ELISA technique

Several heterogeneous electrochemical enzyme immunoassays have been demonstrated. These are based on the enzyme-linked immunosorbent assay (ELISA) technique... 

Fan X, Tyerman K, Ang A, et al. A novel tool for B-cell tolerance research characterization of mouse alloantibody development using a simple and reliable cellular ELISA technique. Transplant. Proc. 2005 37 29-31. 

Enzymes useful for detection purposes in ELISA techniques (Chapter 26) also can be employed in the creation of highly sensitive DNA probes for hybridization assays. The attached enzyme molecule provides detectability for the oligonucleotide through turnover of substrates that can produce chromogenic or fluorescent products. Enzyme-based hybridization assays are perhaps the most common method of nonradioactive detection used in nucleic acid chemistry today. The sensitivity of enzyme-labeled probes can approach or equal that of radiolabeled nucleic acids, thus eliminating the need for radioactivity in most assay systems. 

Incubation periods in excess of 2 h were required before this activity was detected in cell-free supernatants. More recently, the use of cDNA probing of Northern transfers (to detect specific mRNA levels), the use of ELISA techniques (to detect protein levels immunologically) and the development of more specific bioassays (culture techniques in which a biomolecule stimulates proliferation in a particular cell line) have resulted in a more thorough analysis of IL-1 production by neutrophils. IL-1 is only poorly expressed in blood neutrophils because mRNA for this cytokine is detectable only at very low levels (if at all), and protein production is usually below the level of detection of most assays. However, exposure of neutrophils to lipopolysaccharide (LPS), or to cytokines such as GM-CSF, TNF or IL-1 itself, results in a rapid but transient increase in IL-1 expression. 

Fig. 7. Frequency distribution of plasma Lp(a) concentrations (a) and apo(a) alleles (b) in three populations. Plasma Lp(a) levels were measured in 381 subjects by the sandwich ELISA technique apo(a) allele size (number of K-4 repeats) was estimated using pulsed-field gel electrophoresis and genomic blotting. [With permission of Gaw et at. (G11).]...

Immunological methods Methods such as gel-agglutination tests were introduced many years ago (according to Outcherlony) however they are not sufficiently sensitive. Reversed passive latex agglutination kits (SET-RPLA and TST-RPLA) are presently applied for enterotoxin detection and are more sensitive than gel diffusion (Wieneke, 1988). The ELISA technique is also very popular - toxins are detected via VIDAS STAPH ENTEROTOXIN SET and indirect double sandwich ELISA (Meyrand et al., 1998). 

As stated earlier, activation of endothelial cells by pro-inflammatory stimuli leads to the expression of cell adhesion molecules and cytokines such as IL-6 and IL-8. The expression and hence modulation of surface expressed adhesion molecules by e.g. targeted delivery of inhibitors of NFkB, can be measured using flow cytometric analysis or whole cell ELISA techniques. Cytokine production can be measured in the supernatant of cultured cells or in biological fluids. Furthermore, competitive or quantitative RT-PCR analysis of mRNA levels of cell adhesion molecules or cytokines, allows the transcriptional activity of the genes of interest to be estimated. 

Miller MG, McEuen SF, Nasiri M et al. 1991. Application of ELISA techniques to metabolic disposition studies for 1,3-dinitrobenzenes Comparisons with HPLC and radiochemical methods. Chem Res Toxicol 4 324-329... 

VoUer A, Bartlett A, BidweU DE. (1978) Enzyme immunoassays with special reference to ELISA techniques. J Clin Pathol 31, 507-20. 

During in vivo studies under biologically relevant conditions, the cis-Pt loading of the DNA is much lower than for the above-mentioned in vitro studies. It has been calculated that mortality of HeLa cells occurs at an value of 10 5 (i.e., one bound cis-Pt molecule per 105 nucleotides) (64a). This excludes atomic absorption spectroscopy for identification of the in vivo adducts. Immunochemical techniques, however, have shown to be very promising, and high sensitivity and selectivity levels have been reached. At the moment, only a few studies in which antibodies are raised against cis-Pt-treated DNA (64) or against synthetic cis-Pt adducts with mono- or dinucleotides are available (64a). With the latter method, quantitation of the different platinum-DNA adducts formed under in vivo conditions is possible. At the moment, femtomole (10-15 mol) amounts of the adducts can be detected with competitive enzyme-linked immunosorbent assay (ELISA) techniques. It has been demonstrated in this manner that the GG-Pt adduct is also the predominant adduct under in vivo conditions. 

The ELISA techniques offer advantages of longer shelf life of the labelled reagents and elimination of the use of radioisotopes which require expensive scintillation/gamma counters and special disposal needs (38, 56). They are also more sensitive than the RIA s. ranging from pg/ml to pg/ml depending on the size of the molecules, affinity of the antibody and the assay format used (48). 

