Rat Insulin ELISA KIT (H-type)
Research Reagent
For in-vitro laboratory use only
Please, read this instruction carefully before use.
This is an ELISA (Enzyme Linked ImmunoSorbent Assay) kit for measurement of rat linsulin with a proper sensitivity for diabetic model animals using Sandwich assay principle.
Advantage(1) Rapid assay (total reaction time: 3 hours).
(2) A small sample volume (10ml in standard procedure).
(3) An ecologically excellent preservative is used.
(4) Every reagent is provided in liquid form and ready to use.
(5) Excellent precision and reproducibility.
Components
| |
Reagents |
Amounts |
| (A) |
Antibody-coated plate |
96 wells (8x12) / 1 plate |
| (B) |
Standard rat insulin solution (200ng/ml) |
300ml / 1 vial |
| (C) |
Buffer solution |
60ml/1 vial |
| (D) |
Biotin-conjugated anti-insulin |
200ml/ 1 vial |
| (E) |
Peroxidase-conjugated streptavidin |
200ml/ 1 vial |
| (F) |
Chromogenic substrate reagent (TMB) |
12ml/ 1 vial |
| (H) |
Reaction stopper (1M H2SO4) |
12ml/ 1 vial |
| (I) |
Concentrated washing buffer (10x) |
100ml/ 1 bottle |
Assay samplesRat serum or plasma : 10ml/well
Cultured tissue, cells and media
Assay range0.5 ~ 100 ng/ml
Assay operation1. Equipments necessary but not included in the kit.
(1) Micropipette (a micropipette able to deliver sample volume with high precision.), and a pipette for repetitive dispensing.
(2) Microplate washing apparatus (a microplate washer or a flashing bottle with nozzle).
(3) A microplate reader (A densitometer for microplate).
2. Preparation of reagents
(1) Washing buffer: Dilute the concentrated washing buffer (I) to 10X with purified water.
(2) Biotin-conjugated anti-insulin (D) : Dilute to 100X with the buffer solution(C).
(3) HRP-conjugated streptavidin (E): Dilute to 100X with the buffer solution(C).
(4) Other reagents are used as they are.
(5) All the reagent solutions should be used after getting back to room temperature (20-25C).
3. An example of preparing standard solutions
(An example) Prepare the highest standard solution by 1:1 dilution of the original standard solution, then prepare lower standards by a dilution program shown below.
(You can use other mode of dilution for a set of standard solutions.)
| Std. Conc. (ng/ml) |
100 |
50 |
25 |
10 |
5.0 |
1.0 |
0.5 |
0 |
| Std. Sol.(ml) |
100** |
100* |
100* |
100* |
100* |
100* |
100* |
0 |
| Buffer (ml) |
100 |
100 |
100 |
150 |
100 |
400 |
100 |
100 | **Original standard solution, *One rank higher standard solution
4. Assay procedure
Remove the cover sheet of the microplate after getting back to room temperature.
(1) Rinse the antibody-coated wells (A) by filling the washing buffer and discard 4 times, then strike the plate upside-down onto folded several sheets of paper towel, and remove the excess buffer.
(2) Pipette 100ml of biotin-conjugated anti-insulin (D) to all the wells, then shake gently on a plate shaker.
(3) Pipette 10ml of sample into the wells for samples.
(4) Pipette 10ml of the standard solution to the wells for preparing a standard curve.
(5) Shake the plate gently on a plate shaker.
(6) Incubate the plate for 2 hours at room temperature.
(7) Discard the reaction mixture, and then wash the plate 4 times as described in (1), and remove excess washing buffer remaining in the wells as (1).
(8) Pipette 100ml of HRP-conjugated avidin solution to all wells, and shake as (5).
(9) Incubate for 30 minute at room temperature.
(10) Discard the reaction mixture, and then wash the plate 4 times as (2), and remove excess washing buffer.
(11) Pipette 100ml of chromogenic substrate solution to wells, and shake as (5).
