Laboratory Research Human Sex Hormone-binding Globulin ELISA Assay
Kit 96 Wells Size Customized
Cat.No E1011Hu
Assay Procedure
1. Prepare all reagents, standard solutions and samples as
instructed. Bring all reagents to room temperature before use. The
assay is performed at room temperature.
2. Determine the number of strips required for the assay. Insert
the strips in the frames for use. The unused strips should be
stored at 2-8°C.
3. Add 50μl standard to standard well. Note: Don’t add antibody to standard well because the standard solution
contains biotinylated antibody.
4. Add 40μl sample to sample wells and then add 10μl anti-SHBG
antibody to sample wells, then add 50μl streptavidin-HRP to sample
wells and standard wells (Not blank control well). Mix well. Cover
the plate with a sealer. Incubate 60 minutes at 37°C.
5. Remove the sealer and wash the plate 5 times with wash buffer.
Soak wells with at least 0.35 ml wash buffer for 30 seconds to 1
minute for each wash. For automated washing, aspirate all wells and
wash 5 times with wash buffer, overfilling wells with wash buffer.
Blot the plate onto paper towels or other absorbent material.
6. Add 50μl substrate solution A to each well and then add 50μl
substrate solution B to each well. Incubate plate covered with a
new sealer for 10 minutes at 37°C in the dark.
7. Add 50μl Stop Solution to each well, the blue color will change
into yellow immediately.
8. Determine the optical density (OD value) of each well
immediately using a microplate reader set to 450 nm within 10
minuets after adding the stop solution.
Assay Principle
This ELISA Assay Kit is an Enzyme-Linked Immunosorbent Assay
(ELISA). The plate has been pre-coated with human SHBG antibody.
SHBG present in the sample is added and binds to antibodies coated
on the wells. And then biotinylated human SHBG Antibody is added
and binds to SHBG in the sample. Then Streptavidin-HRP is added and
binds to the Biotinylated SHBGantibody. After incubation unbound
Streptavidin-HRP is washed away during a washing step. Substrate
solution is then added and color develops in proportion to the
amount of human SHBG. The reaction is terminated by addition of
acidic stop solution and absorbance is measured at 450 nm.
Intended Use
This sandwich kit is for the accurate quantitative detection of
human Sex Hormone-binding Globulin (also known as SHBG) in serum,
plasma, cell culture supernates, cell lysates, tissue homogenates.
Reagent Provided
Components | Quantity |
Standard Solution (240nmol/L) | 0.5ml x1 |
Pre-coated ELISA Plate | 12 * 8 well strips x1 |
Standard Diluent | 3ml x1 |
Streptavidin-HRP | 6ml x1 |
Stop Solution | 6ml x1 |
Substrate Solution A | 6ml x1 |
Substrate Solution B | 6ml x1 |
Wash Buffer Concentrate (25x) | 20ml x1 |
Biotinylated human SHBG Antibody | 1ml x1 |
User Instruction | 1 |
Plate Sealer | 2 pics |
Zipper bag | 1 pic |
Specimen Collection
Serum Allow serum to clot for 10-20 minutes at room temperature.
Centrifuge at 2000-3000 RPM for 20 minutes.
Plasma Collect plasma using EDTA or heparin as an anticoagulant.
Centrifuge samples for 15 minutes at 2000-3000 RPM at 2 - 8°C
within 30 minutes of collection.
Urine Collect by sterile tube. Centrifuge at 2000-3000 RPM for
approximately 20 minutes. When collecting pleuroperitoneal fluid
and cerebrospinal fluid, please follow the procedures
above-mentioned.
Cell Culture Supernatant Collect by sterile tubes when examining secrete components.
Centrifuge at 2000-3000 RPM for approximately 20 minutes. Collect
the supernatants carefully. When examining the components within
the cell, use PBS (pH 7.2-7.4) to dilute cell suspension to the
cell concentration of approximately 1 million/ml. Damage cells
through repeated freeze-thaw cycles to let out the inside
components. Centrifuge at 2000-3000 RPM for approximately 20
minutes.
Tissue other body fluids Rinse tissues in PBS (pH 7.4) to remove excess blood thoroughly
and weigh before homogenization. Mince tissues and homogenize them
in PBS (pH7.4) with a glass homogenizer on ice. Thaw at 2-8°C or
freeze at -20°C. Centrifuge at 2000-3000 RPM for approximately 20
minutes.
Note
- Sample concentrations should be predicted before being used in the
assay. If the sample concentration is not within the range of the
standard curve, users must contact us to determine the optimal sample for their particular experiments.
- Samples to be used within 5 days should be stored at 2-8°C. Samples
should be aliquoted or must be stored at -20°C within 1 month or
-80°C within 6 months. Avoid repeated freeze thaw cycles.
- Samples should be brought to room temperature before starting the
assay.
- Centrifuge to collect sample before use.
- Samples containing NaN3 can’t be tested as it inhibits the activity
of Horse Radish Peroxidase (HRP).
- Collect the supernatants carefully. When sediments occurred during
storage, centrifugation should be performed again.
- Hemolysis can greatly impact the validity of test results. Take
care to minimize hemolysis.
*Sample can't be diluted with this kit. Owing to the the material we use to prepare the kit, the sample
matrix interference may falsely depress the specificity and
accuracy of the assay.
Standard Curve Range: 0.5nmol/L - 200nmol/L
Sensitivity: 0.28nmol/L
Size: 96 wells
Storage: Store the reagents at 2-8°C. For over 6-month storage refer to
the expiration date keep it at -20°C. Avoid repeated thaw cycles.
If individual reagents are opened it is recommended that the kit be
used within 1 month.
*This product is for research use only, not for use in diagnosis
procedures. It’s highly recommend to read this instruction entirely
before use.
Referances
"Serum concentrations of sex hormone binding globulin are elevated
in kwashiorkor and anorexia nervosa but not in marasmus."
Pascal N., Amouzou E.K., Sanni A., Namour F., Abdelmouttaleb I.,
Vidailhet M., Gueant J.L.
Am. J. Clin. Nutr. 76:239-244(2002)