CDSimple™ TBG Chemiluminescent ELISA Kit

Sample

Human Serum, Plasma or Whole Blood

Species Reactivity

Human serum; Plasma or Whole Blood

Detection Method

Chemiluminescence Immunoassay

Intended Use

The Quantitative Determination of Thyroxine Binding Globulin (TBG) concentration in Human Serum, Plasma or Whole Blood by a Microplate Enzyme Immunoassay, Colorimetric.

Contents of Kit

A. TBG Calibrators - 0.5 ml/vial
Six (6) vials of references TBG Antigen at levels of 1(A), 4(B), 8(C), 16(D), 32(E) and 64(F) μg/ml. A preservative has been added.Store at 2-8°C.
Note: The calibrators, human serum based, were calibrated using a reference preparation, which was assayed against the international reference material (IS 88/638).
B. TBGEnzyme Reagent -5.5ml/vial
One (1) vial containing Enzyme (HRP) labeled TBG in buffer, dye, and preservative. Store at 2-8°C.
C. TBG Antibody Biotin Reagent -5.5ml/vial
One (1) vial of Biotin labeled Anti-TBG polyclonal IgG in buffer, dye and preservatives. Store at 2-8°C.
D. Streptavidin Coated Microplate - 96 wells
One 96-well microplate coated with streptavidin and packaged in an aluminum bag with a drying agent. Store at 2-8°C.
E. Wash Solution Concentrate - 20ml/vial
One (1) vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-8°C.
F. Substrate A-7ml/vial
One (1) vial containing tetramethylbenzidine (TMB) in buffer.
Store at 2-8°C. See "Reagent Preparation."
G. Substrate B - 7ml/vial
One (1) vial containing hydrogen peroxide (H₂O₂) in buffer.
Store at 2-8C. See "Reagent Preparation."
H. Stop Solution -8m/vial
One (1) vial containing a strong acid(1N HCl). Store at 2-8°C.
I. Product Insert
Note: Above reagents are for a single 96-well microplate.

Storage

Do not use reagents beyond the kit expiration date.
Avoid extended exposure to heat and light. Opened reagents are stable for sixty (60) days when stored at 2-8°C. Kit and component stability are identified on the label.

Performance Characteristics

Accuracy
The TBG ELISA Test System was compared against a reference method. Biological specimens (n=167) from population (symptomatic and asymptomatic) were used. The values ranged from 0-97μg/ml. The correlation is presented in Table.

Precision

The within and between assay precision of the TBG ELISA Test System were determined by analyses on three different levels of control sera. The number, mean value, standard deviation and coefficient of variation for each of these control sera are presented in Tables.


*As measured in ten experiments in duplicate.

Sensitivity

The TBG ELISA Test System has a sensitivity of 1.0 μg/ml. The sensitivity was ascertained by determining the variability of the '0' calibrator and using the 2σ (95% certainty) statistic to calculate the minimum dose.

General Description

Thyroxine Binding Globulin (TBG), 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 decrease 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 color.
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.

Standard Curve

A dose response curve is used to ascertain the concentration of TBG in unknown specimens.
1. Record the absorbance obtained from the printout of the microplate reader as outlined in Example 1.
2. Plot the absorbance for each duplicate serum reference versus the corresponding TBG concentration in μg/ml on linear graph paper.
3. Draw the best-fit curve through the plotted points.
4. To determine the concentration of TBG for an unknown, locate the average absorbance of the duplicates for each unknown on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration (in ng/ml) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated). (See Figure 1).
Note: Computer data reduction software designed for ELISA may also be used for the data reduction. If such software is utilized, the validation of the software should be ascertained.


Note: Computer data reduction software designed for ELISA assays may also be used for the data reduction.
*The data presented in Example 1 and Figure 1 is for illustration only and should not be used in lieu of a dose response curve prepared with each assay.
Q.C.PARAMETERS
In order for the assay results to be considered valid the following criteria should be met:
1. The absorbance (OD) of calibrator A should be > 1.3
2. Four out of six quality control pools should be within the established ranges.