CN103558372A

CN103558372A - Preparation method of antigen standard substance and sample diluting solution for ELISA (enzyme-linked immuno sorbent assay)

Info

Publication number: CN103558372A
Application number: CN201310555174.XA
Authority: CN
Inventors: 章登吉; 程禹; 段新伟; 张波
Original assignee: LIGHTARRAY BIOTECH WUXI Co Ltd
Current assignee: Game Biotechnology (Wuxi) Co., Ltd.
Priority date: 2013-11-08
Filing date: 2013-11-08
Publication date: 2014-02-05
Anticipated expiration: 2033-11-08
Also published as: CN103558372B

Abstract

The invention relates to the technical field of immunodetection and provides a preparation method of an antigen standard substance and a sample diluting solution for ELISA (enzyme-linked immuno sorbent assay). The preparation method comprises the following steps: preparing antibody tags; carrying out pouring, closing, washing and drying on a BDNF (brain-derived neurotrophic factor) plate and a prolaction plate respectively, and storing for later use; preparing a standard diluting solution as a blank solution; preparing the sample diluting solution; putting the BDNF plate and the prolaction plate into an incubation vibrator for incubating antibodies; adding detection antibodies and then adding into a kit SA-HRP; reading an optical density (OD) value under a microplate reader; drawing a standard recovery rate curve of BDNF and prolactin, and calculating the recovery rate of a diluted sample. The antigen standard substance and the sample diluting solution effectively solve the problems of high background value, low signal-to-noise ratio and low sensitivity, and have the characteristics of reducing interference generated by a matrix effect and enabling a detection result to be relatively stable and reliable.

Description

The antigen standard items that a kind of ELISA uses and the preparation method of sample dilution

Technical field

The present invention relates to technical field of immunoassay, the antigen standard items that particularly a kind of ELISA uses and the preparation method of sample dilution.

Background technology

ELISA is the abbreviation of enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbnent Assay), is a kind of immunoenzyme technics growing up after immunofluorescence and radioimmunoassay technique.Because the experimental technique of ELISA has sensitivity, the feature such as special, economic, easy, safe, be therefore a kind of detection method the most frequently used in current Immunology Lab.

In ELISA operating process, selecting the reagent of high-quality, good instrument and correct operation is to guarantee ELISA testing result necessary condition accurately and reliably, the operation of ELISA is because of the different difference to some extent of formation of solid phase carrier, and Domestic Medicine check generally all adopts board-like point.

No matter how elisa technique develops, higher sensitivity and accuracy, and higher data repeatability is the target that vast researcher is pursued all the time.The basis of setting up a good ELISA method is antigen and the antibody that first will find, yet this technology still faces many problems and challenge at present: (1) sensitivity has much room for improvement, ELISA method is in hospital, Yao Qihe scientific research institutions are applied to the detection with the various factors of disease association, these factors are all often some endogenic cell factors, chemotactic molecule, adhesion molecule etc., it is low that yet some endogenous molecule is expressed demeanour, be difficult to be detected, highly sensitive kit is conducive to address this problem.(2) matrix effect problem, the matrix species of the sample that ELISA detects is varied, and different substrates composition, physical property difference, can produce and disturb pattern detection, cause the inaccurate or unstable of result; And in the quantitative detection of endogenous factors, the damping fluid of preparation typical curve and the objective difference between the true matrix of sample to be tested also can have influence on last testing result.(3) non-specific adsorption, the reason of non-specific adsorption is complicated, and one of them result is exactly to cause blank background value higher, and signal to noise ratio (S/N ratio) is on the low side, thereby has affected the quantitative sensitivity of whole kit.

Enzyme linked immunoassay is the reaction that a kind of susceptibility is very high, if serum, blood plasma or other matrix sample do not dilute, unavoidably can produce very strong nonspecific reaction, there is false positive, therefore, the complicacy due to sample to be tested mechanism also may cause matrix effect, interference detection results.

Therefore, it is higher that technical field of immunoassay is badly in need of a kind of reduction background value, improves signal to noise ratio (S/N ratio), increases sensitivity, eliminates the interference that matrix effect produces, and makes antigen standard items that the more reliable and more stable ELISA of testing result uses and the preparation method of sample dilution.

