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大鼠糖化血紅蛋白A1c(GHbA1c)ELISA試劑盒使用說明

1
Rat glycated hemoglobin A1c (GHbA1c) ELISA Kit
Catalog Number. CSB-E08140r
For the quantitative determination of rat glycated hemoglobin A1c 
(GHbA1c) concentrations in lysate for RBC.
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 direct competitive inhibition enzyme immunoassay 
technique. Antibody specific for GHbA1c has been pre-coated onto a microplate. 
Standards and samples are pipetted into the wells with  biotin-conjugated 
GHbA1c. A competitive inhibition reaction is launched between  GHbA1c
(Standards or samples) and biotin-conjugated  GHbA1c with the pre-coated 
GHbA1c antibody. After washing, avidin conjugated Horseradish Peroxidase 
(HRP) is added to the wells. Following a wash to remove any unbound reagent, a 
substrate solution is added to the wells and color develops in opposite to the 
amount of GHbA1c bound in the initial step. The color development is stopped 
and the intensity of the color is measured.
DETECTION RANGE
1.25 ng/ml-80 ng/ml.
SENSITIVITY
The minimum detectable dose of rat GHbA1c is typically less than 0.625 ng/ml.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as 
the lowest rat GHbA1c 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
GHbA1c. No significant cross-reactivity or interference between rat GHbA1c and 
analogues was observed.
Note: Limited by current skills and knowledge, it is impossible for us to complete
the  cross-reactivity detection between  rat GHbA1c and all the analogues, 
therefore, cross reaction may still exist.3
PRECISION 
Intra-assay Precision (Precision within an assay): CV%<8%
Three samples of known concentration were tested twenty times on one plate to 
assess.
Inter-assay Precision (Precision between assays): CV%<10%
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 with Sample Diluent and repeat the assay.
? Any variation in  Sample Diluent, 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 (100 x concentrate) 1 x 100 μl
Biotin-conjugate (100 x concentrate)  1 x 60 μl
HRP-avidin (100 x concentrate) 1 x 120 μl
Biotin-conjugate Diluent   1 x 10 ml
HRP-avidin Diluent    1 x 20 ml
Sample Diluent   2 x 20 ml
Wash Buffer (25 x concentrate) 1 x 20 ml
TMB Substrate  1 x 10 ml
Stop Solution   1 x 10 ml
Adhesive Strip (For 96 wells) 4
Instruction manual 1
STORAGE
Unopened 
kit
Store at 2 - 8°C. Do not use the kit beyond the expiration date.
Opened kit
Coated assay 
plate
May be stored for up to 1 month at 2 - 8°C. 
Try to keep it in a sealed aluminum foil bag, 
and avoid the damp.
Standard  May be stored for up to 1 month at 2 - 8° C. 
If don’t make recent use, better keep it store 
at -20°C.
HRP-avidin
Biotin-conjugate
Biotin-conjugate 
Diluent
May be stored for up to 1 month at 2 - 8°C.
HRP-avidin 
Diluent
Sample Diluent
Wash Buffer
TMB Substrate
Stop Solution
*Provided this is within the expiration date of the kit.5
OTHER SUPPLIES REQUIRED
? Microplate reader capable of measuring absorbance at 450 nm, with the 
correction wavelength set at 540 nm or 570 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.
? 100ml and 500ml 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.6
SAMPLE COLLECTION AND STORAGE
? Lysate for RBC     Collect a certain volume of anticoagulant blood sample 
(e.g. 1ml). Centrifuge at 10000rpm for 10min at 4°C to collect the RBC. 
Remove supernate and add  cold pure water to redissolve RBC to the 
previous volume (e.g. 1ml). Then store at -20°C for about 30-60 minutes. 
After three freeze-thaw cycles to break up the cell membranes, the cell 
lysates were centrifuged for 10 minutes at 10000rpm, 4°C. Collect the 
supernatant. RBC 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.
SAMPLE PREPARATION
? Recommend to dilute the lysate for RBC with Sample Diluent(1:5000) 
before test. The suggested 5000-fold dilution can be achieved by adding 
2μl sample to 98μl of Sample Diluent. Complete the 5000-fold dilution by 
adding 3μl of this solution to 297μl of Sample Diluent. The recommended 
dilution factor is for reference only. The optimal dilution factor should be 
determined by users according to their particular experiments.7
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. If the samples are not indicated in the manual, a preliminary experiment to 
determine the validity of the kit is necessary. 
