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Food & Feed Analysis With the exclusive distribution in Belgium of the product lines of : R - Biopharm AG www.r-biopharm.de
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Also Fapas www.fapas.com
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And DSM - Rotronic
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SHBG
ELISA
BL-40-E FOR
IN
VITRO DIAGNOSTIC USE
1 Introduction Sex-hormone-binding
globulin (SHBG) is a ß-globulin that specifically binds steroid hormones. The
major site of SHBG synthesis is thought to be the hepatocytes. Its production is
regulated by androgen/estrogen balance, thyroid hormones, insulin and dietary
factors, among others. SHBG is involved in the transport of sex steroids in
plasma. Its concentration is a major factor regulating their distribution
between protein-bound and free states. Determination
of SHBG concentration is mainly of importance in the evaluation of mild
disorders of androgen metabolism and it allows identification of women with
hirsutism who are likely to respond to estrogen therapy. Testosterone/SHBG
–ratios correlate well with both measured and calculated values for free
testosterone, and help to discriminate between subjects with excessive androgen
activity and normal individuals. 2 PRINCIPLE of the test A
monoclonal antibody specific to SHBG is immobilized on microwell plates, and
another monoclonal antibody, also specific to SHBG, is conjugated with
horseradish peroxidase (HRP). SHBG from
the sample is bound to the plates. After a washing step, HRP conjugate is added.
After a second washing step, enzyme substrate is added. The enzymatic reaction
is proportional to the amount of SHBG in the sample. The reaction is terminated
by adding stopping solution. Absorbance is measured on a plate reader. ·
This kit is for in vitro
diagnostic use only. ·
For information on hazardous
substances included in the kit please refer to Material Safety Data Sheets. ·
All reagents of this test
kit which contain human serum or plasma have been tested and confirmed negative
for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however,
should be treated as potential biohazards in use and for disposal. ·
Avoid contact with Stop
Solution containing 0.5 M H2SO4. It may cause skin
irritation and burns. ·
Never pipet by mouth and
avoid contact of reagents and specimens with skin and mucous membranes. ·
Do not smoke, eat, drink or
apply cosmetics in areas where specimens or kit reagents are handled. ·
Wear disposable latex gloves
when handling specimens and reagents. Microbial contamination of reagents or
specimens may give false results. ·
Handling should be in
accordance with the procedures defined by an appropriate national biohazard
safety guideline or regulation. ·
Do not use reagents beyond
expiry date as shown on the kit labels. ·
All indicated volumes have
to be performed according to the protocol. Optimal test results are only
obtained when using calibrated pipettes. ·
Do not mix or use components
from kits with different lot numbers. It is advised not to exchange wells of
different plates even if the same lot. The kits may have been shipped or stored
under different conditions and the binding characteristics of the plates may
result slightly different. ·
Chemicals and prepared or
used reagents have to be treated as hazardous waste according the national
biohazard safety guideline or regulation. ·
Safety Data Sheets for this
product are available upon request. 4 Kit Components 1. microplate
12x8 (break apart) strips 2. standard curve
N=0 to 4 3.
Assay buffer
1 vial, 80 ml 4.
Enzyme Conjugate 100 x conc. 5.
chromogene
1 vial, 12 ml 6.
stop solution
1 vial, 12 ml 7.
wash solution
40 x conc. 8.
control
N=1, 1 vial, 0.5 ml Note:
Additional Zero Calibrator for
Sample dilution available on request. 4.2 Equipment and material required but not provided 1.
A microtiterplate calibrated
reader (450±10 nm). 2.
Calibrated variable
precision micropipettes (Varipette Eppendorf), Multipette Eppendorf or similar
products. 3.
Absorbent paper. 4.