Summary of the invention

The invention provides antigen standard items that a kind of ELISA uses and the preparation method of sample dilution, technical scheme is as follows:

The antigen standard items of using and a preparation method for sample dilution, is characterized in that, comprises following steps:

The first step, Dispersal risk is coated;

The Brain Derived Neurotrophic Factor BDNF antibody of catching is added in carbonate buffer solution, and added in the hole of elisa plate, be labeled as BNDF plate, be placed in coated 16-24 hour at the temperature of 2-8 ℃;

Further, prolactin Prolactin antibody is added in another group carbonate buffer solution, and added in the hole of elisa plate, be labeled as Prolaction plate, be placed at the temperature of 2-8 ℃ coated 16-24 hour;

Second step, after BDNF plate in the first step and Prolaction plate are poured into a mould respectively, seal, wash, dried, then saves stand-by;

The phosphate buffer PBS that contains 5% bovine serum albumin(BSA) BSA that is 7.4 ± 0.2 by pH value pours into a mould respectively to BNDF plate and Prolaction plate in the first step; Or the 5-10% skimmed milk power that contains that is 7.4 ± 0.2 by pH value is poured into a mould respectively l to BNDF plate and Prolaction plate in the first step;

Further, the BDNF plate of this being poured into a mould and Prolaction plate are placed on respectively sealing at the temperature of 2-8 ℃ and place 16-24 hour;

Further, after the BDNF plate sealing and Prolaction plate being washed, are dried with the PBS solution that contains non-ionics Tween20, be placed at the temperature of 2-8 ℃, preserve stand-by;

The 3rd step, preparation standard dilution, as blank solution;

First 0.05% polyoxyethylene sorbitol mono laurate monoester Tween20 is added in the PBS that contains 5%BSA, and stir;

Further, to the liquid preservative Proclin that adds 15/1000000ths in this solution, after stirring as standard dilution, i.e. blank solution;

The 4th step, preparation sample dilution;

Sample dilution 1: first 0.05%Tween20 is added in the PBS that contains 5%BSA, and stir; Further, in this solution, adding 0.2% surfactant 3-[3-(courage acyl aminopropyl) dimethylamino] propane sulfonic acid inner salt CHAPS stirs; Further, in this solution, add 15/1000000ths Proclin to stir;

Sample dilution 2: first 0.05%Tween20 is added in the PBS that contains 5%BSA, and stir; Further, in this solution, add 0.2%CHAPS to stir; Further, in this solution, add the sequestrant edta edta of 5mmol/l to stir; Further, in this solution, add 15/1000000ths Proclin to stir;

Sample dilution 3: first 0.05%Tween20 is added in the PBS that contains 5%BSA, and stir; Further, in this solution, add 0.2%CHAPS to stir; Further, in this solution, add the EDTA of 5mmol/l to stir; Further, in this solution, add 0.05% cow's serum gamma globulin BGG; Further, in this solution, add 15/1000000ths Proclin to stir;

Sample dilution 4: first 0.05%Tween20 is added in the PBS that contains 5%BSA, and stir; Further, in this solution, add 0.2%CHAPS to stir; Further, in this solution, add the EDTA of 5mmol/l to stir; Further, in this solution, add 0.10%BGG; Further, in this solution, add 15/1000000ths Proclin to stir;

Sample dilution 5: first 0.05%Tween20 is added in the PBS that contains 5%BSA, and stir; Further, in this solution, add 0.2%CHAPS to stir; Further, in this solution, add the EDTA of 5mmol/l to stir; Further, in this solution, add 0.15%BGG; Further, in this solution, add 15/1000000ths Proclin to stir;

Sample dilution 6: first 0.05%Tween20 is added in the PBS that contains 5%BSA, and stir; Further, in this solution, add 0.2%CHAPS to stir; Further, in this solution, add the EDTA of 5mmol/l to stir; Further, in this solution, add 0.20%BGG; Further, in this solution, add 15/1000000ths Proclin to stir;

Sample dilution 7: first 0.05%Tween20 is added in the PBS that contains 5%BSA, and stir; Further, in this solution, add 0.2%CHAPS to stir; Further, in this solution, add the EDTA of 5mmol/l to stir; Further, in this solution, add 0.25%BGG; Further, in this solution, add 15/1000000ths Proclin to stir;

The 5th step, puts into incubation oscillator by BDNF plate and Prolaction plate and hatches antibody;

