Antibody standard curves

In the Hybrid-Capture assay (Digene), a full-length RNA probe is hybridized to denatured HBV DNA in solution and the hybrids are captured on the surface of a tube coated with anti DNA RNA hybrid antibody. The bound hybrids are reacted with antihybrid antibody labeled with alkaline phosphatase. A chemiluminescent substrate is converted to a luminescent compound by the bound alkaline phosphatase. Light emission is measured in a luminometer and the concentration of HBV DNA, in pg/ml, is determined from a standard curve. The concentrations of the standards are determined spectrometrically (A260nm/A280nm). 

Subsequent to a further washing step, to remove any unbound antibody-enzyme conjugate, the activity of the enzyme retained is quantified by a straightforward enzyme assay. The activity recorded is proportional to the quantity of antigen present in the sample assayed. A series of standard antigen concentrations may be assayed to allow construction of a standard curve. The standard curve facilitates calculation of antigen quantities present in unknown samples. 

Once a standard curve has been constructed, the RIA can determine the concentration of hormone in a sample (usually plasma or urine). The values of hormone levels are usually accurate using the RIA, but certain factors (e.g., pH or ionic strength) can affect antigen binding to the antibody. Thus similar conditions must be used for the standard and the sample. 

First of all, a Standard Curve or a Calibration Curve is plotted between the reciprocal value (i.e., 1 x %-J radioactivity bound to the antibody) versus the amount of standard for a series of unknowns. Thus, the amount of hapten present in the unknown sample is measured from the plotted calibration curve conveniently. 

Adrenal Tumours The assay-method is entirely based on the Schwartz-Mann Kit. According to this method, cortisol is first extracted from the plasma using CH2C12 (methylene chloride). In the actual radioimmunoassay the cortisol present in the extract competes with Cortisol-H3 i.e., the radioactive tracer) for the common binding sites on transcortin, which is incidently not an antibody but a cortisol-binding protein. Now, the free cortisol is quantitatively removed by adsorption on dextran-coated charcoal from the one bound to the transcortin. Finally, the bound radioactivity (due to Cortisol-H3) is measured which is then employed to calculate exactly the amount of cortisol present in the sample by the help of a Standard Curve (or Calibration Curve). 

The most commonly used format for quantitation assays is the sandwich assay format. Typically, a monoclonal antibody (MAb) is used to capture the product. It is then detected by another antibody, usually enzyme-labeled. A reference standard is used from which to compare the response of an unknown test sample. There is a relative increase in measured response (optical density) with increasing analyte concentration. Figure 11.2 is an example of a typical ELISA standard curve. 

In most cases, the sample titer curves followed the predicted titer standard curve based upon known concentration ratios. Observed variations in linearity were traced back to inconsistencies in labeling rather than cross-reactivity or sample preparation issues such as pipetting or mixing errors. Overall, antigen arrays performed better than antibody arrays. While the exact reasons for the... 

Radioimmunoassay. Radioimmunoassay (RIA) was first described by Berson and Yalow (34) and Luft and Yalow (35). The assay is based upon the competition for an antibody between a radiolabelled antigen and its unlabelled counterpart. The greater the amount of unlabelled antigen in the test sample, the less radiolabelled antigen bound. The concentration of antigen in a test sample can be determined from comparisons with standard curves. 

Application of the RIA. The RIA was initially used to evaluate the cross-reaction of the STXOL antisera to STX. The antibody was found to have excellent STX cross-reactivity (93%). Subsequent preparation of a logit/log (37) standard curve for STX (Figure 4) demonstrated that the chosen assay format would give good reproducibility and a desirable order of magnitude linear sensitivity range. 

