boiling points will move slowly through the column. The euent passes through a detector that produces an electric signal proportional to the concentration of the volatile components. As in HPLC, the chromatogram is used both to identify the compounds by the retention time and to determine their concentration by the area under the peak (Figure 31.12).

While trying to focus on the application of immunochemical analytical procedures we will postpone our detailed discussion on the nature and types of antibodies, which are immunoglobulins, their structures and functions. This has been discussed in Chapter 23 of Vol. II and will be further elaborated in immunochemistry in the following sections. Whether the laboratory is looking for the antibody in the patient’s serum or circulating antigen (drug or hormone), the laboratory must be able to detect and quantitate the immunologic reaction.

When a specic antibody reacts with a soluble antigen, a suspension of very small particles forms, the concentration of which can be measured by a nephelometer. The use of nephe­lometry principles allows immunological tests to be automated. This is done by passing a beam of light through the suspension (Figure 31.9) resulting from both the antigen–antibody reaction and the resultant precipitate formation. The light gets scaered; the scaered light is then collected electronically and measured for a quantitative report. The greater the amount of light scaered, the more concentrated is the suspension of the particles. This method has largely replaced the RID technique.

The precipitation reaction of antigen and antibody is applied in a radial immunodiusion (RID) test (Figure 31.19) that measures the concentrations of various types of immunoglob­ulins (IgG, IgM or IgA) in patient’s serum. Agar plates, about the size of a microscope slide, are commercially available and contain antihuman globulin or IgG (or antihuman IgA or IgM) diused throughout the agar. The agar also contains small wells. Each well is lled with

a patient’s serum, a serum dilution, or a standard of a known amount of IgG. Aer seing up the plate, it is incubated for several hours. As the IgG in the serum or standard (kept in the well) diuses out of the well and into the agar, it reacts with the anti-IgG in the agar and forms a white ring of precipitation around the well (Figure 31.19). The diameter of the ring is proportional in the concentration of IgG in the sample. A standard curve is constructed using the diameters of the rings produced by the IgG standards. The concentration of the unknown is determined by comparing its precipitation diameter with the standard curve.

31.20–31.22). The elements of an enzyme immunoassay (EIA) are depicted in Figure 31.20. This gure shows the principles behind the application of both EIA and radioimmunoassay (RIA) in the determination of the concentration of various analytes. The uorescent antibody

EIAs use enzyme-labelled antibodies that result in a visible reaction (Figure 31.20). This method utilizes less expensive equipment and therefore is becoming popular in developing countries. The test can be designed to detect either an antibody in a patient’s serum, or an antigen (e.g., viral antigen) in circulation in a patient’s body.

The process goes through four major steps. In the rst step, commercially available antigen-coated beads are taken in a container and incubated with the serum specimen of the patient. The antigen on the bead is sought as the infectious agent. A patient’s serum may have a primary antibody against the antigen sought. If the patient has the antibody against the antigen coated on the bead, it will become aached to the antigen. Subsequent washings in the following step do not take away the aached antibody on the bead. In the second step, a secondary antibody, linked with an appropriate enzyme, is laid on the bead holding the primary antibody. The secondary antibody is an antihuman globulin which reacts with any human antibody and thus gets stuck to the bead. The beads are again washed to remove any free secondary antibody. Now, we have the beads in the tube with the antigen of the pathogen, its primary antibody and the secondary antibody with the linked enzyme, all laid one over the other. Now in the third and nal step, an appropriate amount of a substrate is added upon which the enzyme yields a visible colour. The colour will be proportional to the amount of primary antibody present in the patient’s serum, and therefore, the reported results are considered quantitative in nature.