大鼠性激素結(jié)合球蛋白(SHBG)ELISA試劑盒使用說(shuō)明  

Rat sex hormone-binding globulin (SHBG)  
ELISA Kit 

For the quantitative determination of rat sex hormone-binding globulin 
(SHBG) concentrations in serum, plasma, cell culture supernates, tissue 
homogenates, cell lysates. 
This package insert must be read in its entirety before using this product. 
 
In order to obtain higher efficiency service, please ready to supply the lot number 
of the kit to us (found on the outside of the box).2
PRINCIPLE OF THE ASSAY
This assay employs the competitive inhibition enzyme immunoassay technique. 
The microtiter plate provided in this kit has been  pre-coated with an antibody 
specific to SHBG. Standards or samples are added to the appropriate microtiter 
plate wells with Biotin-conjugated SHBG. A competitive inhibition reaction is 
launched between SHBG (Standards or samples) and Biotin-conjugated SHBG 
with the pre-coated antibody specific for SHBG. The more amount of SHBG in 
samples, the less antibody bound by Biotin-conjugated SHBG. After washing, 
avidin conjugated Horseradish Peroxidase (HRP) is added to the wells. 
Substrate solution is added to the wells and the color develops in opposite to the 
amount of SHBG in the sample. The color development is stopped and the 
intensity of the color is measured. 
DETECTION RANGE
375 ng/ml-6000 ng/ml. 
SENSITIVITY
The minimum detectable dose of rat SHBG is typically less than 187.5ng/ml. 
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as 
the lowest rat SHBG concentration that could be differentiated from zero. It was 
determined the mean O.D value of 20 replicates of the zero standard added by 
their three standard deviations. 
SPECIFICITY
This assay has high sensitivity and excellent specificity for detection of rat SHBG. 
No significant cross-reactivity or interference between rat SHBG and analogues 
was observed. 
Note: Limited by current skills and knowledge, it is impossible for us to complete 
the cross-reactivity detection between rat SHBG and all the analogues, therefore, 
cross reaction may still exist. 3
PRECISION 
Intra-assay Precision (Precision within an assay): CV%<15%
Three samples of known concentration were tested twenty times on one plate to 
assess.  
Inter-assay Precision (Precision between assays): CV%<15%
Three samples of known concentration were tested in twenty assays to assess.  
LIMITATIONS OF THE PROCEDURE
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC 
PROCEDURES. 
The kit should not be used beyond the expiration date on the kit label. 
Do not mix or substitute reagents with those from other lots or sources. 
If samples generate values higher than the highest  standard, dilute the 
samples and repeat the assay. 
Any variation in operator, pipetting technique, washing technique, 
incubation time or temperature, and kit age can cause variation in binding. 
This assay is designed to eliminate interference by soluble receptors, 
binding proteins, and other factors present in biological samples. Until all 
factors have been tested in the Immunoassay, the possibility of 
interference cannot be excluded. 4
MATERIALS PROVIDED
Reagents  Quantity 
Assay plate  1(96 wells) 
Standard  5 x 1 ml 
Conjugate    1 x 6 ml 
HRP-avidin   1 x 6 ml 
Wash Buffer (20 x concentrate) 1 x 15 ml 
Substrate A  1 x 7 ml 
Substrate B  1 x 7 ml 
Stop Solution    1 x 7 ml 
Adhesive Strip (For 96 wells) 4 
Instruction manual  1 
STANDARD CONCENTRATION
Standard S1 S2 S3 S4 S5 
Concentration
(ng/ml) 
375 750 1500 3000 6000 
STORAGE
Unopened kit Store at 2 - 8°C. Do not use the kit beyond the expiration date.
Opened kit May be stored for up to one month at 2 - 8° C. 
*Provided this is within the expiration date of the kit.
OTHER SUPPLIES REQUIRED
Microplate reader capable of measuring absorbance at 450 nm, with the 
correction wavelength set at 600 nm - 630 nm. 
An incubator which can provide stable incubation conditions up to 
37°C±0.5°C. 
Squirt bottle, manifold dispenser, or automated microplate washer. 
Absorbent paper for blotting the microtiter plate. 5
100 mL and 500 mL graduated cylinders. 
Deionized or distilled water. 
Pipettes and pipette tips. 
Test tubes for dilution. 
PRECAUTIONS
The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, 
and clothing protection when using this material. 
SAMPLE COLLECTION AND STORAGE
Serum  Use a serum separator tube (SST) and allow samples to clot for 
two hours at room temperature or overnight at 4°C before centrifugation 
for 15 minutes at 1000 ×g. Remove serum and assay immediay or 
aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw 
cycles. 
