ELISA，also called Enzyme-linked immunosorbent assay, it is a commonly used method for solid enzyme immunoassay. The basic ELISA principle is:
The antigen (or antibody) binds to the surface of a solid phase carrier and maintains its immunological activity.
The antigen (or antibody) is linked to an enzyme to become an enzyme-labeled antigen (or antibody), and the enzyme-labeled antigen (or antibody) retains both its immunological activity and its enzymatic activity.
In the ELISA experiment, the test specimen (antibody or antigen) and the enzyme-labeled antigen (or antibody) are reacted with the antigen or antibody on the surface of the solid phase carrier in different steps. The antigen-antibody complex formed on the solid phase carrier is separated from other substances by a washing method, and finally the amount of the enzyme bound to the solid phase carrier is proportional to the amount of the antibody or antigen to be detected in the specimen. Then, the enzyme reaction substrate is added, and the substrate is catalyzed by the enzyme to become a colored product, and qualitative or quantitative analysis is performed according to the depth of the color reaction to understand the antibody or antigen content in the sample to be tested.
Figure 1. The procedures of ELISA
Types of ELISA: According to the working principle of ELISA, it can be divided into four types: direct ELISA, indirect ELISA, sandwich ELISA and competitive ELISA. For details, please refer to https://www.cusabio.com/c-20659.html
Figure 2. Four Types of ELISA
ELISA technique is widely used to determine various antigens and antibodies. However, ELISA experiment is affected by a variety of factors, and there are certain technical requirements in its operation. Some false results (ie, false positive or false negative results) are often seen in clinical tests and scientific research.
There are three main reasons for the erroneous results of ELISA:
The effects of these three factors on ELISA assays are discussed below:
1. Specimen factor
Specimens that can be used for ELISA assays are extensive: body fluids (such as serum, plasma, cerebrospinal fluid), secretions (saliva), and excreta (such as urine, feces) can be used as specimens to determine an antibody or antigen component. Some specimens can be directly measured (such as serum, urine), while others require pretreatment (such as feces and certain secretions).
Serum is the most commonly used specimen in ELISA assay. Plasma is generally regarded as the same specimen as serum. The false positive and false negative results caused by specimens are mainly caused by interfering substances, which are divided into endogenous substances and exogenous substances:
1.1 Endogenous substances
Some studies have suggested that about 40% of human serum samples contain non-specific interfering substances, which can affect the test results in different degrees.
Common interfering substances: Rheumatoid factor, complement, heterophil antibody, target antigen autoantibody, iatrogenic induced anti-mouse Ig (s) antibody, cross-reacting substances and other substances.
◆ Rheumatoid factor
IgM and IgG-type rheumatoid factor (RF) in human serum can be directly combined with the FC segment in the ELISA system, and the FC segment can capture antibodies and enzyme-labeled secondary antibodies leading to false positives.
In the process of solidoid primary antibody and labeled secondary antibody in the ELISA system, the antibody molecule undergo allostery. The C1q molecule, a complement of the FC segment of the antibody molecule, its binding site is exposed. C1q can join the two, resulting in a false positive.
◆ Heterophil antibody
Human serum contains natural heterophilic antibodies that bind to rodent (eg, murine, etc.) Ig(s), which can link primary and secondary antibodies in an ELISA system and can also cause false positives.
◆ Autoantibodies to target antigens
The autoantibodies of target antigens, such as anti-thyroglobulin and anti-insulin sometimes bind to a target antigen to form a complex, which can interfere with antigen-antibody assay results in an ELISA method.
◆ Iatrogenically induced anti-mouse Ig (s) antibody
The clinical use of monoclonal antibodies such as mouse-derived CD3, radio-isotope labeling of mouse-derived antibody imaging diagnosis, targeted therapy, and other new technologies may lead to the production of anti-mouse antibodies in these patients. In addition, anti-mouse Ig (s) antibodies can also be produced in patients who are bitten by rodents such as rats. These patients can produce false positives when tested by ELISA.
◆ Cross-reactive substance
Digoxin-like, AFP-like substances, etc, can cross-react with the target antigen. When the antigen is measured by the polyclonal antibody, the measurement results are not greatly affected. However, when an antigen is determined by a monoclonal antibody, a false positive result may occur if the cross-antigenic determinant is exactly the target determinant corresponding to the monoclonal antibody used.
