CN102841210A

CN102841210A - Retinol detection kit and preparation method thereof

Info

Publication number: CN102841210A
Application number: CN201210312846XA
Authority: CN
Inventors: 谢兵
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-08-30
Filing date: 2012-08-30
Publication date: 2012-12-26

Abstract

The invention relates to a detection kit for a retinol binding protein. The interior of a detection kit body comprises reagents R1 and reagents R2, wherein the reagents R1 comprise a buffer solution, polyethylene glycol, sodium azide, sodium ethylene diamine tetracetate and bovine serum albumin; and the reagents R2 comprise a buffer solution, latex grains combined with retinol binding protein monoclonal antibodies, tween-20, sodium azide, sodium ethylene diamine tetracetate and bovine serum albumin. A sample and the reagents are mixed at a certain volume ratio and generate a series of reactions, and then a reactant is placed under a semi/full-automatic biochemical analyzer, and the speed of absorbance change in the position of a main wavelength of 340nm is detected, so that the concentration magnitude of the retinol binding protein is measured and calculated. The detection kit has the advantages of accuracy, stability and convenience.

Description

A kind of retinol 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 human retinol-binding protein.

Background technology

RBP ELISA is the combination albumen of specific bond vitamin A in the blood plasma, in the metabolism of vitamin A, plays an important role.

The Clinical detection of RBP ELISA helps the diagnosis of hepatitis, diabetes early diagnosis, kidney trouble.

RBP ELISA is mainly synthetic by the liver rough surfaced endoplasmic reticulum (RER).Be distributed widely in human serum, urine, the cerebrospinal fluid.RBP ELISA exists with composite form in blood.When hepatocellular injury. the synthetic of RBP ELISA is suppressed.Because the half life period of RBP ELISA is 3～12 hours.Its relative molecular weight is little, and relevant with serum total bilirubin, albumin, prothrombin time.So it can reflect the change of liver function sensitively.While acute viral hepatitis patients serum's RBP ELISA content and AST, ALP, ALT negative correlation.So can explain that it is the impaired sensitive index of reflection liver cell that serum retinol combines albumen.

Retinal binding protein is a kind of low molecular protein, and can freely filtering also at glomerular filtration membrane, the overwhelming majority be heavily absorbed and kalabolism by kidney proximal tubule.Under the normal condition, retinal binding protein excretion amount is very little, and when kidney proximal tubule was impaired, retinal binding protein excretion amount obviously increased.Therefore measure the function situation that the retinal binding protein level can reflect renal tubule more delicately, and specificity is higher.

RBP ELISA itself has good stability; Content in vivo is constant relatively; Generally speaking; The content of RBP ELISA is few in the normal person urine, so there is the scholar to think that its excretion in urine can be used as an impaired sensitive index of renal tubular function, can reflect in early days that the infringement of renal tubule asks.Research shows, when diabetes in early days the glomerulus dysfunction do not take place as yet, has had the function damage of renal tubule.And when the diagnosing diabetes tubular injury, retinal binding protein can be used as more responsive diagnosis index of early diabetic nephropathy.For the diagnosis of early diabetic nephropathy provides foundation.So that the patient obtains diagnosis and treatment timely, improve prognosis, improve life quality.

The RBP ELISA assay method is a lot, comprising: (one) radioimmunoassay (RIA), (two) enzymoimmunoassay (ELISA), (three) latex agglutination test and immunoturbidimetry, (four) fluorescence immunoassay, (five) are dripped golden immunization.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 RBP ELISA.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 RBP ELISA 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 RBP ELISA 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 RBP ELISA in the sample 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 RBP ELISA 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

Be combined with the latex particle 5% (v/v) of RBP ELISA monoclonal antibody

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 340nm;

(c) reaction time: 10 minutes, wherein, 5 minutes incubation time, 5 minutes reaction time.

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

(a) get 200 μ L reagent 1 and 10 μ L serum sample mixings;

(b) with the solution behind the mixing 37 ℃ of incubations 5 minutes, read absorbance A1;

(c) add 50 μ L reagent 2 again, react after 5 minutes, read absorbance A2;

(d) calculate △ A=A2-A1.

4. calculate the concentration of RBP ELISA through △ A.

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. Retinal-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 RBP ELISA 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 RBP ELISA monoclonal antibody among the reagent R2 is: the RBP ELISA 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 is diluted to 0.5% with 200-220mmol/L Tris damping fluid again, and adds 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 1:20 between the 1:30.

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 wavelength that uses half/automatic clinical chemistry analyzer to detect is 340nm.