Plasma  Collect plasma using EDTA, or heparin as an anticoagulant. 
Centrifuge for 15 minutes at 1000 ×g at 2-8°C within 30 minutes of 
collection. Assay immediay or aliquot and store  samples at -20°C or 
-80°C. Avoid repeated freeze-thaw cycles. 
Cell Culture Supernates  Remove particulates by centrifugation for 15 
minutes at 1000 x g, 2 - 8°C and assay immediay or aliquot and store 
samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles. 
Tissue Homogenates   100mg tissue was rinsed with 1X PBS, 
homogenized in 1 ml of 1X PBS and stored overnight at -20°C. After two 
freeze-thaw cycles were performed to break the cell membranes, the 
homogenates were centrifuged for 5 minutes at 5000  x g, 2 - 8°C. The 
supernate was removed and assayed immediay. Alternatively, aliquot 
and store samples at -20°C or -80°C. Centrifuge the sample again after 
thawing before the assay. Avoid repeated freeze-thaw cycles. 6
Cell Lysates  (1) Adherent Cell: Remove media and rinse cells once 
with ice-cold PBS (PH7.2-7.4). Scrape cells off the plate and transfer to an 
appropriate tube. Dilute cell suspension with 1xPBS (PH7.2-7.4), until cell 
concentration reached 100 million/ml. Then store overnight at -20°C. After 
two freeze-thaw cycles to break up the cell membranes, the cell lysates 
were centrifuged for 5 minutes at 5000 g, 2 - 8°C. Collect the supernatant. 
Cell lysates should be assayed immediay or aliquotted and stored at 
-20°C. Centrifuge the sample again after thawing before the assay. Avoid 
repeated freeze-thaw cycles.  (2) Suspension Cell: Collect cells with 
appropriate tube, centrifuge for 5 minutes at 1000 g, 2 - 8°C. Remove the 
supernatant and resuspend cells with 1xPBS (PH7.2-7.4). Centrifuge for 5 
minutes at 1000 g, 2 - 8°C. Remove the supernatant. Dilute cell with 
1xPBS (PH7.2-7.4), until cell concentration reached 100 million/ml. Then 
store overnight at -20°C. After two freeze-thaw cycles to break up the cell 
membranes, the cell lysates were centrifuged for 5 minutes at 5000 g, 2 - 
8°C. Collect the supernatant. Cell lysates should be assayed immediay 
or aliquotted and stored at -20°C. Centrifuge the sample again after 
thawing before the assay. Avoid repeated freeze-thaw cycles. 
Note: 
1. CUSABIO is only responsible for the kit itself,  but not for the samples 
consumed during the assay. The user should calculate the possible 
amount of the samples used in the whole test. Please reserve sufficient 
samples in advance. 
2. Samples to be used within 5 days may be stored at 2-8°C, otherwise 
samples must be stored at -20°C (≤1month) or -80°C (≤2month) to avoid 
loss of bioactivity and contamination. 
3. Grossly hemolyzed samples are not suitable for use in this assay. 
4. If the samples are not indicated in the manual, a preliminary experiment to 
determine the validity of the kit is necessary.  
5. Please predict the concentration before assaying. If values for these are 
not within the range of the standard curve, users must determine the 
optimal sample dilutions for their particular experiments. 7
6. Tissue or cell extraction samples prepared by chemical lysis buffer may 
cause unexpected ELISA results due to the impacts of certain chemicals. 
7. Owing to the possibility of mismatching between  antigen from other 
resource and antibody used in our kits (e.g., antibody targets 
conformational epitope rather than linear epitope), some native or 
recombinant proteins from other manufacturers may not be recognized by 
our products. 
8. Influenced by the factors including cell viability, cell number and also 
sampling time, samples from cell culture supernatant may not be detected 
by the kit. 
9. Fresh samples without long time storage are recommended for the test. 
Otherwise, protein degradation and denaturalization may occur in those 
samples and finally lead to wrong results. 
REAGENT PREPARATION
Note:  
Kindly use graduated containers to prepare the reagent.  
Bring all reagents to room temperature (18-25°C) before use for 30min.
Distilled water is recommended to be used to make the preparation for 
reagents or samples. Contaminated water or container for reagent 
preparation will influence the detection result. 
Wash Buffer(1x)- If crystals have formed in the concentrate, warm up to   
room temperature and mix gently until the crystals  have compley 
dissolved. Dilute 15 ml of Wash Buffer Concentrate (20 x) into deionized or 
distilled water to prepare 300 ml of Wash Buffer (1 x). 8
ASSAY PROCEDURE 
Bring all reagents and samples to room temperature before use. Centrifuge 
the sample again after thawing before the assay. It is recommended that all 
samples and standards be assayed in duplicate. 
