CN103558372B - Preparation method of antigen standard substance and sample diluting solution for ELISA (enzyme-linked immuno sorbent assay) - Google Patents
Preparation method of antigen standard substance and sample diluting solution for ELISA (enzyme-linked immuno sorbent assay) Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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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
Technical field
The present invention relates to technical field of immunoassay, particularly to a kind of antigen standard of ELISA and Sample Dilution
The preparation method of liquid.
Background technology
ELISA is the abbreviation of enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbnent Assay),
It is a kind of immunoenzyme technics growing up after immunofluorescence and radioimmunoassay technique.Experimental technique tool due to ELISA
Have sensitivity, special, economical, easy, safe the features such as, be therefore the most frequently used a kind of detection side in current Immunology Lab
Method.
It is to ensure that ELISA detects from the reagent of high-quality, good instrument and correct operation in ELISA operating process
Result accurately and reliably essential condition, the operation of the ELISA difference because the formation of solid phase carrier is different, Domestic Medicine is checked
Generally individually adopt board-like point.
No matter how elisa technique develops, higher sensitivity and accuracy, and higher data repeatability is wide all the time
The target that big researcher is pursued.The basis setting up a good ELISA method is antigen and the antibody first having to find,
But this technology still suffers from many problems and challenge at present：(1) sensitivity has much room for improvement, ELISA method in hospital, medicine enterprise and
Scientific research institutions are applied to the detection of the various factors related to disease, these factors be all often some endogenic cells because
Son, chemotactic molecule, adhesion molecule etc., however some endogenous molecules expression demeanour low it is difficult to detected, highly sensitive examination
Agent box advantageously accounts for this problem.(2) matrix effect problem, the matrix species of the sample that ELISA is detected are varied, no
Same matrix components, physical property are different, can produce interference to pattern detection, cause the inaccurate or unstable of result；And
In the detection by quantitative of endogenous factors, the objective difference prepared between the buffer of standard curve and the true substrate of sample to be tested also can
Have influence on last testing result.(3) non-specific adsorption, complicated the reason non-specific adsorption, one of result is exactly
Lead to blank background value higher, signal to noise ratio is low, thus have impact on the quantitative sensitivity of whole test kit.
Enzyme linked immunoassay is a kind of very high reaction of sensitivity, if serum, blood plasma or other substrate sample do not dilute,
Inevitably produce very strong nonspecific reaction, false positive occurs, therefore, because the complexity of sample to be tested mechanism also may be used
Matrix effect, interference detection results can be caused.
Therefore, a kind of background value that reduces of technical field of immunoassay urgent need is higher, improves signal to noise ratio, increases sensitivity, disappear
Except disturbing produced by matrix effect, make antigen standard and the Sample Dilution of the more reliable and more stable ELISA of testing result
The preparation method of liquid.
Content of the invention
The invention provides the preparation method of a kind of antigen standard of ELISA and Sample dilution, technical scheme is such as
Under：
The preparation method of a kind of antigen standard of ELISA and Sample dilution is it is characterised in that comprise to walk as follows
Suddenly：
The first step, prepares antibody and is coated；
The Brain Derived Neurotrophic Factor BDNF antibody of capture is added in carbonate buffer solution, and is added into
In the hole of elisa plate, it is labeled as BNDF plate, be placed at a temperature of 2-8 DEG C and be coated 16-24 hour；
Further, prolactin antagonist Prolactin antibody is added in another group of carbonate buffer solution, and be added into
In the hole of elisa plate, it is labeled as Prolaction plate, be placed at a temperature of 2-8 DEG C and be coated 16-24 hour；
Second step, after BDNF plate in the first step and Prolaction plate being carried out respectively pour into a mould, close, wash, drying, then
Save stand-by；
By the phosphate buffer PBS containing 5% bovine serum albumin BSA for 7.4 ± 0.2 for the pH value in the first step
BNDF plate and Prolaction plate are poured into a mould respectively；Or by pH value be 7.4 ± 0.2 containing 5-10% defatted milk powder to the first step
In BNDF plate and Prolaction plate pour into a mould respectively；
Further, this is poured the BDNF plate being poured in and Prolaction plate is individually placed to closing at a temperature of 2-8 DEG C and puts
Put 16-24 hour；
Further, with the PBS solution containing nonionic surfactant Tween20 to the BDNF plate closed and
Prolaction plate carries out washing, dry after, be placed at a temperature of 2-8 DEG C preserve stand-by；