Aqua dest. 4.3 Storage and stability of the Kit ·
When stored at 2° to 8°C
unopened reagents will retain reactivity until expiration date. Do not use
reagents beyond this date. ·
Enzyme-Conjugate, Substrate
Solution, Calibrators must be stored at 2° to 8°C. ·
Microtiter wells must be
stored at 2° to 8°C. Once the foilbag has been open care should be taken to
close it tightly again. 4.4 Preparation of Reagents Allow
all reagents and required number of strips to reach room temperature prior to
use. Calibrators,
Control Serum Dilute
Calibrators, Control Serum 1:21 with Assay Buffer, e.g. Dilute 10 µl
calibrators, controls or samples with 200 µl Assay Buffer Enzyme
Conjugate Solution Dilute
the Conjugate Concentrate 1:101 with Assay Buffer as follows:
This
Solution is stable at 2-8°C for 8 weeks. Wash
Solution Dilute
25 ml of concentrated Wash Solution with 975 ml aqua dest. to a final volume of
1000 ml. The diluted Wash Solution is stable for 8 weeks at room temperature. The
disposal of the kit must be made according to the national official regulations.
Special information for this product are given in the Material Safety Data
Sheets. In
case of any severe damage of the test kit or components, Bio-Line have to be
informed written, latest one week after receiving the kit. Severely damaged
single components should not be used for a test run. They have to be stored
until a final solution has been found. After this, they should be disposed
according to the official regulations. Collect blood by venipuncture, allow to clot, and separate serum by
centrifugation at room temperature. Serum and heparin plasma can be used. EDTA-plasma may give slightly lower
results. No interferences resulting from hemolysis, lipemia or bilirubin have
been observed. Specimens should be capped and may be stored
for up to 48 hours at 2-8°C prior to assaying. Specimen held for a longer time
should be frozen only once at -20°C prior to assay. Thawed samples should be
inverted several times prior to testing. Dilute all samples 1:21 with Assay Buffer,
e.g. Dilute 10 µl of all samples with 200 µl Assay Buffer. 6 test procedure ·
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. ·
Use new disposal plastic
pipet tips for each calibrator, control of sample in order to avoid
crossconamination ·
Absorbance is a function of
the incubation time and temperature. Before starting the assay, it is
recommended that all reagents be ready, caps removed, all needed wells secured
in holder, etc. This will ensure equal elapsed time for each pipetting step
without interruption. ·
As a general rule the
enzymatic reaction is linearly proportional to time and temperature. Therefore,
if the Optical Density is too high or too low, the substrate incubation time can
be decreased or increased, respectively. ·
All calibrators, samples,
and controls should be run in duplicate concurrently so that all conditions of
testing are the same. ·
The concentration of the
samples can be read directly from this calibrator curve. 1.
Secure the desired number of Microtiterwells in the holder. 2.
Dilute
calibrators, Control Serum and samples 1:20 with Assay Buffer, e.g. Dilute 10 µl
calibrators, controls or samples with 200 µl Assay Buffer. Dilute Conjugate
1:100 with Assay buffer as described before. 3.
Pipette 100
ml
of Assay Buffer
into each well. 4.
Dispense 25 ml of
diluted Calibrators, controls and samples with
new disposable tips into appropriate wells. 5.
Cover the plate
and incubate for 30 minutes at room
temperature. 6.
Briskly shake out the contents of the wells. 7.
Dispense 100 µl diluted
Enzyme Conjugate (see „Preparation of Reagents“) into each well. 8.
Cover the plate
and incubate for 15 minutes at room
temperature. 9.
Briskly shake out the contents of the wells. 10.
Add 100 µl of TMB Substrate
Solution to each well. 11.
Cover the plate
and incubate for 12 minutes at room
temperature (20-25°C) or for 8 minutes
at room temperature (26°C and more). 12.
Stop the enzymatic reaction by adding 100
µl of Stop Solution to each well. 13.
Read the OD at 450±10 nm with
a microtiterplate reader within 10
minutes after adding the Stop Solution. 6.4 Calculation of Results 1.
Calculate the average
absorbance values for each set of calibrators, controls and patient samples. 2.
Construct a calibrator curve
by plotting the mean absorbance obtained from each calibrator against its
concentration in nmol/l with absorbance value on the vertical(Y) axis and
concentration on the horizontal (X) axis. 3.
