ELISA Protocol-Elisa Experiment Standard Operating Method (Part I)

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The Elisa experiment has been widely used clinically due to its high sensitivity and good specificity. However, each link in the operation has a greater impact on the detection effect of the experiment. If you are not careful, it may lead to incomplete color rendering and patterning. Wait for the result.

ELISA Protocol

The following step by step analysis of the factors that may affect the results of the Elisa test operation.

Step 1: Specimen Selection and Preparation

The most commonly used clinical specimens for ELISA determination are serum (plasma), and sometimes specimens such as saliva, cerebrospinal fluid, urine, and feces are also used for specific testing purposes. At present, the clinically used serum samples to determine the markers generally include antigens and antibodies of infectious pathogens, tumor markers, hormones, special proteins, cytokines, and therapeutic drugs. For the collection of serum samples used for hormone and therapeutic drug determination, it is necessary to pay attention to the collection time or even the body position that may affect the determination results. For example, cortisone will have a peak between 4 and 6 in the morning: growth hormone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) are all released in a paroxysmal manner. Therefore, when measuring these hormones , It is necessary to take several blood samples in closely connected time intervals, and use the median value as the measured value. Another example is when changing from a lying position to a standing position, the serum renin activity will increase significantly. Another example is the detection of therapeutic drugs, the best time after taking the drug should be selected according to the pharmacokinetics for blood sampling. The collection of serum specimens used for the detection of antigens and antibodies of infectious pathogens, tumor markers, and special proteins, etc., has no time and position impact, but the following aspects should be considered in the handling and storage:

  1. Pay attention to avoid severe hemolysis. Hemoglobin contains a heme group, which has a peroxide-like activity. Therefore, in the ELISA assay with HRP as the labeling enzyme, if the concentration of hemoglobin in the serum sample is high, it is easy to adsorb during the incubation process. In the solid phase, it reacts with the HRP substrate added later to develop color.
  2. During sample collection and serum separation, attention should be paid to avoid bacterial contamination as much as possible. For the growth of bacteria, some of the secreted enzymes may decompose proteins such as antigens and antibodies; the other is for some bacterial endogenous enzymes such as The β-galactosidase of Escherichia coli itself can cause non-specific interference with the assay method labeled with the corresponding enzyme.
  3. If the serum specimen is separated by aseptic operation, it can be stored at 2～8℃ for one week, if it is for bacteria operation, it is recommended to freeze preservation. The long-term storage of samples should be below -70°C.
  4. For frozen serum specimens, care must be taken to avoid repeated freezing and thawing caused by power outages. The mechanical shearing force produced by repeated freezing and thawing of the specimen will have a destructive effect on the protein and other molecules in the specimen, thereby causing false negative results. In addition, attention should be paid to the mixing of freeze-thaw specimens. Do not vigorously shake, just invert and mix repeatedly.
  5. If the specimens have turbidity or flocculation caused by bacterial contamination during storage, the supernatant should be centrifuged for testing.

Step 2: Preparation of Reagents

In clinical laboratories, generally little attention is paid to the preparation of reagents. The usual practice is to take the reagents out of the refrigerator and use them during the experiment, ignoring that this method may affect the problem of insufficient incubation time later. The consequence of this is false negatives on some weakly positive specimens. Therefore, the most important thing in the preparation of reagents in the ELISA assay is to take the kit out of the refrigerator before starting the experiment and place it at room temperature for 30-60 minutes before performing the measurement, so that the kit is at room temperature before use. balance. The purpose of this is mainly to enable the temperature in the reaction micropores to reach the required height quickly in the subsequent incubation reaction step to meet the measurement requirements.