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What Should We Pay Attention In ELISA Experiment?

Posted on October 29, 2022 by Zeng YingELISA

ELISA sample processing

The purpose of the ELISA test is to provide an accurate experimental basis for the experiment, and to ensure the reliability of the experimental data, in the experimental process must adhere to comprehensive quality control, and the whole process of quality control, in the collection of specimens must complete plan before.

Catalogue: Equipment For Elisa Kit Manufacturing

ELISA specimen handling notice

ELISA-test
ELISA-test

1. Sample volume collected per enzyme-linked immunosorbent assay = 100ulx assay type, if multiple wells are to be done, specimen volume collected = 100ulx assay type x 2.

2. If the ELISA sample is collected and tested within a week, it can be stored at 2-8°C. If the test is not done in time, please split the sample and freeze it at -20°C or -80°C to avoid repeated freezing and thawing.

3. The detection range of the ELISA kit is not equal to the concentration range of the analyte in the sample. It is recommended to estimate the concentration of the analyte in the model through the relevant literature and determine the selection through pre-experimentation before the experiment.

4. ELISA serum specimens should be collected with care to avoid hemolysis, as erythrocyte lysis releases substances with peroxidase activity, and hemolyzed illustrations may increase non-specific color development.

5. To ensure the accuracy of the urine test, when using ELISA collection, must be properly collected urine specimens and preservation, collection of urine containers must be clean and dry, and it is best to use disposable containers to avoid contamination caused by the use of drugs and clean less, urine samples must be fresh, after retention, should be promptly tested or stored.

6. Specimens should be tested fresh if there is bacterial contamination, the bacterium may contain endogenous HRP, which will also produce a false positive reaction. If stored in the refrigerator for too long, in which polymerization may occur, the indirect method of ELISA in the Lakeview background deepens.

7. In the ELISA specimen processing experiments, repeated freezing and thawing of the specimen will reduce the protein potency, so for the sample to be tested for multiple testing, it is advisable to freeze a small number of portions.

ELISA coating

ELISA specimen types

ELISA-specimen-handling-notice
ELISA-specimen-handling-notice

Common specimens for ELISA

Liquid specimens: serum, plasma, urine, cell supernatant, cerebrospinal fluid, etc.

Cultured cells

Tissue specimens

Storage of ELISA specimens

In general, the ELISA serum specimens measured within 5 days can be placed at 4°C, specimens then stored in the refrigerator for too long will lead to the polymerization of serum IgG, is the reagent background of the indirect method deepened, more than a week to determine the need for -20°C storage, frozen serum dissolved, protein local concentration, divided unevenly, should be fully mixed and avoid the generation of air bubbles.

Cloudy or precipitated serum specimens should be centrifuged or filtered first and then clarified before testing.

The specimens of ELISA sera for antibody measurement should be stored for multiple testing, in small quantities on ice.

Preservation of ELSA sera should only be collected with attention to aseptic operation, and appropriate preservatives can also be added. False-positive specimens caused by incomplete anticoagulation due to the interference of fibrin elements are recommended not to use anticoagulant, especially heparin anticoagulant.

ELISA specimen handling

Serum: ELISA room temperature blood private coagulation for 10-20 minutes, centrifugation for about 20 minutes (2000-3000 rpm). The supernatant should be collected carefully and centrifuged again if precipitation occurs during storage.

Plasma: EDTA or sodium citrate should be selected as anticoagulant according to the requirements of enzyme-linked immunosorbent assay specimens, mixed for 10-20 minutes, centrifuged for about 20 minutes (2000-3000 rpm), and the supernatant should be carefully collected and centrifuged again if precipitate forms

Urine: collected in sterile tubes, and centrifuged for about 20 minutes (2000-3000 rpm), the supernatant was carefully collected, and should be centrifuged again if precipitate formed during preservation. Thoracoabdominal fluid and cerebrospinal fluid refer to the practice.

Cell culture supernatant: When detecting ELISA secretory components, collect in sterile tubes, centrifuge for about 20 minutes (2000-3000 rpm) and carefully collect the supernatant, and when detecting intracellular components, dilute the cell suspension with PBS (PH 7.2-7.4), and the cell concentration reaches about 1 million/ml, by repeated freeze-thawing to destroy the cells and release the intracellular components.

Tissue specimens: After cutting ELISA specimens, weigh them, add a certain amount of PBS, PH 7.4, freeze them quickly with liquid nitrogen, and keep them in reserve. After the specimens are melted, maintain a temperature of 2-8°C at any time, add a certain amount of PBS (PH 7.4), homogenize the specimens fully by hand or homogenizer, centrifuge them for about 20 minutes (2000-3000 rpm), collect the supernatant carefully. The supernatant was carefully collected, one portion was divided for testing, and the rest was frozen and set aside.

ELISA specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature, and the test should be performed as soon as possible after extraction.

ELISA detection methods

ELISA-specimen-types
ELISA-specimen-types

ELISA assay methods are direct, indirect, competition-based double antibody sandwich methods, of which the direct method and competition method are less used, should be the most double antibody sandwich method, which has obvious advantages in sensitivity gene specificity.

Direct ELISA method

The antigen is immobilized on the ELISA class, and then the antigen is detected directly with the enzyme-labeled antibody.

