Homogeneous v/s Heterogeneous Immunoassays

Immunoassays are biochemical tests used to detect the presence or concentration of an analyte in a biological sample via an antibody-antigen reaction. They are widely used to perform screening and semi-quantitative analysis across various fields. These assays offer high sensitivity and specificity by using the unique binding affinity between antibodies and target molecules.

Depending on the assay design, immunoassays can provide qualitative, semi quantitative and fully quantitative results. Immunoassays are also classified based on their format – Homogeneous and Heterogeneous immunoassays. 

While both type of immunoassays have their own characteristics, methods, and applications, which one should you use depends on the context of use. That is why understanding the difference between heterogeneous and homogeneous immunoassays is important. In this guide, we will discuss the same.

What are Homogeneous Immunoassays?

Homogeneous assays can yield accurate read-outs without having to separate the analyte of interest from the biomolecules used to detect it. 

In homogeneous assays, the binding of the analyte to the antibody leads to a measurable change, such as fluorescence, absorbance, or luminescence. The signal is generated only when the antibody-antigen interaction occurs, making the process straightforward and fast.

There are several types of homogeneous immunoassays are used across clinical diagnostics, pharmaceutical research and more, including EMIT, FPIA, CEDIA, LOCI, Nephelometric and Turbidimetric, etc.

What are Heterogeneous Immunoassays?

Heterogeneous assays require multiple steps to separate unbound antibodies. This makes the overall process relatively complex, but this separation improves sensitivity and specificity, which is why these assays provide more precise results. 

These assays usually involve immobilizing either the antigen or antibody on a solid phase (such as a microplate, membrane, or magnetic bead). After the target analyte binds, unbound reagents are washed away, and the detection step follows using enzyme labels, radioactive markers, chemiluminescence, or fluorescence.

Different types of heterogeneous assays are used for clinical diagnostics, biomarker detechtion, infectious disease screening, and more. These assays include ELISA, RIA, CLIA, ECLIA, Wester Blot, IHC, and more.

Difference Between Homogeneous and Heterogeneous Immunoassays

Separation step

Homogeneous Assay - EMIT

EMIT stand for Enzyme Multiplied Immunoassay Technique is a homongeneous assay that is used to detect the presence or the quantity of analyte. It is primarily used in drug testing, therapeutic drug monitoring, and clinical diagnostics.

In EMIT testing kits, the analyte competes with an enzyme-labeled analyte for binding to a specific antibody. When the antibody binds to the enzyme-labeled analyte, it inhibits the enzyme activity. 

Conversely, when the free analyte is present in the sample, it competes for the antibody, leaving more enzyme-labeled analyte unbound and enzymatically active.

The reaction takes place entirely in solution, and the change in enzyme activity directly correlates with the concentration of the analyte. This results in a measurable signal, such as a change in absorbance.

Heterogeneous Assay - ELISA

ELISA stands for Enzyme Linked Immunosorbent Assay is a heterogeneous immunoassay that is used to identify the presence or quantity of an analyte. There are 3 types of ELISA - indirect, sandwich and competitive. 

In ELISA testing kits, the analyte or antibody is immobilized on a solid surface, usually the well of a microtiter plate. When the sample is added, the target analyte binds to the immobilized capture antigen or antibody.

After this, the separation step is performed, which makes ELISA a heterogeneous assay. This step involves washing the plates multiple times to remove any unbound reagents, antibodies, and proteins.

After separation, the enzyme-linked detection antibody is added, which binds to the analyte. A substrate is then introduced that reacts with the enzyme and produces a detectable signal. 

The Bottom Line

Now that you know the difference between homogeneous and heterogeneous assays, what are you waiting for? Choose the assay type for your research based on your goals. And if you want to order high-quality ELISA kits, find a reliable supplier like AAA Biotech.