Enzyme ELISA

Accurate modeling of microbial solubilization of lignocellulose will be dependent on knowledge of the dynamics of microbial cell concentration over the course of bioconversion. While measurement of cell concentrations distinct from the concentration of substrate is trivial for soluble substrates, it is a substantial and not-yet-resolved challenge for fermentation of particulate substrates based on plant cell walls. Cell measurement has been approached on the basis of elemental composition (pellet nitrogen, [25]), concentration of cellular macromolecules (total protein [26] or DNA via quantitative PCR [27]), and estimated by indirect methods, such as off-gas analysis [25] and detection of enzymes (ELISA assays [28]). Future efforts using quantitative proteomics approaches also hold promise. 

Enzyme-linked immunosorbent assays (ELISAs) in kit form are most widely used giving relatively rapid and inexpensive methods for multispecies identification. A typical format for such an ELISA is to coat different strips of the normal 12 x 8-well plate with antisera formed against serum albumin of the various species of interest. An extract of the meat product is added to the antibody-coated wells, incubated to ensure antibody binding of the serum albumin, and, after washing, a second antibody coupled with enzyme is introduced. The sandwich is visualized by addition of a substrate to the enzyme. ELISAs have been developed, also, for meat species identification in cooked meat products. These ELISAs are quite specific and sensitive ( 1% of each species can be detected) but are qualitative, or at best, semiquanti-tative. 

MacroHdes are obtained by controUed submerged aerobic fermentations of soil microorganisms. Although species of Streptomjces have dominated, species of Saccharopoljspora Micromonospora and Streptoverticillium are also weU represented. New techniques such as enzyme-linked immunosorbent assay (ELISA) based assays may prove beneficial for discovering new stmctures (464). 

In each of the assays of potency the amount of the immunoglobulin and the amount of a corresponding standard preparation that are required to neutralize the infectivity or other biological activity of a defined amount of virus or to neutralize a defined amount of a bacterial toxin are determined. The two determined amounts and the assigned unitage of the standard preparation are then used to calculate the potency of the immunoglobulin in International Units (lU). ELISA, enzyme-linked immunosorbent assay. 

HPLC, high-petfomtance liquid chromatogtaphy ELISA, enzyme-linked immunosorbent assay method RIA, radioimmunoassay method. 

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

ELISA Enzyme-linked immunosorbent assay EMS Eosinophilia-myalgia syndrome ENS Enteric nervous system EO Eosinophil... 

H.A. Moye, Enzyme-linked immunosorbent assay (ELISA), in Pesticide Residues in Foods Methods, Techniques, and Regulations, W.G. Fong, H.A. Moye, J.N. Seiber, and J.P. Toth (eds), Wiley, New York, Chapt. 6 (1999). 

Watanabe et al. developed an enzyme-linked immunosorbent assay (ELISA) for the detection of inabenfide, a plant growth regulator, in rice. Specific monoclonal antibody (MAB) is used for this method. The effects of rice matrices on the sensitivity of ELISA can be reduced by adding 0.1% Tween 20. Good reproducibility and accuracy of the proposed ELISA were obtained for rice samples and the recovery was 92% at a fortification level of 5-500 xgkg . ... 

The need to understand the fate of pesticides in the environment has necessitated the development of analytical methods for the determination of residues in environmental media. Adoption of methods utilizing instrumentation such as gas chro-matography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), liquid chromatography/tandem mass spectrometry (LC/MS/MS), or enzyme-linked immunosorbent assay (ELISA) has allowed the detection of minute amounts of pesticides and their degradation products in environmental samples. Sample preparation techniques such as solid-phase extraction (SPE), accelerated solvent extraction (ASE), or solid-phase microextraction (SPME) have also been important in the development of more reliable and sensitive analytical methods. 

Figure 2 Immobilized antigen ELISA format. Antigen is immobilized to a solid phase by passive adsorption. Following removal of unbound antigen, analyte (free H) and antigen (H-protein) compete for a fixed number of primary antibody (Y) binding sites. Unbound materials are removed (dotted line). Secondary antibody-enzyme conjugate (Y-E) is added to bind to primary antibody followed by another wash step. Substrate (A) for the enzyme is added to detect the bound enzyme. The amount of colored product ( ) detected is inversely proportional to the amount of analyte present...

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

Figure 1 Schematic sequence of the direct and indirect competitive ELISA. The principle difference is that for direct competitive immunoassay, the well is coated with primary antibody directly, and for indirect competitive immunoassay, the well is coated with antigen. Primary antibody (Y), blocking protein (Y), analyte (T), analyte-tracer ( ), enzyme labeled secondary antibody ), color development ( J)...

EIAs are more desirable for the measurement of agrochemicals than enzyme-linked immunosorbent assays (ELISAs) for several reasons. EIAs are easier to run, require minimal liquid transfers, and are completed in brief time frames, approximately 40 min for tube assays to 2.5 h for microtiter plate assays. In contrast, ELISAs are more complex, have many steps involving transfer of reagents, and require 6-8 h to complete. Most commercially available immunoassays utilize the EIA format. 

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. 

---