Indirect enzyme linked immunoassay

JO Nyalala, E Livianou, L Leondiadis, GP Evangelatos, DS Ithakissios. Indirect enzyme-linked method for determining biotin in human serum. J Immunoassay 18 1-19, 1997. 

A partially purified Bacillus thurlnglensis var. israelensls (Bti) 6-endotoxin was used to Immunize rabbits. The antisera obtained have an improved specificity towards the mosquito larvacidal activity of the toxin, as opposed to antiserum raised when the whole crystal was used as immunogen. Using a two step/indirect ELISA (enzyme linked immunosorbent assay) procedure developed in our laboratory, fourteen experimental formulations were tested, and the results were compared with bioassays. An average of 69.1 international units 20% c.v. was found to associate with each ug of toxin detected by the ELISA. Our data indicate that when toxin specific antisera are available, Immunoassays can be used to predict the biological activity of Bti samples with reasonable accuracy. 

A companion approach is to form copolymers using pyrrole and pyrrole butyric acid. The copolymer presents available carboxylic acid groups at the surface that may serve to covalently immobilize the IgG. Figure 6 illustrates the covalent tethering of amine-functionalized biotin following EDC/sulfo-NHS activation of the pendant carboxylic acid groups of poly(pyrrole-co-pyrrolylbutyric acid). Subsequent incubation in streptavidin or neutravidin followed by incubation in biotinylated IgG immobilizes the primary antibody to the device surface. This format has been used to build sandwich immunoassays for antigens that are detected by the indirect action of the oxidoreductase enzyme-linked antibodies on the conductivity of the polymer film. 

There are two common forms of heterogeneous assays used to detect and quantify disease markers the direct-labeling method, in which the proteins in a sample are labeled with a detectable tag and isolated from a complex sample by means of a bed of immobilized antibodies, and the indirect-labeling method, in which two antibodies are used for each marker. Sandwich-type immunoassays, such as the enzyme-linked immunosorbent assay (ELISA), require two antibodies an immobilized antibody used to capture a molecule of interest - a protein, a virus, or a small molecule (e.g., a hormone. 

Immunoassays vary by the different labels they use. The most common labels include chromophores, fluorophores, radioisotopes and enzymes. Of those labels, enzyme immunoassay or enzyme-linked immunosorbent assay (ELISA) is the most popular technique. ELISA has as an advantage the amplification of the analytical signal and/or increase of the sensitivity of the immunoassay. There are four types of ELISA direct ELISA, indirect ELISA, sandwich ELISA and competitive ELISA. 

Many of the studies on prostaglandins have been made using either a bioassay, immunoassay, sometimes with the enzyme cyclooxygenase, or using injected or added arachidonic acid or an eicosanoid, so the evidence for a prostaglandin is sometimes indirect. This has sometimes led to a mis-identification.1 0 Both GC—MS and LC—MS have been used for direct identification. Typically identifications are based upon mass spectra, tandem MS (MS/MS), UV spectra, and chromatographic behavior.134 Recent ESI—MS/MS linked to HPLC has provided detection over the range 0.5—50 pg and quantification from 2 to 100 pg.135 Eicosanoids in invertebrates, other than insects, have been reviewed recently.133... 

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