3rd step, prepares standard dilution, as blank solution；
First 0.05% polyoxyethylene sorbitol mono laurate monoester Tween 20 is added in the PBS containing 5%BSA,
And stir；
Further, 15/1000000ths liquid preservative Proclin, conduct after stirring are added in this solution
Standard dilution, i.e. blank solution；
4th step, prepares Sample dilution；
Sample dilution 1：First 0.05%Tween 20 is added in the PBS containing 5%BSA, and stir；Enter
One step ground, adds 0.2% surfactant 3- [3- (gallbladder acyl aminopropyl) dimethylamino] propane sulfonic acid inner salt CHAPS in this solution
Stir；Further, 15/1000000ths Proclin is added to stir in this solution；
Sample dilution 2：First 0.05%Tween 20 is added in the PBS containing 5%BSA, and stir；Enter
One step ground, adds 0.2%CHAPS to stir in this solution；Further, add the chelating of 5mmol/l in this solution
Agent edta edta stirs；Further, 15/1000000ths Proclin is added to stir all in this solution
Even；
Sample dilution 3：First 0.05%Tween 20 is added in the PBS containing 5%BSA, and stir；Enter
One step ground, adds 0.2%CHAPS to stir in this solution；Further, add the EDTA of 5mmol/l in this solution
Stir；Further, add 0.05% Ox blood serum gamma globulin BGG in this solution；Further, in this solution
The Proclin adding 15/1000000ths stirs；
Sample dilution 4：First 0.05%Tween 20 is added in the PBS containing 5%BSA, and stir；Enter
One step ground, adds 0.2%CHAPS to stir in this solution；Further, add the EDTA of 5mmol/l in this solution
Stir；Further, add 0.10%BGG in this solution；Further, 15/1000000ths are added in this solution
Proclin stir；
Sample dilution 5：First 0.05%Tween 20 is added in the PBS containing 5%BSA, and stir；Enter
One step ground, adds 0.2%CHAPS to stir in this solution；Further, add the EDTA of 5mmol/l in this solution
Stir；Further, add 0.15%BGG in this solution；Further, 15/1000000ths are added in this solution
Proclin stir；
Sample dilution 6：First 0.05%Tween 20 is added in the PBS containing 5%BSA, and stir；Enter
One step ground, adds 0.2%CHAPS to stir in this solution；Further, add the EDTA of 5mmol/l in this solution
Stir；Further, add 0.20%BGG in this solution；Further, 15/1000000ths are added in this solution
Proclin stir；
Sample dilution 7：First 0.05%Tween 20 is added in the PBS containing 5%BSA, and stir；Enter
One step ground, adds 0.2%CHAPS to stir in this solution；Further, add the EDTA of 5mmol/l in this solution
Stir；Further, add 0.25%BGG in this solution；Further, 15/1000000ths are added in this solution
Proclin stir；
5th step, BDNF plate and Prolaction plate are put into incubation antibody in incubation oscillator；
Respectively by the standard dilution in the 3rd step, the Sample dilution 1-7 in the 4th step, a normal human serum sample
This, in the hole of the stand-by BDNF plate after drying in second step of being separately added into of repetition and Prolaction plate, then to each reaction
Treatment enzyme is added in hole；
Further, the BDNF plate and Prolaction plate that add good solution are put in incubation oscillator, at 18-25 DEG C
React under room temperature condition；
6th step, adds detection antibody；
With the PBS containing Tween20 as washing liquid, to reacted BDNF plate in the 5th step and Prolaction plate difference
Washing, then add BDNF detection antibody in each reacting hole of BDNF plate accordingly, and accordingly to Prolaction plate
Each reacting hole in add Prolaction detection antibody；
7th step, diluent is added in test kit；
With the PBS containing Tween20 as washing liquid, to reacted BDNF plate in the 6th step and Prolaction plate difference
After washing, it is diluted, then adds the solution after dilution respectively in each hole in test kit SA-HRP；
8th step, is placed on reading optical density OD value under microplate reader；
With the PBS containing Tween20 as washing liquid, reacted test kit SA-HRP in the 7th step is washed respectively, then
Tetramethyl benzidine tmb substrate is added in the test kit SA-HRP that this washs；
Further, the test kit SA-HRP adding good TMB is placed on reading OD value under microplate reader；
9th step, calculates non-specific adsorption data according to OD value, by comparing non-specific adsorption data, further
Determine that Ox blood serum gamma Globulin BGG has direct and positive effect as antigen to reducing background noise.
First, the standard dilution in the 3rd step, the Sample dilution 1-7 in the 4th step and a normal human serum sample
This, the signal results of measured non-specific adsorption NSB on BDNF plate；
Further, the standard dilution in the 3rd step, the Sample dilution 1-7 in the 4th step and a normal human serum
Sample, the signal results of measured non-specific adsorption NSB on Prolactin plate；
Further, find to add the Sample dilution 3-7 of Ox blood serum gamma Globulin BGG in diluent, wrap at two kinds
By the elisa plate of different antibodies, i.e. BNDF plate, the non-specific adsorption NSB all very littles in Prolactin plate, thus illustrate institute
Reasons for its use value is all very low；
Further, because the BGG concentration in Sample dilution 3-7 is different, but all reduce the back of the body in ELISA reaction
Scape signal；
Further, illustrate the Ox blood serum gamma Globulin BGG antigen of any concentration to reduce background noise have directly and
Positive effect.
