Enzyme-linked immunosorbent assay (ELISA) is a labeled immunoassay that is considered the gold standard of immunoassays. This immunological test is very sensitive and is used to detect and quantify substances, including antibodies, antigens, proteins, glycoproteins, and hormones. The detection of these products is accomplished by complexing antibodies and antigens to produce a measurable result. An antibody is a type of protein produced by an individual’s immune system. This protein type has specific regions that bind to antigens. An antigen is a protein that can come from some foreign source and, when bound to an antibody, induces a cascade of events through the body’s immune system. This interaction is utilized in ELISA testing and allows for identifying specific protein antibodies and antigens, with only small amounts of a test sample. ELISA testing is used to diagnose HIV infection, pregnancy tests, and blood typing, among others. This article will discuss the basic principles, procedures, and clinical significance of the ELISA.
Etiology and Epidemiology
Two different research teams invented the direct ELISA simultaneously by scientists Eva Engvall and Peter Perlman and by Van Weemen and Schuurs. The ELISA was developed by the modification of the radioimmunoassay (RIA). This was done by conjugating tagged antigen and antibody with enzymes rather than radioactive iodine 125. The new method was first employed by determining the levels of IgG in rabbit serum. Within the same year, scientists were able to quantify human chorionic gonadotropin in urine by using horseradish peroxidase. Since then, the ELISA method has been used in many different applications and became a routine laboratory research and diagnostic method worldwide.
The first ELISA methodology involved chromogenic reporter molecules and substrates in generating observable color change that monitors the presence of antigen. Further advancement in the ELISA technique leads to the development of fluorogenic, quantitative PCR, and electrochemiluminescent reporters to generate signals. However, some of these techniques do not rely on using enzyme-linked substrates but non-enzymatic reporters that utilize the principle of ELISA.
The latest development, in 2012, was an ultrasensitive enzyme-based ELISA that manipulates nanoparticles as chromogenic reporters. This technique can generate a color signal visible by naked-eye, with blue color for positive results and red color for negative results. However, this method is qualitative and can determine only the presence or absence of an analyte and not its concentration.
Specimen Requirements and Procedure
ELISAs are performed in polystyrene plates, typically in 96-well plates coated to bind protein very strongly. Depending on the ELISA type, testing requires a primary and/or secondary detection antibody, analyte/antigen, coating antibody/antigen, buffer, wash, and substrate/chromogen. The primary detection antibody is a specific antibody that only binds to the protein of interest, while a secondary detection antibody is a second enzyme-conjugated antibody that binds a primary antibody that is not enzyme-conjugated.
There are four main general steps to completing an ELISA immunoassay. These steps are:
- Coating (with either antigen or antibody)
- Blocking (typically with the addition of bovine serum albumin [BSA])
- Final read
Detection is carried out by the addition of a substrate that can generate a color. There are many substrates available for use in ELISA detection. However, the most commonly used horseradish peroxidase (HRP) and alkaline phosphatase (ALP). The substrate for HRP is hydrogen peroxide and results in a blue color change. ALP measures the yellow color of nitrophenol after room temperature incubation periods of 15to 30 minutes and usually uses P-Nitrophenyl-phosphate (pNPP) as its substrate.
Between each of the above four steps is a “wash” of the plate using a buffer, such as phosphate-buffered saline (PBS) and a non-ionic detergent, to remove unbound material. The wells are washed two or more times during each wash step, depending on the specific protocol being followed.
In the ELISA protocol, usually, a serial dilution of concentrations is placed in the wells of the plate. After the results are measured, a standard curve from the serial dilutions data is plotted with a concentration on the x-axis using a log scale and absorbance on the y-axis using a linear scale.
