Practice question

Handling hybridoma cultures demands sensitive high-throughput detection secreted immunoglobulin microgram per mL among abundant serum proteins albumin bovine Ig. ELISA screening provides optimal format 96-well polystyrene plates coated purified antigen 1 ug/mL overnight 4 degrees passive hydrophobic adsorption van der Waals blocked 2 percent BSA 5 percent nonfat milk PBS preventing nonspecific binding plastic. Culture supernatant 50 uL added incubates 1h 37 degrees enabling specific binding Fab antigen interaction affinity association rate 10^5 per molar per second. After three washes PBS 0.05 percent Tween removing unbound proteins immune complex remains due high avidity. HRP conjugated goat anti-mouse IgG secondary binds Fc region captured mouse antibody. Substrate tetramethylbenzidine oxidized peroxidase H2O2 generates blue radical 650 nm converting yellow diimine after sulfuric acid stop absorbance 450 nm proportional titer. Purification Protein A Staphylococcus aureus or Protein G Streptococcus binds Fc CH2-CH3 interface neutral pH 7.4 affinity 10^-8 M eluting pure IgG pH 3.0 glycine followed dialysis concentration ultrafiltration yielding clinical-grade monoclonal reagent therapeutically diagnostically used screening high producers early.