The preparation method of a kind of antigen standard of ELISA and Sample dilution, also comprises as above：Tenth
Step, using the Sample dilution 6 in the 4th step as new standard dilution, only the content of CHAPS is adjusted to 0.1% respectively,
0.15%th, 0.25%, 0.3%, 0.45%, 0.5%, 0.6%, thus forming 7 kinds of new Sample dilution 8-14, other steps
Processed to eight steps fully according to the first step, read OD value, calculate non-specific adsorption coefficient further；And to recording
Non-specific adsorption coefficient is analyzed, and draws in the case that Ox blood serum gamma Globulin BGG content is certain, CHAPS concentration position
When between 0.2%-0.45%, non-specific adsorption NSB of BDNF plate and Prolactin plate all can reduce, so that background
Signal reduces.
The preparation method of a kind of antigen standard of ELISA and Sample dilution, also comprises as above：11st
Step, selects three kinds of signal to noise ratio height from the 4th step and the tenth step, and three kinds of low Sample dilution of non-specific signals are drawing
The response rate standard curve of BDNF and prolactin simultaneously calculates the response rate of dilute sample；Demonstrate to containing according to test result
Have and in the PBS of 5%BSA, be separately added into 0.05%Tween 20, the EDTA of 0.2-0.45%CHAPS, 5mmol/l, any concentration
Ox blood serum gamma Globulin BGG, 15/1000000ths Proclin as diluent, being that a kind of effect is extraordinary is applied to
The Sample dilution of ELISA, concretely comprises the following steps：
First, respectively BDNF and Prolactin standard antigen is added in BDNF and prolactin negative serum；
Further, it is separately added into Sample dilution 3, Sample dilution 10, Sample Dilution in this negative serum sample
In liquid 12, other steps are completely the same to the 8th step with the 5th step, read optical density OD value and the response rate；
Further, two kinds of Sample dilution 3 and Sample dilution 12 are 2 that CHAPS is 0.2% and 0.45% respectively
Critical point, our experiments show that the standard curve of BDNF and Prolactin that three kinds of Sample dilution 3,10,12 are prepared respectively mutually
Parallel, dependency is fine, and the response rate improves compared with negative serum；
Further it was demonstrated that being separately added into 0.05%Tween in the PBS containing 5%BSA being 7.4 ± 0.2 to pH value
20th, the EDTA of 0.2-0.45%CHAPS, 5mmol/l, the Ox blood serum gamma Globulin BGG of any concentration, 15/1000000ths
Proclin, as diluent, is a kind of extraordinary Sample dilution being applied to ELISA of effect.
The invention has the beneficial effects as follows：
1. present invention Ox blood serum gamma Globulin BGG is as antigen, thus playing the effect reducing background noise.
2. present invention determine that in the case that Ox blood serum gamma Globulin BGG content is certain, CHAPS concentration is located at 0.2%-
When between 0.45%, non-specific adsorption NSB of BDNF plate and Prolactin plate all can reduce, and reduces background signal.
3., the invention provides a kind of extraordinary antigen standard being applied to ELISA and Sample dilution, compensate for
The blank of industry, advance science progress.
Brief description
To describe the present invention with reference to the accompanying drawings and detailed description in detail：
Fig. 1 is response rate standard curve in three kinds of diluents for the BDNF of the present invention.
Fig. 2 is response rate standard curve in three kinds of diluents for the Prolactin of the present invention.
Specific embodiment
In order that the technological means of the present invention, creation characteristic, reached purpose and effect are easy to understand, with reference to tool
Body illustrates, and the present invention is expanded on further.
