An Insight into Laboratory Techniques
1. Electrochemiluminescence immunoassay is a highly sensitive and selective method which allows controlled chemiluminescence through a gradable electrode potential. It is versatile, simple and has good temporal and spatial control. A lot of different tests and parameters can be calculated, from picomolar sensitivity to a dynamic range of more than six orders of magnitude. The importance of ECL detection for biological tests is well established, and it is heavily used commercially for many clinical lab applications.
2. Enzyme immunoassay is commonly applied in clinical-laboratory testing. It uses a solid-phase enzyme-immuno reagent to capture antibodies in a liquid phase. When the enzyme-antibody complex is fixed over an immobilized antigen, addition of a substrate catalyses a reaction producing a colour change. The amount of colour denotes the amount of antigen immobilized, which can be read with a suitable reader. The test is highly sensitive and specific, because of the specificity of the antigen-antibody match and of the enzyme and substrate.
3. Immunoturbidimetry involves the reaction between antigen and antibody, where antigen-antibody complexes cause turbidity in the solution. This turbidity — the obstruction of light — can be measured, and corresponds to the quantitative presence of the ligand being tested. It is an important tool in the broad diagnostic field of clinical chemistry.
4. Electrophoresis is the movement of dispersed particles relative to the fluid phase under a uniformly applied electric field, due to the charged interface between particle surfaces and fluid molecules. It is used in analytical chemistry to separate molecules by size, charge, or binding affinity, and is used extensively in DNA, RNA and protein analysis.
5. Immuno-electrophoresis is based on precipitation of an antigen by an antibody. Many diseases with a prevalent monoclonal antibody titre can be qualified and quantified by this technique, e.g. Myeloma and Waldenström macroglobulinemia. The serum is deposited on a gel; on electrophoresis the proteins segregate according to size and charge, and precipitation with antigens specific to each immunoglobulin forms bands for each. This technique — immunofixation — tends to replace protein electrophoresis because it is faster (results within three hours) and somewhat more sensitive, and can reveal an immunoglobulin missed by protein electrophoresis at low concentrations.
6. Chromatography separates a mixture dissolved in a solvent (the mobile phase), carried through a structure holding another material (the stationary phase). Because the mixture's constituents travel at different speeds, they separate due to differential partitioning between the two phases. In common lab practice, chromatography is used to measure quantities and calculate proportions of different molecules in a mixture.
7. Column chromatography is a separation technique in which the stationary bed sits within a tube. Different constituents of a mixture move through the column at different rates according to their differential adsorption, allowing their separation. The technique is quite common because of the range of adsorbents and solvents available, and is relatively cheap, not requiring exceptional expertise or machinery.
8. Nephelometry is used in immunology to determine levels of several blood plasma proteins, e.g. immunoglobulins IgM, IgG, IgA — markers for certain diseases such as multiple myeloma, where quantification of free light chains can indicate disease type and treatment efficacy. Dilute solutions of small particles scatter light rather than absorbing it, and measuring that scattered light at an angle allows estimation of protein levels by comparison with a known control.
9. Haemagglutination: certain viruses cause agglutination of blood cells — the viral envelope protein adsorbs onto the red blood cell membrane, causing cells to clump together into visible clumps, allowing visual evaluation and helping inferential diagnosis.
10. Latex agglutination: adsorption of antigens onto latex or polystyrene particles converts a precipitation reaction into an agglutination reaction, which is more sensitive than precipitation and so more preferred for laboratory and diagnostic purposes.
11. Radial immunodiffusion: a solution containing antibody is mixed into a heated gel medium and allowed to cool in a Petri dish. A solution containing the antigen is placed in a well punched into the gel; diffusion from the well causes a zone of precipitation, whose size estimates the amount of antigen compared with known calibrated controls. The technique is fairly accurate.
12. Indirect immunofluorescence uses two antibodies: an unlabelled primary antibody that binds the target molecule, and a secondary, fluorophore-carrying antibody that binds the primary antibody. Multiple secondary antibodies can bind a single primary antibody, amplifying the signal. This is more complex and time-consuming than the direct method, but allows more flexibility in detection technique.
13. Fluorescence polarization immunoassay is commonly used for therapeutic drug level monitoring. A fluorescein-labelled drug competes with unlabelled drug for the antibody in the test serum; under polarized light, bound labelled molecules emit more light than free ones due to slower rotation — so higher serum drug levels mean less light is emitted.
14. Fluorescent microscopy: the specimen is illuminated with light of a specific wavelength, absorbed by fluorophores that re-emit light at longer wavelengths. A special emission filter isolates this weaker fluorescence, which the microscope's objective focuses onto a detector to produce an observable image.