**I. ELISA Standard Operating Procedures**
To achieve accurate and reliable results in ELISA, it is essential to use high-quality reagents, well-maintained instruments, and proper techniques. The water used in the ELISA process, including the purified water for washing, should be distilled or deionized with a conductivity of less than 1.5 μs/cm. This ensures that no impurities interfere with the reaction.
**1. Sample Collection and Storage**
Most ELISA tests use serum as the sample. When collecting serum, 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. Fresh samples are ideal, but if storage is necessary, they should be kept at 4°C for up to five days. Prolonged storage can lead to IgG polymerization, which increases background in indirect methods. For longer storage, samples should be frozen at -20°C. However, repeated freezing and thawing can reduce antibody titer, so it's best to aliquot samples before freezing.
If the sample is contaminated with bacteria, it may contain endogenous HRP, leading to false positives. To prevent this, samples should be handled aseptically and preservatives may be added if needed. Incomplete anticoagulation can also cause false positives due to fibrinogen interference. Therefore, heparin is often recommended as an anticoagulant.
**2. Sample Loading**
When adding samples to the ELISA plate, ensure that the liquid is placed at the bottom of each well, not on the walls, to avoid spillage and uneven distribution.
**3. Incubation Conditions**
ELISA reactions typically occur at 37°C, where antigen-antibody interactions reach their peak within 1–2 hours. Plates should be covered during incubation to prevent evaporation, using sealing films or plastic wrap. Stacking plates should be avoided to maintain uniform temperature. It’s important to follow incubation time and temperature precisely, as deviations can affect results. Using a water bath instead of an air bath may cause high background or "flower plates." Also, edge effects can lead to higher values on the outer wells, so quality controls should be placed in central positions for accurate readings.
**4. Washing Steps**
Washing is a critical step in ELISA, as it removes unbound enzyme markers and non-specifically adsorbed substances. A neutral buffer containing a non-ionic detergent like Tween 20 (0.05–0.2%) is commonly used. The detergent helps detach proteins from the solid phase by breaking hydrophobic bonds. Too much detergent can strip antigens or antibodies from the plate, reducing sensitivity.
When washing, avoid mixing solutions from different kits and dilute them as instructed. Use water with low conductivity (less than 1.5 μs/cm). If using crystallized reagents, melt them before use. Ensure each well is washed for about 40 seconds, and avoid creating bubbles during manual washing.
**5. Color Development and Reading**
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 buffers (e.g., sulfuric acid) halt the reaction. Some stop solutions keep the blue color for up to 24 hours, making visual assessment easier, while others turn the color yellow, allowing absorbance measurement at 450 nm.
A microplate reader is used to measure absorbance. Key features include reading speed, accuracy, repeatability, and linearity. Ideal readers have a precision of 0.001, ±1% accuracy, and 0.5% repeatability. Avoid placing the reader under direct light, and allow it to warm up for 15–30 minutes before use for stable results.
For optimal readings, select the appropriate wavelength (e.g., 492 nm for OPD). Some readers support dual-wavelength measurements, subtracting background noise caused by scratches or fingerprints.
**II. Causes of Background and False Positives**
**1. Differences Between Genetically Engineered Antigens and Synthetic Peptides**
Genetically engineered antigens are produced through prokaryotic or eukaryotic expression systems, such as *E. coli* or yeast. These antigens differ from synthetic peptides in several ways:
a. **Molecular Weight**: Genetic engineering allows for larger molecular weights compared to synthetic peptides, which are limited in size due to chemical synthesis constraints.
b. **Immunogenicity**: Engineered antigens may better mimic native proteins, offering more specific immune recognition.
c. **Consistency**: Synthetic peptides can vary in purity and sequence, whereas genetically engineered antigens are more consistent in structure and function.
Understanding these differences is crucial when interpreting ELISA results and selecting the most suitable antigen for the test.
Embark on your adventures with Alpenstocks Hiking Poles. Expertly crafted for rugged terrains, these high-performance poles provide exceptional support and stability. With a comfortable grip and precise design, they transform every hike into a seamless journey, ensuring you conquer nature with confidence and ease.
Walking stick ,Folding crutch,Alpenstocks,Hiking Poles
NINGBO JENNY IMPORT & EXPORT CO.,LTD , https://www.jenny-china.com