There are four major types of ELISA:
- Direct ELISA (antigen-coated plate; screening antibody)
- Indirect ELISA (antigen-coated plate; screening antigen/antibody)
- Sandwich ELISA (antibody-coated plate; screening antigen)
- Competitive ELISA (screening antibody)
Both direct and indirect ELISAs begin with the coating of antigen to the ELISA plates. The first binding step involves adding antigen to the plates, which is incubated for one hour at 37 degrees C or can be incubated at 4 degrees C overnight. Once the incubation step is completed, the next step is to wash the plates of any potential unbound antibody and block any unbound sites on the ELISA plate using agents like BSA, ovalbumin, aprotinin, or other animal proteins. This second step is important because it prevents the binding of any non-specific antibodies to the plate and minimizes false-positive results. After adding the buffer, the plate is rewashed, and a selected enzyme-conjugated primary detection antibody is added. The plate is further incubated for one hour.
In a direct ELISA, the primary detection antibody binds directly to the protein of interest. Next, the plate is rewashed to remove any unbound antibody and followed by the addition of a substrate/chromophore, such as alkaline phosphatase (AP) or Horseradish Peroxidase (HRP) to the plate, which results in a color change. The color change of the sample occurs by either the hydrolysis of phosphate groups from the substrate by AP or by the oxidation of substrates by HRP. The advantages of using direct ELISA include eliminating secondary antibody cross-reactivity, and due to fewer steps, it is rapid compared to indirect ELISA. Its disadvantages include its low sensitivity compared to the other types of ELISA and its high cost of reaction.
The steps of the indirect ELISA are identical to the direct ELISA, except for an additional wash step and the types of antibody added after the buffer is removed. Indirect ELISA requires two antibodies, a primary detection antibody that sticks to the protein of interest and a secondary enzyme-linked antibody complementary to the primary antibody. The primary antibody is added first, followed by a wash step, and then the enzyme-conjugated secondary antibody is added and incubated. After this, the steps are the same as the direct ELISA, which includes a wash step, the addition of substrate, and detection of a color change.
The indirect ELISA has a higher sensitivity when compared to the direct ELISA. It is also less expensive and more flexible due to the many possible primary antibodies that can be used. The only major disadvantage with this type of ELISA is the risk of cross-reactivity between the secondary detection antibodies.
Unlike direct and indirect ELISA, the sandwich ELISA begins with a capture antibody coated onto the wells of the plate. It is termed a “sandwich” because the antigens are sandwiched between two layers of antibodies (capture and detection antibodies). After adding the capture antibody to the plates, the plates are then covered and incubated overnight at 4°C. Once the coating step is complete, the plates are washed with PBS, then buffered/blocked with BSA. The buffer washes are carried out for at least 1-2 hours at room temperature. Finally, the plate is washed with PBS once again before the addition of the antigen.
The antigen of interest is then added to the plates to bind to the capture antibody and incubated for 90 min at 37 degrees C. The plate is rewashed, and the primary detection antibody is then added to the plate and incubated for another 1 to 2 hours at room temperature, followed by a buffer wash. Then the secondary enzyme-conjugated antibody is added and incubated for another 1 to 2 hours. The plate is rewashed, and the substrate is added to produce a color change. The sandwich ELISA has the highest sensitivity among all the ELISA types. The major disadvantages of this type of ELISA are the time and expense and the necessary use of “matched pair” (divalent/multivalent antigen) and secondary antibodies.
The competitive ELISA tests for the presence of an antibody specific for antigens in the test serum. This type of ELISA utilizes two specific antibodies, an enzyme-conjugated antibody and another antibody present in the test serum (if the serum is positive). Combining the two antibodies into the wells will allow for a competition for binding to antigen. The presence of a color change means that the test is negative because the enzyme-conjugated antibody bound the antigens (not the antibodies of the test serum). The absence of color indicates a positive test and the presence of antibodies in the test serum. The competitive ELISA has a low specificity and cannot be used in dilute samples. However, the benefits are that there is less sample purification needed, it can measure a large range of antigens in a given sample, can be used for small antigens, and has low variability.
Enzyme-linked immunosorbent assays are applied in many diagnostic tests. Some of the uses of ELISA can include the following:
Detect and Measure the Presence of Antibodies in the Blood
- Autoantibodies (anti-dsDNA, anti-dsg1, ANA, etc.)