The invention provides the preparation method of a kind of antigen standard of ELISA and Sample dilution, concrete steps are such as
Under：
The first step, prepares antibody and is coated；
The Brain Derived Neurotrophic Factor BDNF antibody of capture is added the 0.05mol/l carbonate buffer that pH value is 9.6
In liquid, it is configured to the solution that concentration is 2 μ g/ml, and is added in the hole of elisa plate, be labeled as BNDF plate, be placed in 2-8
It is coated 16-24 hour at a temperature of DEG C；
Further, the 0.05mol/l carbonate that prolactin antagonist Prolactin antibody another group of pH value of addition is 9.6 is delayed
Rush in liquid, be configured to the solution that concentration is 2.5 μ g/ml, and be added in the hole of elisa plate, be labeled as Prolaction
Plate, is placed at a temperature of 2-8 DEG C and is coated 16-24 hour；
Second step, after BDNF plate in the first step and Prolaction plate being carried out respectively pour into a mould, close, wash, drying, then
Save stand-by；
By the phosphate buffer PBS containing 5% bovine serum albumin BSA for 7.4 ± 0.2 for the pH value in the first step
BNDF plate and Prolaction plate are poured into a mould respectively, pour into a mould 300 μ l in each hole；Or by pH value be 7.4 ± 0.2 contain 5-
10% defatted milk powder is poured into a mould respectively to the BNDF plate in the first step and Prolaction plate, pours into a mould 300 μ l in each hole；
Further, this is poured the BDNF plate being poured in and Prolaction plate is individually placed to closing at a temperature of 2-8 DEG C and puts
Put 16-24 hour；
Further, with the PBS solution containing 0.05% nonionic surfactant Tween to the BDNF plate closed
After carrying out with Prolaction plate washing, drying, be placed at a temperature of 2-8 DEG C preserve stand-by；
3rd step, prepares standard dilution, as blank solution；
First 0.05% polyoxyethylene sorbitol mono laurate monoester Tween 20 is added in the PBS containing 5%BSA,
And stir；
Further, 15/1000000ths liquid preservative Proclin, conduct after stirring are added in this solution
Standard dilution, i.e. blank solution；
4th step, prepares Sample dilution；
Sample dilution 1：First 0.05%Tween 20 is added in the PBS containing 5%BSA, and stir；Enter
One step ground, adds 0.2% surfactant 3- [3- (gallbladder acyl aminopropyl) dimethylamino] propane sulfonic acid inner salt CHAPS in this solution
Stir；Further, 15/1000000ths Proclin is added to stir in this solution；
Sample dilution 2：First 0.05%Tween 20 is added in the PBS containing 5%BSA, and stir；Enter
One step ground, adds 0.2%CHAPS to stir in this solution；Further, add the chelating of 5mmol/l in this solution
Agent edta edta stirs；Further, 15/1000000ths Proclin is added to stir all in this solution
Even；
Sample dilution 3：First 0.05%Tween 20 is added in the PBS containing 5%BSA, and stir；Enter
One step ground, adds 0.2%CHAPS to stir in this solution；Further, add the EDTA of 5mmol/l in this solution
Stir；Further, add 0.05% Ox blood serum gamma globulin BGG in this solution；Further, in this solution
The Proclin adding 15/1000000ths stirs；
Sample dilution 4：First 0.05%Tween 20 is added in the PBS containing 5%BSA, and stir；Enter
One step ground, adds 0.2%CHAPS to stir in this solution；Further, add the EDTA of 5mmol/l in this solution
Stir；Further, add 0.10%BGG in this solution；Further, 15/1000000ths are added in this solution
Proclin stir；
Sample dilution 5：First 0.05%Tween 20 is added in the PBS containing 5%BSA, and stir；Enter
One step ground, adds 0.2%CHAPS to stir in this solution；Further, add the EDTA of 5mmol/l in this solution
Stir；Further, add 0.15%BGG in this solution；Further, 15/1000000ths are added in this solution
Proclin stir；
Sample dilution 6：First 0.05%Tween 20 is added in the PBS containing 5%BSA, and stir；Enter
One step ground, adds 0.2%CHAPS to stir in this solution；Further, add the EDTA of 5mmol/l in this solution
Stir；Further, add 0.20%BGG in this solution；Further, 15/1000000ths are added in this solution
Proclin stir；
Sample dilution 7：First 0.05%Tween 20 is added in the PBS containing 5%BSA, and stir；Enter
One step ground, adds 0.2%CHAPS to stir in this solution；Further, add the EDTA of 5mmol/l in this solution
Stir；Further, add 0.25%BGG in this solution；Further, 15/1000000ths are added in this solution
Proclin stir；
5th step, BDNF plate and Prolaction plate are put into incubation antibody in incubation oscillator；
Respectively by the standard dilution in the 3rd step, the Sample dilution 1-7 in the 4th step, a normal human serum sample
This, repeat to be separately added in the hole drying rear stand-by BDNF plate and Prolaction plate in second step with 10, then to each
100 μ l treatment enzyme are added in reacting hole,
Further, the BDNF plate and Prolaction plate that add good solution are put in incubation oscillator, with 500 revs/min
Speed react 2 hours under 18-25 DEG C of room temperature condition.
