Affinity testing of monoclonal antibodies in human blood can be a complex process that requires careful planning and execution to ensure accurate results. Both ELISA and flow cytometry are common techniques for assessing antibody binding, each with its own advantages and limitations. The choice between these methods can depend on the specific characteristics of the antibody and the sample, as well as the resources and expertise available.

Below is a general outline for performing an affinity test using either ELISA or flow cytometry:

ELISA Protocol

ELISA (Enzyme-Linked Immunosorbent Assay) is a plate-based assay that can be used to quantify the binding of antibodies to their target antigen.

Materials Needed:

• Microplate coated with the antigen of interest
• Monoclonal antibody to be tested
• Human donor blood samples
• Washing buffer (PBS-Tween)
• Secondary antibody conjugated to an enzyme (e.g., HRP or alkaline phosphatase)
• Substrate for the enzyme (e.g., TMB or pNPP)
• Stop solution (e.g., sulfuric acid or hydrochloric acid)
• Plate reader for absorbance measurement

Protocol:

Flow Cytometry Protocol

Flow cytometry is a powerful tool for analyzing the physical and chemical characteristics of cells or particles in suspension.

Materials Needed:

• Human donor blood samples
• Monoclonal antibody to be tested
• Fluorescently-labeled secondary antibody or streptavidin conjugate if using biotinylated primary antibody
• Isolation reagents for specific blood cells (if needed)
• Washing buffer (PBS or PBS + FBS)
• Fixative (e.g., paraformaldehyde) if performing a fixation step

Protocol:

Remember, these protocols are general guidelines and should be optimized for the specific monoclonal antibody and target antigen. The concentration of the monoclonal antibody, the incubation times, and the washing steps should be carefully optimized to ensure that the binding is specific and that background noise is minimized. It’s also important to include appropriate controls, such as isotype controls and non-specific binding controls, to interpret the results accurately.