Description

A kind of cardic fatty acid binding protein detection kit and preparation method thereof

Technical field

The invention belongs to the biological medicine technology field, relate in particular to a kind of detection kit that detects popular feeling type fatty acid binding protein.

Background technology

Cardic fatty acid binding protein (H-FABP) is that molecular weight is the solubility endochylema albumen of l4～15kd; Mainly be distributed in the cardiac muscular tissue; Account for 4%～8% of cardiac muscle cell's soluble protein, a small amount of distribution is also arranged in skeletal muscle, renal tubule, brain tissue, mammary gland, placenta tissue.H-FABP forms acidic protein by 132 amino acid residues, and its isoelectric point (PI) is 5, and its assignment of genes gene mapping is in No. 1 chromosome.

Under normal circumstances, LCFA is cardiac muscle cell's a main energy source.H-FABP combines with intramyocardial LCFA as the carrier protein of fatty acid, it is transported to mitochondria from cytoplasma membrane, thereby get into oxygenolysis in the energetic supersession system, generates atriphos, for myocardial contraction provides energy.In addition, also participate in by-passing signal conduction: as through fatty acid signal transposition to peroxisome Proliferator-activated receptor indirect adjustments and controls gene expression, and be considered to when myocardial ischemia causes local LCFA gathering, H-FABP has protective effect to cardiac muscle.

Under the physiological condition, do not contain H-FABP or content in blood plasma and the urine seldom, can detect the H-FABP of low concentration in the normal human blood.Sex, age and circadian rhythm can cause that all the variation of H-FABP content such as male sex's muscle ratios are higher than the women, so male blood plasma H-FABP concentration is higher than the women; H-FABP is mainly removed by kidney in the blood, and blood plasma H-FABP concentration raise with the age.

H-FABP has very strong tissue specificity, after myocardial damage, is released into blood rapidly, begins in 1 hour to raise in acute myocardial infarction AMI (AMI) morbidity, and peaking fast can be used as the biochemical marker of myocardial damage early detection such as AMI.

Blocking behind the AMI is the severe complication of myocardial infarction again, but in clinical position, still lacks effective monitoring means at present.The concentration of biomarker in blood commonly used will continue a couple of days and just return to normally, can not block behind the early detection AMI again.H-FABP surpasses the fault value in AMI outbreak 3h, the peaking time is short, and in 12～24h, is got rid of fully by kidney and fall after rise to baseline.AMI takes place can to obstruct by the early detection cardiac muscle through continuous detecting blood H-FABP after 10 hours again.Because the Hemodynamics characteristic that H-FABP is unique, early stage, continuous detecting H-FABP concentration helps finding early that AMI obstructs again, takes the measure of treating timely and effectively, improves patient's prognosis.

H-FABP is a kind of metastable protein, and existing at present multiple qualitative and quantitative detection method can be used.Be the radioimmunology that Ockner proposed in 1974 the earliest, specific radioactivity comes detection by quantitative FABP in the immunoprecipitation through measuring.After develop assay methods such as immunoturbidimetry mensuration that sandwich ELISA method, particulate strengthen, immunosensor method again.These methods have the shortcoming of complex operation, length consuming time, excess waste resource, are inappropriate for routine inspection.

Summary of the invention

The detection kit that the purpose of this invention is to provide a kind of cardic fatty acid binding protein.This kit can directly apply on the automatic clinical chemistry analyzer, accuracy is strong as a result, reagent stability good, easy to use, be convenient to large-scale promotion.

Another object of the present invention provides the production preparation and the method for application of above-mentioned detection kit.

For realizing above-mentioned purpose, kit provided by the invention consists of:

Reagent R1: damping fluid, polyglycol, Sodium azide, sodium ethylene diamine tetracetate, bovine serum albumin(BSA);

Reagent R2: damping fluid, the latex particle that is combined with the cardic fatty acid binding protein monoclonal antibody, Tween-20, Sodium azide, sodium ethylene diamine tetracetate, bovine serum albumin(BSA).

It is following that the preparation method of mentioned reagent box provided by the invention and behaviour do step:

(a) prepare reagent in following ratio:

Reagent R1:

Tris damping fluid 200-220mmol/L

Polyglycol 5-10 mmol/L

Sodium azide 1-5 mmol/L

Sodium ethylene diamine tetracetate 1-5mmol/L

Bovine serum albumin(BSA) 1-5mmol/L;

Reagent R2:

Tris damping fluid 200-220mmol/L

Be combined with the latex particle 3.5-5% (v/v) of cardic fatty acid binding protein monoclonal antibody

Tween-20 1-5mmol/L

Sodium azide 0.5-3 mmol/L

Sodium ethylene diamine tetracetate 1-5mmol/L

Bovine serum albumin(BSA) 1-5mmol/L;

(b) reagent is mixed with sample to be tested by a certain percentage, make its complete reaction;

(c) measure the absorbance changing value with half/automatic clinical chemistry analyzer;

(d) calculate the concentration of center of a sample's type fatty acid binding protein according to the absorbance changing value.