FIGURE 23-3 Radioimmunoassay (RIA). (a) A low concentration of radiolabeled hormone (red) is incubated with (T) a fixed amount of antibody specific for that hormone or (2) a fixed amount of antibody and various concentrations of unlabeled hormone (blue). In the latter case, unlabeled hormone competes with labeled hormone for binding to the antibody the amount of labeled hormone bound varies inversely with the concentration of unlabeled hormone present, (b) A radioimmunoassay for adrenocorticotropic hormone (ACTH). A standard curve of the ratio [bound] to [unbound radiolabeled ACTH] vs. [unlabeled ACTH added] is constructed and used to determine the amount of (unlabeled) ACTH in an unknown sample. If an aliquot containing an unknown quantity of unlabeled hormone gives, say, a value of 0.4 for the ratio [bound]/[unbound] (see arrow), the aliquot must contain about 20 pg of ACTH. 

In the tubes containing higher concentrations of hormone, the labeled hormone has been diluted more, and the amount bound to antibody is less than in tubes with lower concentrations of hormone. The tubes of known concentrations are used to construct a standard curve from which the unknown concentrations can be read. As little as a femptomole of hormone (i.e., the amount present in 1 ml of a 10 12 M solution can be detected).1 Methods are available for virtually every pure hormone.c... 

Calculate the concentration of antibody-toxm conjugate in sample wells by comparison with a standard curve (see Note 23). 

The concentration of antibody-toxin conjugate in samples is determined from the optical density values at 492 nm, given by a standard curve. The standard curve is obtained by the same procedure using dilutions of the stock antibody-toxin solution having final concentrations between 0.125 and 20 ng/mL in casein buffer. 

Deduct from the standard curve the nanomoles of biotin corresponding to the observed change in absorbance. The ratio between nanomoles of biotin and nanomoles of antibody used to displace HABA represents the degree of biotinylation, as seen from the following equation ... 

Plot the peak positions against the manufacturer s specified antibody binding capacity for each bead in the mixture to create a standard curve. Figure 3 illustrates curves for Simply Cellular beads stained with a PE-labeled antibody in a direct assay (A), and m an indirect assay with a PE-labeled antimouse IgG (B). The increased sensitivity of the indirect assay is evident from the rightward shift of the curve. 

A similar method is used for beads with defined fluorochrome content (e g, Quantum beads, Flow Cytometry Standards Corporation), except that the beads are not subjected to the antibody-staining procedure The number of fluorochrome molecules per cell is deduced from the standard curve, and is converted to bound antibody molecules from knowledge of the fluorochrome protein ratio of the conjugate. 

Key Words ELISA competition binding substrate antibody pairs standard curve. 

Read the plate at 450/540 nm. Make a 4PL curve fit and generate a standard curve with both x-axis and y-axis linearly. Determine concentration of collagen-specific antibodies based on the standard curve. 

Fig. 6.4 A four-parameter logistic standard curve depicting a competitive assay. In this format the antibody (Ab) is present in a limited amount. The known amount of labeled drug competes with the drug analyte in the sample for the limited sites of the Ab. The response factor produced by the label antigen-antibody complex (Ag-Ab) is inversely proportional to the concentration of the drug analyte in the sample. In this example the % B/B0 (% label bound at concentration X divided by...

ProteinChip arrays with preactivated surfaces are also available for covalently coupling of a specific bait molecule (protein, DNA, RNA). This allows the investigation of specific protein interactions such as DNA-protein, receptor-ligand, and antibody-antigen (Ab-Ag), the latter of which permits the generation of a standard curve and hence quantitation studies (21). 

The most common, but by no means the only or even the most promising, immunochemical assay for small molecules is radioimmunoassay (R1A). As an overview, an immunoassay involves chemically attaching the small molecule of interest (or a derivative of it) to a carrier protein and raising specific antibody titers to it in the serum of an animal. Very dilute antibody solutions are then used to bind the small molecule which has been radiolabeled. The competition of varying known concentrations of unlabeled material is measured and the resulting standard curve used to determine unknown concentrations (Table 1). The steps leading to the development of an R1A are outlined below followed by a description of other immunochemical procedures and an analysis of the attributes and limitations of immunoassay. 

An increasing amount of unlabeled antigen displaces a constant amount of labeled antigen from a constant amount of antibody. Separation of antibody bound and free material results in a standard curve that can be used to determine the amount of unlabeled antigen in unknown samples. 

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