How to Use Multiplex Immunoassays Effectively?


What is an Immunoassay?

Immunoassay is a biochemical test introduced in the early 1960s which is used to check the presence of antigen or antibody in the sample, which may be body fluids or blood. 

There are numbers of applications of an Immunoassay. Mostly immunoassay is used to study the antibodies, proteins and hormones. 

In the food industry, it is used to detect the contamination in food, water and monitor certain molecules during the processing of the products.

Immunoassays are a broadly accepted tool in laboratory, hospitals, clinical diagnosis and research studies to enhance the health and well-being of humans.

Conventional immunoassay has more advantage but also have many drawbacks like the cost is more, requires more labour, time-consuming, and requires a large number of samples. 

This led to the discovery of Multiplex Immunoassays. Roger Ekins, a British biophysicist (in 1989), described microarray technology principles. This leads to the opening of new opportunities in the field of diagnostic and research.

What is Multiplex Immunoassays?

One of the most efficient and widely used classical serological tests for antigen or antibody detection is the Enzyme-Linked Immunosorbent Assay (ELISA) test. 

The standard ELISA test allows you to obtain 1 test and 1 answer. ELISA test depends on an enzymatic reaction.

The ELISA test is a very flexible test, healthcare people and laboratory technicians can easily perform the test in the laboratory. 

The blood tests of ELISA can be performed to detect the hormones, allergens, antigens, bacterial antigens for Tuberculosis, COVID-19, Hepatitis B antigens, and other infectious diseases.

The most innovative approach of immunoassay is the Multiplex Immunoassays which is used to analyze multiple analytes together at the same time in a single experiment using coloured magnetic beads. 

Using this immunoassay, a range of biomarkers can be identified and quantified for health and disease.

Multiplex Immunoassays allows you to obtain in 1 test up to 100 different answers. It depends on fluorescent probes.

In this immunoassay, very less quantity of the sample volume (25–50 µL) is required to run the test multiple markers at a very short time. That is why this method of immunoassay is beneficial for precious expensive samples.

The coloured magnetic beads allow for the simultaneous detection of multiple analytes in a single well or reaction.

The idea of Multiplex immunoassay for diagnostics purposes was conceived in the year 1963 by A.W. Wheeler and Joseph G. Feinberg.

Working of Multiplex Immunoassays?

The technology facilitates multiplex immunoassays in which one antibody to a specific analyte is bound together to a set of magnetic beads with the identical colour, and the second antibody to the analyte is impregnated with the dye mixture.

The different coloured magnetic beads enable multiplex detection at the same time for many other analytes in the same sample. 

Immunoassay detection scheme

A double detection flow cytometer categorizes the different assays by the colour of the beads in one channel and determines the concentration of the analyte by measuring the reporter dye fluorescence in another channel.

Bead readers or high‐resolution scanners can be used efficiently in this technology. The contents of each microplate well are drawn and dyed beads are pushed through a detection chamber in a single file or magnetically immobilized. 

A flow cytometer is used for data acquisition. It sends a stream of coloured magnetic beads through the laser beams like one bead at a time and the beads are stimulated individually by two laser beams. One laser is red and the other is the green colour.

The red colour classification laser (635 nm) stimulates the dyes in each bead, the spectral address gets identified. The green colour reporter laser (532 nm), stimulates the reporter molecule associated with the beads, which allows to measure the concentration of the analyte.

The latest multiplex immunoassay is the Invitrogen™ Cytokine Human Magnetic 35-Plex Magnetic multiplex kit. It measures 35 analytes all together with just 25 µL of blood plasma, and serum in just 4 hours.

12 Application of Multiplex Immunoassays

  1. The antigen can be selected among a large number of different possibilities
  1. Screening for relevant biomarkers of a given disease.
  1. Monitoring therapy and vaccine efficacy.
  1. Reduce costs: Multiplex immunoassays are with reduced cost than singleplex ELISA. It has greater output per sample, and the higher output predicates more resolute, detailed diagnostics and enable personalised medicine. 
  1. It is used to study in details of antibodies, proteins and hormones.
  1. To detect malaria antibodies in the blood samples of the patients.
  1. Saves time: Multiplex Immunoassays helps scientists and researchers to gather more information and results in a very short time.
  1. Multiplex Immunoassays is useful for the detection of nucleic acids.
  1. Require less sample: Very less quantity of the sample volume (25–50 µL) is required to run the test multiple markers. Thus, this method of immunoassay is useful for precious and expensive samples.
  1. Multiplex Immunoassays is perfectly designed for the diagnosis of the disorders of the immune system (allergies, and autoimmune diseases).
  1. COVID-19 kit, named TaqPath, is a multiplex immunoassay real-time RT-PCR diagnostic kit which allows hospitals, public health laboratories and clinics to quickly diagnose COVID-19. 

It displays the result very fast and is a highly sensitive multiplex diagnostic for detection of SARS-CoV-2 virus.

  1. New pediatric vaccines that are administered during the first few months after birth, which evoke immune responses are evaluated.

The bottom line:

Nowadays, increasing awareness of the combined nature of pathological states and various diseases like sepsis, cancer, etc. calls for concurrent, cost-effective and time‐saving measurement of multiple analytes.

Research companies like Invitrogen, Perkin Elimer, Bio-Rad, Qiagen, Millipore, EMD, Origene, started their diagnostic kits globally. 

These diagnostic kits are based on Multiplex Immunoassays which are extensively used for clinical diagnostics, drug discovery, drug analysis, biomarker discovery, infectious disease, biomedical research, and HLA testing.

Lately, Bio-Rad company develop a multiplex immunoassay named 53-plex. It measures 53 different variety of protein all together in the sample. This helps the researcher for better diagnose of the disease.

Multiplex immunoassay will help to speed up the development of new drug and discover new possibilities.

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