Free Testosterone ELISA kit
|Category Name||Steroid ELISA kits|
|Method||ELISA method: Enzyme Linked Immunosorbent Assay|
|Principle||Competitive Enzyme Immunoassay|
|Detection Range||0.06 - 100 pg/ml|
|Sample||20 ul serum|
|Total Time||~ 75 min|
|Shelf Life||12-14 Months from the manufacturing date|
Materials provided with Free Testosterone ELISA Test Kit:
1.Free Testosterone EIA Kit Standards 6x STD 0 â€“STD 5 (1 vial = 1 mL)
2.Conjugte (1 bottle) 15 mL Testosterone-HRP conjugate.
3.Control ( 1 vial = 1ml)
4.Free Testosterone Test Coated Microplate (1 microplate breakable)
Anti-Testosterone IgG adsorbed on microplate
5.TMB-substrate (1 bottle) 15 mL
H2O2-TMB 0.26gr/L (avoid any skin contact)
6.Free Testosterone EIA Test Stop solution (1 bottle) 15 mL
Sulphuric acid 0.15 M (avoid any skin contact)
7. Free Testosternone Kit Conc. Wash Solution 10X (1 bottle = 50mL)
NaCI 160 g/L; tween-20 10 g/L 20 mM Phosphate buffer, pH 7.4
Materials & Instrumentation required but not included:
Auxiliary materials and instrumentation
2.ELISA kit Microplate Washer
3.ELISA kit Microplate Reader at 450nm & 620 nm wavelength
Diagnostic Automation Free Testosterone Elisa test is a steroid hormone from the androgen group.
SUMMARY AND EXPLANATION
It is a competitive immunoenzymatic colorimetric method for quantitative determination of Free Testosterone concentration in serum and plasma.
Testosterone is primarily secreted in the testes of males and the ovaries of females although small amounts are secreted by the adrenal glands.
It is the principal male sex hormone and an anabolic steroid. In both males and females, it plays key roles in health and well-being. Only 1-2% of circulating testosterone exists as unbound or free testosterone.
The majority, approximately 60%, is bound to SHBG with high affinity, while the remainder is loosely bound to albumin.
Both the albumin-bound and free fractions may be biologically active, while SHBG effectively inhibits testosterone action.
Testosterone effects can be classified as virilizing and anabolic effects. Anabolic effects include growth of muscle mass and strength,
increased bone density and strength, and stimulation of linear growth and bone maturation. Virilizing effects include maturation of the sex organs.
Testosterone levels decline gradually with age in men. Measurement of the free or unbound fraction of serum testosterone has been proposed as a means of estimating the physiologically bioactive hormone.
Free testosterone levels are elevated in women with hyperandrogenism associated with hirsutism in the presence or absence of polycystic ovarian disease.
In addition, free testosterone measurements may be more useful than total testosterone in situations where SHBG is increased or decreased (e.g. hypothyroidism and obesity).
FREE TESTOSTERONE ELISA TEST PRINCIPLE
Free Testosterone (antigen) in the sample competes with horseradish peroxidase testosterone (enzyme labeled antigen) for binding onto the limited number of anti- testosterone (antibody) sites on the microplates (solid phase).
After incubation the bound/free separation is performed by a simple solid-phase washing. The enzyme substrate (H2O2) and the TMB-Substrate (TMB) are added. After an appropriate time has elapsed for maximum color development,
the enzyme reaction is stopped and the absorbance is determinated. Free Testosterone concentration in the sample is calculated based on a series of standard.
The color intensity is inversely proportional to the Free Testosterone concentration of in the sample. Testosterone in the blood is bound to SHBG (60 %) and in lower quantity to other protein.
Only the measurement of Free Testosterone (< 1% of Total Testosterone) permits the estimating of the hormone biologically active.
FREE TESTOSTERONE EIA PERFORMANCE AND CHARACTERISTICS
1. Intra Assay Variation
Within run variation was determined by replicate determination (15x) of three different serum samples in
the same assay. The within assay variability is <10%.
2. Inter Assay Variation
Between run variations was determined by replicate measurements of three different control sera and
two serum samples in 10 different lots. The between assay variability is <10%.
The minimum detection limit (MDL) was calculated by linear regression from average abs standard zero
and standard 1 then was dosed the 2nd s.d. of standard zero abs. The lowest detectable concentration of
Free Testosterone is 0.06 pg/ml.
The specificity was assessed by measuring the apparent response of the assay to the following
potentially cross-reactive analytes and interfering substances (Anticoagulants).
The cross reaction of the antibody calculated at 50% according to Abraham are shown in Free testosterone Elisa insert, specificity.
The Free Testosterone ELISA was compared to present Free Testosterone RIA. Serum samples of 24
females and 17 males were analyzed according in both test systems.
The linear regression curve was calculated
Diagnostic Automation/ Cortez Diagnostic, Inc. = 0.957* (FT RIA) + 0.953
r2 = 0.937