6th step, adds detection antibody；
With the PBS containing 0.05%Tween20 as washing liquid, to reacted BDNF plate and Prolaction in the 5th step
Plate washs 4 times respectively, then adds the BDNF detection antibody of 100 μ l in each reacting hole of BDNF plate accordingly, and accordingly
Each reacting hole to Prolaction plate in add the Prolaction detection antibody of 100 μ l；
7th step, diluent is added in test kit；
With the PBS containing 0.05%Tween20 as washing liquid, to reacted BDNF plate and Prolaction in the 6th step
Plate washs 4 times respectively, then with 1:10000 pairs its be diluted after, respectively in each hole in test kit SA-HRP add 100
Solution after the dilution of μ l；
8th step, is placed on reading optical density OD value under microplate reader；
With the PBS containing 0.05%Tween20 as washing liquid, reacted test kit SA-HRP in the 7th step is washed respectively
Wash 4 times, then in the test kit SA-HRP washing to this, add tetramethyl benzidine tmb substrate；
Further, the test kit SA-HRP adding good TMB is placed on reading OD value under the microplate reader that wavelength is 450nm；
9th step, calculates non-specific adsorption data according to OD value, by comparing non-specific adsorption data, further
Determine that Ox blood serum gamma Globulin BGG has direct and positive effect as antigen to reducing background noise；
First, the standard dilution in the 3rd step, the Sample dilution 1-7 in the 4th step, a normal human serum sample,
The signal results of measured non-specific adsorption NSB on BDNF plate, such as table one：
Table one
Further, the standard dilution in the 3rd step, the Sample dilution 1-7 in the 4th step, a normal human serum
Sample, the signal results of measured non-specific adsorption NSB on Prolactin plate, such as table two：
Table two
It is analyzed according to the result in above-mentioned table one and table two, find to add Ox blood serum gamma Globulin in diluent
The Sample dilution 3-7 of BGG, is coated the elisa plate of different antibodies at two kinds, i.e. BNDF plate, non-specific in Prolactin plate
Property absorption NSB all very littles, thus explanation produced by background value all very low, be between 0.02-0.05；
Further, because the BGG concentration in Sample dilution 3-7 is different, but all reduce the back of the body in ELISA reaction
Scape signal；
Further, illustrate that Ox blood serum gamma Globulin BGG antigen has direct and positive effect to reducing background noise；
Tenth step, using the Sample dilution 6 in the 4th step as new standard dilution, only by the content difference of CHAPS
It is adjusted to 0.1%, 0.15%, 0.25%, 0.3%, 0.45%, 0.5%, 0.6%, thus forming 7 kinds of new Sample dilution
8-14.Other steps are processed to eight steps fully according to the first step, read OD value；
11st step, calculates the value of non-specific adsorption NSB according to OD value, thus obtaining in Ox blood serum gamma Globulin
In the case that BGG content is certain, when CHAPS concentration is located between 0.2%-0.45%, the non-spy of BDNF plate and Prolactin plate
Opposite sex absorption NSB can reduce, so that the reduction of background signal；
Sample dilution 6 in tenth step, Sample dilution 8-14 and a normal human serum sample institute on BDNF plate
The signal results of non-specific adsorption NSB recording, such 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, such as table four：
Table four
EDTA, Tween20 are all additive commonly used in the art, but the additive that CHAPS is not everybody to be commonly used.Cause
This, be analyzed according to the result in above-mentioned table three and table four, finds in the certain situation of Ox blood serum gamma Globulin BGG content
Under, when CHAPS concentration is located between 0.2%-0.45%, non-specific adsorption NSB of BDNF plate and Prolactin plate all can drop
Low, so that the reduction of background signal；
12nd step, selects three kinds of signal to noise ratio height from the 4th step and the tenth step, three kinds of low samples of non-specific signals
This diluent is drawing the response rate standard curve of BDNF and prolactin, and calculates the response rate of dilute sample；Fig. 1 is this
Invention response rate standard curve in three kinds of diluents for the BDNF, Fig. 2 is Prolactin of the present invention returning in three kinds of diluents
Yield standard curve；The response rate according to recording demonstrate be separately added into in the PBS containing 5%BSA 0.05%Tween 20,
The EDTA of 0.2-0.45%CHAPS, 5mmol/l, the Ox blood serum gamma globulin BGG of any concentration, 15/1000000ths
Proclin, as diluent, is a kind of extraordinary Sample dilution being applied to ELISA of effect.
