Immunoassays & Analyzers: Methods, Applications, & Equipment Types

The biotech industry has made major advances in developing efforts to diagnose diseases and monitor disease outcomes. Approaches that tailor treatments for each patient's care spearhead efforts to make precision medicine widely available. 

There also exists a need to ensure environmental health by monitoring pollutants and other contaminants in the environment. Both facets require tracking the concentrations of diverse molecules from samples over time.

For decades, researchers have leveraged immunoassays to quantify molecules in diverse samples. The immunoassay’s success lies in harnessing immune system proteins to measure multiple molecules accurately. 

Many kinds of immunoassays exist, along with immunoanalyzers that automate the experimental pipeline. Researchers are also improving existing immunoassays by allowing more molecules to be analyzed concurrently and precisely. Altogether, immunoassays are a powerful tool to diagnose disease and sustain the environment. 

What Is an Immunoassay?

What makes immunoassays such a powerful technique for detecting molecules? Innately, immunoassays are designed to measure the concentrations of target molecules, or analytes. Antibodies are attached to molecules called detection labels within an immunoassay. The antibodies with the attached detection labels are then called detection antibodies. These labels allow the amount of antibody that binds to a given analyte to act as a proxy for analyte concentrations.

The antibody’s binding activity originates in the immune system, acting as the foundation for all immunoassays. A mammalian immune system normally produces antibodies – Y-shaped proteins – as it encounters a foreign substance called an antigen. 

Antibodies comprise two components: the core region and the hypervariable region. The core region comprises the antibody’s backbone. The hypervariable region allows antibodies to bind to all kinds of antigens. Such binding occurs through electrostatic interactions at an antigen’s epitopes, molecular regions that the antibodies target. It’s these properties that the immunoassay harnesses to target all kinds of analytes.

How Antibodies Are Made & Used In an Immunoassay

In an immunoassay, antibodies that target analytes are produced by exposing animal models to the analytes, acting as antigens. Once the animal produces antibodies against the analyte, the antibodies are isolated and purified from the animal’s serum. Preparing antibodies for an immunoassay requires multiple steps to be optimized, including:

• Analyte purification: For an antibody to bind to a specific analyte, the animal models must only be exposed to that analyte. Ensuring that a pure analyte is produced will reduce the risk of the antibody binding to molecules other than the target analyte.
• Antibody purification: Purifying antibodies can range from crude preparations to specific preparations, but any robust method requires high-quality antibodies to be isolated to minimize non-specific binding.
• Antibody characterization: Many sets of antibodies are available for targeting specific analytes, such as those from commercially prepared kits. Determining which antibodies have been validated for your needs is an important first step in preparing a successful immunoassay.

Within these considerations, two kinds of antibodies can be prepared for conducting an immunoassay:

• Primary antibodies: These antibodies bind directly to the analytes of interest. They are mainly used in direct immunoassays to minimize the risk of non-specific binding to other biomolecules. However, this approach provides minimal signal amplification, making it difficult to detect lowly abundant analytes.
• Secondary antibodies: These antibodies address the issue of signal amplification by binding to primary antibodies. Used in indirect immunoassays, these antibodies increase the immunoassay’s sensitivity by enhancing signal strength. This same advantage also increases the risk of cross-reactivity, which occurs when the secondary antibodies bind to molecules other than the primary antibodies.

Kinds of Immunoassays

Many kinds of immunoassays exist for researchers to quantify analytes in solution. The detection label attached to the antibodies differentiates these assays from each other. The three most common immunoassays used are:

