How to control the large inter batch difference in ELISA using biological reagent AB?

If the slope difference affects quantification, 1-2 known concentrations of quality control or calibration samples can be added to each plate, and the recovery rate of the quality control samples can be used to determine whether the inter batch is acceptable. Don't just focus on the slope number, if the quality control recovery rate is within the preset range, the data can still be compared. If the deviation of the recovery rate exceeds the acceptable standard in the laboratory, adjust the coating concentration or consider changing batches. This way, even if there are fluctuations between batches, there is evidence available for tracing and explanation.

In our lab, when performing cytokine ELISA, we try to lock in the same lot number of coating antibodies within the project period when purchasing. We confirm with the distributor in advance whether they can provide the same lot number, COA, and aliquoting date. Lead time generally requires 3–4 weeks, and for commonly used amounts, we can stock up for six months to a year at once. After arrival, aliquot according to the instructions and keep proper lot number records to avoid mixing old and new batches. Although locking in a lot number may lengthen the lead time, it is very useful for reducing lot-to-lot variation.

Don't rush to change batches in large quantities for now. It is recommended to use both the old and new batch numbers for parallel titration on the same plate with the same set of standard samples and quality control serum. Repeat the wells with the old batch working concentration and the new batch dilution series. Focus not only on slope, but also on blank absorbance, high-value platform, and low point signal-to-noise ratio. If the main difference is in potency, re titrating the package concentration is usually more stable than directly changing batches. Leave a small amount of old batches as bridging samples before each batch change, which will make the data more comparable.

The inter batch differences may not necessarily come entirely from the antibodies themselves. The pH, ionic strength, enzyme-linked immunosorbent assay (ELISA) plate batch, and even washing solution replacement of the coating buffer may cause changes in slope. Suggest fixing the packaging buffer and color system, using the same manufacturer and product number for the board, and keeping the packaging temperature and time consistent. It is best to quantify antibodies first and then dilute them, not just by volume ratio; The concentration of different batches of raw materials may vary, and calculating based on protein content can better restore the working concentration.