Instruction manual for human blocking antibody (BA) elisa kit This kit is for research use only.
Detection range: 96T
4ng / L-120ng / L
purpose of usage:
This kit is used to determine the content of blocking antibody (BA) in human serum, plasma and related liquid samples.
Experimental principle The kit uses the double antigen sandwich method to determine the level of blocking antibody (BA) in the specimen. The microplate is coated with purified antigen to prepare a solid-phase antigen, and blocking antibody (BA) is added to the microwells coated with mAb in sequence, and then combined with the HRP-labeled antigen to form an antigen-antibody-enzyme-labeled antigen complex After thorough washing, add substrate TMB to develop color. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with the blocking antibody (BA) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human blocking antibody (BA) in the sample was calculated by a standard curve.
Kit composition
1 30 times concentrated washing solution 20ml × 1 bottle 7 stop solution 6ml × 1 bottle
2 Enzyme label reagent 6ml × 1 bottle 8 standard (240ng / L) 0.5ml × 1 bottle
3 Enzyme label coating plate 12 wells × 8 strips 9 Standard diluent 1.5ml × 1 bottle
4 Sample diluent 6ml × 1 bottle 10 Instructions 1 copy
5 Developer A solution 6ml × 1 bottle 11 sealing film 2 sheets
6 Developer B liquid 6ml × 1 / bottle 12 sealed bag 1 specimen requirement
1. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided
2. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
Steps
1. Dilution of standard products: This kit provides one original standard product. The user can perform dilution in a small test tube according to the following chart.
120ng / L No. 5 standard, 150μl original standard, add 150μl standard dilution
60ng / L No. 4 standard 150μl No. 5 standard added 150μl standard dilution
30ng / L No. 3 standard 150μl No. 4 standard added 150μl standard dilution
15ng / L No. 2 standard 150μl No. 3 standard added 150μl standard dilution
7.5ng / L No. 1 standard 150μl No. 2 standard added 150μl standard dilution
2. Sample addition: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the operation is the same), standard wells, sample wells to be tested. Accurately add 50μl of the standard on the enzyme-coated plate, add 40μl of sample diluent to the sample well, and then add 10μl of the sample to be tested (the final dilution of the sample is 5 times) Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix.
3. Incubation: Seal the plate with a sealing plate and incubate at 37 ° C for 30 minutes.
4. Mixing solution: Dilute 30 times concentrated washing liquid with distilled water 30 times and reserve
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds and then discard, repeat this 5 times, pat dry.
6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 15 minutes.
10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.
Summary of operating procedures:
Calculate the standard concentration as the abscissa and the OD value as the ordinate. Draw a standard curve on the coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample; multiply by the dilution factor; or use the standard The linear regression equation of the standard curve is calculated from the concentration and the OD value, the OD value of the sample is substituted into the equation, the sample concentration is calculated, and then multiplied by the dilution factor, which is the actual concentration of the sample.
Precautions
1. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme label coated plate is unopened, the strip should be stored in a sealed bag.
2. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.
3. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.
4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the test substance in the specimen is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please first dilute it with a certain multiple (n times) of the sample diluent and then determine it. When calculating, please multiply the total dilution Multiple (× n × 5).
5. The sealing film is limited to one-time use to avoid cross-contamination.
6. Please keep the substrate away from light.
7. Strictly follow the instructions, and the test results must be determined by the microplate reader.
8. All samples, washing liquids and various wastes should be treated as infectious agents.
9. The components of different batches of this reagent shall not be mixed.
10. If there is any difference with the English manual, the English manual shall prevail.
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months
Human blocking antibody (BA)
FOR RESEARCH USE ONLY
Assay range: 4ng / L-120ng / L 96 determinations
Purpose
This kit allows for the determination of BA concentrations in Human serum, cell culture supernates and other biological fluids
Principle of the assay
The kit assay Human BA level in the sample, use Purified antigen to coat microtiter plate wells, make solid-phase antigen, then add BA to wells, Combined antigen which With HRP labeled, become antigen – antibody-enzyme- antigen complex, after washing Completely, Add TMB substrate solution, TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Human BA in the samples is then determined by comparing the OD of the samples to the standard curve.
Materials provided with the kit
1 wash solution 20ml × 1bottle 7 Stopp Solution 6ml × 1 bottle
2 HRP-Conjugate reagent 6ml × 1 bottle 8 Standard (240ng / L) 0.5ml × 1 bottle
3 Microelisa stripplate 12well × 8strips 9 Standard diluent 1.5ml × 1bottle
4 Sample diluent 6ml × 1 bottle 10 Instruction 1
5 Chromogen Solution A 6ml × 1 bottle 11 Closure plate membrane 2
6 Chromogen Solution B 6ml × 1 bottle 12 Sealed bags 1
Specimen requirements
1. extract as soon as possible after Specimen collection, and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can't, specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze -thaw cycles.
2. Can't detect the sample which contain NaN3, because NaN3 inhibits HRP active.
Assay procedure
1. Dilute and add sample: Dilute Original density Standard as follow table:
120ng / L 5 Standard 150μl Original density Standard + 150μl Standard diluent
60ng / L 4 Standard 150μl 5 Standard + 150μl Standard diluent
30ng / L 3 Standard 150μl 4 Standard + 150μl Standard diluent
15ng / L 2 Standard 150μl 3 Standard + 150μl Standard diluent
7.5ng / L 1 Standard 150μl 2 Standard + 150μl Standard diluent
2.add sample: Set blank wells separately (blank comparison wells don't add sample and HRP-Conjugate reagent, other each step operation is same). Testing sample well. Add Sample dilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is 5-fold), add sample to wells, don't touch the well wall as far as possible, and Gently mix.
3.Incubate: After closing plate with Closure plate membrane, incubate for 30 min at 37 ℃.
4.Configurate liquid: 30-fold (or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.
5.washing: Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.
6.add enzyme: Add HRP-Conjugate reagent 50μl to each well, except blank well.
7.incubate: Operation with 3.
8.washing: Operation with 5.
9.color: Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37 ℃
10.Stop the reaction: Add Stop Solution50μl to each well, Stop the reaction (the blue color change to yellow color).
11.assay: take blank well as zero, Read absorbance at 450nm after Adding Stop Solution and within 15min.
Steps description
Standard, Sample diluent
Add Standard, Sample diluent, incubate for 30 min at 37 ℃.
Wash 5 time, Add HRP-Conjugate reagent, incubate for 30 min at 37 ℃.
Wash 5 times, Add Chromogen Solution A and B, incubate for 30 min at 37 ℃.
Add Stopp Solution
Read absorbance at 450nm within 15 min
calculate
Calculate
Take the standard density as the horizontal, the OD value for the vertical, draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value, with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.
Important notes
1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.
2. washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute. Washing does not affect the result.
3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. Add sample within 5 min, if the number of sample is much, recommend to use Volley.
4. if the testing material content is excessively higher (The sample OD is bigger than the first standard well), please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor. (× n × 5).
5. Closure plate membrane only limits the Disposable use, to avoid cross-contamination.
6. The substrate evade the light preservation.
7. Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.
8. All samples, washing buffer and each kind of reject should according to infective material process.
9. Do not mix reagents with those from other lots.
Storage and validity
1. Storage: 2-8 ℃.
2. validity: six months
Henan Hanchen Medical Technology Co., LTD , https://www.tchanchen.com