3. 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.
4. 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.
5. 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.
6. 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.8
REAGENT PREPARATION
Note: 
? Kindly use graduated containers to prepare the reagent. Please don't 
prepare the reagent directly in the Diluent vials provided in the kit.
? Bring all reagents to room temperature (18-25°C) before use for 30min.
? Prepare fresh standard for each assay. Use within 4 hours and discard 
after use.
? Making serial dilution in the wells directly is not permitted. 
? 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.
1. Biotin-conjugate (1x) - Centrifuge the vial before opening. 
Biotin-conjugate requires a 100-fold dilution. A suggested 100-fold 
dilution is 10 μl of Biotin-conjugate + 990 μl of Biotin-conjugate Diluent.
2. HRP-avidin (1x) - Centrifuge the vial before opening.
HRP-avidin requires a 100-fold dilution. A suggested 100-fold dilution is 10 
μl of HRP-avidin + 990 μl of HRP-avidin Diluent.
3. 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 20 ml of Wash Buffer Concentrate (25 x) into deionized or 
distilled water to prepare 500 ml of Wash Buffer (1 x).9
4. Standard 
Centrifuge the standard vial at 6000-10000rpm for 30s. 
Dilute the  Standard(100x) with  Sample Diluent. A suggested 100-fold 
dilution is 5 μl of Standard(100x) + 495 μl of Sample Diluent. This diluted 
Standard (S7) serves as the high standard (80 ng/ml). 
Do not substitute other diluents. Mix the standard to ensure complete 
dilution and allow the standard to sit for a minimum of 15 minutes with 
gentle agitation prior to making dilutions.
Pipette 150 μl of Sample Diluent into each tube (S0-S6). Use the stock 
solution to produce a 2-fold dilution series (below). Mix each tube 
thoroughly before the next transfer. Sample Diluent serves as the zero 
standard (0 ng/ml).
Tube S7 S6 S5 S4 S3 S2 S1 S0
ng/ml 80 40 20 10 5 2.5 1.25 010
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, working standards, and samples as directed in the 
previous sections.
2. Refer to the Assay Layout Sheet to 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 and sample per well. 
5. Add 50μl of Biotin-conjugate (1x) to each well(not to Blank well). Cover 
with a new adhesive strip. Incubate for  40 minutes at 37°C. 
(Biotin-conjugate (1x) may appear cloudy. Warm up to room temperature 
and mix gently until solution appears uniform.)
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 2 minutes, 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 100μl of HRP-avidin (1x) to each well(not to Blank well). Cover the 
microtiter plate with a new adhesive strip. Incubate for 40 minutes at 37°C.
8. Repeat the aspiration/wash process for five times as in step 6.
9. Add 90μl of TMB Substrate to each well. Incubate for 20 minutes at 37°C.
Protect from light.
10. Add 50μl of Stop Solution to each well, gently tap the plate to ensure 
thorough mixing.11
11. Determine the optical density of each well within 5 minutes, using a 
microplate reader set to 450 nm. If wavelength correction is available, set 
to 540 nm or 570 nm. Subtract readings at 540 nm or 570 nm from the 
readings at 450 nm. This subtraction will correct for optical imperfections 
in the plate. Readings made directly at 450 nm without correction may be 
higher and less accurate. 
*Samples may require dilution. Please refer to Sample Preparation section.
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 use the freshly prepared Standard. 
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 2 minutes soak 
period following the addition of wash buffer, and/or rotating the plate 180 
degrees between wash steps may improve assay precision.12
5. Controlling of reaction time: Observe the change of color after adding TMB 
Substrate (e.g. observation once  every 10 minutes), TMB Substrate
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. TMB Substrate is easily contaminated. TMB Substrate 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 TMB 
Substrate. 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 TMB Substrate.13
ASSAY PROCEDURE SUMMARY
*Samples may require dilution. Please refer to Sample Preparation section.14
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 GHbA1c 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.

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