Using the mean absorbance
value for each sample determine the corresponding concentration of SHBG in
nmol/l from the calibrator curve. Depending on experience and/or the
availability of computer capability, other methods of data deduction may be
employed. 4.
Automated method: Computer programs using cubic spline, 4 PL (4 Parameter
Logistics) or Logit-Log can generally give a good fit. 5.
The concentration of the samples can be read directly from this
calibrator curve. Samples with SHBG concentration higher than that of the
highest calibrator have to be diluted with zero calibrator. For the calculation
of the concentrations this dilution factor has to be taken into account. 7
Assay Characteristics It is
strongly recommended that each laboratory should determine its own normal and
abnormal values. Serum
samples from apparently healthy women and men were assayed using the SHBG ELISA,
with the following results:
7.2 Specificity Specificity of the SHBG ELISA was studied by
measuring apparent SHBG response caused by high levels of TBG (Thyroxine Binding
Globulin) and CBG (Cortisol Binding Globulin). No cross-reactions were found
when testing up to 500 mg/l of TBG and 500 mg/l of CBG. 7.3 Sensitivity The minimum
detectable concentration of SHBG by this assay is estimated to be 0.2
nmol. Quality
Control It
is recommended to use control samples according to state and federal
regulations. The use of control samples is advised to assure the day to day
validity of results. Use controls at both normal and pathological levels. The
controls and the corresponding results of the QC-Laboratory are stated in the QC
certificate added to the kit. The values stated on the QC sheet always refer to
the current kit lot and should be used for direct comparison of the results. It
is also recommended to make use of national or international Quality Assessment
programs in order to ensure the accuracy of the results. Employ
appropriate statistical methods for analysing control values and trends. If the
results of the assay do not fit to the established acceptable ranges of control
materials patient results should be considered invalid. In
this case, please check the following technical areas: Pipetting and timing
devices; photometer, expiration dates of reagents, storage and incubation
conditions, aspiration and washing methods. 7.6.1
Intra Assay Variation The
within assay variability is shown below:
7.6.2
Inter Assay Variation The
between assay variability is shown below:
A
known amount of SHBG was added to three patient sera and the quantitities
recovered were measured. The results are shown in the following table:
Any
improper handling of samples or modification of this test might influence the
results. Interferences caused by improper sample handling are explained in the
chapters ‘Specimen - Collection’. No
hook effect was observed in this test up to 10000
nmol/l of SHBG. The
test must be performed exactly as per the manufacturer’s instructions for use.
Moreover the user must strictly adhere to the rules of GLP (Good Laboratory
Practice) or other applicable national standards and/or laws. This is especially
relevant for the use of control reagents. It is important to always include,
within the test procedure, a sufficient number of controls for validating the
accuracy and precision of the test. The
test results are valid only if all controls are within the specified ranges and
if all other test parameters are also within the given assay specifications. 9.2 Therapeutical Consequences Therapeutical
consequences should never be based on laboratory results alone even if all test
results are in agreement with the items as stated under point 9.1. Any
laboratory result is only a part of the total clinical picture of a patient. Only
in cases where the laboratory results are in acceptable agreement with the
overall clinical picture of the patient should therapeutical consequences be
derived. The
test result itself should never be the sole determinant for deriving any
therapeutical consequences. Any
modification of the test kit and/or exchange or mixture of any components of
different lots from one test kit to another could negatively affect the intended
results and validity of the overall test. Such modification and/or exchanges
invalidate any claim for replacement. Claims
submitted due to customer misinterpretation of laboratory results subject to
point 9.2. are also invalid. Regardless, in the event of any claim, the
manufacturer’s liability is not to exceed the value of the test kit. Any
damage caused to the test kit during transportation is not subject to the
liability of the manufacturer. 1.
Moore, J.W. and
Bulbrook R.D. (1988) The epidemiology and
function of sex hormone binding globulin. IN: Oxford Reviews of Reproductive
Biology, 10: 180-236. 2. Selby, C. (1980) Sex hormone binding globulin: origin, function and clinical significance. Ann Clin Biochem 27: 532-541 SHBG Flow chart
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