Enzyme assay plate types

To demonstrate the ability of the system to perform a matrix experiment as described above, concentrations of enzyme, substrate, and ATP were varied across the 24 wells in a row of an SBS 384-well microtiter plate. Results of these types of evaluations for the optimization of an assay for a protein kinase A and Kemptide system were presented by Wu et al.12 All the reactions were carried out in lOOmM HEPES, pH 7.4, lOmM MgCl2, lOmM DTT, and 0.015% Brij-35. No quenching agent was used. A sample from each of the 24 wells was analyzed in parallel every 6.5 min as the 24 enzymatic reactions progressed. 

We (K1) attempted to develop a noncompetitive assay based on the anti-idiotype antibodies for a conjugated bile acid metabolite, ursodeoxycholic acid 7-A-acetyl-glucosaminide (UDCA 7-NAG), which is expected to serve as a diagnostic index for an autoimmune disease, primary biliary cirrhosis. In our assay, the hapten UDCA 7-NAG, a /3-type antibody, and a biotin-labeled a-type antibody were simultaneously added to a microtiter plate coated with an F(ab )2 fragment of a specific anti-UDCA 7-NAG antibody, then incubated at room temperature for 8 h. Bound biotin was then detected with HRP-labeled streptavidin, whose enzyme activity was measured using o-phenylenediamine/H202 as a substrate. This noncompetitive assay system provided a subfemtomole-order sensitivity (detection limit 118 amol) that was 7 times lower than the competitive immunoassay using the same anti-hapten antibody (K2), even with a common colorimetric detection (Fig. 13). Somewhat improved specificity was also obtained namely, better... 

The principle approach to immunoassay is illustrated in Figure 1, which shows a basic sandwich immunoassay. In this type of assay, an antibody to the analyte to be measured is immobilized onto a solid surface, such as a bead or a plastic (microtiter) plate. The test sample suspected of containing the analyte is mixed with the antibody beads or placed in the plastic plate, resulting in the formation of the antibody—analyte complex. A second antibody which carries an indicator reagent is then added to the mixture. This indicator may be a radioisotope, for RIA an enzyme, for EIA or a fluorophore, for fluorescence immunoassay (FIA). The antibody-indicator binds to the first antibody—analyte complex, free second antibody-indicator is washed away, and the two-antibody—analyte complex is quantified using a method compatible with the indicator reagent, such as quantifying radioactivity or enzyme-mediated color formation (see Automated instrumentation, clinical chemistry). 

The method may also be used the other way round with GOx as the enzyme label, glucose as substrate, and [Eu(Tc)] as the fluorescent indicator. In that type of assay, the presence of enzyme-labeled antibodies is indicated by an increase in the observed fluorescence intensity. It should be noted that commercially available fluorescence microplate readers, preferable equipped with time resolution, are also suited for the screening of all the microwell plate-based assays presented in this chapter. Nevertheless, the imaging process is much faster, accomplished in the order of one second, and enables ratiometric measurements. 

Polyclonal antibodies of different types are known to show affinity for specific compounds. Thus, antibodies that can bind to a specific substance to be analyzed are immobilized to the walls of the test tubes, plates, or microwells. Such test tubes and plates are commercially available and supplied in the test kit. A measured amount (between 10 and 50 pL) of sample or sample extract is added to one such test tube containing an assay diluent (a phosphate buffer). An equal volume of analyte-enzyme conjugate (commercially available and supplied in the kit) is then added to the test tube. The enzyme conjugate is a solution containing the same analytes covalently bound to an enzyme. The solution mixture is incubated or allowed to stand for a specific amount of time. During this period, the enzyme conjugate competes with the analyte molecules for a limited number of antibody binding sites in the test tube. 

Wells in a microtiter plate are coated with 0.1 /i,g of hAFP per milliliter in 0.1 M NaHCOa. The plate is washed twice, and all wells are filled with 100 p.1 of incubation buffer. Fifty microliters of standard hAFP and unknown samples are added to a set of wells and then titrated by threefold dilutions. One hundred microliters of antiserum diluted 1 300 are then added to each well. The plate is incubated at room temperature for 3 hr. After another three washings, substrate is added and the enzyme is inactivated after 15 min. Figure 7 shows a standard curve for hAFP obtained in this assay. The inhibition curve has all the features of a regular standard curve in a competitive type assay. 

A sandwich ELISA is used to search for a desired analyte in a test solution, as follows the solid phase is coated with analyte-specific capture antibodies to pull the analyte out of the test sample. After washing, the amount of analyte bound to the solid phase can be determined by adding an excess of enzyme-labelled analyte-specific antibody. The specificity of the method can be improved by using a sandwich-type, two-site assay, in which the capture and labelled antibodies have specificities for different parts of the analyte, as mentioned above. The performance of an immunoassay in a standard microtitre plate requires several hours. Such long incubation times are mostly linked to inefficient mass transport from the solution to the surface, whereas the immunocapture itself is a rapid process. 

A number of assays for the hydrolase-type of Hyals were developed in the last decade that facilitated their characterization. These include microtiter-based ELISA-like assays, in which a highly specific HA-binding protein substitutes for the antibody component [186]. Biotinylated HA bound to microtiter plates is subjected to Hyal activity, and the remaining HA quantified by an avidin-enzyme color reaction [107]. HA substrate gel zymography procedures were also formulated that facilitated additional studies of these enzymes [187]. 

Read optical absorbance at 510 nm in an enzyme-linked immunosorbent assay type plate reader. Use the three empty wells to set the background. Calculate an average absorbance value for the nine untreated cultures on a plate. Express the absorbance of dye for the treated cultures on the same plate as a percentage of that value. Repeat for each individual plate. 

To assess the activity of a-gal-mannose glycoconjugates binding to the anti-Gal antibodies, a previously reported inhibition-type enzyme-linked immunosorbent assay (ELISA) was conducted [21]. Mouse laminin having a-gal epitopes were fixed on ELISA plate as solid phase antigens. Test glycoconjugates were then incubated with human anti-Gal antibodies on the ELISA plate. The plate was washed and incubated with horseradish peroxidase (HRP) conjugated anti-human IgG antibody. 

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