Respectively by the sample dilution 1-7 in the standard dilution in the 3rd step, the 4th step, a normal human serum sample, that repeats adds respectively in the hole of drying rear stand-by BDNF plate and Prolaction plate in second step, then adds treatment enzyme in each reacting hole;

Further, the BDNF plate and the Prolaction plate that have added solution are put into incubation oscillator, under the room temperature condition of 18-25 ℃, react;

The 6th step, adds detection antibody;

With the PBS that contains Tween20 as washing lotion, reacted BDNF plate and Prolaction plate in the 5th step are washed respectively, in each reacting hole of BDNF plate, add BDNF to detect antibody accordingly again, and in each reacting hole of Prolaction plate, add Prolaction to detect antibody accordingly;

The 7th step, adds dilution in kit;

With the PBS that contains Tween20, as washing lotion, reacted BDNF plate and Prolaction plate in the 6th step are diluted it after washing respectively, then in each hole in kit SA-HRP, add the solution after dilution respectively;

The 8th step, is placed on and under microplate reader, reads optical density OD value;

With the PBS that contains Tween20, as washing lotion, reacted kit SA-HRP in the 6th step is washed respectively, then add tetramethyl benzidine tmb substrate in the kit SA-HRP washing to this;

Further, the kit SA-HRP that has added TMB is placed on and under microplate reader, reads OD value;

The 9th step, calculates non-specific adsorption data according to OD value, by comparing non-specific adsorption data, further determines cow's serum gamma Globulin BGG and to reducing background noise, has direct and positive effect as antigen.

First, the sample dilution 1-7 in the standard dilution in the 3rd step, the 4th step and a normal human serum sample, the signal results of measured non-specific adsorption NSB on BDNF plate;

Further, the sample dilution 1-7 in the standard dilution in the 3rd step, the 4th step and a normal human serum sample, the signal results of measured non-specific adsorption NSB on Prolactin plate;

Further, discovery adds the sample dilution 3-7 of cow's serum gamma Globulin BGG in dilution, at the elisa plate of two kinds of coated different antibodies, the non-specific adsorption NSB in BNDF plate, Prolactin plate is very little, thereby the background value that explanation produces is all very low;

Further, because the BGG concentration in sample dilution 3-7 is different, but all reduced the background signal in ELISA reaction;

Further, the cow's serum gamma Globulin BGG antigen that any concentration is described has direct and positive effect to reducing background noise.

The antigen standard items that a kind of ELISA as above uses and the preparation method of sample diluting liquid, also comprise: the tenth step, using the sample dilution 6 in the 4th step as new standard dilution, only the content of CHAPS is adjusted into respectively to 0.1%, 0.15%, 0.25%, 0.3%, 0.45%, 0.5%, 0.6%, thereby form 7 kinds of new sample dilution 8-14, other step is processed according to the first step to eight step completely, reads OD value, further calculates non-specific adsorption coefficient; And the non-specific adsorption coefficient recording is analyzed, draw in the situation that cow's serum gamma Globulin BGG content is certain, CHAPS concentration is between 0.2%-0.45% time, and the non-specific adsorption NSB of BDNF plate and Prolactin plate can reduce, thereby background signal is reduced.

The antigen standard items that a kind of ELISA as above uses and the preparation method of sample diluting liquid, also comprise: the 11 step, from the 4th step and the tenth step, select three kinds of signal to noise ratio (S/N ratio)s high, three kinds of sample dilutions that non-specific signal is low are drawn the recovery typical curve of BDNF and prolactin and are calculated the recovery of dilute sample; According to test result proved to adding respectively the EDTA of 0.05%Tween20,0.2-0.45%CHAPS, 5mmol/l in the PBS that contains 5%BSA, the cow's serum gamma Globulin BGG of any concentration, 15/1000000ths Proclin as dilution, be the sample dilution of the extraordinary ELISA of being applicable to of a kind of effect, concrete steps are:

First, respectively BDNF and Prolactin standard antigen are added in BDNF and prolactin negative serum;

Further, to adding respectively in this negative serum sample in sample dilution 3, sample dilution 10, sample dilution 12 other steps and the 5th step to the eight steps in full accord, read optical density OD value and the recovery;

Further, two kinds of sample dilutions 3 and sample dilution 12 are respectively that CHAPS is 2 critical points of 0.2% and 0.45%, our experiments show that three kinds of sample dilutions, 3,10,12 BDNF of preparation and the typical curve of Prolactin are parallel to each other respectively, correlativity is fine, and compares the recovery with negative serum and improve;

Further, in the PBS that contains 5%BSA that proof is 7.4 ± 0.2 to pH value, add respectively the EDTA of 0.05%Tween20,0.2-0.45%CHAPS, 5mmol/l, the cow's serum gamma Globulin BGG of any concentration, 15/1000000ths Proclin, as dilution, are the sample dilutions of the extraordinary ELISA of being applicable to of a kind of effect.

