ELISA inhibition standard

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

Viral encephalitides 10-100 organisms Serum Viral isolation, Serology ELISA or hemagglutination inhibition Standard Precautions (mosquito control)... 

RIAs are highly sensitive and quantitative, capable of detecting small amounts of Ag or Ab. As a result, they are often used to measure the quantities of hormones or drugs present in a patient s serum. In this case, RIAs are performed in a manner similar to the competitive ELISA. The presence of the hormone in the serum sample inhibits binding of the radiolabeled hormone. Thus, the amount of radioactivity present in the test is inversely proportional to the amount of hormone in the serum sample. A standard curve using increasing amounts of known concentrations of the hormone is used to determine the quantity in the sample. 

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. 

Assay Development and Validation. Reproducibility of this ELISA assay was determined based on a set of clomazone standards that were run on different plates on the same day (intra-assay) and on different days (inter-assay). The intra-assay coefficient of variation of the standards changed from 1.5 at the highest clomazone concentration (250 ppb) to 22 at the lowest concentration of 1.4 ppb. The coefficient of variation(CV) at clomazone rate of 12.5 ppb was 10. Similar values were obtained for the inter-assay variability, with the CV of the 1.4 ppb concentration being 22.5, and the CV for the 250 ppb concentration being 2.7. The CV for the 10 ppb concentration of clomazone was about 5 between tests. Analysis of the data for this range of clomazone concentrations indicates that there is good correlation (r =0.97) between the log of the concentration of clomazone and percent inhibition in the assay when the linear regression equation was used. Based on these results, the limit of the test s sensitivity was defined as 2 ppb (10 ppb in soil) and the limit of detection was set at 1 ppb. 

Figure 2. Inhibition ELISA analysis of alachlor in deionized water. Means and standard deviations were calculated from 20 separate analyses.

The evaluation of a number of immunoassay diagnostic kits was undertaken to determine their usefulness in a regulatory analytical laboratory environment in the food, feed and pesticide areas. Four rapid enzyme immunoassay tests for the detection of aflatoxin residues at the 20 ppb level in animal feeds were compared to the official HPLC procedure. In the pesticide area, a commercial pentachlorophenol competitive inhibition assay for residues in water was investigated as to its applicability to poultry and pork liver matrices. In addition, an ELISA screening procedure for the herbicide fusilade was developed. Modifications were incorporated into the rapid immunoband 1-2 Test procedure for the detection of motile Salmonella in various food and animal feed products resulting in quicker analysis than the standard culture method. Also, a comparative evaluation of a Quik-Card Test for sulphamethazine drug residues in pork urine, liver and muscle tissue, is described. 

To evaluate the amount of acetaminophen bound to proteins, utilizing the competitive A-B ELISA or PCFIA, inhibition by unknown samples was compared with an assay standard prepared by derivatizing protein with NAPQI. For some experiments, 3-(N-acetyl-L-cystein-S-yDacetaminophen was used as an assay standard, in which case, the values obtained were corrected for differences in the relative inhibitory potency of 3-(N-acetyl-L-cystein- yl)acetaminophen and 3-Cys-A protein adduct (120 fmol/well and 2300 finol/well, respectively) (14). After dialysis, unknown samples were diluted to a final concentration of approximately 4 / g protein/assay well, assayed in duplicate, and expressed as nmoles of 3-Cys-A per mg of protein. The ELISA and PCFIA were shown to have similar limits of detection (20 pmole/mg protein) and to recognize the same itope as demonstrated by similar relative inhibitory potencies for N-acetylcysteine-acetaminophen, acetaminophen-bound... 

Since our intent was to use the competitive A-B ELISA to quantify 3-Cys-A adducts formed in biological samples at unknown and perhaps variable levels of protein modification, experiments were conducted to determine the effect of adduct substitution level on quantification. Standards of known substitution level were prepared by derivatizing 9,000 g liver supernatant with various concentrations of [3H]NAPQI. After extensive dialysis to remove noncovalently bound materials, protein concentrations were determined and the substitution level of each standard was determined by scintillation counting. These synthetic standards, which ranged from 0.5 to 30 nmol 3-(cystein-S-yl)[3H]acetaminophen per mg protein, were analyzed in the competitive immunoassay. When the data were plotted with percent inhibition as a function of protein concentration, the results show an ordered family of inhibition curves where the most highly substituted proteins were the... 

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