1. Prepare all reagents and samples as directed in the previous sections. 
2. Determine the number of wells to be used and put any remaining wells 
and the desiccant back into the pouch and seal the ziploc, store unused 
wells at 4°C. 
3. Set a Blank well without any solution.  
4. Add 50µl of Standard or Sample per well. Standard need test in duplicate.  
5. Add 50µl of Conjugate to each well (not to Blank well). Mix well and then 
incubate for 60 minutes at 37°C.  
6. Aspirate each well and wash, repeating the process two times for a total of 
three washes. Wash by filling each well with Wash Buffer (200µl) using a 
squirt bottle, multi-channel pipette, manifold dispenser, or autowasher, 
and let it stand for 10 seconds, complete removal of liquid at each step is 
essential to good performance. After the last wash, remove any remaining 
Wash Buffer by aspirating ordecanting. Invert the plate and blot it against 
clean paper towels. 
7. Add 50µl of HRP-avidin to each well (not to Blank well). Mix well and then 
incubate for 30 minutes at 37°C.  
8. Repeat the aspiration/wash process for three times as in step 6. 
9. Add 50µl of Substrate A and 50µl of Substrate B to each well, mix well. 
Incubate for 15 minutes at 37°C. Keeping the plate away from drafts and 
other temperature fluctuations in the dark. 
10. Add 50µl of Stop Solution to each well, gently tap the plate to ensure 
thorough mixing.  
11. Determine the optical density of each well within 10 minutes, using a 
microplate reader set to 450 nm. 9
Note: 
1. The final experimental results will be closely related to validity of the 
products, operation skills of the end users and the experimental 
environments.  
2. Samples or reagents addition: Please carefully add samples to wells and 
mix gently to avoid foaming. Do not touch the well wall as possible. For 
each step in the procedure, total dispensing time for addition of reagents 
or samples to the assay plate should not exceed 10  minutes. This will 
ensure equal elapsed time for each pipetting step,  without interruption. 
Duplication of all standards and specimens, although not required, is 
recommended. To avoid cross-contamination, change pipette tips 
between additions of each standard level, between sample additions, and 
between reagent additions. Also, use separate reservoirs for each 
reagent. 
3. Incubation: To ensure accurate results, proper adhesion of plate sealers 
during incubation steps is necessary. Do not allow wells to sit uncovered 
for extended periods between incubation steps. Once reagents have been 
added to the well strips, DO NOT let the strips DRY at any time during the 
assay. Incubation time and temperature must be observed. 
4. Washing: The wash procedure is critical. Complete removal of liquid at 
each step is essential to good performance. After the last wash, remove 
any remaining Wash Solution by aspirating or decanting and remove any 
drop of water and fingerprint on the bottom of the  plate. Insufficient 
washing will result in poor precision and falsely elevated absorbance 
reading. When using an automated plate washer, adding a 10 second 
soak period following the addition of wash buffer, and/or rotating the plate 
180 degrees between wash steps may improve assay precision. 
5. Controlling of reaction time: Observe the change of color after adding 
Substrates (e.g. observation once every 10 minutes). Substrates should 
change from colorless or light blue to gradations of blue. If the color is too 
deep, add Stop Solution in advance to avoid excessively strong reaction 
which will result in inaccurate absorbance reading.
6. Substrates are easily contaminated. Substrates should remain colorless or 
light blue until added to the plate. Please protect it from light. 
7. Stop Solution should be added to the plate in the same order as the 
Substrates. The color developed in the wells will turn from blue to yellow 
upon addition of the Stop Solution. Wells that are green in color indicate 
that the Stop Solution has not mixed thoroughly with the Substrates.10
ASSAY PROCEDURE SUMMARY 11
CALCULATION OF RESULTS 
Using the professional soft "Curve Expert 1.3" to make a standard curve is 
recommended, which can be downloaded from our web. 
Average the duplicate readings for each standard and sample and subtract the 
average optical density of Blank.  
Create a standard curve by reducing the data using computer software capable 
of generating a four parameter logistic (4-PL) curve-fit. As an alternative, 
construct a standard curve by plotting the mean absorbance for each standard 
on the x-axis against the concentration on the y-axis and draw a best fit curve 
through the points on the graph. The data may be linearized by plotting the log of 
the SHBG concentrations versus the log of the O.D. and the best fit line can be 
determined by regression analysis. This procedure will produce an adequate but 
less precise fit of the data.  
If samples have been diluted, the concentration read from the standard curve 
must be multiplied by the dilution factor. 12
Notes 13141516