Inside an ELISA Lab: Understanding the Technology Behind Diagnostics

For centuries, doctors have been using laboratory examinations to confirm disease diagnosis. Before the discovery of tools such as microscopes, doctors conducted macroscopic examinations by observing multiple specimens with bare eyes. However, macroscopic investigations cannot reveal changes at the microscopic levels, such as the form and type of parasitic cells. After the discovery of the light microscope in 1590, the magnification of small objects paved the way for research and discoveries in medicine, biology, and other sciences. With time, methods for studying laboratory samples have evolved. 

In microscopic examinations, the ELISA method has emerged as a significant solution. The discovery of ELISA analysis for substances in complex biological matrices has now become a routine method in research and diagnostic laboratories. The current article discusses ELISA immunoassays and the technology behind it.

Understanding ELISA labs and the technology in ELISA assay development

The ELISA assay was first used to determine IgG antibody levels in rabbit blood serum. This method was eventually patented, after which it was widely used by the scientific community. Over time, ELISA assays were used for multiple applications, such as to diagnose malaria, identify trichinosis, and infections caused by parainfluenza, influenza, and mumps viruses. Later, ELISA analyses were used to identify and quantify compounds such as proteins, peptides, and hormones in complex study samples. Today, ELISA assays have advanced applications such as hepatitis screening, virus antigen testing, parasite analysis, hormone level measurement, ADA study, etc.

ELISA analysis involves two working principles: antigen-antibody reaction and enzyme and substrate reactions. These reactions take place in a microplate well. The well is coated with specific antibodies specific to the target antigen. When a sample with the target antigen is added to the microplate well, the antibodies react with the antigen and form an antigen-antibody complex. However, samples may contain other components that are eliminated through washing steps. The antigen-antibody complex is complexed with secondary antibodies conjugated with an enzyme label. The antigen-antibody complex is determined by adding a substrate to the enzyme, which reacts and develops a color product. Finally, a stop solution such as HCl or NaOH is added to stop the reaction, and the absorbance is measured using a plate reader.

There are three primary ELISA assay types

1. Direct ELISA: in this method, the antigen directly binds to the enzyme-labeled antibody. This approach is employed to determine antigen concentrations.

2. Indirect ELISA: in this format, instead of direct binding of enzyme-labeled antibody, the target antigen first binds to a primary antibody. This method is ideal for determining the total antibodies in the study sample.

3. Sandwich ELISA: unlike direct and indirect methods, sandwich ELISA coats the assay well with capture antibodies. These antibodies capture the target analyte and are then attached by direct and indirect antibodies, capturing the target analyte between two antibodies. This format can measure antigen concentrations in complex matrices such as cytokines and hormones.

ELISA assays are easy and rapid. They have multiple advantages, such as:

• High efficiency

• Simple procedure

• High sensitivity and specificity

• Environmentally friendly and safe

However, they do have certain limitations, such as:

• Antibody instability

• Possibility of false negative or false positive results

• Need expensive culture media and complex techniques

• High antibody preparation cost

• Need storage and transportation at cold temperatures

In ELISA aalysis, it is critical to verify qualitative and semi-quantitative results before using them for healthcare decisions. The laboratory must have a quality control system for ELISA analysis. Besides, before establishing an ELISA program, laboratories should set policies, assign responsibilities, train staff, and ensure resource availability. Ensure that all quality control data is recorded and reviewed by the quality control manager and lab director.

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Positive and negative controls are necessary for all bioanalytical tests, including protocols with endpoints such as color change or agglutination. These controls should be employed with each assay run. Moreover, study controls help validate new kit lots or reagents, evaluate testing protocols, and check storage conditions and temperatures.

However, while using traditional negative and positive controls, researchers should evaluate the control materials in the same manner as patient samples. They should use control samples, ideally once each day or as recommended by the guidelines. Also, choose positive controls close to cut-off values to ensure the assay detects weak positive data points.

Conclusion

Since the initial development of ELISA assays, it has undergone massive changes and alterations, giving rise to complex and advanced formats such as multiplexed ELISA assays. These modifications and advances have significantly contributed to biological research, medicine, disease diagnosis, and other scientific domains.

ELISA analysis has multiple advantages, such as high efficiency, sensitivity, and simple protocols. However, they do face challenges. Nevertheless, the ELISA method is one of the most common and robust laboratory techniques with applications across health monitoring, disease diagnosis, and research. With advances in technology and sample preparation, the ELISA method will continue to grow and evolve and help us understand core concepts in biomedical and bioanalytical sciences.