• Enzyme-linked immunosorbent assays (ELISAs): All ELISAs feature an enzymatic reaction whose activity indicates analyte concentrations. ELISAs typically use one of two enzymes whose reactions can be monitored: horseradish peroxidase and alkaline phosphatase. The former uses hydrogen peroxide as an oxidizing reagent, causing the solution to emit a blue color whose intensity indicates analyte concentrations. The latter mediates a reaction that takes off a phosphate molecule from a molecule called adamantyl 1,2-dioxetane phosphate. As the 1,2-dioxetane decomposes further degrades, the solution emits a blue color whose intensity increases with increasing analyte concentrations.
• Fluorescence immunoassays (FIA): In an FIA, the detection antibodies are conjugated with compounds called dyes that emit a fluorescent color. This emission occurs through the photoelectric effect (insert link to the confocal microscopy) after the dyes are exposed to light at the maximum absorption wavelength. Many fluorescent dyes have been attached to the detection antibodies, including fluorescein and rhodamine B. The color dye used for a given antibody can help distinguish specific analytes within the same solution.
• Chemiluminescent immunoassays (CLIA): In a CLIA, detection antibodies are tagged with a molecule that lights up after a chemical reaction. These assays are different from ELISAs because the chemical reactions take place with a catalyst that’s not an enzyme. These assays also differ from FIAs because the light is emitted not from light absorption, but from a chemical reaction. These differences allow CLIAs to have a high range of quantification and improved sensitivity for detecting analytes.

 Across each of these assays, four types can be conducted:

• Direct: Here, the antibody that binds to the analyte has a detection label attached, allowing analytes to be quantified directly.
• Indirect: Indirect immunoassays have primary antibodies that bind to the analyte and a secondary antibody that binds to the primary antibody. The detection label is attached to the secondary antibody for quantification.
• Sandwich: In a sandwich immunoassay, the bottoms of the assay wells are coated with antibodies that bind to a specific analyte. Once incubated with a sample, the bound analytes are attached to the antibodies with the detection label. This approach allows improved signal sensitivity while retaining analyte specificity.  
• Competitive: The competitive immunoassay also uses antibodies coated at the bottoms of assay wells. However, the analytes of interest are conjugated with the detection label instead of the antibody. This approach allows non-conjugated molecules and conjugated analytes to compete, ensuring that only the analytes of interest are detected.

Life Sciences Applications

Immunoassays help scientists measure the concentrations of all kinds of molecules in diverse sample types. Its flexibility has allowed researchers to apply immunoassays to enhance human health and ensure environmental health as follows:

• Diagnosing disease: Immunoassays can detect molecular regions specific to different kinds of infectious disease agents and cancers. For one, researchers have developed a modified one-step FIA for detecting influenza A and B viruses. Scientists have also developed immunoassays that detect molecules within blood that are associated with tumor emergence.
• Improving precision medicine: Precision medicine represents a growing field of research that adjusts medical treatments depending on the patient. Immunoassays push precision medicine forward by helping researchers find biomarkers associated with a person’s health status and response to treatment. For example, Thermo Fisher has recently received FDA clearance to use immunoassays to identify pregnant women with an increased risk of preeclampsia. Immunoassays can also enhance point-of-care testing by monitoring a patient’s hormone levels.
• Monitoring chemical contaminants: Immunoassays can also track chemical pollutants in the environment, from food to water. Through immunoassays, researchers can find pollutants in food that disrupt normal reproductive development by interfering with hormonal activity. Researchers can also monitor many classes of waterborne and soilborne contaminants, such as heavy metals, toxins, and drugs using immunoassays.

Advances in Immunoassay Research

Being assured of immunoassay data requires antibodies to specifically bind to their analytes. Realizing this, researchers have developed new immunoassays that improve analyte specificity while allowing hundreds of analytes to be measured:

• Somalogic: The Colorado-based company developed the SomaScan to address issues of cross-reactivity encountered in conventional immunoassays. The technology quantifies thousands of proteins from microvolumes of blood/serum with SOMAmers, single-stranded DNA reagents that bind strongly to specific analytes in solution. A special molecular label called biotin is then used to measure analyte abundances. Washing away non-target proteins bound to the SOMAmer complexes with competitor molecules ensures strong signal specificity.  
• OLink Proteomics: The Sweden-based company also reduces cross-reactivity by using an approach of its own. The proximity extension assay (PEA) employs two antibodies connected at their core regions by two DNA sequences that complement each other. When the DNA sequences hybridize, the resulting DNA is an oligonucleotide tag. Researchers can then use quantitative PCR to measure the number of oligonucleotide tags formed as a proxy of analyte concentrations.  

