Key Points in ELISA Operation

High-quality reagents, good instrumentation, and correct operation are essential for accurate and reliable ELISA test results. ELISA procedures vary depending on the solid phase carrier used. Plate-based ELISAs are generally used in domestic medical testing. This article will describe the key points for each step in the plate-based ELISA procedure. Bead-based, tube-based, and magnetic bead-based ELISAs are all used with specialized instrumentation. Detailed instructions are provided for each type of ELISA. Strict adherence to these instructions will ensure accurate results.

Specimen Collection and Storage

A wide range of specimens can be used for ELISA assays. Body fluids (such as serum), secretions (saliva), and excreta (such as urine and feces) can all be used to assay for specific antibody or antigen components. Some specimens can be assayed directly (such as serum and urine), while others require pretreatment (such as feces and certain secretions). Most ELISA tests use serum as the specimen. With the exception of fibrinogen and anticoagulants, plasma is similar in composition to serum. Plasma specimens require the use of an anticoagulant, while serum specimens can be obtained after the serum has naturally coagulated and the clot has retracted. Except in special circumstances, serum is used as the specimen for testing in medical laboratories. Plasma and serum can be used equally in ELISAs. Serum specimens can be collected using conventional methods, but care should be taken to avoid hemolysis. Lysis of red blood cells releases substances with peroxidase activity. In HRP-labeled ELISAs, hemolyzed specimens may increase nonspecific color development.

Serum specimens should be tested fresh. Bacterial contamination may cause endogenous HRP, which can also produce false-positive reactions. Prolonged storage in the refrigerator can lead to polymerization, which can increase the background in indirect ELISAs.

Generally speaking, serum specimens tested within 5 days can be stored at 4°C; those tested over a week should be kept on ice. After thawing, frozen serum will have locally concentrated proteins and uneven distribution. Mix thoroughly and gently to avoid air bubbles. Mix by inverting the sample, but avoid vigorous shaking on a blender. Turbid or precipitated serum samples should be centrifuged or filtered to clarify before testing. Repeated freezing and thawing can cause antibody titers to drop. Therefore, if serum samples for antibody testing need to be stored for multiple tests, they should be aliquoted and stored on ice. Serum should be stored aseptically from the time of collection, and appropriate preservatives may be added.

Reagent Preparation

Prepare the reagents required for the experiment according to the kit instructions. Distilled or deionized water used in ELISA, including that used for washing, should be fresh and of high quality. Homemade buffers should be calibrated using a pH meter. Reagents removed from the refrigerator should be allowed to equilibrate to room temperature before use. Any unused portion of the kit should be promptly returned to the refrigerator.

Sample Addition

An ELISA typically involves three sample addition steps: sample addition, enzyme conjugate addition, and substrate addition. When adding samples, they should be placed at the bottom of the ELISA plate wells, avoiding the upper walls. Be careful not to splash or create bubbles.

Specimens are generally added using a micropipette, with the prescribed amount added to the wells. The pipette tip should be changed each time to prevent cross-contamination. Disposable plastic tubes can also be used for sample addition. For assays (such as indirect ELISA) that require diluted serum, dilute it in a test tube to the prescribed dilution before adding the sample. Alternatively, add the diluent to the wells, then add the serum sample, and shake on a microshaker for 1 minute to ensure mixing. When adding the enzyme conjugate solution and substrate solution, a quantitative multichannel pipette can be used to expedite the addition process.

Incubation
In ELISA, there are typically two antigen-antibody reactions: after sample addition and after enzyme conjugate addition. The completion of the antigen-antibody reaction requires a specific temperature and time. This incubation process is called incubation. Some people call it "incubation," which seems inappropriate in the context of ELISA.

ELISA is a solid-phase immunoassay, in which antigen-antibody binding occurs only on the solid surface. For example, in the antibody-coated sandwich method, when a sample is added to a plate well, not all antigens present have an equal chance of binding to the solid phase. Only the antigen in the layer of solution closest to the well wall comes into direct contact with the antibody. This is a gradual equilibrium process, requiring diffusion to reach the endpoint of the reaction. The same applies to the binding of the enzyme-labeled antibody added later to the solid phase antigen. This is why ELISA reactions always require a certain incubation time.

Common incubation temperatures include 43°C, 37°C, room temperature, and 4°C (refrigerator temperature). 37°C is a commonly used incubation temperature in the laboratory and is the ideal temperature for most antigen-antibody binding. When developing ELISA methods for reaction kinetics studies, experiments have shown that product formation typically reaches peak after one to two hours at 37°C for two antigen-antibody reactions. To accelerate the reaction, the reaction temperature can be increased; some experiments are conducted at 43°C, but higher temperatures are not recommended. Antigen-antibody reactions are more thorough at 4°C. In radioimmunoassays, reactions are often refrigerated overnight to maximize precipitation. However, this is generally not used in ELISA because it takes too long.

A water bath is generally used for heat preservation, except for some ELISA instruments equipped with a dedicated heating block. The ELISA plate can be placed in a water bath with the bottom of the plate touching the water surface to allow for rapid temperature equilibration. To prevent evaporation, the plate should be covered. Alternatively, the wells can be covered with plastic wrap or plastic wrap, allowing the plate to float on the water surface. If an incubator is used, the ELISA plate should be placed in a humidified chamber made of a material with good heat conductivity, such as metal. A damp gauze should be placed on the bottom of the chamber, and the plate should be placed on top of the damp gauze. The humidified chamber should be preheated to the specified temperature in the incubator, especially in colder temperatures. Whether incubating in a water bath or a humidified chamber, the reaction plates should not be stacked to ensure rapid temperature equilibration. For room temperature incubation, the operating temperature should be strictly within the specified range. The standard room temperature is 20-25°C, but specific operation can be controlled according to the instructions in the instructions. During room temperature incubation, the ELISA plate can simply be placed flat on the workbench. Care should be taken to ensure that the incubation temperature and time are accurately specified. To ensure this, one person should not test more than two plates at a time.