Contents of the kit Assay procedure 1. 1 bottle TRACER,32 ml, ready for use. SHBG [125I] IRMA KIT
Contains less than 740 kBq of 125I labelled
Label coated tubes in duplicate for each standard (S0-S5), control serum(C) and
anti-SHBG and biotin labelled anti-SHBG in buffer containing proteins, 0.1% sodium
Pipette 10 µl each of STANDARD(S0- 2. 6 vials STANDARD(S0-S5), ready for use.
0.5ml, human serum containing 0.1% NaN .
direct quantitative determination of SHBG in
Pipette 300 µl of TRACER into each
range of 0-250 nmol/l using 10 µl serum
3. 1 vial CONTROL SERUM, lyophilized
samples. Each kit contains materials sufficient
0.5 ml human serum, containing 0.1% NaN3.
with a plastic foil. If optional total counts
construction of one standard curve and the
specified in the quality certificate enclosed
4.. 2 boxes COATED TUBES, ready for use.
2X50 plastic tube, coated with streptavidin. Introduction 5. 1 bottle WASH BUFFER
adjust an adequate speed such that liquid
Sex hormone-binding globulin (SHBG), also
is constantly rotating or shaking in each
Dilute with 700 ml distilled water before use.
tube. Incubate tubes for 2 hours at room
temperature. (Note: The efficient rotation
protein (SBP), is a circulating glycoprotein
is a critical factor to achieve good
with a molecular weight of around 86000. It is
performance. An uneven or incomplete
thought to be synthesized in the liver, and in
Materials, tools and equipment shaking may result in a serious error.
the circulation its biological function is the
required Never use a rack type with open hole.)
transport of sex steroid hormones.It has a high
tube and decant the supernatant from all
disposable tips (10µl, 300µl, 2ml), shaker,
tubes by the inversion of the rack. In the
plastic foil, adsorbent tissue, gamma counter
birth, increase to high levels during infancy,
Recommended tools and equipment
then decrease during puberty. The highest
repeating pipettes, dispenser with reservoir
physiologic levels of SHBG are observed in
Specimen collection and storage
Abnormal serum SHBG levels have been reported in number of conditions, including
Calculation of results
Serum samples can be prepared according to
common procedures used routinely in clinical
Calculate the average CPM for each pair of
(including polycystic ovary dicease). Serum
laboratory practice. Samples can be stored at
SHBG levels are inversely related to free,
2-8 °C if the assay is carried out within 48
plotting mean CPM of each standard level
hours, otherwise aliquots should be prepared
concentracions, a “free testosterone index”
and stored deep frozen (-20°C). Do not store
concentration, except for 0 standard (abscissa)
based on the ratio of total testosterone to
serum samples longer than 4 months. Do not
use lipemic, hemolyzed or turbid specimens.
Obtain sample concentration by interpolation
Principle of method
thoroughly mixed before assaying. Repeated
For computerised calculations and/or quality
values, rather than cpm values are used.
Preparation of reagents, storage
Specific binding values can be calculated for
each standard and sample according to the
antibodies react simultaneously with the
opening. At this temperature reagent is stable
antigen present in standards or samples which
until expiry date. The actual expiry date is
Assay Protocol, Pipetting Guide
leads to the formation of a capture antibody -
given on the package label and in the quality
Add 0.5 ml distilled water to the lyophilised control serum, and mix gently with shaking or
immobilised on the reactive surface of test
Ensure that complete dissolution is achieved,
tubes. Reaction mixture is then discarded, test
and allow the solution to equilibrate at room
radioactivity is measured in a gamma counter.
Add the wash buffer concentrate to 700 ml
The concentration of antigen is directly
distilled water. The diluted solution can be
proportional to the radioactivity measured in
stored at 2-8 °C until expiry date of the kit.
test tubes. By constructing a calibration curve
CAUTION!
plotting binding values against a series of
Equilibrate all reagents and serum samples to
Decant the fluid and blot on filter paper
Decant the fluid and blot on filter paper
Typical assay data Chemical hazard
antimicrobial agent. Dispose of waste by
flushing with copious amount of water to
avoid build-up of explosive metallic azides in
copper and lead plumbing. The total azide
Recovery
increase expressed as per cent of expected
increase upon spiking serum samples with
known amount of SHBG. Values for 8 serum
pooles spiked with SHBGat 3 levels were as
Typical standard curve Dilution test 4 samples were measured in a series of
dilution (2, 4, 8, 16-fold) with zero-standard.
The following equation obtained for measured (Y) versus expected (X) concentration
y =0.9301x - 0.5552, R=0.9962, n=16 Expected values
establish its own reference intervals. In a population (n=134) of adult female blood
donors the mean (±SD) serum concentration
Characterization of assay
In a population (n=139) of adult male blood donors the mean (±SD) serum concentration
Calibration
Standards are calibrated against the WHO
Results obtained should only be interpreted in
Assay parame ters
the context of the overall clinical picture.
None of in vitro diagnostic kits can be used as
the one and only proof of any disease or
Analytical sensitivity
The analytical sensitivity is 0.11nmol/lAdditional information
It is defined as the concentration of SHBG
Components from various lots or from kits of
equivalent to the mean CPM of 20 replicates
different manufacturers should not be mixed
Functional sensitivity Precaution
The value of functional sensitivity is found to
Radioactivity
It is defined as the value extrapolated to 20 %
This product contains radioactive material. It
of the inter-assay imprecision profile obtained
is the responsibility of the user to ensure that
from 10 independent runs on patient samples
local regulations or code of practice related to
the handling of radioactive materials are
Hook effect
There is no high dose “hook effect” up to a
Biohazard
Human blood products used in the kit have been obtained from healthy human donors.
Specificity
Cross reactivity of the SHBG antiserum has
been measured against human IgG (10 g/l)
negative, for the presence of both Human
In both cases cross reactivity non-detectable.
Immunodeficiency Virus antibody (Anti-HIV-
WEB site: http://www.izotop.hu Technical e -mail: immuno@izotop.hu Precision and reproducibility
(HBsAg).Care should always be taken when
Commercial e-mail:commerce@izotop.hu
Samples with 20 replicates in 1 assay run, and
handling human specimens to be tested with
with duplicates in 12 runs were measured to
diagnostic kits. Even if the subject has been
precision, respectively. Values obtained are
Immunodeficiency Virus (HIV-1), or other
infectious agents are absent. Human blood
Tel.: (+36) 1-392-2577, Fax: (+36) 1-395-9247
potentially infectious materials.
HE CALLER SAYS THAT SHE IS BEING ATTACKED by invisible mites. The attack has been going on for Tmonths and she has visited a half dozen physicians, but none was able to help her. Two prescribed Kwell lotion (see Glossary), but the sensations persist. She has treated her skin with alcohol, vinegar, Lysol, bleach, kerosene, and various home remedies. She has boiled her bed linens and clothing da
Typical assay data