ELISA assays

In order to measure the exact amount of a specific protein (analyte) by IHC signal intensity, a critical requirement is the availability of a standard reference material (present in a known amount by weight) that can be used to calibrate the assay (IHC stain). It is then possible to determine the amount of test analyte (protein) by a translation process from the intensity of IHC signals. In this respect it is helpful to consider the IHC stain as a tissue based ELISA assay (Enzyme Linked ImmunoSorbent Assay), noting that ELISA is used in the clinical laboratory as a standard quantitative method for measuring protein by weight in fluids, by reference to a calibrating reference standard. 

In summary - IHC should no longer be regarded a just a special stain but rather as a standardized controlled quantitative tissue based ELISA assay - quantification of IHC is coming, it is just a matter of how and when.11,22... 

E. Eteshola and D. Leckband, Development and characterization of an ELISA assay in PDMS microfluidic channels. Sens. Actuators B. 72, 129—133 (2001). 

Fig. 8.7 CTB-GM1-ganglioside binding ELISA assay. Plates, coated first with GMrganglioside and bovine serum albumin (BSA), respectively, were irrigated with total soluble plant protein from chloroplast transgenic lines (3 and 7) and 300 ng of purified bacterial CTB. The absorbance of the GM1-ganglioside-CTB-antibody complex in each case was measured at 405 nm. Total soluble protein from untransformed plants was used as the negative control.

Blood Alkylation of ELISA assay Sulphur Low cost Monoclonal antibody... 

There exists a wide variety in the setup of ELISA assays (direct binding or competition setups) and the enzymatic reaction utilized [148]. A similar principle to enhance sensitivity by enzymatic coupling is realized after gel electrophoretic separation of proteins. Here proteins are transferred to nitrocellulose ( western blot ) and detected by antibody-coupled enzymes. 

Cell-based ELISA assay pGSK3 3 IC50 = 1.9 pM... 

Fig. 20c. 1. ELISA assay, (a) Antibodies to the drug of interest are secured to a solid substratum such as a test tube or micro-well plate. The sample containing the analyte antigen is added to the reaction surface, (b) After the analyte has bound to the antibody, the vessel is rinsed to remove unbound antibody. A second antibody to the analyte is added. This antibody has a bound enzyme which has been chosen because its reaction produces a colored product which can be detected spectrophotometrically. (c) After this second antibody has bound to the first antibody-antigen complex, the surface is again rinsed to remove unbound-antibody enzyme. The enzyme substrate is added in sufficient excess such that the rate of product formed is proportional to the amount of enzyme present. The enzyme-linked assays are very sensitive, since each enzyme can rapidly catalyze thousands of substrate to product reactions.

In summary, these are the clinically relevant questions about the immunogenicity of rDNA species-specific proteins will antibody be induced in the recipient that will neutralize the therapeutic effect or lead to immune complex disease What is the class (e.g., IgG or IgE) and specificity (i.e., reactivity against specific protein or contaminant) of the antibody induced The former antibody type could potentially neutralize the product and produce immune complex disease, while the latter could result in an anaphylaxis response. It is possible that the antibody induced is of insignificant health consequence, and its presence is known only because of improvements made in the sensitivity of detection methods with the introduction of the enzyme-linked immunosorbent (ELISA) assay. 

Immune detection is a key utility of antibodies in biotechnology [3, 5]. Antiden-drimer sera efficiently detect dendrimers in multiple assay formats, including enzyme-linked immunosorbent assays (ELISA), and in Western and dot blots [3, 5], ELISA assays are commonly used to quantitate proteins, and a quantitative ELISA could be developed for dendrimers using our sera, though doing so would require development of dendrimer standards of known concentration that could be used for calibration. 

A competitive ELISA assay for Lp(a) was recently described (Y4) in which the microtiter plate was coated with Lp(a) purified from a pool of donors. The method is simple and easy to perform, with satisfactory analytical parameters. A good stability and a reproducible coating of plates with the large Lp(a) lipoprotein is, however, critical in this type of assay. Wang et al. (W6) described an indirect sandwich assay for the measurement of Lp(a) in plasma and in dried blood spots, which can be applied to screening elevated Lp(a) levels in newborns (V3, V4). 

The main features of the immunological assays for Lp(a) quantification are summarized in Table 5. RIA and ELISA assays are among the most sensitive, while RID and EIA are less sensitive and less suited to the measurement of low Lp(a) levels. The reference values reported for Lp(a) are lowest with the EIA techniques and highest with the turbidimetric assays (R15). 

The results of international Lp(a) surveys carried out in collaboration with the Centers for Disease Control (Atlanta) (L4) confirmed the general use of ELISA assays (Table 6). Among laboratories using ELISA assays, 60% of the participants relied upon commercial-available kits. 

False Positives. There are individuals who are not HIV infected but who test antibody-positive in the ELISA assay. False positives (0.1%) are a... 

Because of the significant false positive rate for the ELISA test, a second, more specific test for HIV antibodies is also used the Western blot test. This technique has a lower incidence of false positives than the ELISA assay. In practice, serum samples that score antibody positive by the ELISA test are generally retested by the Western blot procedure. Serum samples are considered positive if they are found to contain HIV-specific antibodies by both tests. 

Some patients will have repeatedly nonspecific reactivity in an ELISA assay, since the HIV-1 antigen used for the antibody assays is produced in cultured human T cells. It is not unexpected that occasional false positive assays occur in human sera from individuals with autoimmune diseases a history of multiple pregnancies or multiple transfusions or antibodies to certain class II histocompatibility antigens (especially HLA-DR4). Block reagents have been added to specimen diluents to minimize cross-reactions in these sera. This necessitates the use of confirmatory tests, especially the Western blot. With the use of both ELISA and Western blots, false positives decrease to less than 1 per 100,000. 

ELISA assays may be competitive or noncompetitive. As the name imphes, in a competitive ELISA, enzyme-labeled antigen competes with free antigen (the analyte of interest) for a fixed and limited quantity of immobihzed antibody binding sites. After incubation, the microtiter plate (sohd support) is rinsed to remove all unbound species and the enzyme substrate is added in saturating concentration. The conversion of substrate to produce can be measured continuously (kinetic assay) or, more commonly. 

Such thermoplastics have also been used as DNA microarray substrates (Matson et al., 1995 Shchepinov, 1997 Beier and Hoheisel, 1999) and in the construction of protein microarrays in microwells (Matson et al., 2001 Moody, 2001). Pierce (see searchlight perbio.com) introduced the Search-Light series of microarray-based ELISA assays immobilizing capture antibodies in a low density array format into polystyrene microwells. 

Non-HIV Infected individuals There is a theoretical risk that enfuvirtide use may lead to the production of antienfuvirtide antibodies that cross react with HIV gp41. This could result in a false positive HIV test with an ELISA assay a confirmatory western P.1126... 

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