Indirect ELISA

Immunochemical methods have been developed and placed on the market to analyze tetracycline residues (see Table 4). Thus, a qualitative EIA has been developed and used to analyze tetracyclines in honey samples with a detection level of 20 pg/kg-1 [96]. A microplate-based indirect ELISA has been developed to analyze tetracyclines using polyclonal antibodies. The assay could measure tetracycline in the range between 0.1 and 6 ng mL L Other tetracycline antibiotics such as chlortetracycline, rolitetracycline, or minocycline are also highly recognized in this assay [98]. Several immunoassay kits are commercially available for the analysis of tetracyclines although, to our knowledge, none of them... 

Beyond polyclonal antibodies, monoclonal antibodies to isoxazolyl penicillins were recently produced by immunization of mice with a cloxacillin-human serum albumin conjugate prepared by a mixed anhydride procedure (35). Sensitivity and specificity of these antibodies were tested in an indirect ELISA in which a cloxacillin-glucose oxidase conjugate prepared by an activated ester procedure served as a coating agent. It was found diat die prepared antibodies could be... 

Sulfamethazine has been further detected in milk without prior sample treatment (66). Using antibodies raised against sulfamethazine-ovalbumin C, an indirect ELISA was developed that allowed a detection limit of 0.05 ppb to be readily attained. 

Indirect ELISA Procedure Using Antigen-Coated Plates... 

Several variations of indirect ELISA procedures have been described.25-38... 

Fig. 1. Quantitative measurement of antigenic cross-reactivity between several tobamo-viruses assessed by indirect ELISA on antigen-coated plates. A, Homologous reactions , A, , and O, heterologous reactions with increasingly distantly related viruses. The SDI value separating the homologous reaction (A) from the most distant heterologous reaction (O) was 14.5 — 8.8 5.7. [From M. Jaegle and M. H. V. Van Regenmortel, J. Virol. Methods 11,189 (1985).]...

The main advantage of the ELISA over Western blotting is its ability to analyze many samples at one time. In addition, modifications to the general ELISA protocol can be used to quantify the exact concentration of antigen in an unknown solution, the concentration of a specific antibody in an unknown solution, the dissociation constant for a particular antigen-antibody interaction, or the relative affinity of two different antibodies for the same antigen. An example of an indirect ELISA, and its use in the determination of the concentration of an antigen in an unknown solution, is the subject of Experiment 17. 

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. 

Solid-phase support Figure 10.9 Diagrammatic overview of indirect ELISA. 

We will consider two among the several types of ELISA. The indirect ELISA is used to detect the presence of... 

Figure 4.35. ludirect ELISA and Sandwich ELISA (A) In indirect ELISA, the production of color indicates the amount of an antibody to a specific antigen. (B) In sandwich ELISA, the production of color indicates the quantity of antigen. [After R. A. Goldsby, T. J. Kindt, B. A. Osborne, Kuby Immunology, 4th ed. (W. H. Freeman and Company,... 

There are several modifications to the procedure that allow the target to be identified and quantified. Since the topic is immunocytochemistry, the procedures described deal with ELISAs performed on intact cells fixed onto the wells of 96-well plates. There are two forms of the procedure direct and indirect. The direct method calls for linking the enzyme directly to the antibody of interest. Depending on the availability of the antibody and its activity, direct conjugation of the enzyme to the antibody may interfere with specificity or success of binding with the target. The preferred method is the indirect ELISA. The antigen or cell of interest is immobilized onto the well. The primary antibody (often a... 

The competition assay was designed which followed the standard indirect ELISA format (17-18 . The methoprene conjugate was bound to a solid support in the form of a microtiter plate. Free methoprene in methanol (5 fib) was added to the pre-coated wells followed by methoprene-specific antiserum. The antibodies were allowed to compete for both immunogen-bound and free methoprene. Enzyme-conjugated, goat-antirabbit antibody was added, followed by substrate, and the color was allowed to develop. The absorbance of substrate over a range of methoprene concentrations can be drawn as a standard curve, which is presented as percent inhibition of the assay (Figure 6). The 50% inhibition (I5g) of methoprene was at a concentration of approximately 50 ng/mL. 

Figure 3. Indirect ELISA inhibition standard curve using the rabbit antiserum to partially purified BT israelensis toxin. — israelensis proteins — kurstaki proteins.

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