Nephelometry immunoassay

Turbidimetry and Nephelometry. In contrast to classical absorbance methods, immunoassay reactions frequently involve agglutination in which the optical scatter signal of the agglutinated particles is measured by turbidimetric or nephelometric means. The principles of light scattering as it relates to analytical methods is discussed in reference 6. 

Forerunners of nonisotopic immunoassay had already appeared before radioimmunoassay was developed. For example, nephelometry is based on precipitation, which is known as the classical immune reaction, and the ideas of particle immunoassay and viroimmunoassay seem to have developed from the hemagglutination test. The principles of enzyme and fluorescence immunoassay had already been used as enzyme and fluorescence antibody techniques in histochemical analysis. In 1971, two groups reported use of an enzyme immunoassay (E5, V2). Leute et al. reported spin immunoassay, which has spurred recent development of nonisotopic immunoassays (L5). 

The term homogeneous immunoassay may be defined as an immunoassay in which the extent of the antigen-antibody reaction can be determined without physical separation of the free and bound forms. This term is usually used for immunoassays such as enzyme and fluorescence immunoassays in which labeled substances (markers) are used. It does not include immunoassay systems such as nephelometry in which no labeled substances are used. Homogeneous immunoassays are widely used as routine tests because the procedures involved are simple. The principle of homogeneous immunoassay is based on changes in signals of the indicators by the antigen-antibody reaction (N5, U2). 

Light scattering is a physical phenomenon resulting from the interaction of light with particles in solution. Nephelometry and turbidimetry are analytical techniques used to measure scattered light. Light-scattering measurements have been applied to immunoassays of specific proteins and haptens. Specific applications are described in Chapters 9,20, and 26. 

These two optical techniques are particularly applicable to methods measuring the precipitate formation in antigen antibody reactions (see Chapter 9). Turbidimetry is used in several chemistry immunoassay systems for therapeutic drug monitoring and specific protein assays. Details of turbidimetry and nephelometry are discussed in Chapter 3. 

Immunochemical methods are also used for clinical assays because they are rapid and easily automated. Because of the differences in molecular size and corresponding diffusion rates, gel diffusion techniques, such as radial immunodiffusion (RID) require correction for phenotype and are therefore time consuming and inconvenient. Immunoassays in solution, such as nephelometry and turbidimetry, are influenced slightly by size as well, but the differences are relatively insignificant. 

Quantitative assay of specific proteins is necessary for estimates of glomerular selectivity and for evaluation of tubular proteinuria. Nephelometry and turbidimetry are sufficiently sensitive for most of the analytes (see Chapters 3 and 9). More sensitive immunoassays are required for BMG because of the low levels that are typically excreted. In addition, this protein is susceptible to degradation at low pH, and specimens should be stored at a pH greater than 6.0 during the collection period. In assessment of the nephrotic syndrome, glomerular selectivity is estimated usually by measuring two individual proteins of different molecular size, such as albumin or transferrin plus IgG. A random urine sample and a corresponding serum are collected and analyzed for the two proteins. The ratio of the individual clearances is then calculated... 

Specific quantitative assays of particular proteins by immunochemical methods (see Chapter 9) using specific antisera and measurement of the antigen antibody complexes by nephelometry, turbidimetry, RID, or electroim-muno assay or, if present in very low concentrations, by RIA, enzyme immunoassay (EIA), fluorescence immunoassay, or chemiluminescence. 

Many different immunoassays can be used for the quantification of immunoglobulin isotypes and subclasses, varying from ELISA/ FEIA and radial immunodiffusion (RID) to nephelometry/turbid-imetry (Rose et al., 2002). The immunoglobulin isotypes IgA, IgG, and IgM in particular are present in relatively high concentrations in the circulation (0.5-20 g/l). These isotypes are often quantified by... 

To our surprise, hyperapobetalipoproteinemia is found more commonly in people who do not have coronary heart disease. With our technique (solubilization of LDL precipitated with heparin at pH 5.12, determination of cholesterol, and immunoassay of Apo-B by kinetic nephelometry) we find hyperapobetahpoproteinemia not to be a risk factor. 

A large number of latex agglutination immunoassays have recently been adopted from clinical chemistry. These assays are based on the visualization of antigen-antibody complexes by the attachment of latex particles or gold colloids. Entities of this type with dimensions in the nanometer or micrometer range can be quantified by turbi-dimetry, nephelometry, light scattering techniques, and particle counters [22] - [25]. 

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