◆ Other substances
Excessive serum lipids, bilirubin, hemoglobin, and excessive blood viscosity all have an interference effect on ELISA results.
1.2 Exogenous substance
The effects of exogenous substances are often caused by improper collection and storage of blood samples used for ELISA determination. Such as specimen hemolysis, specimen contamination by bacteria, specimen storage for too long, incomplete agglutination of specimens and the influence of the addition in blood collection tube.
◆ Specimen hemolysis
The hemolysis of specimens caused by various human factors can release a large amount of hemoglobin with peroxidase activity when red blood cells are destroyed. In an ELISA assay labeled with horseradish peroxidase, non-specific coloration is caused, which interferes with the assay results. In order to overcome the above interference effects, the specimen must be collected to avoid hemolysis.
◆ Specimens are contaminated by bacteria
Since the bacteria may contain endogenous horseradish peroxidase, specimens contaminated with bacteria, like the hemolyzed specimens, can also produce non-specific coloration and interfere with the measurement results.
◆ Improper preservation of specimens
Specimens that have been stored in the refrigerator for a long time, IgG in serum can be polymerized into multimers, and AFP can form dimers, which can lead to excessive ELISA background and even false positives in indirect ELISA. Specimens are placed for too long (for more than one day), sometimes immunoreactivity of antigen or antibody is weakened, and false negatives may occur.
In order to overcome the above interference, serum samples determined by ELISA should be freshly collected. If not immediately determined, serum samples measured within 5 days can be stored at 2-8 ° C, -20 °C no more than 1 month, -80 °C no more than 2 month. Specimens dissolved after cryopreservation, the protein is partially concentrated and unevenly distributed. The protein should be mixed thoroughly before determination. However, it should be gentle when mixing, and should not be strongly oscillated.
◆ Incomplete agglutination of specimens
In the absence of procoagulant and anticoagulant, normal blood begins to clot at 0.5 to 2 h after collection and completely coagulates at 18 to 24 h. If the serum is centrifuged when the blood is not completely coagulated, some of the fibrinogen remains in the serum, and a fibrin block visible to the naked eye can be formed during the ELISA measurement, which is likely to cause false positive results. Therefore, after blood sample collection, serum must be isolated after sufficient coagulation, or the specimens are collected with a blood collection tube with a separation gel or a suitable coagulant added to the blood collection tube.
◆ Effect of added substances in the specimen tube
Anticoagulants (such as heparin), enzyme inhibitors (such as NaN3 can inhibit horseradish peroxidase activity in ELISA system), and rapid serum separation gels can interfere ELISA determination.
In summary, the false positive or false negative results in the ELISA assay should be analyzed from the specimen factors before considering the reagent factors and operational factors, and corresponding measures should be taken to eliminate the interference, so as to obtain correct and reliable results.
2. Reagent factor
At present, the quality of ELISA kits on the market varies greatly. The quality of the kits will seriously affect the accuracy of the ELISA test results. So how to choose the right kit is very important, you can refer to it: 11 tips for choosing your right ELISA kit.
The uses of ELISA kit, such as the detection range, test samples, application species; the quality control of the kit, such as specificity, sensitivity, and reproducibility; the customer verification of the kit, such as literature references, as well as the expiration date of the kit, is a major factor we need to consider.
2.1 Detection range
The detection range of the kit is the range of the standard curve. Pay special attention to whether the concentration of the sample to be tested falls within the range of the standard curve. For samples with high concentrations, you can perform a preliminary experiment to find a good dilution factor and then measure the sample in large quantities. Prior to this, we can compare the sample values given in the ELISA kit with the reference values of protein expression given in the NCBI literature, uniprot or pax-dab.
2.2 Application species
In general, ELISA kits of different species are not universal, except as specified in the kit.
2.3 Test sample
For ELISA kits, common sample types to be tested are: serum and plasma (different anticoagulants), cell supernatants, and cell lysates. In the ELISA kit instructions, a detailed description of the type of sample to be tested and the diluent is given, please note that the dilutions of different samples are not mixed.
The specificity of the ELISA kit is related to the key component of the kit, the antibody (pair). The kit is used to screen targeted antibody materials, which are strictly controlled to ensure the specificity of the kit.