- Antibodies against infectious disease (antibacterial, antiviral, antifungal)
- Hepatitis A, B, C, HIV, etc.
Detect and Estimate the Levels of Tumor Markers
- Prostate-specific antigen (PSA)
- Carcinoembryonic Antigen (CEA)
Detect and Estimate Hormone Levels
- Luteinizing hormone
- Follicular stimulating hormone
- Human chorionic gonadotropin (hCG)
Tracking Disease Outbreaks
Detecting Past Exposures
- Lyme disease
Screening Donated Blood for Possible Viral Contaminants
Detecting Drug Abuse
Factors that can interfere with appropriate ELISA testing can occur at any phase of the testing process, beginning with specimen collection. The quality and integrity of the assay plate, coating buffer, capture antibody, blocking buffer, target antigen, detection antibody, enzyme conjugate, washes, substrate, signal detection can all interfere with proper ELISA testing. Some of the factors that can interfere in testing are the following:
- Plate Assay: the shape and quality of the wells, the material of the plate, potential pre-activation, even or uneven coating
- Buffer: pH, contamination
- Capture and detection antibody: incubation time, temperature, specificity, titer, affinity
- Blocking Buffer: cross-reactivity, concentration, contamination
- Target antigen: conformation, stability, epitopes
- Enzyme conjugate: type, concentration, function, cross-reactivity
- Washes: contamination, frequency, volume, duration, composition
- Substrate: quality/manufacturer
- Detection: instrument dependent factors
- Reader/human error
Results, Reporting, and Critical Findings
Data gathered from ELISA tests can be quantitative, qualitative, or semiquantitative. The quantitative concentration results are plotted and compared to a standard curve. The qualitative results confirm or deny the presence of a particular antigen/antibody in a sample. The semiquantitative results compare the intensity of the signals, which can compare relative antigen levels in a sample.
Once color changes are measured from the assay, the results are graphed either on paper or software. Typically, the graph compares optical density to log concentration, which gives a sigmoidal curve. Known concentrations give the graph's standard curve, and measurement of unknowns can then occur when sample values are compared to the linear portion of the graphed standard curve.
ELISAs can be used in many settings, including rapid antibody screening tests for Human immunodeficiency virus (HIV), detection of other viruses, bacteria, fungi, autoimmune diseases, food allergens, blood typing, presence of the pregnancy hormone hCG, laboratory and clinical research, forensic toxicology and many other diagnostic settings.
In HIV testing, a blood or saliva specimen is collected for testing, typically using indirect ELISA-based tests. The ELISA is a screening tool for HIV detection, but not diagnostic. Diagnosis requires further testing by Western blot due to potential false positives. Another virus, Molluscum contagiosum virus (MCV), which commonly infects the skin of children and young adults, can be detected by ELISA testing. ELISA testing in this setting is currently being evaluated for the assessment of global MCV seroprevalence.
ELISA has also been used to detect desmogleins 1 and 3 and bullous pemphigoid antigen 180 autoantibodies, which are implicated in pemphigus and bullous pemphigoid autoimmune blistering diseases, respectively. In food allergy, the evolution of the ELISA has played an important role in allergy research and diagnosis. Ultrasensitive ELISA variations have been developed to detect quantities of allergens in the scale of picograms. This is important because of the life-threatening role that food allergies can have on a public health scale.
Enhancing Healthcare Team Outcomes
ELISA testing is an important part of medical care and scientific research. Collaboration between scientists, laboratory technicians, phlebotomists, physicians, nurses, and other medical professionals is necessary for appropriate specimen collection, testing, interpretation, diagnosis, and effective patient education and treatment planning. ELISA technologies continue to grow and play a major role in clinical research allowing for the development of more diagnostic and screening tests. The continued evolution of ELISA testing is promising for the future of medicine and has allowed for the improvement of early diagnosis of HIV and pregnancy detection.