**I. ELISA Standard Operating Procedures**
Ensuring accurate and reliable ELISA results requires high-quality reagents, well-maintained instruments, and proper technique. The water used in ELISA procedures, including purified water for washing, should be distilled or deionized with a conductivity of less than 1.5 μs/cm to prevent interference.
**1. Specimen Collection and Storage**
Most ELISA tests use serum as the sample. Serum should be collected using standard techniques, and care must be taken to avoid hemolysis, as red blood cell lysis can release peroxidase-like substances that may cause non-specific background in HRP-labeled assays. Hemolyzed samples can lead to false positives. Fresh serum is ideal, but if not available, it can be stored at 4°C for up to five days. For longer storage, freezing at -20°C is recommended. However, repeated freeze-thaw cycles can reduce antibody titer, so samples should be aliquoted before freezing. If bacterial contamination occurs, it may introduce endogenous HRP, leading to false positives. To prevent this, samples should be handled under sterile conditions, and preservatives can be added if necessary.
In cases where anticoagulation is incomplete, fibrinogen can interfere and cause false positives. Therefore, it's advisable to use anticoagulants like heparin whenever possible.
**2. Sample Addition**
When adding samples to ELISA plates, ensure that the liquid is dispensed to the bottom of each well, avoiding contact with the sides to prevent spillage and cross-contamination.
**3. Incubation**
ELISA reactions typically occur at 37°C, as this temperature optimizes antigen-antibody binding. The reaction reaches its peak within 1–2 hours. To maintain consistent temperature, plates should be covered with sealing film or plastic wrap to prevent evaporation. Avoid stacking plates, as this can affect temperature uniformity. Incubation time and temperature must be strictly followed. Using a water bath instead of an air incubator may result in inconsistent readings or "flower plates." Additionally, edge effects can cause higher values on the plate’s outer wells, so quality controls should be placed in central positions for more accurate results.
**4. Washing**
Washing is a critical step in ELISA, as it removes unbound enzymes and non-specifically adsorbed substances. A neutral buffer containing a nonionic detergent, such as Tween 20 (typically 0.05%–0.2%), is commonly used. The detergent helps detach proteins from the solid phase by breaking hydrophobic bonds. Excessive detergent concentration may strip antigens or antibodies from the plate, reducing sensitivity.
During washing, ensure that different kit solutions are not mixed. If dilution is needed, follow the manufacturer's instructions. Water used for washing must have low conductivity. Plates should be washed for about 40 seconds, and the liquid should be completely removed. Manual washing should be done carefully to avoid bubble formation in the wells.
**5. Color Development and Readout**
After adding TMB substrate, the color develops rapidly, peaking around 40 minutes and fading after two hours. Stop solutions, such as sodium azide or acidic solutions, halt the reaction. Acidic stop solutions turn the blue color yellow, allowing absorbance measurement at 450 nm. Microplate readers are essential for quantifying the results. They should be operated in a room with stable temperature (15–30°C) and preheated for 15–30 minutes before use. High-quality readers offer accuracy up to 0.001 and repeatability of 0.5%.
For optimal results, select the appropriate wavelength based on the chromogen used. Some readers support dual-wavelength analysis, which improves accuracy by minimizing interference from scratches or fingerprints on the plate.
**2. Common Causes of Background and False Positives**
**1. Differences Between Genetically Engineered Antigens and Synthetic Peptides**
Genetically engineered antigens are produced by expressing the target gene in prokaryotic or eukaryotic systems, such as *E. coli* or yeast. These antigens differ from synthetic peptides in several ways:
a. **Molecular Weight**: Genetically expressed antigens tend to have a larger molecular weight compared to synthetic peptides, which are limited in size due to chemical synthesis constraints.
b. **Structural Complexity**: Engineered antigens often retain native conformation, while synthetic peptides may lack proper folding, affecting antibody recognition.
c. **Immunogenicity**: The full-length protein structure of genetically engineered antigens can elicit stronger immune responses than shorter synthetic peptides.
Understanding these differences is crucial for selecting the right antigen type based on the intended application.
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