Working Of Elisa Kits 

Working Of Elisa Kits 

According to one market research report, ELISA (Enzyme-linked immunosorbent assay) kits dominate the immunoassay market with a 60% market share. An immunoassay is a highly selective bioanalytical technique that detects the presence or measures the concentration of target immune system components using biorecognition.  

ELISA or enzyme immunoassay (EIA) kits are vital components in disease diagnostics and seroepidemiology (surveys conducted to determine how infectious diseases impact specific populations). Although other immunoassay kit types are available, ELISA kits are preferable for their high sensitivity and specificity. Therefore, click on ELISA test kits to discover how they work or learn more below. 

What Is An Elisa Kit? 

An ELISA or EIA test is one Immunoassay tests can detect a target molecule within a crude mixture, isolate a target molecule from an organism’s fluid sample, and measure the volume of the target molecule. Such tests also use reporter/detection systems and separation systems to quantify the target molecule in an analyte.   

However, immunoassay tests vary in innate analytical capacities because they apply unique principles to yield an immunoassay report. For instance, ELISA immunoassay testing kits build on the principle of ELISA. The principle of ELISA leverages the highly selective antigen-antibody interaction exhibited during immunological responses to test analytes. Below is a breakdown of the ELISA principle and its application in ELISA test kits. 

The Principle Of Elisa 

As stated earlier, immunoassays detect and measure the presence and volume of immune system molecules in an analyte like blood and other bodily fluid samples. Antibodies and antigens are the immune system molecules that the principle of ELISA exploits to yield immunoassay reports. 

The relationship between antibodies and antigens stems from their recognition capacity during an adaptive immune response. An adaptive immune response occurs when disease-causing pathogens like bacteria and viruses (antigens) enter the body. When the body detects such pathogens, it activates special immune cells (B cells) that release corresponding antibodies to neutralize the invading antigens. 

Antigens weaken the body by attaching themselves to healthy cells causing cell damage that affects physiological functions. Therefore, antibodies bind to the antigens to prevent the latter from attacking and killing healthy cells. Antigens feature a particular binding site called an epitope, while antibodies feature a corresponding binding site called a paratope. 

The epitope and paratope on corresponding antigens and antibodies have a high affinity and form covalent bonds. Therefore, the principle of ELISA relies on these covalent bonds and the recognition capacity antigens and antibodies exhibit to detect antigen presence in an analyte. 

ELISA test kits feature healthy plates with antigens or antibodies that function as biorecognition agents. The most common ELISA well tissue culture plates are the 96-well and the 384-well plates; the primary difference between the two plates is the volume. 

Commercially-produced ELISA kit types, including direct ELISA, direct ELISA, sandwich, and competitive ELISA kits, are available, but researchers can design custom kits. Nonetheless, the well plates in commercial and custom testing kits feature a coating of immobilized enzyme-conjugated antibodies. Conjugated enzymes constitute the detection system in ELISA kits. 

Besides the ELISA principle, ELISA test kits utilize a specific protocol to test for antibodies or antigens. Below is an overview of the ELISA protocol. 

The Elisa Protocol 

The ELISA protocol summarizes the step-by-step procedure for running an ELISA assay. Below is an overview of each step. 

Coating 

The coating is the first ELISA kit preparation stage. It entails adding a diluent buffer to an antigen or antibody and leaving the solution to incubate overnight. The diluent or coating buffer maximizes the biorecognition body’s absorption.  However, some commercially-produced ELISA kits come pre-coated, allowing you to skip this preparation step. 

Washing 

Rinsing with a washing buffer occurs after the biorecognition agents incubate into the well plates. The rinsing removes the excess or unadsorbed mixture from the well plates. 

Capture Antibody 

The incubation of a second antibody called a capture antibody specific to the target antigen on the well plate post-detection antigen adsorption is possible. However, the caption antigen is specific to the sandwich ELISA test kit. A rinsing step is necessary to rid the plate of excess capture antibodies. 

Blocking 

Blocking entails using blocking buffers, protein solutions like casein and aprotinin, to passively absorb into the unoccupied portions of the ELISA well plate. The protein buffers diminish background interference, enhancing the test’s sensitivity. Another rinsing stage is also necessary to eliminate excess blocking protein solution. 

Analyte/Sample Fluid Incubation 

Incubating the well plates with the sample fluid (blood, urine, saliva) allows the incubated antibodies to recognize the target antigens within the analyte sample. The incubation duration depends on the ELISA test type. A thorough washing step is necessary once the demarcated incubation duration lapses to eliminate unbound elements away from the incubation plate. 

Conjugated Enzyme Incubation 

Conjugated enzymes used in assay tests, like horseradish peroxidase (HRP), oxidize a substance to release a quantifiable color change, luminescence, or fluorescence. High intensity in the color change, luminescence, or fluorescence indicates that the capture antibodies arrest a large target antigen volume.   

Detection 

Detection entails using a plate reader to map out the target antigen presence within the analyte sample. The plate reader typically features a negative control to measure background noise and a positive plate to amplify the signal.  

Different ELISA kits have varying signal-to-noise ratios and low limit detection levels. Therefore, consider using kits with a high signal-to-noise ratio and the lowest limit detection levels. 

Conclusion: 

While all ELISA test kits apply the principle of ELISA, the ELISA protocol steps, test sensitivity, and specificity may vary slightly. Therefore, due diligence is necessary to identify a test kit that best meets your assay needs.