Chemiluminescence Immunoassay (CLIA)
In the presence of complimentary antigen and antibody, the paratope of the antibody binds to the epitope of the antigen to form an antigen-antibody or an immune complex. Estimating the levels of such immune complex by use of labeled antibodies form the basis of CLIA. It involves use of stationary solid particles coated either with the antigen or antibody of interest. Post incubation, which ensures intact immune complexes are formed, substrate is added. This results in generation of light, the intensity of which is directly proportional to the amount of labeled complexes present and which indirectly aids in quantification of the analyte of interest. The intensity of light is measured in terms of Relative Light Units (RLU).
The main advantage of this technology includes sensitivity and its ability to be unaffected by background signals. Also, the analyzers working under this principle are simple in design and operation.
Enzyme Linked Immunosorbent Assay (ELISA)
ELISA in an immunoassay which majorly deals with analyzing antigen-antibody complexes using enzyme labeled antibodies in a 96 well microtitre plate. The commercial plates are pre-coated with specific antigens, and on addition of patient serum, antibodies if present against the antigen, will bind and form immune complex. Post incubation and washing, a secondary labeled antibody is added, followed by substrate to aid in color development. The intensity of the color can then be measured using an ELISA plate reader.
The main advantage of this technique involves, its versatility to be designed and used for multitude of infections diagnosis. The tests can also be performed both in qualitative as well as quantitative fashion requiring simple instruments.
High Performance Liquid Chromatography (HPLC)
HPLC is an automated version of column chromatography, which involves use of a stationary phase in the form of a column, a mobile phase, complete with a pump and a detector. The sample is injected within the column and mixed with the mobile phase followed by being pumped under high pressure. The analytes in the sample mixture interact with the stationary phase within the column differently depending on their chemical nature. Some might be retained for a longer time as compared to others and will be hence eluted at a later stage. Finally, all the eluted components are recorded by the detector and expressed in the form of a chromatogram. This chromatogram depicts each analyte within the mixture in the form of a peak plotted against the retention time (RT). The area under the curve (AUC) is generally depictive of the concentration of the analyte.
The main advantage of this technique is the platform is open and can be exploited to develop and standardize any protocol of interest. The sensitivity is very high, and with an appropriate choice of mobile phase, column and detector, multiple analytes can be identified and quantified in a single assay run.
Ion Selective Electrode (ISE)
This technology involves use of a pH meter or an ion selective probe to identify and quantify each single dissolved ion in the solution. It works on the principle of Nernst equation and can measure both positive as well as negative ions in a solution without being affected by any interference of other dissolved components.
The major advantage include ease of operation and the cost associated is cheap as compared to another analytical platforms which can also be used.
Nephelometry / Immuno Turbidity Metric
This technology is popularly used for quantification of immunoglobulin concentration in the sera of patients. During reaction, antigen-antibody complexes are generated in solution which is then quantified with the aid of a light beam. The light when incident on the immune complex, is scattered which is captured by lens fit at right angles to the incident light. This scattered light is then measured to determine concentration of the analyte of interest. This is popularly used for estimation of immunoglobulins like IgG, IgM, IgA, etc. which aids in diagnosis of allergy and autoimmune disorders.
The basic principle of this technology involves measurement of quantity of light absorbing analyte in a solution. This can only be however applied to solutions which follow the Beer Lambert’s law. Analytes which have the tendency to absorb light, when exposed to a beam of incident light, will absorb some. This results in reflection of a light of lower intensity. The intensity of the reflected light is then considered inversely proportional to the concentration of the analyte of interest in the solution.
The main advantage of this technology involves the ease of operation and the wide variety of parameters which can be covered by this assay.