The cross-reaction of the antibody calculated at 50% according to Abraham, are shown in the table:
% Cross reactivity
FREE TESTOSTERONE ELISA
The Free Testosterone ELISA test system is an enzyme linked immunosorbent assay (ELISA) for the measurement of
SUMMARY AND EXPLANATION
Testosterone is a steroid hormone from the androgen group. Testosterone is a primarily secreted in the testes of males
and the ovaries of females although small amounts are secreted by the adrenal glands. Testosterone is the principal
male sex hormone, is an anabolic steroid, and, for both males and females, plays a key role in health and well-being.
Testosterone within the circulation is principally bound to proteins, the most important of which, is sex hormone binding
globulin (SHBG). Measurement of the free or unbound fraction of serum testosterone has been proposed as a mean 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
McCann D, Kirkish L. Evaluation of Free Testosterone in serum. J.Clin. Immunoassay 1985; 8:234-236.
testosterone measurements may be more useful than total testosterone in situations where SHBG is increased or
Ekins R.P. Free hormones in blood J. Clin. Immunoassay 1984; 7(2): 163-180.
decreased (e.g. hypothyroidism and obesity).
Paulson JD, et al. Free Testosterone concentration in serum: elevation is the hallmark of hirsutism. Am.J.Obst. Gynecol 1977; 128:851-857.
PRINCIPLE OF THE TEST
Odlind V. et al. Plasma androgenic acitivity in women with acne vulgaris and in healthy gils before, during and after puberty. Clin.Endocrinology 1982; 16:243-249.
The free Testosterone ELISA KIT is based on the principle of competitive binding between testosterone in the test
specimen and Testosterone-HRP conjugate for a constant amount of rabbit anti-Free Testosterone. In the incubation,
Green PJ. Free Testosterone determination by ultrafiltration and comparison with dialysis.Clin.Chem. 1982;28:163-180.
goat anti-rabbit IgG-coated wells are incubated with 25µl of Testosterone standards, patient samples, 50µl
testosterone-HRP conjugate reagent and 50µl rabbit anti-free testosterone reagent at room temperature for 60 minutes.
Wu Ch. Plasma free and protein-bound testosterone in hirsutism. Obstet.Gynecol 1982; 60:188-194.
During the incubation, a fixed amount of HRP labeled testosterone competes with the endogenous testosterone in the
Abraham, G.E. (1969) Solid-phase radioimmunoassay of estradiol -17b./clin. Endocr.Metab. 29, 866-870.
standard and sample, for a fixed number of binding sites of the specific fee testosterone antibody. Thus, the amount of
testosterone peroxidise conjugate immunologically bound to the well progressively decreases as the concentration of
free testosterone in the specimen increases. Unbound testosterone peroxidise conjugate is then removed and the wells
washed. Next, 100µl of TMB Reagent is added and incubated at room temperature for 15 minutes, resulting in the
development of blue color. The color development is stopped with the addition of 50µl stop solution, and the
All of BQ ELISA Kits have not been tested for clinical use and are not approved in the United States by the FDA for
absorbance is measured spectrophotometrically at 450nm. A standard curve is prepared relating color intensity to the
diagnostic clinical use. They are components or reagents made solely for research use, further manufacturing and
export use. It is the commitment of BQ customers to receive its products solely for the purpose of exportation or
research, and not for the purposes of clinical diagnostic use.
Microwells coated with Goat anti-rabbit IgG
BQ KITS DOES NOT MAKE ANY OTHER WARRANTY OR REPRESENTATION WHATSOEVER, WHETHER
EXPRESS OR IMPLIED, WITH RESPECT TO THESE PRODUCTS. IN PARTICULAR BQ KITS, INC. DOES NOT
MAKE ANY WARRANTY OF SUITABILITY, NON-INFRINGEMENT, MERCHANTABILITY OR FITNESS FOR ANY
PARTICULAR PURPOSE OF ANY PRODUCT
Rabbit Anti-Testosterone Reagent (ready to use)
MATERIAL NOT PROVIDED
ELISA reader capable of reading absorbance at 450nm
STORAGE AND STABILITY OF THE KIT
Calculate the average absorbance values for each set of standards, controls and patient samples
Construct a standard curve by plotting the mean absorbance obtained from each standard against its concentration in
Keep microwells sealed in a dry bag with desiccants.
pg/ml with absorbance value on the vertical(Y) axis and concentration on the horizontal (X) axis
Opened stardares are stable for 6 months at 2 – 8 C all other reagents are stable until expiration of the kit.