First, respectively the BDNF and Prolactin standard antigen of 4000pg/ml is added BDNF and prolactin negative
In serum；
Further, respectively with 1:2,1:4,1:8 pairs of negative serums preparing are diluted, and are separately added into sample after dilution
In this diluent 3, Sample dilution 10, Sample dilution 12, other steps are completely the same to the 8th step with the 5th step, read light
Density OD value, further calculates the response rate, and draws standard curve according to the response rate, and Fig. 1 is BDNF of the present invention three
Plant the response rate standard curve in diluent, Fig. 2 is that response rate standard in three kinds of diluents for the Prolactin of the present invention is bent
Line；
The optical density OD value of the prepared BDNF standard curve of three kinds of Sample dilution and the response rate, such as table five：
Table five
The optical density OD value of the prepared Prolactin standard curve of three kinds of Sample dilution and the response rate, such as table six：
Table six
Further, using negative serum sample as comparison, the average recovery rate of comparative sample diluent；
Average recovery rate in three kinds of Sample dilution for the BDNF, such as table seven：
Table seven
Average recovery rate in three kinds of Sample dilution for the prolactin, such as table eight：
Table eight
According to the analysis to table five, six, seven, eight, two kinds of Sample dilution 3 and Sample dilution 12 are that CHAPS is respectively
0.2% and 0.45% 2 critical points；Our experiments show that BDNF and Prolactin that three kinds of Sample dilution 3,10,12 are prepared
Standard curve respectively parallel to each other, dependency is fine；Diluted respectively with these three Sample dilution containing BDNF and
The serum sample of Prolactin, compared with negative serum sample, its response rate brings up to 85-115%, the response rate from 65-77%
Have and greatly improve, result precision improves, thus meeting the requirement of detection；
Further it was demonstrated that being separately added into 0.05%Tween in the PBS containing 5%BSA being 7.4 ± 0.2 to pH value
20th, the EDTA of 0.2-0.45%CHAPS, 5mmol/l, the Ox blood serum gamma Globulin BGG of any concentration, 15/1000000ths
Proclin, as diluent, is a kind of extraordinary Sample dilution being applied to ELISA of effect.
Ox blood serum gamma Globulin BGG is as antigen, thus playing the effect reducing background noise for the present invention.
Present invention determine that in the case that Ox blood serum gamma Globulin BGG content is certain, CHAPS concentration is located at 0.2%-
When between 0.45%, non-specific adsorption NSB of BDNF plate and Prolactin plate all can reduce, and reduces background signal.
The invention provides a kind of extraordinary antigen standard being applied to ELISA and Sample dilution, compensate for going
The blank of industry, advance science progress.
Ultimate principle, principal character and the advantages of the present invention of the present invention have been shown and described above.The technology of the industry
, it should be appreciated that the present invention is not restricted to the described embodiments, the simply explanation described in above-described embodiment and description is originally for personnel
Invention principle, without departing from the spirit and scope of the present invention the present invention also have various changes and modifications, these change
Change and improvement both falls within scope of the claimed invention.Claimed scope by appending claims and its
Equivalent defines.
Claims (3)
1. the preparation method of a kind of antigen standard of ELISA and Sample dilution is it is characterised in that comprise the steps of：
The first step, prepares antibody and is coated；
The Brain Derived Neurotrophic Factor antibody of capture is added in carbonate buffer solution, and is added into the hole of elisa plate
In, it is labeled as BNDF plate, be placed at a temperature of 2-8 DEG C and be coated 16-24 hour；
Further, prolactin antagonist antibody is added in another group of carbonate buffer solution, and is added in the hole of elisa plate,
It is labeled as prolactin antagonist plate, be placed at a temperature of 2-8 DEG C and be coated 16-24 hour；
Second step, the Brain Derived Neurotrophic Factor plate in the first step and prolactin antagonist plate carried out respectively pour into a mould, close, wash,
After drying, then save stand-by；
The phosphate buffer containing 5% bovine serum albumin that pH value is 7.4 ± 0.2 is neural to the brain source property in the first step
Trophic factors plate and prolactin antagonist plate are poured into a mould respectively；Or by pH value be 7.4 ± 0.2 containing 5-10% defatted milk powder to the first step
In Brain Derived Neurotrophic Factor plate and prolactin antagonist plate pour into a mould respectively；
Further, this is poured the Brain Derived Neurotrophic Factor plate being poured in and prolactin antagonist plate is individually placed at a temperature of 2-8 DEG C
16-24 hour is placed in closing；
Further, with the PBS solution containing nonionic surfactant to the Brain Derived Neurotrophic Factor plate closed
After carrying out with prolactin antagonist plate washing, drying, be placed at a temperature of 2-8 DEG C preserve stand-by；
3rd step, prepares standard dilution, as blank solution；
First 0.05% polyoxyethylene sorbitol mono laurate monoester is added the phosphate-buffered containing 5% bovine serum albumin
In liquid, and stir；
Further, add 15/1000000ths liquid preservative in this solution, as standard dilution after stirring,
I.e. blank solution；
4th step, prepares Sample dilution；
Sample dilution 1：First 0.05% polyoxyethylene sorbitol mono laurate monoester is added and contain 5% bovine serum albumin
Phosphate buffer in, and stir；Further, add 0.2% surfactant 3- [3- (gallbladder in this solution
Acyl aminopropyl) dimethylamino] propane sulfonic acid inner salt stirs；Further, add 15/1000000ths liquid in this solution
Body preservative stirs；
