Is the linear range of BCA assay kit sufficient for precise protein quantification before antibody conjugation?

If precision is required, do not use only a BSA standard curve to quantify antibodies. BCA is sensitive to protein amino acid composition, and the colorimetric response of IgG and BSA may differ significantly; directly calculating antibody concentration using a BSA standard curve can sometimes be 10%-30% too high or too low. A more reliable approach is to use an IgG standard of the same species with a known concentration, or to measure the concentration of a batch of antibody by A280 and use it as an internal standard, then use BCA for routine process quantification. For intermediate purification steps, if only relative concentration or yield is being assessed, using a BSA standard curve is acceptable; however, before conjugation, the final feed ratio must be confirmed by switching to an IgG standard curve or an orthogonal method.

The conventional BCA linear range is generally 0.02-2 mg/mL, and antibodies with a concentration of 0.5-8 mg/mL must be diluted. It is recommended not to only do one dilution point, but to do two independent dilutions for each sample, ideally within the range of 0.5-1.5 mg/mL in the middle section of the standard curve. For example, estimating a sample of 2 mg/mL at 1:2 and 1:4, and estimating an 8 mg/mL sample at 1:8 and 1:16. Calculate the concentration of the original solution with two dilutions, and the difference should preferably be within 5% -10%. Otherwise, check the pipette, dilution buffer, and sample mixing. High concentration antibodies have viscosity, and before sampling, they should be centrifuged at low speed and equilibrated at room temperature to avoid nozzle sticking to the wall and bubbles. The buffer used for dilution should be consistent with the sample matrix, and solutions containing interfering components such as Tris and glycine should be avoided.

Reducing dilution errors is more practical than choosing improved reagents. High concentration samples should be diluted using the weight method first: the sample is weighed on an analytical balance, and then dilution buffer is added according to the mass to avoid volume errors caused by high concentration liquid hanging on the wall and sticking to the gun head. The pipette should use a calibrated low residue pipette tip and be cleaned two or three times for suction, discharge, and lubrication. Add 10% -15% more working fluid according to the actual number of holes, and do not get stuck in the exact amount. When adding samples, the sample and standard curve are arranged alternately on the same plate to reduce the position effect of the 96 well plate. Before reading the plate, gently tap the orifice plate to eliminate bubbles, read at 562 nm, and record 450 nm or 590 nm as background reference if necessary.

BCA is suitable for rapid quantification during intermediate purification steps, but for final quantification before conjugation, it is recommended to cross-validate with methods such as A280 or SEC. A280 requires an accurate extinction coefficient and a buffer with low UV interference; if the sample contains nucleic acids, Tween, or other UV-absorbing impurities, A280 will be affected. BCA and A280 results can be compared: if the deviation is stable, it indicates that BCA can be used for within-batch comparison; if the deviation fluctuates widely, it often suggests buffer changes, aggregate formation, or sample degradation. Do not rely solely on a single BCA reading to determine the conjugation ratio, especially for high-concentration antibody stock solutions.