Embodiment

Following embodiment is for the more detailed the present invention of explanation, is confined to this but should not be construed as the present invention.

Embodiment 1:

Kit of the present invention is double reagent for example, wherein:

Reagent R1:

Tris damping fluid 200mmol/L

Polyglycol 8 mmol/L

Sodium azide 5 mmol/L

Sodium ethylene diamine tetracetate 5mmol/L

Bovine serum albumin(BSA) 5mmol/L;

Reagent R2:

Tris damping fluid 200mmol/L

Be combined with the latex particle 5% (v/v) of cardic fatty acid binding protein monoclonal antibody

Tween-20 5mmol/L

Sodium azide 3 mmol/L

Sodium ethylene diamine tetracetate 5mmol/L

Bovine serum albumin(BSA) 5mmol/L.

Embodiment 2: the kit method of application.

1. reagent is prepared, and reagent is liquid double reagent, and uncork i.e. usefulness, wherein:

Reagent R1:

Tris damping fluid 200mmol/L

Polyglycol 8 mmol/L

Sodium azide 5 mmol/L

Sodium ethylene diamine tetracetate 5mmol/L

Bovine serum albumin(BSA) 5mmol/L;

Reagent R2:

Tris damping fluid 200mmol/L

Tween-20 5mmol/L

Sodium azide 3 mmol/L

Sodium ethylene diamine tetracetate 5mmol/L

Bovine serum albumin(BSA) 5mmol/L.

2. the full-automatic biochemical instrument parameter is provided with

(a) detected temperatures: 37 ℃

(b) detect wavelength: predominant wavelength is 500nm; Commplementary wave length is 700 nm;

(c) reaction time: 8 minutes 20 seconds, wherein, 3 minutes incubation time, 100 seconds reaction time, 220 seconds detection times.

3. detect step (all in automatic clinical chemistry analyzer, accomplishing)

(a) get 150 μ L reagent 1 and 10 μ L serum sample mixings;

(b) with the solution behind the mixing 37 ℃ of incubations 3 minutes;

(c) add 50 μ L reagent 2, react after 100 seconds, the absorbance that under the 500nm condition, detects 220 seconds changes.

4. the concentration that calculates cardic fatty acid binding protein through the conjunction absorbance that reads.

The advantage that the present invention has:

Accuracy is strong as a result, reagent stability good for this kit, easy to use, be convenient to large-scale promotion.Required detecting instrument (Biochemical Analyzer) generally uses at various big hospital and inspection center.

Claims

1. cardic fatty acid binding protein detection kit, it consists of: reagent R1, reagent R2, wherein:

A, reagent R1 comprise: 200-220mmol/L Tris damping fluid, 5-10 mmol/L polyglycol, 1-5 mmol/L Sodium azide, 1-5mmol/L sodium ethylene diamine tetracetate, 1-5mmol/L bovine serum albumin(BSA);

B, reagent R2:200-220mmol/L Tris damping fluid, the latex particle that is combined with the cardic fatty acid binding protein monoclonal antibody, 1-5mmol/L Tween-20,0.5-3 mmol/L Sodium azide, 1-5mmol/L sodium ethylene diamine tetracetate, 1-5mmol/L bovine serum albumin(BSA).

2. the kit of claim 1; It is characterized in that: the preparation method of the latex particle of the cardic fatty acid binding protein monoclonal antibody among the reagent R2 is: the cardic fatty acid binding protein monoclonal antibody of 10.5mg/ml and 10% latex particle (diameter 50-100nm); Join in the 200-220mmol/L Tris damping fluid concussion reaction 4 hours, the centrifugal supernatant that goes; Be diluted to 0.5% with 200-220mmol/L Tris damping fluid again, and add 0.1-0.3 mmol/L Sodium azide.

3. the preparation of mentioned reagent box and method of operating, key step is:

(a) prepare reagent in following ratio:

Reagent R1:

Tris damping fluid 200-220mmol/L

Polyglycol 5-10 mmol/L

Sodium azide 1-5 mmol/L

Sodium ethylene diamine tetracetate 1-5mmol/L

Bovine serum albumin(BSA) 1-5mmol/L

Reagent R2:

Tris damping fluid 200-220mmol/L

Tween-20 1-5mmol/L

Sodium azide 0.5-3 mmol/L

Sodium ethylene diamine tetracetate 1-5mmol/L

Bovine serum albumin(BSA) 1-5mmol/L

4. the method for claim 3 is characterized in that, sample and ratio of reagents should be controlled at 1:15 between the 1:25.

5. the method for claim 3 is characterized in that, temperature of reaction is 37 (± 1) ℃.

6. the method for claim 3 is characterized in that, the reaction time is 8-12 minute.

7. the method for claim 3 is characterized in that, the predominant wavelength of using half/automatic clinical chemistry analyzer to detect is 500nm, and commplementary wave length is 700 nm.