Sample dilution 2：First 0.05% polyoxyethylene sorbitol mono laurate monoester is added and contain 5% bovine serum albumin
Phosphate buffer in, and stir；Further, add 0.2% 3- [3- (gallbladder acyl aminopropyl) in this solution
Dimethylamino] propane sulfonic acid inner salt stirs；Further, add the chelating agen ethylenediamine tetrem of 5mmol/l in this solution
Acid stirs；Further, 15/1000000ths liquid preservative is added to stir in this solution；
Sample dilution 3：First 0.05% polyoxyethylene sorbitol mono laurate monoester is added and contain 5% bovine serum albumin
Phosphate buffer in, and stir；Further, add 0.2% 3- [3- (gallbladder acyl aminopropyl) in this solution
Dimethylamino] propane sulfonic acid inner salt stirs；Further, add the ethylenediaminetetraacetic acid stirring of 5mmol/l in this solution
Uniformly；Further, add 0.05% Ox blood serum gamma Globulin in this solution；Further, million are added in this solution
/ ten five liquid preservative stirs；
Sample dilution 4：First 0.05% polyoxyethylene sorbitol mono laurate monoester is added and contain 5% bovine serum albumin
Phosphate buffer in, and stir；Further, add 0.2% 3- [3- (gallbladder acyl aminopropyl) in this solution
Dimethylamino] propane sulfonic acid inner salt stirs；Further, add the ethylenediaminetetraacetic acid stirring of 5mmol/l in this solution
Uniformly；Further, add 0.10% Ox blood serum gamma Globulin in this solution；Further, million are added in this solution
/ ten five liquid preservative stirs；
Sample dilution 5：First 0.05% polyoxyethylene sorbitol mono laurate monoester is added and contain 5% bovine serum albumin
Phosphate buffer in, and stir；Further, add 0.2% 3- [3- (gallbladder acyl aminopropyl) in this solution
Dimethylamino] propane sulfonic acid inner salt stirs；Further, add the ethylenediaminetetraacetic acid stirring of 5mmol/l in this solution
Uniformly；Further, add 0.15% Ox blood serum gamma Globulin in this solution；Further, million are added in this solution
/ ten five liquid preservative stirs；
Sample dilution 6：First 0.05% polyoxyethylene sorbitol mono laurate monoester is added and contain 5% bovine serum albumin
Phosphate buffer in, and stir；Further, add 0.2% 3- [3- (gallbladder acyl aminopropyl) in this solution
Dimethylamino] propane sulfonic acid inner salt stirs；Further, add the ethylenediaminetetraacetic acid stirring of 5mmol/l in this solution
Uniformly；Further, add 0.20% Ox blood serum gamma Globulin in this solution；Further, million are added in this solution
/ ten five liquid preservative stirs；
Sample dilution 7：First 0.05% polyoxyethylene sorbitol mono laurate monoester is added and contain 5% bovine serum albumin
Phosphate buffer in, and stir；Further, add 0.2% 3- [3- (gallbladder acyl aminopropyl) in this solution
Dimethylamino] propane sulfonic acid inner salt stirs；Further, add the ethylenediaminetetraacetic acid stirring of 5mmol/l in this solution
Uniformly；Further, add 0.25% Ox blood serum gamma Globulin in this solution；Further, million are added in this solution
/ ten five liquid preservative stirs；
5th step, Brain Derived Neurotrophic Factor plate and prolactin antagonist plate are put into incubation antibody in incubation oscillator；
Respectively by the standard dilution in the 3rd step, the Sample dilution 1-7 in the 4th step, a normal human serum sample, weight
Multiple is separately added in the hole drying rear stand-by Brain Derived Neurotrophic Factor plate and prolactin antagonist plate in second step, then to each
Treatment enzyme is added in reacting hole,
Further, the Brain Derived Neurotrophic Factor plate and prolactin antagonist plate that add good solution are put in incubation oscillator, in 18-
React under 25 DEG C of room temperature condition；
6th step, adds detection antibody；
With the PBS containing Tween20 as washing liquid, to reacted Brain Derived Neurotrophic Factor plate and prolactin antagonist in the 5th step
Plate washs respectively, then adds Brain Derived Neurotrophic Factor detection antibody in each reacting hole of BDNF plate accordingly, and
The corresponding addition prolactin antagonist detection antibody in each reacting hole of prolactin antagonist plate；
7th step, diluent is added in test kit；
With the PBS containing Tween20 as washing liquid, to reacted Brain Derived Neurotrophic Factor plate and prolactin antagonist in the 6th step
Plate is diluted to it after washing respectively, then adds the solution after dilution respectively in each hole in this test kit；
8th step, is placed on reading optical density value under microplate reader；
With the PBS containing Tween20 as washing liquid, reacted test kit in the 7th step is washed respectively, then washs to this
Test kit in add tetramethyl benzidine substrates；
Further, the test kit adding good TMB is placed on reading optical density value under microplate reader；
9th step, calculates non-specific adsorption data according to optical density value, by comparing non-specific adsorption data, further
Determine that Ox blood serum gamma Globulin has direct and positive effect as antigen to reducing background noise；
First, the standard dilution in the 3rd step, the Sample dilution 1-7 in the 4th step, a normal human serum sample, in brain
The signal results of measured non-specific adsorption NSB on derived neurotrophic factor plate；
Further, the standard dilution in the 3rd step, the Sample dilution 1-7 in the 4th step, a normal human serum sample,
The signal results of measured non-specific adsorption NSB on prolactin antagonist plate；
Further, find to add the Sample dilution 3-7 of Ox blood serum gamma Globulin in diluent, be coated at two kinds different anti-
The elisa plate of body, i.e. non-specific adsorption NSB all very littles in neurotrophic factor plate and prolactin antagonist plate, thus illustrate to be produced
Raw background value is all very low, is between 0.02-0.045；
Further, because the Ox blood serum gamma Globulin concentration in Sample dilution 3-7 is different, but it is anti-all to reduce ELISA
Background signal in answering；
Further, illustrate that Ox blood serum gamma Globulin antigen has direct and positive effect to reducing background noise.