The invention has the beneficial effects as follows:

1. the present invention uses cow's serum gamma Globulin BGG as antigen, thereby plays the effect that reduces background noise.

2. the present invention has determined in the situation that cow's serum gamma Globulin BGG content is certain, and CHAPS concentration is between 0.2%-0.45% time, and the non-specific adsorption NSB of BDNF plate and Prolactin plate can reduce, and has reduced background signal.

3. the invention provides antigen standard items and the sample dilution of a kind of extraordinary ELISA of being applicable to, made up the blank of industry, progress advances science.

Accompanying drawing explanation

Below in conjunction with the drawings and specific embodiments, describe the present invention in detail:

Fig. 1 is the recovery typical curve of BDNF of the present invention in three kinds of dilutions.

Fig. 2 is the recovery typical curve of Prolactin of the present invention in three kinds of dilutions.

Embodiment

In order to make technological means of the present invention, creation characteristic, reach object and effect is easy to understand, below in conjunction with concrete diagram, further set forth the present invention.

The invention provides antigen standard items that a kind of ELISA uses and the preparation method of sample dilution, concrete steps are as follows:

The first step, Dispersal risk is coated;

It is in 9.6 0.05mol/l carbonate buffer solution that the Brain Derived Neurotrophic Factor BDNF antibody of catching is added to pH value, be mixed with the solution that concentration is 2 μ g/ml, and added in the hole of elisa plate, be labeled as BNDF plate, be placed in coated 16-24 hour at the temperature of 2-8 ℃;

Further, prolactin Prolactin antibody is added in the 0.05mol/l carbonate buffer solution that another group pH value is 9.6, be mixed with the solution that concentration is 2.5 μ g/ml, and added in the hole of elisa plate, be labeled as Prolaction plate, be placed at the temperature of 2-8 ℃ coated 16-24 hour;

The phosphate buffer PBS that contains 5% bovine serum albumin(BSA) BSA that is 7.4 ± 0.2 by pH value pours into a mould respectively to BNDF plate and Prolaction plate in the first step, pours into a mould 300 μ l in each hole; Or the 5-10% skimmed milk power that contains that is 7.4 ± 0.2 by pH value is poured into a mould respectively to BNDF plate and Prolaction plate in the first step, pours into a mould 300 μ l in each hole;

Further, after the BDNF plate sealing and Prolaction plate being washed, are dried with the PBS solution that contains 0.05% non-ionics Tween, be placed at the temperature of 2-8 ℃, preserve stand-by;

The 3rd step, preparation standard dilution, as blank solution;

The 4th step, preparation sample dilution;

Respectively by the sample dilution 1-7 in the standard dilution in the 3rd step, the 4th step, a normal human serum sample, with 10, repeat to add respectively in the hole of drying rear stand-by BDNF plate and Prolaction plate in second step, in each reacting hole, add 100 μ l treatment enzyme again

Further, the BDNF plate and the Prolaction plate that have added solution are put into incubation oscillator, with the speed of 500 revs/min, under the room temperature condition of 18-25 ℃, react 2 hours.

The 6th step, adds detection antibody;

With the PBS that contains 0.05%Tween20 as washing lotion, reacted BDNF plate and Prolaction plate in the 5th step are washed respectively 4 times, in each reacting hole of BDNF plate, add the BDNF of 100 μ l to detect antibody accordingly again, and in each reacting hole of Prolaction plate, add the Prolaction of 100 μ l to detect antibody accordingly;

The 7th step, adds dilution in kit;

With the PBS that contains 0.05%Tween20 as washing lotion, reacted BDNF plate and Prolaction plate in the 6th step are washed respectively 4 times, after it being diluted with 1:10000 again, respectively to the solution adding in each hole in kit SA-HRP after the dilution of 100 μ l;

With the PBS that contains 0.05%Tween20, as washing lotion, reacted kit SA-HRP in the 6th step is washed respectively to 4 times, then add tetramethyl benzidine tmb substrate in the kit SA-HRP washing to this;

