I. Scope of Application
This protein A detection ELISA kit is used for the quantitative detection of recombinant alkali-resistant and other non-natural protein A. It can only be used for scientific research and production and should not be applied to the diagnosis and treatment of human or animal diseases. This kit provides an acidification treatment method for samples to separate protein A from IgG products. When the concentration of humanized monoclonal antibodies is as high as 2mg/mL, the detection limit of protein A is as low as below 78pg/mL.
 
Ii. Detection Principle
This kit adopts the technical method of double-antibody sandwich labeled with biotin system. The specific monoclonal antibody of protein A is pre-coated on the microplate. The sample containing protein A is diluted with the sample diluent and then mixed with the acidification solution to separate protein A from the antibody product. The sample reacts with the protein A capture antibody pre-coated on the microplate. Then, biotin-labeled detection antibodies are added to react and form a sandwich complex. Then, wash to remove the substances that did not participate in the reaction, add avidin-labeled HRP, incubate briefly, and then perform TMB color development. Then, add the stop solution to terminate the color development reaction, and measure the absorbance value at 450nm. The OD value is directly proportional to the sample content.
 
Iii. Items needed but not provided in the reagent kit
Pipettes with diameters ranging from 10 to 1000μL, available in single and multiple channels
2. 100mL and 1000mL beakers
3. Deionized water
4. Centrifuge tubes of various models
5. Microplate reader (for 450nm measurement
6. Data processing and analysis software

Iv. Components of the Kit
 

Component	Product #	Storage
1. Protein A Standard（proteinA Protein standard） Recombinant Alkali-tolerant Protein A in a protein matrix with preservative, 50μg/mL, 1x0.1mL	Bk031	2-8°C
2. Biotinylated Antibody（Biotin-labeled antibodies）                                     Biotinylated anti-protein A monoclonal antibody in a protein matrix with preservative, 1x13mL	Bk016	2-8℃
3. Sample Diluent    （Sample diluent）                                     Tris buffered saline with a protein matrix and preservative, 1x30mL	Bk017	2-8℃
4. Denaturing Buffer   （Acidification buffer solution）                                Citrate buffer with detergent and preservative, 1x20mL	Bk018	2-8℃
5. 10x Assay Buffer      （10x）Washing buffer solution                                   phosphate buffer with preservative, 1x30mL	Bk004	2-8℃
6. Streptavidin labeled HRP   Avidin-labeled HRP       Streptavidin labeled HRP in a protein matrix with preservative, 1x13mL	Bk019	2-8℃
7. TMB Substrate    （TMB chromogenic solution）                                     3,3’,5,5’Tetramethylbenzidine, 1x13mL	Bk007	2-8℃
8. Stop Solution        （Termination solution）                                   2M sulphuric acid, 1x13mL	Bk008	2-8℃
9. Monoclonal anti-Protein A Coated plate of 96 wells （96-well microplate coated with proteinA antibody）                12x8 well strips in a bag with desiccant	Bk032	2-8℃

 




























































V. Reagent Preparation
Please leave this kit at room temperature (20-25 degrees) before use.
2. Preparation of washing solution: Add 270 ml of deionized water to 30mL of washing solution (10x), mix well to prepare 300mL of working washing solution. If there are crystals inside, please dissolve and mix well before use
3. Other reagents can be used directly without dilution
 
Vi. Sample Processing Procedure:
If the sample needs to be diluted, use the sample diluent in the kit for dilution, which can be done in a 96-well plate or EP tube.
2. Ensure that the volume of each well (tube) containing the sample is 100μL.
3. Additionally, add 100μL of the standard and quality control substances to the Wells.
4. Add another 50μL of acidification solution to each well, pipette to mix well, and incubate at room temperature for 10 minutes (to acidify and separate proteinA and antibodies in the sample).
 
Vii. Precautions
This kit is only for scientific research and production and must not be used for in vitro diagnosis.
2. The termination solution is 2M sulfuric acid. Avoid contact with eyes, skin and clothing. In addition, the reagents in this kit will not cause any harm to the human body.
3. High or low pH values, detergents, urea, high salt concentrations and organic reagents are all influencing factors of the ELISA method. pH has a significant impact. The PH value of the sample should be controlled between 7.0 and 7.4.
4. If you encounter any problems during use, please contact our technical department.