Light-scattering immunoassay

With appropriate sample dilution, the light-scattering immunoassays will provide precise results within an interval of O.lmg/L to 20g/L. Choice of an immunoinhibition assay wiU ensure that the antigen excess does not lead to an erroneously reported result (see Chapter 9), although sample dilution wiU be required to give an accurate result at high protein concentration. Values for imprecision of less than 5% within run and 8% between runs are attainable over a concentration range of 0.1 mg/L to 20 g/L with the appropriate choice of reaction conditions. 

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. 

Liu, X., Dai, Q., Austin, L., Courts, J., Knowles, G., Zou, J. H., Chen, H., and Huo, Q. (2008). A one-step homogeneous immunoassay for cancer biomarker detection using gold nanoparticle probes coupled with dynamic light scattering. J Am. Chem. Soc. 130 2780-2782. 

The front surface approach shows similar sensitivity to the right angle detectors hut is more susceptible to background light scatter. Front surface fluorometry has been widely applied to heterogeneous solid-phase fluorescence immunoassay systems. 

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. 

Operationally, the optical components used in turbidimeters and nephelometers are similar to those used in fluorometers or photometers. For example, the light sources commonly used are quartz halogen lamps, xenon lamps, and lasers. He-Ne lasers, which operate at 633 nm, have typically been used for light-scattering applications, such as nephelometric immunoassays and particle size and shape determinations. The laser beam is used specifically in some nephelometers because of its high intensity in addition, the coherent nature of laser light makes it ideally suited for nephelometric applications. 

The FPIA method can be potentially used either independently or as a complementary method to enzyme-linked immunosorbent assay (ELISA) and fluorescence polarization immunoassays (FPIA) technique. FCIA does not need polarization equipment and is not markedly influenced by light scattering effects. The FCIA technique can be expanded for analysis of enzymes and receptors including adaptation to fibro-optic techniques. 

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]. 

In space-resolved immunoassays, a smooth metal slide is coated with an antibody monolayer, and a parallel laser beam is used to quantitate surface bound fluorophore.1(37,38) Scattered light is low since the excitation is reflected into a different space, although scatter still remains the principal source of background. 

This method was modified by Lisi et al. (L8) using antibody-coated microspheres [Ab— -]. Added unlabeled antigen [Ag] and fluorescein-labeled antibody [F-Ab] bind to the antibody-coated microspheres as in the conventional sandwich immunoassay [F-Ab Ag Ab - -]. After the reaction is complete the suspension is introduced into a flow cytometer with a laser light source. By gating fluorescence light accumulation on scattered light pulses,... 

Heterogeneous fluorescent immunoassays for T4 based on lanthanide rare earth ions and time-resolved fluorescence were also developed. The use of europium chelates as fluorescent probes is particularly attractive because of their extraordinarily long Stokes shifts and long fluorescence decay times. Thus the sharp emission peak of europium (613 nm) can be easily separated fr om scattering caused by excitation light (340 nm) or by interfering substances in... 

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