The ELISA sensitivity reflects the ability of the kit to detect the minimum amount of the substance under test. You can select the appropriate kit according to the amount of indicators to be tested in your sample. If the amount of the indicator to be tested is very low, the general kit cannot meet the requirements, you can choose high-sensitivity ELISA kit.
Scientific experiments focus on repeatability. For general ELISA kits, the intra- and inter-plate variation coefficients should be controlled within 15%. In CUSABIO ELISA kit, the coefficient of variation within most plates is less than 8%, and the coefficient of variation between plates is less than 10%.
2.7 Literature citation
Product literature citations, especially those of high-quality SCI articles, are a matter of concern to many customers. This is a very important indicator for product recognition, and it has certain reference function for the research of related users. The customer reference literature of CUSABIO ELISA kit has reached more than 4,500, and the number is increasing by hundreds every year.
2.8 Validity period
In general, the kit is valid for half a year, and the validity of the kit needs to be considered when preparing the sample.
3. Operating factor
ELISA technology has high sensitivity and specificity, and has been widely recognized in biological research, and is also widely used in clinical testing. For beginners, if you do not pay attention to all ELISA steps in the process of experimental operation, it may have a relatively large impact on the final experimental results, such as the white plate, weak color, low sensitivity, flower plate phenomenon, no gradient and high ELISA background. Specific problems and solutions that may arise, you can refer to: Do you often trouble in these problems of ELISA?
In summary, we should strictly follow the ELISA protocol in the ELISA experiment, and pay attention to the following issues:
a. Reagent preservation: Protein antibody reagents can be stored at -20 ℃when not in use for a short period of time, and the wash solution of color-substrate solution is stored at 2-8 ℃.
b. Try to avoid air bubbles in the micropores when terminating the reaction.
c. The microplate reader reading should be completed within 5 minutes after the termination of the reaction.
3.1 Prepare reagents in advance
a. The pre-coated enzyme label slabs and each reagent component need to be equilibrated at room temperature for more than 30 min.
b. Reagents should be thoroughly mixed before use to ensure the uniformity and accuracy of reagents.
c. Common washing liquid is a concentrated liquid, which needs to be diluted in advance, and the pH value should be kept between 7.2 and 7.4.
d. The general detection antibody and the enzyme-labeled secondary antibody need to be diluted to the working concentration with the corresponding dilution.
e. Reagents of different batches cannot be mixed and the validity of each component should be checked.
3.2 Add the test sample
a. If there are too many samples to be tested, it is recommended to operate in batches.
b. The samples that need to be diluted shall be diluted in advance. Please refer to the instruction manual to select the appropriate diluent.
c. Control the sample loading time to avoid errors caused by too long sample loading time.
d. Pay attention to the angle of adding sample. Add to the bottom of the plate vertically, and do not touch the ELISA plate wall.
e. One sample, one pipette tip. Prevent cross contamination.
3.3 Incubation and washing
a. Incubation time and temperature are carried out according to the instructions in the kit.
b. Incubate in a water bath or wet box to avoid “edge effects”.
c. Pay attention to the number of washings, the amount of washing liquid, the length of time the washing solution is soaked, the strength of washing.
d. When hand-washing the ELISA plate, make the plate vertical to avoid cross contamination; the force cannot be too strong to prevent the antigen-antibody complex from disengaging.
e. When washing the plate with the washing machine, always check if the flushing head is unobstructed.
3.4 Color development
The color reaction needs to be protected from light and reacted at 37 °C or room temperature for 15 to 30 minutes.
3.5 Reading and data analysis
Try to avoid air bubbles in the micropores when terminating the reaction.
The microplate reader reading should be completed within 5 minutes after the termination of the reaction.
The microplate reader should be preheated for 15 to 30 minutes in advance.
Data analysis is generally performed using four parameters and straight line fitting method; the correlation coefficient (R2) of the standard curve can be used to determine which fitting method to choose. R2 generally needs to be larger than 0.99, you can click here to view the ELISA data analysis method.
3.6 Reagent preservation
a. Proteins and antibody reagents not used in the short term are kept at -20 ℃.
b. The coloring solution and washing solution are stored at 2-8 ℃.