Further, the kit SA-HRP that has added TMB is placed under the microplate reader that wavelength is 450nm and reads OD value;

The 9th step, calculates non-specific adsorption data according to OD value, by comparing non-specific adsorption data, further determines cow's serum gamma Globulin BGG and to reducing background noise, has direct and positive effect as antigen;

First, the sample dilution 1-7 in the standard dilution in the 3rd step, the 4th step, a normal human serum sample, the signal results of measured non-specific adsorption NSB on BDNF plate, as table one:

Table one

Further, the sample dilution 1-7 in the standard dilution in the 3rd step, the 4th step, a normal human serum sample, the signal results of measured non-specific adsorption NSB on Prolactin plate, as table two:

Table two

According to the result in above-mentioned table one and table two, analyze, discovery adds the sample dilution 3-7 of cow's serum gamma Globulin BGG in dilution, elisa plate two kinds of coated different antibodies, be that non-specific adsorption NSB in BNDF plate, Prolactin plate is very little, thereby illustrate that produced background value is all very low, between 0.02-0.05;

Further, illustrate that cow's serum gamma Globulin BGG antigen has direct and positive effect to reducing background noise;

The tenth step, using the sample dilution 6 in the 4th step as new standard dilution, is only adjusted into respectively 0.1%, 0.15%, 0.25%, 0.3%, 0.45%, 0.5%, 0.6% by the content of CHAPS, thereby forms 7 kinds of new sample dilution 8-14.Other step is processed according to the first step to eight step completely, reads OD value;

The 11 step, according to OD value, calculate the value of non-specific adsorption NSB, thereby obtain in the situation that cow's serum gamma Globulin BGG content is certain, CHAPS concentration is between 0.2%-0.45% time, the non-specific adsorption NSB of BDNF plate and Prolactin plate can reduce, thereby makes the reduction of background signal;

The signal results of sample dilution 6 in the tenth step, sample dilution 8-14 and a normal human serum sample measured non-specific adsorption NSB on BDNF plate, as table three:

Table three

Further, the sample dilution 6 in the tenth step, sample dilution 8-14, a normal human serum sample, the signal results of measured non-specific adsorption NSB on Prolactin plate, as table four:

Table four

EDTA, Tween20 is all this area typical additives, but the adjuvant that CHAPS is not everybody generally to be used.Therefore, according to the result in above-mentioned table three and table four, analyze, find in the situation that cow's serum gamma Globulin BGG content is certain, CHAPS concentration is between 0.2%-0.45% time, the non-specific adsorption NSB of BDNF plate and Prolactin plate can reduce, thereby makes the reduction of background signal;

The 12 step selects three kinds of signal to noise ratio (S/N ratio)s high from the 4th step and the tenth step, and three kinds of sample dilutions that non-specific signal is low are drawn the recovery typical curve of BDNF and prolactin, and calculates the recovery of dilute sample; Fig. 1 is the recovery typical curve of BDNF of the present invention in three kinds of dilutions, and Fig. 2 is the recovery typical curve of Prolactin of the present invention in three kinds of dilutions; According to the recovery recording, proved to adding respectively the EDTA of 0.05%Tween20,0.2-0.45%CHAPS, 5mmol/l in the PBS that contains 5%BSA, the cow's serum gamma globulin BGG of any concentration, 15/1000000ths Proclin, as dilution, are the sample dilutions of the extraordinary ELISA of being applicable to of a kind of effect.

First, respectively the BDNF of 4000pg/ml and Prolactin standard antigen are added in BDNF and prolactin negative serum;

Further, respectively with 1:2,1:4,1:8 dilutes the negative serum preparing, after dilution, add respectively in sample dilution 3, sample dilution 10, sample dilution 12, other steps and the 5th step to the eight steps are in full accord, read optical density OD value, further calculate the recovery, and according to recovery drawing standard curve, Fig. 1 is the recovery typical curve of BDNF of the present invention in three kinds of dilutions, and Fig. 2 is the recovery typical curve of Prolactin of the present invention in three kinds of dilutions;

Three kinds of sample dilutions are prepared optical density OD value and the recovery of BDNF typical curve, as table five:

Table five

Three kinds of sample dilutions are prepared optical density OD value and the recovery of Prolactin typical curve, as table six:

Table six

Further, with negative serum sample in contrast, the average recovery rate of comparative sample dilution;

The average recovery rate of BDNF in three kinds of sample dilutions, as table seven:

Table seven

The average recovery rate of prolactin in three kinds of sample dilutions, as table eight:

Table eight

According to the analysis of his-and-hers watches five, six, seven, eight, two kinds of sample dilutions 3 and sample dilution 12 are respectively that CHAPS is 2 critical points of 0.2% and 0.45%; Our experiments show that three kinds of sample dilutions, 3,10,12 BDNF of preparation and the typical curve of Prolactin are parallel to each other respectively, correlativity is fine; With these three kinds of sample dilutions, dilute respectively the serum sample that contains BDNF and Prolactin, compare with negative serum sample, its recovery is brought up to 85-115% from 65-77%, and the recovery has had raising greatly, result precision improves, thereby meets the requirement detecting;

The present invention uses cow's serum gamma Globulin BGG as antigen, thereby plays the effect that reduces background noise.

The present invention determined in the situation that cow's serum gamma Globulin BGG content is certain, and CHAPS concentration is between 0.2%-0.45% time, and the non-specific adsorption NSB of BDNF plate and Prolactin plate can reduce, and has reduced background signal.

The invention provides antigen standard items and the sample dilution of a kind of extraordinary ELISA of being applicable to, made up the blank of industry, progress advances science.

More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that in above-described embodiment and instructions, describes just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.

Claims

1. the antigen standard items that ELISA uses and a preparation method for sample dilution, is characterized in that, comprises following steps:

The first step, Dispersal risk is coated;

The Brain Derived Neurotrophic Factor antibody of catching is added in carbonate buffer solution, and added in the hole of elisa plate, be labeled as BNDF plate, be placed in coated 16-24 hour at the temperature of 2-8 ℃;

Further, prolactin antibody is added in another group carbonate buffer solution, and added in the hole of elisa plate, be labeled as prolactin plate, be placed at the temperature of 2-8 ℃ coated 16-24 hour;

Second step, after the Brain Derived Neurotrophic Factor plate in the first step and prolactin plate are poured into a mould respectively, seal, wash, dried, then saves stand-by;

The phosphate buffer that contains 5% bovine serum albumin(BSA) that is 7.4 ± 0.2 by pH value is poured into a mould respectively to Brain Derived Neurotrophic Factor plate and prolactin plate in the first step; Or the 5-10% skimmed milk power that contains that is 7.4 ± 0.2 by pH value is poured into a mould respectively to Brain Derived Neurotrophic Factor plate and prolactin plate in the first step;

Further, the Brain Derived Neurotrophic Factor plate of this being poured into a mould and prolactin plate are placed on respectively sealing at the temperature of 2-8 ℃ and place 16-24 hour;

Further, after the Brain Derived Neurotrophic Factor plate sealing and prolactin plate being washed, are dried with the PBS solution that contains non-ionics, be placed at the temperature of 2-8 ℃, preserve stand-by;

The 3rd step, preparation standard dilution, as blank solution;

First 0.05% polyoxyethylene sorbitol mono laurate monoester is added in the phosphate buffer that contains 5% bovine serum albumin(BSA), and stir;

Further, to the liquid preservative that adds 15/1000000ths in this solution, after stirring as standard dilution, i.e. blank solution;

The 4th step, preparation sample dilution;

Sample dilution 1: first 0.05% polyoxyethylene sorbitol mono laurate monoester is added in the phosphate buffer that contains 5% bovine serum albumin(BSA), and stir; Further, in this solution, adding 0.2% surfactant 3-[3-(courage acyl aminopropyl) dimethylamino] propane sulfonic acid inner salt stirs; Further, to the liquid preservative that adds 15/1000000ths in this solution, stir;

Sample dilution 2: first the phosphate buffer of 0.05% bovine serum albumin(BSA) is added in the phosphate buffer that contains 5% bovine serum albumin(BSA), and stir; Further, to 3-[3-(the courage acyl aminopropyl) dimethylamino that adds 0.2% in this solution] propane sulfonic acid inner salt stirs; Further, in this solution, add the sequestrant ethylenediamine tetraacetic acid of 5mmol/l to stir; Further, to the liquid preservative that adds 15/1000000ths in this solution, stir;

