Antibodies binding site calibration

Other types of calibration help may be available in the form of a different type of calibrated microsphere. Beads can be obtained that possess a known number of binding sites for immunoglobulin molecules. They can be treated as if they were cells and stained in the routine way with the antibody stain (direct or indirect) in question. The intensity of the beads with known numbers of antibody binding sites can be used to calibrate the scale, converting ADC channels to... 

Labeled-Analogue Methods, In a typical one-step RIA for FT4, endogenous free hormone and a I-labeledT4 analogue compete for solid-phase antibody binding sites in the presence of serum protein the amount of labeled analogue bound by the specific antibody is inversely related to the amount of FT, in the specimen. Test results are expressed relative to secondary serum calibrators that have been independently calibrated using equilibrium dialysis or ultrafiltration techniques. A number of one-step RIAs have been developed and are commercially available. 

This example assumes that RIA was chosen. The principle behind RIA is the competition between the analyte A and a radioactively tagged control C (e.g., a /-marked ester of the species in question) for the binding site of an antibody specifically induced and harvested for this purpose. The calibration function takes on the shape of a logistic curve that extends over about three orders of magnitude. (Cf. Fig. 4.38a.) The limit of detection is near the B/Bo = 1 point (arrow ) in the upper left corner, where the antibody s binding sites are fully sequestered by C the nearly linear center portion is preferrably used for quantitation. 

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). 

Beads Beads are particles (made, usually, of polystyrene) that can be used as stable and inert standards for flow cytometric analysis. Beads can be obtained conjugated to various fluorochromes in order to standardize fluorescence detection settings and optical alignment or to calibrate fluorescence scales. They can also be conjugated to antibodies in order to calibrate the scale in terms of number of binding sites. More recently, in multiplexed assays, beads with capture molecules have been used to determine the concentration of soluble analytes. 

Binding of antibody to T4-E decreases enzyme activity. The unbound T4-enzyme conjugate, on the other hand, is active and catalyzes the oxidation of glucose-6-phosphate with simultaneous reduction of NAD-t to NADH. The rate of increase of absorbance at 340 nm is inversely proportional to the number of unsaturated TBP binding sites. Test results are expressed as percent of T uptake using a calibration curve or a mathematical function. Manual and automated versions of this assay are commercially available. ... 

Modulation of the activity response by competing, unlabeled immu-noreactants may be achieved by two approaches (i) the labeled and unlabeled ligand compete simultaneously for the available binding sites of the antibodies (equilibrium techniques) and, (ii) the unlabeled ligand (test or calibration sample) is reacted first with the available binding sites and the number of occupied binding sites is estimated by the subsequent addition of the labeled reactant (sequential saturation). These methods are conceptually different and... 

It is wise to load the antigen with a filtration device (Schleicher and Schiill). Afterward, you fix the proteins (e.g., with ethanol/acetic acid solutions, TCA, or heating up) and block the protein binding sites of the membrane (BSA, milk powder, and so on, see Section 1.6.2). This is followed by incubations with anti-antigen and peroxidase conjugated anti-IgG antibody. You wash between the individual steps (Table 6.2). Afterward, you punch the dots on the nitrocellulose membrane into the depressions (wells) of microtiter plates. With a suitable substrate solution, a peroxidase-catalyzed color reaction develops in the wells. The color solution is transferred into new microtiter plates (to get rid of the confetti) and finally measured in the ELISA reader. A calibration curve with defined amounts of antigen quantifies the results (Becker et al. 1989). 

TBG tests that use labeled T4 have also been developed. In one commercial assay, a two-site IRMA or sandwich assay is used. TBG antibody bound to glass particles is added in excess to the patient specimen and binds virtually aU the endogenous TBG present. I-T4 is then added it binds to the TBG, forming an anti-TBG-TBG- I T4 sandwich. After a short 15-minute incubation period at room temperature, the precipitate is collected by centrifugation and its radioactivity is counted. Test values are interpreted from a calibration curve run simultaneously with the patient specimens. Unlike the typical RIA procedure, radioactivity in this IRMA method is directly proportional to the amount of TBG present in the specimen. Patients with molecular variants of TBG may have discordant results, but the assay is not affected by excess thyroxine or phenytoin. ... 

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