13 successful tips for ELISA troubleshooting (part one)

As a classic immunoassay, ELISA seems pretty usual, but it is not easy to do. What are the secrets to make a successful ELISA assay? In this article, we collect 11 ELISA tips for you.

  1. Make sure your sample type and kit are compatible

Common sample types include serum, plasma, and cell culture supernatant, if it is verified by the manufacturer’s experiment, it can be purchased according to the product description.

Note: Anticoagulants used in plasma preparation include EDTA, citrate, heparin, etc., and the corresponding plasma properties are different, and compatibility must be confirmed separately.

Special samples, such as urine, pleural effusion, cerebrospinal fluid, lavage fluid, tears, tissue homogenate, etc., may lack the support of manufacturer data. Therefore, you’d better to consult relevant expert or make a self-experimental verification of feasibility.

  1. The kit detection range needs to match the concentration of the marker in the sample.

The quantitative detection range of the ELISA kit needs to refer to the standard curve, which is a linear range in the lowest and highest concentration range of the standard curve, and the values ​​are credible and quantifiable. Samples with concentrations above the linear range are diluted to a linear range for measurement, while samples with concentrations below the linear range cannot be used to calculate concentrations and can only be used as a reference. A common situation is that the concentration of many markers in healthy human samples is low and the measured value is lower than the lowest value of the standard curve.

Many companies’ product data sheet provides you with a comparison of the results of a healthy person sample to confirm the reliability of your results. It should be noted that due to differences in race, disease and preservation methods, even if similar samples in the reference documents such as the manual can be detected, the testability of your sample cannot be guaranteed. This sample concentration below the measurement range can be identified by ND (not detectable), or <” lowest point concentration of standard curve ” (eg <3.1pg/ml, IL-6 Quantikine) and statistically analyzed.

  1. Five useful tricks to sample collection

Take serum as an example, the process can refer to the kit instructions, but pay attention to the following points.

  1. Samples should be collected in sterile tubes as much as possible to avoid the effects of bacterial enzymes and metabolic products on the results.
  2. Hemolysis should be avoided during the collection process because the active substances released by red blood cell lysis may affect the detection.
  3. If there is a precipitate during the preservation process, the supernatant should be taken after centrifugation.
  4. If the sample is not measured immediately, it is recommended to store it at -20 °C according to the amount of each use.
  5. One tube per test is used to avoid cross-contamination and repeated freezing and thawing. It can ensure the parallel comparability of the test results.
  6. Prepare the appropriate reagents before the experiment

All reagents are equilibrated to room temperature in advance, which takes about half an hour. The washing liquid is a concentrated liquid, and if it is crystallized, it needs to be completely dissolved and then diluted. A and B chromogenic substrate were mixed in a ratio of 1:1 before 15 minutes of use. In order to ensure the quality of the protein standard solution preparation, the protein standard is lyophilized powder. Before reconstitution, the tube should be centrifuged, and the buffer specified in the instruction manual should be added. Shake it slowly on the shaker or mix it by inversion for at least 15 minutes. If it needs to be stored after dissolving, it should be stored according to the temperature required by the instructions after being packed according to the amount of each use. When using a gradient-diluted protein standard, it is recommended to use a polypropylene tube (low adsorption for protein), mix thoroughly at each step and replace the new tip to ensure gradient accuracy.

  1. Preparation of equipment is also essential

Related equipment includes pipettes, washers/shakers, and microplate readers. The accuracy of the pipette is critical to the reliability of the ELISA results and requires regular calibration maintenance. The use of a shaker is to allow the reaction system to quickly reach equilibrium and achieve stable, repeatable results. The usual effect of not using a shaker on the experiment is that the OD value is low and the CV is large. Equipment such as microplate readers are preheated 15 minutes before use.

  1. Pre-experiment: Usually the pre-experiment includes all standard curves and small samples.

Pre-experiment has two main purposes, one is to familiarize with the experimental process and find problems that may be ignored. The second is to test whether the sample and the kit match, and if there is a mismatch, to make sure no waste of too many samples. In addition, a general understanding of the abundance of the analyte of interest in the sample is made to determine the appropriate dilution.

…to be continued in PART TWO.