Shanghai Korain Biotech Co., Ltd |
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96 Well Plate Rat High Precision and Sensitivity Fibrinogen
Immunoassays Test Kit For Research
Cat.No E0685Ra
Standard Curve Range: 0.05mg/ml - 20mg/ml
Sensitivity: 0.018mg/ml
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.
Precautions
Assay Principle
This ELISA Test Kit is an Enzyme-Linked Immunosorbent Assay
(ELISA). The plate has been pre-coated with Rat Fbg antibody. Fbg
present in the sample is added and binds to antibodies coated on
the wells. And then biotinylated Rat Fbg Antibody is added and
binds to Fbg in the sample. Then Streptavidin-HRP is added and
binds to the Biotinylated Fbg antibody. 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 Rat Fbg. The reaction is terminated by addition of acidic
stop solution and absorbance is measured at 450 nm.
Precision
Intra-Assay Precision (Precision within an assay) Three samples of
known concentration were tested on one plate to assess intra-assay
precision.
Inter-Assay Precision (Precision between assays) Three samples of
known concentration were tested in separate assays to assess
inter-assay precision.
CV(%) = SD/mean x 100
Intra-Assay: CV<8%
Inter-Assay: CV<10%
Reagent Preparation
All reagents should be brought to room temperature before use.
Standard Reconstitute the 120μl of the standard (24mg/ml) with
120μl of standard diluent to generate a 12mg/ml standard stock
solution. Allow the standard to sit for 15 mins with gentle
agitation prior to making dilutions. Prepare duplicate standard
points by serially diluting the standard stock solution (12mg/ml)
1:2 with standard diluent to produce 6mg/ml, 3mg/ml, 1.5mg/ml and
0.75mg/ml solutions. Standard diluent serves as the zero standard(0
mg/ml). Any remaining solution should be frozen at -20°C and used
within one month. Dilution of standard solutions suggested are as
follows:
12mg/ml | Standard No.5 | 120μl Original Standard + 120μl Standard Diluent |
6mg/ml | Standard No.4 | 120μl Standard No.5 + 120μl Standard Diluent |
3mg/ml | Standard No.3 | 120μl Standard No.4 + 120μl Standard Diluent |
1.5mg/ml | Standard No.2 | 120μl Standard No.3 + 120μl Standard Diluent |
0.75mg/ml | Standard No.1 | 120μl Standard No.2 + 120μl Standard Diluent |
Standard Concentration | Standard No.5 | Standard No.4 | Standard No.3 | Standard No.2 | Standard No.1 |
24mg/ml | 12mg/ml | 6mg/ml | 3mg/ml | 1.5mg/ml | 0.75mg/ml |
Wash Buffer Dilute 20ml of Wash Buffer Concentrate 25x into deionized or
distilled water to yield 500 ml of 1x Wash Buffer. If crystals have
formed in the concentrate, mix gently until the crystals have
completely dissolved.
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-Fbg
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.
Calculation of Result
Construct a standard curve by plotting the average OD for each
standard on the vertical (Y) axis against the concentration on the
horizontal (X) axis and draw a best fit curve through the points on
the graph. These calculations can be best performed with
computer-based curve-fitting software and the best fit line can be
determined by regression analysis.