What are the main points of the ELISA test operation?

High-quality reagents, advanced instrumentation, and proper technique are essential for obtaining accurate and reliable ELISA results. This guide provides a detailed overview of best practices in specimen handling, reagent preparation, incubation, washing, color development, and result interpretation.

1. Specimen Collection and Storage

ELISA can use various biological specimens such as serum, plasma, urine, or other body fluids. While some samples can be tested directly (like serum or urine), others require pretreatment (e.g., feces). Serum is the most commonly used specimen, obtained by allowing blood to clot naturally. Plasma requires anticoagulants. It's important to avoid hemolysis, as it may release peroxidase activity that interferes with HRP-based assays. Hemolyzed samples may lead to non-specific background staining. Fresh serum should be used if possible. If stored, it should be kept at 4°C for up to five days or frozen for longer periods. Repeated freeze-thaw cycles can reduce antibody titer, so it’s advisable to aliquot serum before storage. Always handle specimens aseptically and consider adding preservatives when necessary.

2. Reagent Preparation

Follow the manufacturer’s instructions carefully when preparing reagents. Use high-quality distilled or deionized water for all steps, including washing. Buffers should be pH-checked using a meter. Avoid using reagents that have been taken out of the refrigerator without being brought to room temperature first. Unused portions should be returned to the fridge promptly to maintain stability.

3. Sample Loading

When adding samples to the ELISA plate, ensure that the liquid is added to the bottom of the well to prevent splashing or bubble formation. Use a micropipette for precise dosing, and replace tips between samples to avoid cross-contamination. For dilutions, mix thoroughly on a micro-vibrator after adding the sample. Enzyme conjugates and substrates can be added using multi-channel pipettes for efficiency and consistency.

4. Incubation

Incubation is critical for antigen-antibody binding. The standard temperature is 37°C, but some tests may use 43°C for faster reactions or 4°C for overnight incubation. The incubation time typically ranges from 1 to 2 hours. Ensure even temperature distribution by placing plates in a water bath or wet box. Avoid stacking plates during incubation. At room temperature, maintain a stable environment between 20–25°C. Accurate timing is essential to ensure reproducible results.

5. Washing

Washing is crucial for removing unbound components and reducing background noise. Use a buffer containing a non-ionic detergent like Tween 20 (typically 0.05–0.2%) to help displace non-specifically bound proteins. Manual washing involves immersion and aspiration, while running water methods can be used for bead-based systems. Perform 3–4 washes, ensuring each step is thorough. Proper washing improves specificity and sensitivity.

6. Color Development and Readout

After substrate addition, the enzyme catalyzes the reaction to produce a colored product. The reaction time and temperature must be controlled to avoid overdevelopment. For example, OPD may stop developing after 20–30 minutes, and excess time can increase background. TMB is more stable and can be read visually or with a microplate reader. After stopping the reaction, measure absorbance at the appropriate wavelength (e.g., 492 nm for OPD, 450 nm for TMB). A microplate reader ensures accurate and consistent readings.

7. Result Interpretation

Qualitative results are reported as positive or negative based on comparison with controls. For semi-quantitative analysis, use a cut-off value derived from the negative control. In competitive ELISA, a higher inhibition rate indicates a stronger signal. Quantitative analysis requires a standard curve, typically plotted on semi-logarithmic paper. Ensure that the standard curve covers the expected range of the analyte and that the linear portion is used for calculations.