2. the preparation method of a kind of antigen standard of ELISA according to claim 1 and Sample dilution, also wraps
Contain：Tenth step, using the Sample dilution 6 in the 4th step as new standard dilution, only by this 3- [3- (gallbladder acyl aminopropyl)
Dimethylamino] propane sulfonic acid inner salt content be adjusted to 0.1% respectively, 0.15%, 0.25%, 0.3%, 0.45%, 0.5%,
0.6%, thus forming 7 kinds of new Sample dilution 8-14, other steps are processed to eight steps fully according to the first step, read
Optical density value, calculates non-specific adsorption coefficient further；And the non-specific adsorption coefficient recording is analyzed, draw
In the case that Ox blood serum gamma Globulin content is certain, in 3- [3- (gallbladder acyl aminopropyl) dimethylamino] propane sulfonic acid, salinity is located at
When between 0.2%-0.45%, the non-specific adsorption of brain source property neural factor plate and prolactin antagonist plate all can reduce, so that the back of the body
Scape signal reduces.
3. the preparation method of a kind of antigen standard of ELISA according to claim 2 and Sample dilution, also wraps
Contain：11st step, selects three kinds of signal to noise ratio height from the 4th step and the tenth step, three kinds of low Sample Dilution of non-specific signals
Liquid is drawing the response rate standard curve of BDNF and prolactin and to calculate the response rate of dilute sample；Demonstrate,proved according to test result
Understand and be separately added into 0.05% polyoxyethylene sorbitol mono laurate list in the phosphate buffer containing bovine serum albumin
Fat, 3- [3- (gallbladder acyl aminopropyl) dimethylamino] the propane sulfonic acid inner salt of 0.2-0.45%, the ethylenediaminetetraacetic acid of 5mmol/l, appoint
The Ox blood serum gamma Globulin of meaning concentration, 15/1000000ths liquid preservative, as diluent, are that a kind of effect is extraordinary
It is applied to the Sample dilution of ELISA, concretely comprise the following steps：
First, respectively brain source property neural factor and prolactin antagonist standard antigen are added brain source property neural factor and prolactin antagonist feminine gender blood
In clear；
Further, respectively the negative serum preparing is diluted, after dilution, is separately added into three kinds of signal to noise ratio height, non-specific
In the property low Sample dilution of signal 3, Sample dilution 10, Sample dilution 12, other steps are complete to the 8th step with the 5th step
Entirely consistent, read optical density value and the response rate；
Further, Sample dilution 3 and Sample dilution 12 are 3- [3- (gallbladder acyl aminopropyl) dimethylamino] propane sulfonic acid respectively
Inner salt is 0.2% and 0.45% 2 critical points, our experiments show that the brain source property god that three kinds of Sample dilution 3,10,12 are prepared
Standard curve through the factor and prolactin antagonist is parallel to each other respectively, and dependency is fine, and the response rate improves compared with negative serum；
Further it was demonstrated that being separately added in the phosphate buffer containing bovine serum albumin being 7.4 ± 0.2 to pH value
0.05% polyoxyethylene sorbitol mono laurate monoester, 3- [3- (the gallbladder acyl aminopropyl) dimethylamino] propane sulfonic acid of 0.2-0.45%
Inner salt, the ethylenediaminetetraacetic acid of 5mmol/l, the Ox blood serum gamma Globulin of 0.05%-0.2%, 15/1000000ths liquid are prevented
Rotten agent, as diluent, is a kind of extraordinary Sample dilution being applied to ELISA of effect.
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