Recombinant proteins NY-ESO-1, Her-2/neu, MAGE-A4 (Origene, Rockville, US), CA125 (R&D Systems) and MAGE-A3, MAGE-A10 (Abnova, Taipei, Taiwan) were diluted in Carbonate Coating Buffer (Invitrogen, Prague, Czech Republic) to a final concentration of 1 μg/ml and coated to 96-well plates overnight at 4°C. Plates were blocked for 1 hour with Assay Buffer (Invitrogen, Carlsbad, CA). Patients and control sera diluted to 1:50, 1:100 and 1:200 were incubated in the antigen-coated wells for 2 h. Plates were then incubated with secondary antibody (goat polyclonal antibody to human IgG, Abcam, Cambridge, UK) for 1 hour. TMB (Invitrogen) was used as a substrate. Reaction was stopped after 20 minutes by adding Stop Solution (Invitrogen). Plates were immediately read with absorbance at 450 nm. As a positive control the Cytomegalovirus Glycoprotein B protein was used. The cutoff value designating positive reaction was assessed as the mean OD of 15 sera obtained from healthy controls (NHS) + 3SD.
Carbonate coating buffer
Manufactured by Thermo Fisher Scientific
Carbonate Coating Buffer is a laboratory reagent used to coat the surface of microplates or other solid supports in various immunoassay and enzyme-linked immunosorbent assay (ELISA) applications. The buffer provides a stable and uniform coating surface to facilitate the immobilization of proteins, antibodies, or other target molecules.
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Lab products found in correlation
Stop solution, by Thermo Fisher Scientific (2 mentions)
Goat polyclonal antibody to human igg, by Abcam (2 mentions)
Mage a4, by OriGene (2 mentions)
Ca125, by R&D Systems (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Thermo Fisher Scientific (1 mentions)
Tetramethylbenzidine substrate, by Thermo Fisher Scientific (1 mentions)
2 protocols using carbonate coating buffer
1
Multiplex Serum Autoantibody Detection
2
Detecting Tumor Antigens in NSCLC Serum
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The presence of antibodies against the three tumor antigens HER2/neu, NY-ESO-1, and MAGE-A4 in serum of patients with NSCLC and controls was detected by ELISA as adopted from Gnjatic et al., 13 Long et al., 14 and Stockert et al. 15 The recombinant proteins NY-ESO-1, HER-2/neu, and MAGE-A4 (Origene) were diluted in carbonate coating buffer (Invitrogen, Carlsbad, CA) to a final concentration of 1 mg/mL and coated to 96-well plates overnight at 4 C. Plates were blocked for 1 hour with Assay Buffer (Invitrogen). Human sera diluted to 1:100 and 1:200 were incubated in the antigen-coated wells for 2 hours. Plates were then incubated with secondary antibody (goat polyclonal antibody to human IgG [Abcam, Cambridge, United Kingdom]) for 1 hour. Tetramethylbenzidine substrate (Invitrogen) was added and incubated for 20 minutes. The reaction was stopped by adding Stop Solution (Invitrogen). Plates were immediately read with absorbance at 450 nm. As a positive control, the cytomegalovirus glycoprotein B protein was used.
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