Mix the antibody solution with the enzyme solution at a ratio of 1 1 (v/v). Since an equal mass of antibody and enzyme is present in the final solution, this will result in a 3.75 molar excess of HRP over the amount of IgG. For conjugates consisting of greater enzyme-to-antibody ratios, proportionally increase the amount of enzyme solution as required. Typically, molar ratios of 4 1 to 15 1 (enzyme antibody) give acceptable conjugates useful in a variety of ELISA techniques. 

For the present open observation on sublingual immunotherapy (SLIT), nonspecific inflammatory markers were chosen, which are supposed to be involved in the allergic cascade and which can be measured by common ELISA techniques soluble intercellular adhesion molecule-1 (sICAM-1), sE-selectin, soluble interleukin-2 receptor (sIL-2R), interleukin-12 (IL-12) and specific IgG4. Earlier investigations in this field were mostly performed on a smaller number of patients and the results are sometimes contradicting. 

The methodology and assay protocols described hereinafter will be limited to the primary applications of the present microfluidic platform and will thus concentrate mainly on the ELISA technique, where the affinity reaction incorporates an antibody to recognise the diagnostic molecule. 

In the example of a-human atrial natriuretic peptide (ANP), found at increased plasma levels in patients with heart failure, Numata et al. [70] demonstrated how IPCR sensitivity accelerated conventional assay procedures. For individual treatment of the cardiac patients, a prompt detection of atrial distension by the presence of the ANP marker would be desirable. Common ANP tests, however, take 2-3 days for the quantification of plasma by radiometric or ELISA techniques. With sandwich IPCR, the assay time could be shortened to 5 hours. A good correlation between IPCR and radiometric detection was maintained, combined with an additional improvement of the detection limit to 2 ng/L ANP. The average level of ANP in plasma for 25 patients with heart failure was found to be 117 100 ng/L, significantly higher than the typical level of 20 14 ng/L for healthy subjects. 

Voller, A., A. Bartlett, and D.E. Bidwell (1978). Enzyme immunoassay with special reference to ELISA techniques. J. Clin. Path., 31 507-520. 

Measurement of the serum concentrations of administered antibodies is a general tool to evaluate their persistence in circulation. This is usually performed by introducing a sufficient amount of the test antibody either by the intravenous or by the intraperi-toneal routes (see Note 3), in a quantity that can be easily detected and quantified in serum samples, even after a two log reduction in concentration. The antibody tracer can be labeled with radioisotope which permits direct quantification in serum samples. To minimize radioactive isotope use, we use an antibody tracer that is unmodified or labeled with biotin or other derivation chemistries, and then determine its serum concentrations by ELISA techniques. We commonly inject 100 xg of the test antibody in a 200 xl volume of phosphate-buffered saline into each mouse intraperitoneally (i.p.) (see Note 4). This amount can vary depending on the goals of the experiment and the sensitivity of the detection method. A minimum of five inbred mice, sex-matched and age-matched, at 8-16 weeks of age are recommended for each antibody to be tested. 

The ELISpot assay has been adapted for the detection of individual cells secreting specific cytokines or other antigens. ELISpot assays employ the quantitative sandwich ELISA technique. A monoclonal antibody specific for the cytokine is precoated onto a microplate. Cells are pipetted into the wells of the microplates. During the incubation period, the immobilized antibody (in the immediate vicinity... 

Fluorescence may by induced using labeled mono- or polyclonal antibodies raised against the compound of interest. This technique has been used successfully to detect the presence of okadaic acid in cultures of Prorocentrum lima, and, further, to estimate quantities of the compound in individual cells.110 Immunofluoresence in combination with thin layer chromatography and ELISA techniques have also been used to detect multiple haptens in mycotoxin families.111112... 

Following successful recovery of peptide/protein molecule from the microspheres, a simple spectrophotometric method does not always allow discrimination between the monomeric protein form and its aggregates. However, HPLC might separate these species and thus provides more accurate qualitative data [96], But HPLC cannot quantify exclusively the amount of active protein antigen, as is the case with ELISA techniques [97], Nowadays, Fourier transform infrared (FTIR) spectroscopy has become a popular, noninvasive method, as it is able to characterize the secondary structure of entrapped proteins [26, 95, 98-101], Only recently, the integrity of their primary structure was evaluated, thanks to a new matrix-assisted laser... 

Spandidos D A., Field, J K, Agnatis, N J, Evan, G I, and Moore, J. P (1989) High levels of c-myc protein in human breast tumours determined by a sensitive ELISA technique. Anbcancer Res. 9, 821-826. 

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