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Checkerboard titration method

AE Asmaa M. El-Rasikh
HF Haithem A. M. Farghali
HA Hisham A. Abdelrahman
ME Mostafa Elgaffary
SA Shaymaa Abdelmalek
IE Ibrahim A. Emam
MG Magdy A. Ghoneim
SS Salah A. Selim
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The first step was the standardization of indirect Enzyme-Linked Immunosorbent Assay (ELISA) by checkerboard titration as described in previous studies19–22 using the following recombinant proteins as coating antigens: TP53 (ABIN1046804), MUC1 (ABIN1877158), MYC (ABIN2130698), and PCNA (ABIN622005). All the antigens were purchased from Antibodies Online (https://www.antibodies-online.com/) (Germany) and they are of human origin. Antigens were subjugated to Lasergene software version 3. 18 (DNAStar, Madison, WI) analysis to evaluate their identity % with feline antigens (UniProtKB/ Swiss- Prot: P41685.1, NCBI Reference Sequence: XP_023103255.1, UniProtKB/ Swiss- Prot: P68271.1, and NCBI Reference Sequence: XP_003983789.1. The checkerboard titration was conducted to optimize antigen concentration and serum dilution. Antigen concentrations were 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 ng/well and serum dilutions were 1/40, 1/80, 1/160, 1/320, 1/640, 1/1280, 1/2560, 1/5120, 1/10,240, 1/20,480.

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