Protein A Residue Detection Kit

I. self prepared materials:

1, 10-1000ul pipette, single channel and multi-channel

2, 100ml and 1000ml beakers

3, deionized water

4, various types of centrifuge tubes

5, microplate reader (measuring 450nm)

6, data processing and analysis software

II. Reagent preparation:

1. Please place this kit at room temperature (20-25 ℃) before use
2. Preparation of washing solution: add 270ml deionized water to 30ml of washing solution (10x) and mix well to prepare 300ml of working washing solution. If there are crystals in it, please dissolve and mix before use
3. Other reagents can be used directly without dilution

III. sample processing:

1. If the sample needs to be diluted, use the sample diluent in the kit to dilute it, which can be carried out in a 96 well plate or EP tube
2. Ensure that the volume of each hole (tube) containing the sample is 100ul
3. Add 100ul of standard and quality control into the well
4. Add 50ul acidification solution to each well, blow and mix well, and incubate at room temperature for 10min (acidify and separate proteinA and antibody in the sample)

IV. operation steps of the kit:

1. Dilution of standard: mark 8 centrifuge tubes with 1#- 8#, add 990ul of sample diluent to 1#and 2#, and add 500ul of sample diluent to 3#- 8#. Take another 10ul of sample standard and add it to the 1 × tube, vortex and mix it well, then add 10ul to the 2 × tube and vortex and mix it well. Then add 500ul liquid from 2 × tube to 3 × tube for vortex mixing, and then take 500ul liquid from 3 × tube to 4 × tube for vortex mixing until 8 × tube

after the dilution is complete, the concentration of protein a standard in the 2 × 8 × centrifuge tube is 5, 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078ng/ml respectively. Mark another tube as 0ng/ml and add sample diluent. After the standard and sample are ready, enter the sample processing procedure.

1. Add 100ul/ well biotin label to the enzyme plate coated with protein A antibodyOf the binding antibody
2. Add 25ul acidified standard, sample, quality control sample and blank into appropriate wells and incubate at room temperature for 1H
3. After drying and washing for 4 times, 100ul/ well of avidin labeled HRP was added and incubated for 10min at room temperature
4. After drying and washing for 4 times, add 100ul/ well TMB color developing solution for 10min, add 100ul/ well stop solution, and then detect the absorbance at 450nm

v. data processing and result analysis:

use software (such as curve expert or ELISA Calc) to establish the detection standard curve through the concentration gradient of the standard, and use 4-parameter logical fitting or cubic polynomial linear fitting to synthesize the corresponding relationship between the OD value and the concentration of the standard, Then the OD value of the test sample is brought into the corresponding curve equation to calculate the corresponding sample concentration

VI. indicators of analytical method:

1. Precision: when the sample concentration is greater than 0.3ng/ml, the coefficient of variation of detection is less than 10%; When the sample concentration is less than 0.3ng/ml, the detection coefficient of variation is slightly greater than 10%
2. It is suggested that when testing specific samples, the method of adding quality control materials should be used to investigate the influence of sample matrix effect on the test results
3. The sensitivity of this kit is 0.05ng/ml
4. Hook effect: this kit is a double antibody sandwich one-step method, and the concentration of hook effect is 10ug/ml.

This protein a detection ELISA kit is used to quantitatively detect non natural protein a such as recombinant alkali resistance. It can only be used in scientific research and production, and cannot be applied to the diagnosis and treatment of human or animal diseases. This kit provides a sample acidification method to separate protein A from IgG products. When the concentration of humanized monoclonal antibody is up to 2mg/ml, the detection limit of protein A is lower than 78pg/ml

product components:

1. Protein a standard

recombinant alkali tolerant protein A in a protein matrix with conservative, 50ug/ml, 1x01ml

1. Biotinylated anti protein a monoclonal antibody in a protein matrix with preserved, 1x13ml
2. Sample diluent

tris buffered saline with a protein matrix andpreserved, 1x30ml

4Denaturing buffer (acidification buffer)

citrate buffer with reagent and preserved, 1x20ml

5, 10x assay buffer (10x)

washing buffer with preserved, 1x30ml

6, streptavidin labeled HRP in a protein matrix withprservative, 1x13ml

7TMB substrate

3,3’, 5,5’ Tetramethylbenzidine, 1x13ml

8, stop solution

2m sulphuric acid, 1x13ml

9, monoclonal anti protein a coated plate of 96 wells

12x8 well strips in a bag with destructive