Sample dilution 3: first 0.05% polyoxyethylene sorbitol mono laurate monoester is added in the phosphate buffer that contains 5% bovine serum albumin(BSA), and stir; Further, to 3-[3-(the courage acyl aminopropyl) dimethylamino that adds 0.2% in this solution] propane sulfonic acid inner salt stirs; Further, in this solution, add the ethylenediamine tetraacetic acid of 5mmol/l to stir; Further, in this solution, add 0.05% cow's serum gamma Globulin; Further, to the liquid preservative that adds 15/1000000ths in this solution, stir;

Sample dilution 4: first 0.05% polyoxyethylene sorbitol mono laurate monoester is added in the phosphate buffer that contains 5% bovine serum albumin(BSA), and stir; Further, to 3-[3-(the courage acyl aminopropyl) dimethylamino that adds 0.2% in this solution] propane sulfonic acid inner salt stirs; Further, in this solution, add the ethylenediamine tetraacetic acid of 5mmol/l to stir; Further, in this solution, add 0.10% cow's serum gamma Globulin; Further, to the liquid preservative that adds 15/1000000ths in this solution, stir;

Sample dilution 5: first 0.05% polyoxyethylene sorbitol mono laurate monoester is added in the phosphate buffer that contains 5% bovine serum albumin(BSA), and stir; Further, to 3-[3-(the courage acyl aminopropyl) dimethylamino that adds 0.2% in this solution] propane sulfonic acid inner salt stirs; Further, in this solution, add the ethylenediamine tetraacetic acid of 5mmol/l to stir; Further, in this solution, add 0.15% cow's serum gamma Globulin; Further, to the liquid preservative that adds 15/1000000ths in this solution, stir;

Sample dilution 6: first 0.05% polyoxyethylene sorbitol mono laurate monoester is added in the phosphate buffer that contains 5% bovine serum albumin(BSA), and stir; Further, to 3-[3-(the courage acyl aminopropyl) dimethylamino that adds 0.2% in this solution] propane sulfonic acid inner salt stirs; Further, in this solution, add the ethylenediamine tetraacetic acid of 5mmol/l to stir; Further, in this solution, add 0.20% cow's serum gamma Globulin; Further, to the liquid preservative that adds 15/1000000ths in this solution, stir;

Sample dilution 7: first 0.05% polyoxyethylene sorbitol mono laurate monoester is added in the phosphate buffer that contains 5% bovine serum albumin(BSA), and stir; Further, to 3-[3-(the courage acyl aminopropyl) dimethylamino that adds 0.2% in this solution] propane sulfonic acid inner salt stirs; Further, in this solution, add the ethylenediamine tetraacetic acid of 5mmol/l to stir; Further, in this solution, add 0.25% cow's serum gamma Globulin; Further, to the liquid preservative that adds 15/1000000ths in this solution, stir;

The 5th step, puts into incubation oscillator by Brain Derived Neurotrophic Factor plate and prolactin plate and hatches antibody;

Respectively by the sample dilution 1-7 in the standard dilution in the 3rd step, the 4th step, a normal human serum sample, that repeats adds respectively in the hole of drying rear stand-by Brain Derived Neurotrophic Factor plate and prolactin plate in second step, in each reacting hole, add treatment enzyme again

Further, the Brain Derived Neurotrophic Factor plate and the prolactin plate that have added solution are put into incubation oscillator, under the room temperature condition of 18-25 ℃, react;

The 6th step, adds detection antibody;

With the phosphate buffer that contains Brain Derived Neurotrophic Factor as washing lotion, reacted Brain Derived Neurotrophic Factor plate and prolactin plate in the 5th step are washed respectively, in each reacting hole of BDNF plate, add Brain Derived Neurotrophic Factor to detect antibody accordingly again, and in each reacting hole of prolactin plate, add prolactin to detect antibody accordingly;

The 7th step, adds dilution in kit;

With the phosphate buffer that contains Brain Derived Neurotrophic Factor as washing lotion, reacted Brain Derived Neurotrophic Factor plate and prolactin plate in the 6th step are diluted it after washing respectively, then in each hole in this kit, add the solution after dilution respectively.

The 8th step, is placed under microplate reader and reads optical density value;

With the phosphate buffer that contains Brain Derived Neurotrophic Factor, as washing lotion, reacted kit in the 6th step is washed respectively, then add tetramethyl benzidine substrate in the kit washing to this;

Further, the kit that has added TMB is placed under microplate reader and reads optical density value;

The 9th step, calculates non-specific adsorption data according to optical density value, by comparing non-specific adsorption data, further determines cow's serum gamma Globulin and to reducing background noise, has direct and positive effect as antigen.

