Dilution linearity assays, calibration curves

Following a 10-fold dilution of the sample with 0.6 M perchloric acid, the product of this reaction absorbs maximally at 420 nm. The original assay, developed by Kissane and Robins,12 uses DABA dihydrochloride for depurination (no perchloric acid was used) and employs fluorescence measurements at 520 nm after excitation at 420 nm. More recent work shows that the assay with perchloric acid can be conducted with 10-pL DNA sample volumes and provides a linear fluorescence calibration curve over the 10-500-ng total DNA range (1-50 pg/mL).13 The DABA method has since been modified for colorimetric measurements at 420 nm, and has a detection limit of 25 pg.14 The DABA reaction is specific for DNA, but... 

The concentration of analyte in the unknown sample is extrapolated from the calibration curve. To obtain an accurate and precise quantitative value, the optical density (OD) for the sample solutions must fall on the linear portion of the calibration curve. If the sample OD is too high, the sample solution must be diluted until the OD falls within the quantitative range of the assay. The concentration of the analyte in the original sample is calculated by correcting for any dilution factor that was introduced in preparing the sample for application to the microplate. 

Because of the possibility that the herbicide alachlor could adulterate food if either poultry or livestock consumed contaminated materials, Lehotay and Miller evaluated three commercial immunoassays in milk and urine samples from a cow dosed with alachlor. They found that milk samples needed to be diluted with appropriate solvents (1 2, v/v) to eliminate the matrix effect. One assay kit (selected based on cost) was also evaluated for use with eggs and liver samples from chickens. Egg and liver samples were blended with acetonitrile, filtered, and diluted with water. Linear calibration curves prepared from fortified egg and liver samples were identical... 

Fig. 18.6. Calibration curve obtained by the immuno-assay procedure in the detection of anti-E2 using the ITO-Poly (pyrrole-benzophenone) coated optical fibers. The linear range of the calibration curve was obtained for titer 1 64,000 and lower. The curve was fitted according to the equation y = A+B(x), where x is the human sera (anti-E2 antibodies) dilution value and y is the chemiluminescence response. The obtained correlation coefficient was R2 = 0.988.

Determine the concentration of host-cell proteins from the calibration curve. If the samples do not show linear dilution then the capture antibody was not in excess and the assay results are invalid. 

In a linear dilution series the concentrations are separated by an equal amount, e.g. a series containing cadmium at 0, 0.2, 0.4, 0.6, 0.8, l.OmmolL" might be used to prepare a calibration curve for atomic absorption spectroscopy (p. 170) when assaying polluted soil samples. Use [Ci]F] =[C2]f2 to calculate the volume of standard solution for each member of the series and pipette or syringe the calculated volume into an appropriately sized volumetric flask as described above. Remember to label clearly each diluted solution as you prepare it, since it is easy to get confused. The process is outlined in Box 4.3. 

Procedurally, dilutional linearity should not be confused with the MRD of an assay. The MRD is a designed integral part of an analytical method and involves a predefined dilution of test samples, QC samples and, often, calibrators usually with a buffer-based matrix. In contrast, dilution linearity is used only to support analysis of study samples that exceed the assay s ULOQ and involves dilution(s) intended to result in an analyte concentration within the standard curve s validated range. Another notable difference is that, while MRD is usually performed in buffer, dilutional linearity is performed in matrix, often the same one used to prepare the standard curve. 

Some workers will interpret this as the ability to reproduce calibration curves for a particular assay that are parallel to each other when plotted graphically whether linear or nonlinear, as opposed to a demonstration of analyte recovery without bias, when sample matrix is diluted to assay samples whose concentrations lie above the analytical range of the method. Parallelism and other potential matrix effects should be investigated separately. 

Linearity has been described by some workers in a way which, by the current authors, would be interpreted as matrix parallelism, whereas others will use the term to describe the extent to which a calibration curve is linear in nonligand-binding assays. For the purpose of this chapter, the term linearity or dilution linearity is used to describe the results of experiments conducted using spiked samples to demonstrate the potential for high-concentration samples to be able to be diluted into the analytical range and read with acceptable accuracy and precision. It is often used to give an indication that matrix effects will not cause a problem upon sample dilution in circumstances where incurred or volunteer samples are not available with concentrations of analyte sufficiently high to conduct parallelism experiments. 

The sensitivity of an immunoassay is typically defined by its LLOQ, which can only be determined when a reference standard is available. Alternatively, a detection limit of qualitative assays is used where a distinction is essentially made between positive and negative results only. Typically, the antibody data are reported as titer, the titer being the reciprocal of the highest dilution of a sample in which the instrument response is greater than the cutoff response [22]. At least two positive control analytes should be used. If a positive control antibody is available, a pseudocalibration curve can be generated by a series of dilutions. However, one should keep in mind that true quantitation is impossible because of the lack of a true reference compound. Determination of the dilutional linearity is not so important when the result is reported as a titer. However, if the determination of positive samples is based on the interpolation from a reference standard curve, it is essential to demonstrate that the QC samples fall within the (limited) linear range of the calibration curve and not on a plateau or a region that may include a hook effect. 

Fig. 4. Typical BART curves from (a) a positive and a negative sample (b) BART assay run in different volumes showing the same peaking time (c) dilution series of target DNA amplified by LAMP (d) linear calibration for DNA amplified by LAMP (e) dilution series of target DNA amplified by RDC (f) linear calibration DNA...

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