Antigen concentration determination

If another antigen than PT is used, it can be a good idea to determine the optimal antigen concentration prior to the array experiment. 

To determine the effect of spot size, various photolithographic masks were used to create arrays of square patterns at spot widths from 80 to 1145 p. The Kjjpp ( 1 standard deviation) did not vary over the spot size range of 80 to 1145 p under constant flow conditions at fixed levels of antigen concentration, and little effect of variation in spot size was noted on mean binding. 

In addition to successful linking of target antigen and DNA marker, as discussed in the previous chapter, the subsequent amplification of the DNA is the second key factor for efficient IPCR. Similar to many protocols developed for quantitative PCR [2], the DNA amplification product has to be converted into a detectable signal. Typically, a simple yes/no decision on the presence of the DNA marker is not sufficient, and a quantitative readout dependent on the antigen concentration is needed. Therefore, in many IPCR applications the cycle number in PCR-amplification is limited to the exponential phase of the amplification for example, 30 or fewer cycles [10, 24-26, 29, 31, 33, 37]. Alternatively, successful applications of 40 cycles were also reported [34-36, 38, 39, 41], underlining the relative flexibility of PCR conditions for the amplification step. The need for an optimized cycle number is only important for end point determinations such as gel electrophoresis (Section 2.2.1) or PCR-ELISA (Section 2.2.2). Recently, the... 

Incubate environmental samples in step 1 in place of antigen. Process through step 5. Determine concentration of antigen in samples by using the standard curve to convert signal intensity (step 5) to antigen concentration. 

The potentiometric determination of estradiol-1 P in solution provides an example of a solid-phase competitive immunoassay (316). The anti-estradiol-17P antibodies are immobilized on a pig skin gelatin membrane. After incubation with HRP-labeled steroid and estradiol, the membrane is mounted over an iodide-selective electrode to measure the enzymatic activity. The electrode potential is a function of antigen concentration at levels ranging from 57 pM to 9.2 nM. 

Among a number of procedures that were proposed for quantitative determinations, only some which make use of the measurements of a variable and its comparison to a calibration curve will be considered. The results of all but one of the procedures below are expressed in arbitrary units the concentration of the antigen in a solution chosen as standard. The concentration determined may be corrected by calculation and expressed in weight of antigen per unit volume if such a concentration in the standard solution is known. 

Labeled-antibody methods rely on the presence of a large excess of the labeled antibody. This antibody is incubated with standard concentrations of added antigen (the analyte), and the concentration of bound antibody is determined. The resulting calibration curve is linear, as shown in Figure 6.2(a), and linearity extends upward to concentrations where Ab is no longer in excess. Even for very low antigen concentrations, if an excess of Ab is present, some complex will form in principle, even a single Ag molecule can be detected.10... 

Perform coating with the optimal antigen concentration as determined in test A. 

Concentration of the antigen can be readily determined from a standard dose-response curve. The range of antigen concentration rarely exceeds two logic dilutions and is, therefore, much smaller than for different antisera. Microcomputers or desk-top programmable calculators, interfaced with microplate readers, may help to linearize rapidly the dose-response curves and to transform the values obtained for the test-samples into concentrations. In contrast, dose-response curves for antibodies are complicated by a variety of factors ... 

Fig. 15.13. Response error relationship (A) and precision profiles (B) to optimize (particularly AM-)EIA according to Ekins (1979). The immunoreactant concentrations which will give minimum variance, will give also minimum variance when small amounts of sample are added and allows the highest detectability. First, the R is measured at different responses (reliability is not high with a few replicates. Section 15.1). The error in dose (AC) or the relative error (AC/C coefficient of variation) can then be determined by the AR (corresponding to the R of that dose) and the sensitivity of the dose-response curve as shown in Fig. 15.1. A change in, e.g., the antibody concentration may change the precision profile from (a) to (b), which has a greater detectability, but less precision at high antigen concentrations.

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