(12) Let the plate stand for 30 minutes at room temperature.
(13) Add 100 ml of the reaction stopper (H) to all wells and shake.
(14) Measure the absorbance of each well at 450 nm (sub-wave length, 620nm) by a plate reader within 30 minutes.
Summary of Assay Procedure
Antibody-coated 96 well plate
¯
Washing 4 times
¯
Biotin-conjugated anti-insulin antibody 100ml
¯Shaking
Sample or Standard 10ml
¯
Shaking and reaction for 2 hour at 20~25C
¯
Washing 4 times
¯
Peroxidase-conjugated avidin 100ml
¯
Shaking, and reaction for 30 min at 20~25C
¯
Washing 4 times
¯
Chromogenic substrate solution 100ml
¯
Shaking, and reaction for 30 min. at 20~25C
¯
Reaction stopper (1M H2SO4) 100ml
¯
Shaking and measurement of absorbance at 450nm(sub. 620nm)
Calculation of insulin concentration
(1) Prepare a standard curve using normal or semi-logarithmic or bi-logarithmic section paper by plotting absorbance* (Y-axis) against standard concentration (ng/ml) on X-axis. For the manual reading from the standard curve, we recommend the use of bi-logarithmic section paper.
*Absorbance at 450nm minus absorbance at 620nm.
(2) Read insulin concentration of a sample from its absorbance. Though the assay range is wide enough, in case the absorbances of some samples are higher than that of the highest standard, please repeat the assay after proper dilution of samples with the buffer solution.
* We recommend the use of 3rd order regression curve or 4 parameter method in computer calculation. If you use logarithm transformation for insulin concentration and absorbance, the fitness of the 3rd order regression curve will be improved.
Important notice in the treatments1. Treatment of assay samples
(1) Use serum or plasma samples obtained by ordinary standard method. We recommend heparin for obtaining plasma samples.
(2) Turbid samples or those containing insoluble materials should be centrifuged before assay and remove those materials.
(3) Measure the samples as soon as possible after sampling.
(4) It would be also convenient to dilute the assay samples first in test tubes, and pipette 50ml of the diluted sample to a well.
2. Storage of assay samples.
If assay samples have to be stored for a long period, freeze samples and store below -35C.
Avoid repeated freezing and thawing.
3. Influence of interfering substances
If presence of interfering substances is suspected, examine by a dilution test using more than 2 points.
Assay rangeA model standard curve
Statements and precaution
(1) The reagents included in this assay kit should be used only for research works.
(2) The reagent solutions of the kit should be used principally immediately after reconstitution. Otherwise, keep them in a dark place with the temperature 2-8C,Aand use them within 3 days.
(3) The reagents were prepared to give accurate results by their combination within the kit. So, do not combine the reagents in the kit of other lot number. Even the lot number is the same, do not mix the reagents with those that have been preserved for some period.
(4) Pipetting and dilution of the reagent solutions should be made accurately because these steps influence the assay precision.
(5) Do not dry the assay plate to avoid denaturation of the coated antibody.
(6) Measurement of the reaction time should be started from the pipetting of reagent to the first well.
(7) Prepare the standard curve in each assay.
(8) Dilution of the assay sample must be carried out using the buffer solution attached to the kit.
(9) Storage condition for the kit should be strictly followed.
(10) Be careful not to allow the reagent solutions of the kit to touch the skin and mucus. Especially be careful for the stopping solution because it is 1M sulfuric acid.
(11) HRP-conjugated reagent solution, chromogenic substrate solution, and reaction stopper must be avoided from contacting with any metal.
(12) In treating assay samples of animal origin, be careful for possible biohazards.
(13) As the antibody-coated plate is module type of 8wells x 12 rows, each row can be separated by a cutter and used independently.
Storage conditionStore the kit at 2~8C. Do not freeze.
Term of validitySix months from production. Expiration date is indicated on the container.
Unit of package96-wells/1 plate
Product codeAKRIN-010H
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