This is an ELISA (Enzyme Linked ImmunoSorbent Assay) kit for measurement of mouse C-peptide with high sensitivity using Sandwich assay principle.
[Advantage]
(1) Rapid assay (total reaction time: 5 hours.).
(2) A small sample volume (10µl in standard assay 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) Anti-C-peptide-coated plate 96 wells(8x12) / 1 plate
(B) Standard mouse C-peptide solution (6000pg/ml) 500µl / 1 vial
(C) Buffer solution 60ml/1 vial
(D) Biotin-conjugated anti-C-peptide 100µl/ 1 vial
(E) Peroxidase-conjugated streptavidin 100µl/ 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 sample]
Mouse serum or plasma 10 µl/well (Standard procedure)
[Assay purpose]
Measurement of C-peptide in mouse serum or plasma samples, and in culture media.
[Assay range]
Assay range of the standard curve is 30 ~ 3000 pg/ml
According to the standard procedure where the samples are diluted 5 times, practical assay range is 150~15,000pg/ml
1. 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-C-peptide (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
Dilute the original standard solution (B) with the buffer solution to prepare 3000ng/ml, then prepare lower standard solutions by a dilution program shown below.
Conc.(pg/ml) 3,000 1,500 600 300 150 60 30 0
Std.solution. (µl) 150** 150* 150* 150* 150* 150* 150* 0
Buffer (µl) 150 150 225 150 150 225 150 150
**Original standard soloution, *One rank higher standard solution
4. Assay procedure
1) Remove the cover sheet of the microplate after getting back to room temperature.
2) Rinse the anti-body coated wells (A) by filling the washing buffer and discard 3 times, then strike the plate upside-down onto folded several sheets of paper towel, and remove the excess buffer.
3) Pipette 40µl of buffer solution into the wells for samples, then add 10µl of sample.
Alternatively, if samples are already diluted to 5X, pipette 50µl of the diluted sample to each well, skipping the addition of buffer solution.
4) Pipette 50µl of the standard solution to the wells for preparing a standard curve.
5) Shake the plate gently on a plate shaker.
6) Incubate for 2 hour at room temperature (20-25C).
7) Discard the reaction mixture, and then wash wells as described in (2).
8) Pipette 50µl of biotin-conjugated anti-C-peptide solution to all wells. Then shake gently on a plate shaker.
9) Incubate the plate for 2 hour at room temperature.
10) Discard the reaction mixture, and then wash the plate as (2).
11) Pipette 50µl of HRP-conjugated avidin solution to all wells, and shake as (5).
12) Incubate for 30 minute at room temperature.
13) Discard the reaction mixture, and wash the plate as (2).
14) Pipette 50µl of chromogenic substrate solution to wells, and shake as (5).
15) Let the plate stand for 20 minutes at room temperature.
16) Add 50 µl of the reaction stopper (H) to all wells and shake.
17) Measure the absorbance of each well at 450 nm (sub-wave length, 620nm) by a plate reader within 30 minutes. |