Nephelometry is a technique used in immunology to determine the levels of several blood plasma proteins. For example the total levels of antibodies isotypes or classes: Immunoglobulin M, Immunoglobulin G, and Immunoglobulin A. It is important in quantification of M-proteins in diseases such as multiple myeloma. Quantification is important for disease classification and for disease monitoring once a patient has been treated (increased M-protein after a patient has been treated is an indication of disease recurrence).
It is performed by measuring the turbidity in a water sample by passing light through the sample being measured. In nephelometry the measurement is made by measuring the light passed through a sample at an angle.
This technique is widely used in clinical laboratories because it is relatively easily automated. It is based on the principle that a dilute suspension of small particles will scatter light (usually a laser) passed through it rather than simply absorbing it. The amount of scatter is determined by collecting the light at an angle (usually at 30 and 90 degrees).
Antibody and the antigen are mixed in concentrations such that only small aggregates are formed that do not quickly settle to the bottom. The amount of light scatter is measured and compared to the amount of scatter from known mixtures. The amount of the unknown is determined from a standard curve.
Nephelometry can be used to detect either antigen or antibody, but it is usually run with antibody as the reagent and the patient antigen as the unknown. (Clinical Immunology and Seriology 3rd Ed., Stevens, pg 127, F.A. Davis Company) In the Immunology Medical Lab, two types of tests can be run: "end point nephelometry" and "kinetic (rate) nephelometry".
End point nephelometry tests are run by allowing the antibody/antigen reaction to run through to completion (until all of the present reagent antibodies and the present patient sample antigens that can aggregate have done so and no more complexes can form). Unfortunately, the large particles will fall out of the solution and cause a false scatter reading, thus kinetic nephelometry was devised.
In kinetic nephelometry, the rate of scatter is measured right after the reagent is added. As long as the reagent is constant the rate of change can be seen as directly related to the amount of antigen present.
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