(i) Sandwich ELISA:
Suitable for detecting large molecular antigens (e.g., proteins, viral particles). It requires the target antigen to have at least two epitopes capable of simultaneously binding both the capture antibody and the detection antibody. Its advantages include strong specificity and simple sample processing, making it suitable for complex matrix samples like serum and plasma. It is commonly used for tumor marker and cytokine detection.

(ii) Indirect ELISA:
Primarily used for detecting antibodies (e.g., autoantibodies, pathogen antibodies) or small molecular antigens. It requires only one epitope on the target substance and amplifies the signal using enzyme-labeled secondary antibodies. It is easy to operate and cost-effective but susceptible to interference from nonspecific antibodies in samples, making it suitable for large-scale screening scenarios.

(iii) Competitive ELISA:
Designed for small molecular antigens (e.g., hormones, drugs, toxins) that cannot simultaneously bind two antibodies. Quantification is achieved by competition between the antigen in the sample and enzyme-labeled antigen for binding to the solid-phase antibody. It exhibits strong anti-interference capability and is suitable for samples with complex matrices or structurally simple target substances.

Selection should be based on the molecular weight of the target substance, the number of epitopes, sample type, and detection throughput.

After collection, serum samples should first be left at room temperature for 30 minutes to 1 hour to allow natural blood coagulation. The serum is then separated by centrifugation at 4°C and 3000 rpm for 10 minutes, avoiding vigorous centrifugation that may cause hemolysis. If testing can be completed within 24 hours, the separated serum can be stored at 4°C. For long-term storage, the serum should be aliquoted into small volumes and quickly frozen at -80°C, avoiding repeated freeze-thaw cycles. During thawing, samples should be slowly brought to room temperature and thoroughly mixed.

Protease inhibitors can be added during storage to prevent target protein degradation by endogenous proteases. For easily degradable substances such as hormones and cytokines, 0.02% sodium azide can be added to the serum as a preservative. During transportation, dry ice should be used to maintain temperatures below -20°C, avoiding fluctuations that could lead to loss of target substance activity and ensuring accurate detection results.

(i) Use a Signal Amplification System:
Employ the biotin-avidin amplification system, which leverages the high-affinity binding between biotin-labeled antibodies and enzyme-labeled avidin to achieve cascading signal amplification. This can enhance sensitivity by 10–100 times, making it suitable for detecting low-abundance substances at the pg/mL level.

(ii) Optimize Sample Pretreatment:
Concentrate samples using methods such as ultrafiltration centrifuge tubes for serum or cell culture media to increase target substance concentration. If inhibitors are present in the sample, add appropriate neutralizing reagents to reduce interference.

(iii) Select High-Activity Substrates:
Prefer high-sensitivity substrates, such as enhanced TMB substrates or chemiluminescent substrates. However, note the signal stability of chemiluminescent substrates, and ensure readings are completed within the specified time.

(iv) Optimize the Solid Phase Carrier:
Use surface-modified microplates to enhance the immobilization efficiency of antigens or antibodies, reduce nonspecific adsorption, and improve the signal-to-noise ratio.

Cross-reactivity in ELISA mainly stems from antibody cross-binding to non-target antigens or nonspecific adsorption of interfering substances in the sample. At the antibody level, if the antibody lacks sufficient specificity, it may cross-react with structurally similar proteins in the sample. In this case, switch to a more specific monoclonal antibody or optimize polyclonal antibodies through antigen affinity purification. At the sample level, interfering substances such as rheumatoid factors, complement, and heterophilic antibodies in serum may directly bind to enzyme-labeled secondary antibodies or solid-phase antibodies, leading to false positives. To resolve this, add 10% fetal bovine serum or 5% skimmed milk to the sample dilution buffer to block binding sites of interfering substances. Alternatively, pretreat the sample with specific blocking reagents. Furthermore, if the target antigen shares homologous sequences with other proteins, design antibodies targeting unique epitopes or use competitive methods to reduce cross-binding. Simultaneously, optimize washing steps to enhance the elution of nonspecifically bound substances.