Enzyme-linked immunosorbent assay procedure

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. 

The amino acid composition of purified SCP2 is also compared in Table 4 with the amino acid composition of FABP [48]. Marked differences exist in the abundance of threonine, alanine, valine and leucine. The correlation coefficient between SCPj and FABP is 0.732. Clearly, SCP and FABP are separate and distinct proteins. Furthermore, there is no immunological cross-reactivity between SCP2 and FABP, in an extremely sensitive enzyme-linked immunosorbent assay procedure. 

FIGURE 3.10 Schematic representation of the sandwich enzyme-linked immunosorbent assay procedure used with the magnetoelastic E. coli 0157 H7 sensor. 

Enzyme-linked immunosorbent assay (ELISA). Li and Li developed an ELISA procedure for imidacloprid to determine its residues in coffee cherry and bean extracts. A 25-g amount of sample extracted with 300 mL of methanol and 1% sulfuric acid (3 1, v/v) for 3 min. An aliquot of the sample extract (0.5 mL) is mixed with 1 mL of water and a gentle stream of nitrogen is used to evaporate methanol. The solution is then extracted with 1 mL of ethyl acetate, the extract is reconstituted in 1 mL of PBST (phosphate-buffered saline containing 0.05% Tween 20) and competitive ELISA is performed to quantify imidacloprid in the extract. Eor methanol extracts of coffee cherries and beans fortified with imidacloprid at 0.5 mgL recoveries of imidacloprid by the ELISA method were 108 and 94, respectively. 

Diagnostic procedures include dark-field microscopy12, non-treponemal exams10 (i.e., the Venereal Disease Laboratory and the rapid plasma reagin test), and treponemal exams (i.e., enzyme immunoassay, the T. pallidum hemagglutination test, the fluorescent treponemal antibody test, and the enzyme-linked immunosorbent assay). 

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

Diagnosis Blood cultures require a prolonged period of incubation in the acute phase. Bone marrow cultures produce a higher yield. Confirmation requires phage-typing, oxidative metabolism, or genotyping procedure. Enzyme-Linked Immunosorbent Assays... 

An earlier attempt by Berger and Berger (29) to develop an enzyme-linked immunosorbent assay (ELISA) procedure with sheep-ant i-ciguatoxin was unsuccessful. In part, this may be attributable to the approaches taken and to the insufficient number of samples of documented toxic and non-toxic fishes examined. Except that the putative toxic fish samples came from Tahiti, evidence for their toxicity was not presented in the report (29). 

Enzyme-linked immunosorbent assay is a heterogenous immunoassay. Reactions involve a solid phase to which components are sequentially presented and successively bound. This method is very effective in the determination of the total alkaloid content. The positive characteristics of this method are the use of non-toxic reagents and basic equipment with low costs, a small sample volume and the ability to measure alkaloids in crude sample extracts. According to the literature, compared with results obtained from GLC, the precision of ELISA for quinolizidine alkaloids is not as high as that of the gas chromatography procedure, but is adequate for plant breeding purposes. The use of enzymes in developing the methods of quinolizidine alkaloids analysis looks likely to increase in the future. 

Among the many immunological assay methods, the enzyme-linked immunosorbent assay methods (ELISA) are the most popular methods. ELISA can detect both antigen molecules and antibody molecules with only a slight modification of the procedure. The direct-binding and sandwich methods that are used for the... 

Antibody Assays Monoclonal antibodies can be analyzed by the solid-phase assay techniques such as enzyme linked immunosorbent assays (ELISA) or radioimmuno assays (RIA). The typical assay procedures are as follows (Figure 5.9)... 

An application example of Immuchip for the analysis of Interleukin IB by enzyme-linked immunosorbent assay is described in Procedure 50 (see in CD accompanying this book). 

LaFrance, P, O. Banton, H. Sabik, S. Cooper, P. Brousseau, and M. Fournier (1996). Comparison between an enzyme-linked immunosorbent assay and a gas chromatographic procedure for the determination of diluted fractions of atrazine in runoff water. Analusis, 24 133-138. 

Schraer, S.M., D.R. Shaw, M. Boyette, et al. 2000. Comparison of enzyme-linked immunosorbent assay and gas chromatography procedures for the detection of cyanazine and metolachlor in surface water samples. J. Agric. Food Chem. 48 5881-5886. 

Cliquet, P., E. Cox, W. Haasnoot, et al. 2003. Extraction procedure for sulfachloropyridazine in porcine tissues and detection in a sulfonamide-specific enzyme-linked immunosorbent assay (ELISA). Anal. Chim. Acta 494 21-28. 

Enzyme-linked Immunosorbent Assay. A promising alternative to the RIA procedure is an enzyme-linked immunosorbent assay (ELISA) which depends upon the conjugation of a functional enzyme to either an antigen or antibody. The amount of enzyme present in a competitive binding assay is quantitated instead of the amount of radiolabeled compound. The concentration of the enzyme can be determined through its subsequent reaction with a substrate which results in a measurable spectroscopic change. 

The enzyme-linked Immunosorbent assay (ELISA) is a rapid Immunochemical procedure which can be used for trace analysis. We have applied the procedure to paraquat and other compounds difficult to analyze by the more classical methods. The Immunoassay for paraquat shows the practicality of the method for fortified and actual residue samples, and Is being compared with a gas chromatography procedure. Our work with the ELISA Illustrates that the Immunochemical technology can be used to solve problems encountered In pesticide residue analysis. 

The cultured root segments were divided into proximal, distal and newly developed lateral root parts and their endogenous lAA levels were periodically analyzed using enzyme-linked immunosorbent assay (ELISA) kits for lAA (PHYTODETEK iAA, Idetek Inc., USA) according to the procedure of Weiler et al. [39]. The lAA levels in the root segments are indicated in Fig. (26). The lAA levels in the roots cultured on HF B5 medium that initiated shoot formation increased once at day 9 and consequently decreased in both proximal and distal parts. On the other hand, lAA levels in proximal part of the roots cultured in the presence of 0.5 mg/1 TIBA increased along with the culture period. The roots cultured on the MS medium containing 0.5 mg/1 NAA (source for the experiments) accumulated low amounts of lAA and the lateral roots did not accumulate detectable amounts of lAA in all the cases. 

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