Compared with other types of ELISA experiments, direct ELISA experiments have fewer steps, fast detection, no need to use secondary antibodies, avoid cross-reactivity, and test results are not prone to error.

However, because the antigen of direct ELISA is not specifically fixed, the target protein and other impurity proteins in the sample will bind to the ELISA version, the experimental background will be higher, and in direct ELISA each target protein needs to prepare a primary antibody that can bind specifically to it, the experiment is less flexible. In addition, because no secondary antibody is used, the signal is not amplified, reducing the sensitivity of the assay.

Indirect ELISA method

The antigen is immobilized on the ELISA class, and then the antigen is detected directly with the enzyme-labeled antibody.

Compared with other types of ELISA experiments, direct ELISA experiments have fewer steps, fast detection, no need to use secondary antibodies, avoid cross-reactivity, and test results are not prone to error.

However, because the antigen of direct ELISA is not specifically fixed, the target protein and other impurity proteins in the sample will bind to the ELISA version, the experimental background will be higher, and in direct ELISA each target protein needs to prepare a primary antibody that can bind specifically to it, the experiment is less flexible. In addition, because no secondary antibody is used, the signal is not amplified, reducing the sensitivity of the assay.

Sandwich ELISA method

The antigen being tested is encapsulated between two antibodies, one of which immobilizes the antigen on a carrier, i.e., the capture antibody, and the other is the detection antibody, which can be enzyme-labeled for direct determination of the amount of antigen, or unlabeled and then measured by enzyme-labeled secondary antibodies, which must be carefully selected to avoid cross-reacting cargoes competing for the same amount of antigen binding sites.

Competition ELISA method

If the antigen is an antigen, the antigen will compete with the enzyme-labeled antibody to bind the enzyme-labeled antibody on the solid-phase carrier. If the substance to be detected is an antibody, the antibody to be detected will compete with the enzyme-labeled antibody in the system to bind to the antigen wrapped on the re-solidified carrier, wash off the competitively bound enzyme-labeled antibody by washing, and finally add the substrate to develop the color.

Competition ELISA is a bit more complicated compared to the above three methods, but the above three ELISA types are suitable for the form of competition ELISA, its main point is to detect impure samples and high reproducibility of data, but there is the problem of poor overall sensitivity and specificity.

Procedure of double antibody ELISA sandwich assay

Principle of ELISA experiment

EncapsulationAntigens or antibodies can be physically adsorbed on the surface of the solid-phase carrier, because the hydrophobic part between the protein and polystyrene surface adsorption, after adsorption can maintain the antigenic reaction and other immune activity.
Labeling Antigens or antibodies can be covalently bonded to enzymes to form enzyme conjugates which still maintain their immune activity and enzyme catalytic characteristics.
Color developmentAfter the enzyme conjugate binds to the corresponding antigen or antibody encapsulated in the solid phase carrier, it is also immobilized on the solid phase carrier, and the color development reaction can occur after adding the enzyme-substrate, and the relative content of the antigen or antibody can be calculated according to the color shade of the reaction.

How to choose ELISA kits?

Direct-ELISA-method
Direct-ELISA-method

Specificity: The specificity of the ELISA kit is related to the key component of the kit, the antibody pair, if one of the antibody pairs is multiple antibodies, the other must be a monoclonal antibody, it is recommended to use a double monoclonal antibody.

Sensitivity: Sensitivity reflects the ability of the kit to detect the minimum amount of the substance to be detected, the user can choose the appropriate kit according to the number of indicators to be detected in their samples, if the amount of indicators to be detected is very low, the general kit can not meet the requirements, you can choose a highly sensitive ELISA kit.

Repeatability: scientific experiments require repeatability, general ELISA kits, and a period within the plate and the interplate coefficient of variation should be controlled within 15%.

Simplicity: The shorter the experimental time and the more convenient the operation, the more likely the kit will be welcomed by the user, under the condition of ensuring high-quality data.

Common problems and solutions of ELISA

QuestionPossible causesSolution
High background or negative control is only high
Antibody non-specific binding
Enzyme conjugate is too concentrated
Incubation temperature too high
Exposure before reuse of substrate
Too long dwell time before plate reading
Negative control wells are contaminated by positive control samples
The blocking solution is not suitable, replace the blocking solution
Please check whether the enzyme conjugate is diluted according to the instructions
Check whether the incubator temperature is correct and stable
Readings should be taken within 3 minutes after addition of termination solution in a dark place, protected from light.
Do not spill wash solution out of the wells when washing
The standard curve of standard curve is good, but the sample has no detection signal
Other substances in the sample affect/obscure the detection
False low values due to HOOK effect due to high concentrations of detectors in the sample
No or very low level of detection in the sample
Appropriate dilution to reduce the interference of other substances, or serial dilution, detection recovery
Dilute the sample in appropriate multiples to keep the concentration of the test within the detection range of the kit
Set up internal reference and re-experiment
Poor repeatability
Reagent not mixed
Impurities or precipitates in the sample
cut by the pipette tip
Air bubbles in the wells
Ensure adequate mixing of reagents
Before use; centrifuge
Be careful when adding liquid
Use a needle tip to pick out air bubbles
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