In ELISA testing, both positive and negative controls play a crucial role in ensuring the accuracy and reliability of the results. These control samples are not only used to validate the test itself but also serve as reference points for interpreting the outcomes. To ensure consistency, the composition of the reference material—especially the negative control—should closely resemble that of the actual test sample. When working with human serum as the specimen, it's ideal for the control materials to also be based on human serum. This is because normal human serum can generate varying levels of background signal depending on the ELISA format being used. However, since obtaining large quantities of human serum is often challenging, many foreign ELISA kits use recalcified human plasma as the base. This involves adding calcium ions to plasma, which causes fibrin to form and then removing the clot. The resulting liquid has a composition very similar to serum, making it a suitable alternative.
The negative control should contain no target analyte, such as HBsAg in the case of an HBsAg detection assay. Ideally, it should also be negative for other related markers like anti-HBs. On the other hand, the positive control typically consists of a buffer solution containing a protein stabilizer, along with a known amount of the target substance. The concentration of this substance is usually specified in the kit’s instructions and should be appropriate for the sensitivity of the reagent. By comparing the absorbance values obtained from the sample with those from the positive control, you can estimate the concentration of the target substance in the specimen. For example, if the sensitivity of the ELISA kit for HBsAg is around 0.5 ng/ml, the positive control might contain approximately 10 ng/ml of HBsAg.
To ensure long-term stability, preservatives and sometimes antibiotics are added to the control solutions. In quantitative ELISA kits, a set of standard concentrations is provided to create a standard curve. These standards usually consist of 4–5 different concentrations that span the detectable range of the assay. They are typically prepared in a buffer containing protein protectants and preservatives to maintain their integrity over time. This setup allows for accurate quantification of the target analyte in the test samples.
Lip Brush,Retractable Lip Brush,Lip Care Brush,Lip Brush Retracted
SAMINA FORAM (SHENZHEN) CO., LIMITED. , https://www.saminabrush.com