MONOCLONAL ANTIBODIES REQUIRING COATING BUFFER WITH LOW PH FOR EFFICIENT ANTIGEN CAPTURE IN SANDWICH ELISA: THE RARITIES OR PRACTICALLY IMPORTANT PHENOMENA?

Abstract

This article reexamines some opinions concerning pH requirements for optimal immobilization of monoclonal antibodies (mAbs) by passive adsorption in antigen capture ELISA. It was discovered that substitution of “classical” sodium phosphate (pH 7.5) and carbonate (pH 9.5) coating solutions by acid (pH 2.8) buffers maximized antigen capture 4 out of 10 different tested anti-HBsAg mAbs, resulting in a 1.5–2.5 increase of binding curve coefficients. By measuring both mAbs amounts and functionality, the enhancement effect was attributed to the better preservation of solid phase antibodies activity.

Keywords:

• analytical sensitivity
• antibody adsorption
• coating buffer pH
• immunoassay optimization
• monoclonal antibodies
• sandwich ELISA

Notes

The wells of immunoplates were coated with different anti-HBsAg capture mAbs at 3 µg/mL using the following coating buffers: carbonate pH 9.5, phosphate pH 7.5, and glycine pH 2.8. The coated surfaces were reacted with a dilution series of HRP-labeled recombinant HBsAg (of ayw subtype). The analytical sensitivity of the assays was evaluated by comparing curve coefficients (× 10), relatively to binding curve linear region (i.e., “response/concentration ratio”). For other experimental conditions see Figure 2 and the section entitled “Procedure to Measure Antigen Capture Capacity of Adsorbed Antibodies.”

*Curve coefficient after antibody coating at pH 2.8 was statistically higher (p 95%) if compared to coating at pH 7.5 and 9.5.

n =  number of experiments.

100 µL aliquots of the solution of anti-HBsAg mAbs 18C8 (3 µg/mL), labeled with fluorescent due Cy5, were incubated overnight in different coating buffers in the wells of Nunc Maxisorp immunoplates, at 8 replicates for each coating buffer. This was followed by the measurements of the remaining fluorescence in these aliquots after adsorption. These measurements were compared with the fluorescence signals of eight 100 µL aliquots of the solutions of Cy5-labeled mAbs 18C8 in the same buffers (3 µg/mL), which did not contact with polystyrene. As concentration of Cy5-labeled antibodies is proportional to fluorescence signal, these tests allowed to estimate IgG concentration changes in the given samples after adsorption. The resulting surface antibody densities were calculated, assuming that coated area for 100 µL coating volumes constituted 0.94 cm². For further experimental details see the sections entitled “Labeling of Antibodies with Cy5 Succinimidyl Ester Dye” and “Estimation of Total Surface Antibody Densities at Different Coating Buffer pH.”

The surface concentration of biologically active antibodies on a solid phase (in ng/cm²) was estimated from saturation binding curve (Figure 7), taking the concentration of immune complexes at a large antigen excess (where the “plateau” is achieved) as a measure of active antibodies (binding sites) on a solid phase, obtained after adsorption. These transformations were made under assumptions that each biologically active solid phase mAb bound one antigen molecule. By comparing these data with total amounts of adsorbed IgG (Table 2) the proportions of biologically active molecules at different coating conditions was calculated. For other details of experimental design and calculations see the section entitled” The Measurements of Total Surface Antibody Densities and the Amounts of Biologically Active Antibodies Obtained after Adsorption at Different pH.”