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|Method & Line||Sample & Target||Product||Package Info||Product||Package Info|
|Chemiluminescence MicroELISA||Whole blood,Plasma,Serum||CLIAgen TBG Kit||Tests per Package: 96|
|CLIAgen||CLIAgen TBG Kit is a microplate chemiluminescence assay for quantitative determination of TBG Thyroxine Binding Globulin concentration in Human Serum, Plasma or Whole Blood.||Code: CLI4049||Package: 1 Microplate|
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CLIAgen TBG Kit is a microplate chemiluminescence assay for quantitative determination of TBG Thyroxine Binding Globulin concentration in Human Serum, Plasma or Whole Blood.
TBG (Thyroxine Binding Globulin) a 54 kD liver glycoprotein is the principal binding protein for T4 and T3 in circulation. Electrophoretic analyses indicate that T4 is bound, in decreasing order, to TBG, to a T4 binding prealbumin (TBPA) and to albumin. By virtue of its intense affinity for T4, TBG is by far the major determinant of overall binding capacity. The interaction between T4 and its binding proteins conforms to a reversible binding equilibrium in which the majority of the hormone is bound and a very small portion (≤ 0.05%) is free. T3 is not bound by TBPA and is bound by TBG less firmly than is T4. As a consequence proportion of free T3 is normally 8-10 times greater than T4. Only free (T3/T4) hormones are available to the tissues, therefore the metabolic state of the patient will correlate more closely with the free than with the total concentration of the hormones.
The diagnostic accuracy of the total hormone measurements would be equal to the free hormone if all the patients had similar binding protein concentrations. Unfortunately, serum TBG abnormalities that distort the total: free relationship, are commonly encountered in clinical practice. Additionally the presence of antibodies to thyroid hormones, in some patients, renders total hormone measurements unreliable. Considerable confusion still exists regarding the validity of free hormone testing. There is controversy regarding the clinical utility of free hormone testing in conditions associated with binding protein abnormalities of pregnancy and non-thyroidal illness. Methods that are sensitive to albumin concentrations, the effect of certain drugs, high free fatty acid and levels of hormones binding inhibitors are considered inadequate by some researchers. However, the techniques for physically separating the exceedingly small amounts of free hormones from the dominant protein bound moiety are too technically demanding, inconvenient and expensive for a routine clinical laboratory. Such methods that employ equilibrium dialysis, ultrafilteration and gel-filtration are typically used by researchers. In routine analysis the clinical laboratories rely on direct measurements of free and total hormones and their binding proteins, mainly TBG.
Based on their serum concentrations, familial TBG variants are divided into four major categories: excess, normal, partial deficiency and complete absence. The studies show that estrogens – pregnancy and oral contraceptives – acute intermittent porphyria and chronic liver disease increase TBG concentrations while, androgenic and anabolic steroids, large doses of glucocorticoids and nephrosis decreases TBG levels.
In this method, TBG calibrator, patient specimen or control is first added to a streptavidin coated well. Biotinylated polyclonal antibody (highly specific for TBG) and enzyme labeled TBG are added, in sequence, and the reactants mixed. Reaction between the TBG antibodies, enzyme labeled TBG and native TBG forms a complex that binds with the streptavidin coated to the well.
After the completion of the required incubation period, the excess enzyme conjugate is separated from the bound fraction via a wash step. The activity of the enzyme present on the surface of the well is quantitated by reaction with a suitable substrate to produce light.
The employment of several serum references of known TBG levels permits construction of a dose response curve of activity and concentration. From comparison to the dose response curve, an unknown specimen's activity can be correlated with TBG concentration.
The essential reagents required for an enzyme immunoassay include high affinity and specificity antibody in, enzyme labeled antigen and the native antigen. In this procedure, the immobilization takes place during the assay at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated polyclonal anti-TBG antibody. Upon mixing polyclonal biotinylated antibody with the enzyme-labeled antigen and a serum containing the native antigen, a competition reaction results between the native antigen and the enzyme labeled antigen for a limited number of specific binding sites on the antibody. The antigen bound antibody attaches to the surface of the plastic wells because of the biotin label on it and the streptavidin that is present on the plastic well.
Simultaneously, the complex is deposited to the well through the high affinity reaction of streptavidin and biotinylated antibody.
After equilibrium is attained, the excess serum proteins, antibodies and the enzyme labeled antigen are removed via a wash step. The enzyme activity in the antibody‑bound fraction is inversely proportional to the native antigen concentration. By utilizing several different serum references of known antigen values, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained.
The CLIAgen TBG Kit contains sufficient reagent for 96 wells. Each kit contains the following reagents:
Wash Buffer Concentrate 50x
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