Using the mean absorbance value for each sample determine the corresponding concentration of Free Testosterone
Do not expose test reagents to heat, sun, or strong light.
from the standard curve. Depending on experience and/or the availability of computer capability, other methods of data
WARNINGS AND PRECAUTIONS
Automated method: Computer programs using cubic spline, 4 PL (4 Parameter Logistics) or Logit-Log can generally give
The standards contain human source components which have been tested and found non-reactive for hepatitis B
The concentration of the samples can be read directly from this standard curve. Samples with Free Testosterone
surface antigen as well as HIV antibody with FDA licensed reagents. However, as there is no test method that
concentration higher than the concentration of the highest standard have to be diluted with zero standard. For the
can offer complete assurance that HIV, Hepatitis B virus or other infectious agents are absent, these reagents
calculation of the concentrations this dilution factor has to be taken into account.
should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Control/National
Institutes of Health manual, "Biosafety in Microbiological and Biomedical Laboratories." 1984
Example of Standard Curve
This kit is designed for research use only.
Optimal results will be obtained by strict adherence to this protocol. Accurate and precise pipetting, as well as
following the exact time and temperature requirements prescribed are essential. Any deviation from this may yield invalid data.
The components in this kit are intended for use as an integral unit. The components of dif erent lots should not be
mixed. It is recommended that serum samples be run in duplicate.
This test kit is designed for Research and Development use only.
Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which specimens or kit reagents are handled.
SPECIMEN COLLECTION HANDLING
Serum: Collect blood by venipuncture, allow to clot, and separate serum by centrifugation at room temperature.
Plasma: Whole blood should be collected into centrifuge tubes containing anti-coagulant and centrifuged
Do not use haemolytic, icteric or lipaemic serum.
It is recommended that each laboratory establish its own normal ranges based on a representative sampling of the local
Testosterone can be determined in plasma as well as in serum of patients who have been fasting. The clinical
population. The following values for TSH may be used as initial guideline ranges only:
significance of the determination of Free Testosterone can be invalidated if the patient was treated with cortisone
Conc. Range (pg/ml)
Specimens which are not used at the same day of collection have to be frozen only once at -20°C prior to assay. Thawed samples should be inverted several times prior to testing
Samples with values greater than the highest standard should be diluted with standard 0 and reassayed.
PREPARATION OF REAGENTS
Prepare 1X Wash Buffer by adding the contents of the bottle (25ml, 20X) to 475 ml of distilled or
LIMITATIONS OF THE TEST
deionized water. Store at room temperature (18-26 °C).
Do not use sodium azide as preservative. Sodium azide inhibits HRP enzyme activities.
All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed
without foaming. Once the test has been started, all steps should be completed without interruption.
Secure the desired number of microwell strips in the holder.
Dispense 20 µl Testosterone Standards, controls and samples with new disposable tips into appropriate wells.
Correlation with a Reference ELISA kit:
A total of 74 sera were tested by this ELISA and a commercially available free-testoserone reference ELISA kit.
Dispense 50µl anti-testosterone reagent into each well.
The linear regression curve was calculated as:
Dispense 50 µl Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.
Incubate for 1 hour at room temperature.
Briskly shake out the contents of the wells. Rinse the wells 3 times with diluted wash solution. Strike the wells
sharply on absorbent paper to remove residual water droplets. NOTE:
The sensitivity and precision of this assay is markedly influenced by the correct performance of the
Add 100 µl of Substrate Solution to each well.
Incubate for 15 minutes at room temperature in the dark.
10. Stop the enzymatic reaction by adding 50 µl of Stop Solution to each well.
11. Read absorbance on ELISA Reader at 450 nm within 10 minutes after adding the stop solution. CALCULATION OF RESULTS
Project L.E.A.P. Transcript Brain Injury Association of Florida presents Project L.E.A.P. Law Enforcers as Partners with Individuals with Traumatic Brain Injury The State of Florida Department of Health Brain & Spinal Cord Injury Program Slide 1 - Brain Injury Association of Florida presents Project L.E.A.P. - Law Enforcers as Partners with Individuals with Traumatic Br
Orientation Welcome to Tabir! You're going to have a wonderful time and, potentially, a life-changing (in the best sense of the word) experience. Please read this, though, before you go, so you can prepare properly for your "adventure". Although we have a dispensary at the Tabir, our range of medical supplies is quite limited. We will have some help from a camp physician, and I will be