First, the sample dilution 1-7 in the standard dilution in the 3rd step, the 4th step, a normal human serum sample, the signal results of measured non-specific adsorption NSB on Brain Derived Neurotrophic Factor plate;

Further, the sample dilution 1-7 in the standard dilution in the 3rd step, the 4th step, a normal human serum sample, the signal results of measured non-specific adsorption NSB on prolactin plate;

Further, discovery adds the sample dilution 3-7 of cow's serum gamma Globulin in dilution, elisa plate two kinds of coated different antibodies, be that non-specific adsorption NSB in neurotrophic factor plate and prolactin plate is very little, thereby illustrate that produced background value is all very low, between 0.02-0.045;

Further, because the cow's serum gamma Globulin concentration in sample dilution 3-7 is different, but all reduced the background signal in ELISA reaction;

Further, illustrate that cow's serum gamma Globulin antigen has direct and positive effect to reducing background noise.

2. the antigen standard items that a kind of ELISA according to claim 1 uses and the preparation method of sample diluting liquid, also comprise: the tenth step, using the sample dilution 6 in the 4th step as new standard dilution, only by this 3-[3-(courage acyl aminopropyl) dimethylamino] content of propane sulfonic acid inner salt is adjusted into respectively 0.1%, 0.15%, 0.25%, 0.3%, 0.45%, 0.5%, 0.6%, thereby form 7 kinds of new sample dilution 8-14, other step is processed according to the first step to eight step completely, read optical density value, further calculate non-specific adsorption coefficient; And the non-specific adsorption coefficient recording is analyzed, draw in the situation that cow's serum gamma Globulin content is certain, 3-[3-(courage acyl aminopropyl) dimethylamino] when salinity is between 0.2%-0.45% in propane sulfonic acid, the non-specific adsorption of brain source property neural factor plate and prolactin plate all can reduce, thereby background signal is reduced.

3. the antigen standard items that a kind of ELISA according to claim 2 uses and the preparation method of sample diluting liquid, also comprise: the 11 step, from the 4th step and the tenth step, select three kinds of signal to noise ratio (S/N ratio)s high, three kinds of sample dilutions that non-specific signal is low are drawn the recovery typical curve of BDNF and prolactin and are calculated the recovery of dilute sample; According to test result, having proved to 3-[3-(the courage acyl aminopropyl) dimethylamino that adds respectively 0.05% polyoxyethylene sorbitol mono laurate monoester, 0.2-0.45% in the phosphate buffer that contains bovine serum albumin(BSA)] propane sulfonic acid inner salt, the ethylenediamine tetraacetic acid of 5mmol/l, the liquid preservative of cow's serum gamma Globulin, 1,000,000/15 of any concentration be as dilution, be the sample dilution of the extraordinary ELISA of being applicable to of a kind of effect, concrete steps are:

First, respectively brain source property neural factor and prolactin standard antigen are added in brain source property neural factor and prolactin negative serum;

Further, respectively the negative serum preparing is diluted, add respectively in sample dilution 3, sample dilution 10, sample dilution 12 after dilution, other steps and the 5th step to the eight steps are in full accord, read optical density value and the recovery;

Further, two kinds of sample dilutions 3 and sample dilution 12 are respectively 3-[3-(courage acyl aminopropyl) dimethylaminos] propane sulfonic acid inner salt is 2 critical points of 0.2% and 0.45%, our experiments show that three kinds of sample dilutions, 3,10, the 12 brain source property neural factors of preparation and the typical curve of prolactin are parallel to each other respectively, correlativity is fine, and compares the recovery with negative serum and improve;

Further, 3-[3-(the courage acyl aminopropyl) dimethylamino that adds respectively 0.05% polyoxyethylene sorbitol mono laurate monoester, 0.2-0.45% in the phosphate buffer that contains bovine serum albumin(BSA) that proof is 7.4 ± 0.2 to pH value] propane sulfonic acid inner salt, the ethylenediamine tetraacetic acid of 5mmol/l be, the liquid preservative of cow's serum gamma Globulin, 1,000,000/15 of any concentration as dilution, is the sample dilution of the extraordinary ELISA of being applicable to of a kind of effect.