Immunoassay solid phase

Liquid-phase and solid-phase reactions Antibody or antigen reactions can occur where both components are in the same liquid phase (in solution), or one component is in the solid phase and the other is in the liquid phase (solid-liquid interface). The former liquid phase assays were the first type used but have since largely been superseded by various forms of solid-phase immunoassay. Solid-phase assays... 

Table 5. Solid-Phase Automated Immunoassay Analyzers ...

GC = gas chromatography ECD = electron capture detector EIA = enzyme-immunoassay GPC = gel permeation chromatography HPLC = high-performance liquid chromatography ITMS = ion trap mass spectrometer LSE = liquid solid extraction MS = mass spectrometry RSD = relative standard deviation SPE = solid phase extraction... 

E. S-Layers as Novel Matrix for Dipstick-Style Solid-Phase Immunoassays... 

Figure 1 Schematic of the quasi-equihbria using heterologous haptens in coating antigen immunoassay formats. Ka represents the equilibrium constant for binding of antibody (Y) to target analyte (A). Kh is the equilibrium constant for the binding of antibody to hapten-protein conjugate (H-) immobihzed on a solid phase...

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

Figure 4 Sandwich immunoassay. A capture antibody (Y) is passively adsorbed on a solid phase. The target protein contained in the sample and the enzyme-labeled reporter antibody (Y-E) are added. Both the capture antibody and enzyme-labeled reporter antibody bind to the target protein at different sites, sandwiching it between the antibodies. Following a wash step, the substrate (<>) is added and colored product ( ) formed. The amount of colored product is directly proportional to the amount of target protein captured...

Specific extraction methods are used to prepare the analyte for immunoassay by freeing the analyte fromboth specific and nonspecific interferences. Supercritical fluid extraction has been used to decrease the amount of solvent waste generated. Solid-phase extraction has gained popularity, and many different supports are available. One promising extraction and concentration method is immunoaffinity chromatography, which will be addressed later. 

Kennedy et al. developed a lasalocid immunoassay for application to residues in chicken meat and liver samples. The antibody was specific and did not cross-react with salinomycin, maduramicin, or monensin. Sample preparation consisted of homogenization in aqueous acetonitrile, removal of fat from an aliquot of the aqueous acetonitrile by hexane extraction, and evaporation of acetonitrile. The sample was then reconstituted with assay buffer. Liver required an additional solid phase extraction step. The LOQ was 0.02 xgkg for muscle and 0.15 agkg for liver. These workers were able to use the system to determine the half-life of lasalocid in the tissues. 

Figure 1 Schematic of an enzyme immunoassay. (1, 2) The test solution and enzyme conjugate are added to a tube or well pre-coated with anti-anal) e antibodies. (3) After the inhibition step, the solid phase is washed, and only antibody-bound material is retained. (4A-C) Colorless substrate is added and is converted to a visible color in inverse proportion to the amount of analyte in the sample...

This method was used, for example, for the solid-phase immunoassay of thyroxine (affinity chromatography). Various activation methods (CDI, periodate, and cyanogen bromide procedures) were compared with each other for coupling antibodies to magnetizable cellulose/iron oxide solid-phase particles. 211]... 

Hiroshima, 0., et al. (1986). Basal level of prostaglandin D2 in rat brain by a solid-phase enzyme immunoassay. Prostaglandins 32, 63-80. 

Butler, J.E. (2000a) Solid supports in enzyme-linked immunosorbent assay and other solid-phase immunoassays. Methods 22, 4-23. 

Butler, J.E. (2000b) Solid supports in enzyme-linked immunosorbent assay and other solid-phase immunoassays. In Methods in Molecular Medicine Molecular Diagnosis of Infectious Diseases. (J. Decker, and Reischl, eds.), Vol. 94, pp. 333-372. Humana Press, Inc., Totowa, NJ. 

Cantarero, L.A., Butler, J.E., and Osborne, J.W. (1980) The adsorptive characteristics of proteins for polystyrene and their significance in solid-phase immunoassays. Anal. Biochem. 105, 375-382. 

Salmain, M., Fischer-Durand, N., Cavalier L., Rudolf, B., Zakrzewski, J., and Jaouen, G. (2002) Transition metal-carbonyl labeling of biotin and avidin for use in solid-phase carbonyl metallo immunoassay (CMIA). Bioconjugate Cbem. 13, 693-698. 

Varenne, A., Salmain, M., Brisson, C., and Jaouen, G. (1992) Transition metal carbonyl labeling of proteins. A novel approach to a solid-phase two-site immunoassay using Fourier transform infrared spectroscopy. Bioconjugate Chem. 3, 471-476. 

H. Zhang and M.E. Meyerhoff, Gold-coated magnetic particles for solid-phase immunoassays enhancing immobilized antibody binding efficiency and analytical performance. Anal. Chem. 78, 609-616 (2006). 

H.C.B. Graves, The effect of surface-charge on non-specific binding of rabbit immunoglobulin-G in solid-phase immunoassays. J. Immunol. Methods 111, 157—166 (1988). 

An example of an immunoassay format is shown in Figure 3. This immunoassay format relies on partial saturation of the solid-phase antibody (Ab) by the antigen (Ag) and on its competition with the labeled antigen (Ag-L) for the available antibody sites. At low antibody and tracer concentrations the sensitivity of... 

Figure 3 Layout of competitive solid-phase antibody immunoassay format in an incubation step antigen (Ag) and labeled antigen (Ag-L) compete for the solid-phase antibodybinding sites (Ab) after the solid-phase antibodies are washed, the activity of the bound labeled antigen is measured, and a calibration curve constructed. The measured signal is inversely proportional to the antigen concentration.

Pankey et al.21 described a rapid, reliable, and specific enzyme multiplied immunoassay technique (EMIT ) for amitriptyline, nortriptyline, imipramine, and desipramine in sera. To overcome crossreactivity, solid phase extraction was included in sample pretreatment. Disposable 1 mL columns packed with covalently labeled silica gel were conditioned with HPLC-grade methanol (1 mL) and then with de-ionized or distilled water (1 mL). Serum (calibrator, control, or patient sample, 500 L) was applied onto the column, eluted to waste, washed with 900 /uL of wash solution containing acetonitrile (236.1 g/L) and ion-pairing reagent in acetate buffer, pH 4.2, washed with 500 fiL of mobile phase solution containing acetonitrile (393.5 g/L) in methanolic phosphate buffer, pH 7.0,... 

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