Immunoassay Analyzers

Scientists have also developed many types of immunoanalyzers that allow hundreds of analytes to be measured in a single run. Some of the companies manufacturing  immunoanalyzers include:

• Beckman Coulter: Beckman Coulter offers four kinds of immunoanalyzers that provide reproducible data by automating staining and data analysis workflows. These immunoassays provide varying high-throughput capacities. On the one hand, the Access 2 Immunoassay System allows 50 tests to be done per hour while ensuring portability as a benchtop machine. Conversely, the DxI 800 Access Immunoassay System allows up to 400 tests per hour, ensuring high-throughput data analysis. Each of these systems allows multiple clinical biomarkers to be detected for many kinds of diseases, from anemia to cardiac disease.
• Bio-Rad: Researchers eager to reduce reaction volumes and increase high-throughput analyses will want to try the Bio-Plex 200 system. The system measures the abundance of up to 100 analytes in a single sample. Bio-Rad accomplishes such a high-throughput rate by using 100 different color-coded bead sets that have incremental ratios of two different colors in a mixture. Each of these beads is attached to antibodies specific to a given analyte. Once the analytes bind to their respective antibodies, antibodies with a fluorescent detection label bind to the antibody-analyte complexes, allowing analyte concentrations to be measured.     
• Roche Diagnostics: Roche has tapped into the immunoassay market with its immunoanalyzer, the cobas e 601 module, which harnesses the power of electrochemiluminescence (ECL). ECL uses an electrical current to produce unstable intermediate compounds from an electrode, resulting in their excitation. These intermediates emit light once they return to a ground state after excitation.
• Siemens: The ADVIA Centaur XPT Immunoassay system allows up to 240 tests per hour without a pause for loading reagents, consumables, and samples. The system uses a class of compounds called acridinium esters to conduct chemiluminescence assays. These molecules are small compared to the enzymes used to conduct ELISAs. When cleaved, these molecules emit light that allows researchers to measure analyte concentrations. Their smaller size and mechanism of activity enable increased specificity and sensitivity for the analytes being measured.

Excedr’s Pricing Model

At Excedr, we have a unique lease pricing model for our customers; we don’t just consider your financial capabilities when leasing to your company. We also want to lease you the equipment that will most likely  help you discover and validate novel biomarkers. 

To that end, we have provided a series of factors to consider. Each factor can affect the type of equipment you will want to use, as well as the terms of your lease:

• How many biomarkers do you want to discover or assess? If you only have a small select group of markers, consider a portable immunoanalyzer such as the Beckman Coulter Access 2. If you want to assay hundreds of biomarkers in a single sample, consider leasing a Bio-Rad Bio-Plex 200 system.
• Are you looking at established or novel biomarkers? If you’re looking for the latter, consider a company that offers you the ability to develop biomarkers for your needs. Bio-Rad and OLink can provide you with custom-made panels, for instance. On the other hand, Siemens and Beckman Coulter immunoanalyzers come with a diverse menu of panels for specific diseases, from chronic kidney disease to metabolic function.  
• What kind of samples are you working with? Different sample types are distinct sets of biomolecules. Some specimens may contain compounds that reduce how effectively an antibody binds to its analyte, a phenomenon called interference. Your immunoassay protocols should consider protocols that minimize interference and maximize signal strength. Finding an immunoanalyzer that enables you to develop suitable protocols will be an important factor in ensuring success in your research.

Speak with an Excedr Representative Today

Immunoassays help scientists assess hundreds of analytes as biomarkers within a single run. They allow researchers to validate disease mechanisms , maintain environmental health, and develop therapeutics to restore patient well-being.

If you want to take a big step in biomarker development, speak with us today. We boast a wide array of immunoanalyzers that allow hundreds of analytes to be measured simultaneously. 

Use this article as a resource to learn about immunoassays and consider important factors as you decide on an immunoanalyzer. Then speak with a representative today to see how our leasing program can help you select an immunoanalyzer that suits your experimental needs. Are you interested in leasing an immunoanalyzer? Let us know!