What are the main points of the ELISA test operation?

High-quality reagents, advanced instrumentation, and proper technique are essential for achieving accurate and reliable ELISA results. This guide outlines key steps to ensure success in ELISA procedures, from specimen handling to result interpretation.

1. Specimen Collection and Storage

ELISA can utilize a variety of biological specimens such as serum, plasma, urine, or other body fluids. While some samples like serum and urine can be directly used, others such as feces may require pretreatment. Plasma is obtained by adding anticoagulants, while serum is collected after blood clotting. It's important to avoid hemolysis, as red blood cell lysis can release peroxidase-like substances that interfere with HRP-based assays. Fresh samples should be used when possible, and if stored, they should be kept at 4°C for up to 5 days or frozen for longer periods. Repeated freeze-thaw cycles can reduce antibody titer, so it’s best to store samples in small aliquots.

2. Reagent Preparation

Follow the manufacturer's instructions for reagent preparation. Use high-quality distilled or deionized water for all washes. Buffer solutions should be pH-adjusted using a calibrated meter. Always allow reagents to reach room temperature before use and return unused portions to the refrigerator promptly.

3. Sample Loading

When loading samples, add them to the bottom of the wells to prevent splashing and bubble formation. Use a micropipette and replace tips between each sample to avoid cross-contamination. For dilutions, mix thoroughly on a micro-vibrator for 1 minute. Enzyme conjugates and substrates can be added using a multi-channel pipette for efficiency.

4. Incubation

Incubation is critical for antigen-antibody binding. The optimal temperature is typically 37°C, though some protocols use 43°C for faster reactions or 4°C for overnight incubation. Ensure even heat distribution by placing plates in a water bath or wet box. Avoid stacking plates during incubation to maintain consistent conditions.

5. Washing

Washing removes unbound components and reduces background noise. Use a buffer containing non-ionic detergents like Tween 20 (0.05–0.2%) to enhance washing efficiency. Immersion or running water methods can be used, with immersion being more common for microtiter plates. Wash 3–4 times, ensuring thorough removal of excess reagents.

6. Color Development and Measurement

Color development depends on enzyme-substrate interactions. For OPD, color intensity peaks within 20–30 minutes, after which background may increase. TMB is stable under light and develops to a peak in ~40 minutes. Stop solutions like sulfuric acid or SDS terminate the reaction. Read absorbance at the appropriate wavelength (e.g., 492 nm for OPD, 450 nm for TMB) using a microplate reader.

7. Result Interpretation

Qualitative results are determined as positive or negative based on comparison with controls. Quantitative analysis requires a standard curve, with absorbance values plotted against known concentrations. Different ELISA formats (e.g., sandwich vs. competitive) have distinct interpretation methods. Always use a negative control to set thresholds and ensure accuracy.

Following these guidelines ensures consistency and reliability in ELISA experiments. Proper training, attention to detail, and adherence to protocol are key to obtaining meaningful data.