Dear Sir. Concerning your issue about washing procedure in elisa insulin kit 96-well plate. If an automated machine is used for the assay, follow the manufacturer’s instructions for all washing steps described in this protocol. 3. Add 20 µL Assay Buffer to the NSB (Non-Specific Binding) wells and each of the sample wells (refer to IX for suggested well orientations). 4. If samples to be assayed are serum or plasma, add 20 µL Matrix Solution to the NSB, Standard, and Control wells. If samples are free of significant serum matrix components, add 20 µL Assay Buffer instead. 5. Add in duplicate 20 µL Human Insulin Standards in the order of ascending concentration to the appropriate wells. 7. Add 20 µL QC1 and 20 µL QC2 to the appropriate wells. 8. Add sequentially 20 µL of the unknown samples in duplicate to the remaining wells. 9. Add 20 µL Detection Antibody to all wells. For best result all additions should be completed within 30 minutes. Cover the plate with plate sealer and incubate at room temperature for 1 hour on an orbital microtiter plate shaker set to rotate at moderate speed (approximately 400 to 500 rpm). 9. Remove plate sealer and decant solutions from the plate. Tap as before to remove residual solutions in the wells. 10. Wash wells 3 times with diluted HRP Wash Buffer, 300 µL per well per wash. Decant and tap after each wash to remove residual buffer. 11. Add 100 µL Enzyme Solution to each well. Cover the plate with sealer and incubate with moderate shaking at room temperature for 30 minutes on the microtiter plate shaker. 12. Remove sealer, decant solutions from the plate, and tap plate to remove the residual fluid. 13. Wash wells 5 times with diluted HRP Wash Buffer, 300 µL per well per wash. Decant and tap after each wash to remove residual buffer. 14. Add 100 µL of Substrate Solution to each well, cover plate with sealer and shake on the plate shaker for approximately 5 to 20 minutes. Blue color should be formed in wells of insulin standards with intensity proportional to increasing concentrations of insulin I think the following below links may help you in your analysis:

https://sceti.co.jp/images/psearch/pdf/LIN_EZHI-14K_p.pdf

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/ERINS.pdf

http://www.abcam.co.jp/ps/products/100/ab100578/documents/ab100578%20Insulin%20Human%20ELISA%20Kit_v%209%20(website).pdf

http://www.abcam.com/ps/products/100/ab100578/documents/ab100578%20Insulin%20Human%20ELISA%20Kit_v4%20(website).pdf

https://www.raybiotech.com/files/manual/ELISA/ELR-Insulin.pdf

Thanks