Immunoassay techniques are bio-analytical methods used for the quantitation of analyte depending on reaction with a specific antibody. These methods have wide applications in clinical research and pharmaceuticals as well. These methods are useful for quantifying a wide range of compounds including low molecular weight drugs, metabolites, macromolecular bio-molecules, and various biomarkers which act as indicative of certain diseases or infection. When it comes to pharmaceutical analysis such as diagnosis of diseases, pharmacokinetics, therapeutic drug monitoring, and drug discovery, immunoassays are of vital importance.
The Principle Of Immunoassay Techniques
Immunoassays techniques for quantification of analyte depend on the reaction of an antigen (analyte) and an antibody. The underlying principle of the technique is the competitive binding reaction between an analyte and highly-specific antibody molecules. A fixed amount of labeled analyte (labeled with radiation, fluorescence or enzymes) competes with a variable amount of unlabeled sample analyte in order to bind to a limited amount of highly specific anti-analyte antibody.
In the procedure, a mixture of immune-analytical reagents is prepared and incubated. When it binds to the antibody molecules, it generates immune complexes. The complexes are separated from unbound content with the help of a physical or chemical separation technique. Labeling activity is measured. A standard curve is plotted which represents the measured signal as a function of the concentration of the unlabeled analyte. Thus, the unknown analyte concentration is determined using this calibration curve.
Applications Of Immunoassay Techniques
Immunoassay techniques mainly radioimmunoassay (RIA) and fluroimmunoassay (FIA) have wide applications in clinical research, diagnosis, prognosis and other areas of pharmaceuticals including:
• Drug discovery
• Therapeutic drug monitoring,
• Clinical pharmacokinetics
• Bioequivalence studies radioimmunoassay-ria
• Biomarker indications or identification
What makes immunoassay a significant research and analysis tool is its inherent specificity, high-throughput, and high sensitivity for the analysis of a wide variety of analytes in different biological samples.
In clinical research, where detection of various biological samples is required, ranging from larger amounts to as minute concentrations (femtomole to attomole etc), immunoassays are the methods of choice due to their better specificity and sensitivity.
Reagents Required For Immunoassay
For an immunoassay technique, these reagents are required;
• Antibodies- the key reagents
• Signal-generating labels such as radioactive 125I, 3H, and 14C and also non-radioactive such as fluorescent probes and enzymes
• Separation matrices such as polyethylene glycol and microbeads
Additionally, specific physical or chemical methods are used. Automated methods can also be applied depending on the type of immunoassay and the availability of resources.
Types Of Immunoassays For Pharmaceutical Analysis
There are various types of immunoassay techniques depending on the reagents or labeled and also according to the certain analyte to be analyzed. A few are:
1. Radio-Immunoassay (RIA)
As the name suggests, this technique involves radioactive isotopes. Through this assay, the detection and estimation of immune molecule complexes, such as antibodies, hormones, and related substances is possible. Another similar technique is radioallergosorbent (RAST). The radioallergosorbent technique is particularly useful for the detection and estimation of allergens in the blood.
RIA methods have been widely used for the determination of a vast number of pharmaceutically significant compounds in biological fluids. Most of these techniques are now automated. Thus, there are lesser chances of man-made error. Furthermore, it saves time and gives results with great precision and extreme sensitivity.
2. Flouroimmunoassay (FIA)
Flouroimmunoassay is similar to radioimmunoassay in technique. The major difference is that it involves fluorophore labels rather than a radioisotope. FIA can be for heterogeneous or homogeneous assays, depending on the requirement of separation steps.
FIA can analyze and detect numerous pharmaceutical compounds in body fluids. These include aminoglycoside antibiotic, morphine-3-glucuronide- the key metabolite of heroin and morphine in urine. This same technique applies to the determination of the total thyroxine in human serum. One major advantage of fluoro immunoassay is that it does not involve radioactive isotopes for labeling purposes which radioimmunoassay applies.
Subtypes Of Fluoroimmunoassay
• Fluorescence Polarization Fluoroimmunoassay (FPFIA): One of the most common types of assays technique which involves fluorescent-labeled analyte. The label is excited with polarized light. Polarization signals are measured which vary according to the concentration of the analyte. Generally, the small molecules such as drugs decrease the polarization signals. On the other hand, larger molecules such as antibodies complexes tend to increase the polarization signals.
• Time-Resolved Fluoroimmunoassay (TRFIA): This is another type of fluoro immunoassay which is a highly sensitive technique and applied for the analysis of various pharmaceutical compounds in biological fluids
1. Enzyme Immunoassay (EIA)
The principle of this technique is again similar to radio immune assay, whereas the major difference is the use of enzymes for labeling rather than radioactive isotopes. Furthermore, the addition of related substrate molecules to the reaction helps in subsequent monitoring. The substrate when converted to a product often gives measurable physical or chemical differences. For instance, colorless chromogenic substrates convert into colored products by the activity of the enzyme label.
This type of immunoassay is useful for the analysis of antibiotics such as amikacin, gentamicin, cyclosporines, netilmicin and tobramycin in biological fluids.
2. Chemiluminescence Immunoassay (CLIA)
This assay technique involves a chemiluminescent substance as a label. The technique is a growing success in the pharmaceutical analysis due to its high performance and good precision.
Limitations Of Immunoassays
Despite the fact that there are numerous advantages of immunoassay techniques, they have some limitations. The underlying principle of immunoassays reaction is the affinity between the analyte and a biological antibody. This may have more inherent ambiguity than other methods employed in pharmaceutical analysis, such as chromatography.
Some of these techniques are not highly selective, and they might show similar responses to a group of compounds such as aminoglycosides, pesticides and few others. Thus, when it comes to pharmaceutical analysis, care must be taken to ensure the safety and precision of the technique used for better characterization and detection of biological substances or analytes in a given sample. No doubt, marked improvement has been achieved in the field of immunoassay development for the purposes of pharmaceutical analysis.