DRG Leptin ELISA Kit Instructions

DRG Leptin ELISA Kit Instructions

( Germany DRG: EIA-2395)

1 , description

DRG 's leptinase-free kit can be used for the quantitative detection of leptin in human serum and plasma. This test kit is for in vitro diagnostic use only.

2 , the principle of experiment

   DRG 's leptinase-free assay is a solid-phase enzyme-free adsorption assay ( ELISA ) based on the sandwich principle . The micro-detection well on the reaction plate is coated with a monoclonal antibody that binds to a unique antigenic site on the leptin molecule. A patient sample containing endogenous leptin is incubated with a specific rabbit anti-leptin antibody in a coated well, after which a sandwich complex is formed. After the incubation, the unbound material was washed away with a washing solution, and a peroxidase-labeled anti-rabbit antibody was added to detect the amount of leptin bound to the coated plate. After the addition of the substrate solution, the apparent color intensity is proportional to the concentration of leptin in the patient sample.

3 , kit components

1)        Coated plate, 12 × 8 well, detachable, microporous coated with anti-leptin antibody (monoclonal)

2)        Standard (0-5), 6 Lyophilizable 0.5mL, concentration: 0,2,5,25,50,100ng / ml, containing 0.3% of a preservative Proclin

3)        Quality control, 2 , 200ul , ready to use, 2 concentrations (low and high), please refer to the reagent bottle label or QC quality control sheet for specific values ​​and ranges . Containing 0.3% Proclin preservatives.

4)        Assay buffer, 1, 11 ml of, i.e. use, containing 0.3% of a preservative Proclin

5)        Antisera, one, 11 ml of, i.e. use, leptin monoclonal antisera, containing 0.3% of a preservative Proclin.

6)        Enzyme complex, 1, 11 ml of, i.e. with, horseradish peroxidase labeled anti-rabbit enzyme complex, containing 0.3% of a preservative Proclin

7)        TMB substrate solution, 1 branch, 14ml , ready to use

8)        Stop solution, 1 branch, 14ml , ready to use, containing 0.5M The H ₂ SO _4, to avoid contact with stop solution, to avoid skin irritation or burning of the skin.

9)        Washing solution (40 ×), 1 30ml branched

Note: Zero standards should be determined as needed.

4 , the equipment required for the experiment (but the kit does not provide)

1)        Microplate reader ( 450 ± 10nm ) (such as DRG 's microplate reader)

2)        Precision sampling gun

3)        Absorbent paper.

4)        Distilled water

5 , storage conditions  

   Unopened reagents at 2 — 8 °C Under storage, it can remain active during the period of validity. Do not use expired reagents. Enzyme conjugates, substrate solutions, standards, and zero standards must be in 2 — 8 °C Store under.

   Microporous reaction plates must be in 2 — 8 °C Store under. Once the bag is opened, it must be carefully sealed.

6 , sample collection

1)   Serum: Whole blood was collected by venipuncture, and after coagulation, serum was separated by centrifugation at room temperature. Do not centrifuge before incomplete coagulation. Patients who have received anticoagulant therapy may require longer clotting times.

2)   Plasma: Whole blood was collected into a centrifuge tube containing an anticoagulant and centrifuged immediately after collection.

3)   Storage of samples: Seal the sample with a lid before the start of the experiment, 2 -8 °C It can be stored for 24 hours. If the sample needs to be stored for a longer period of time before the experiment, the sample should be stored frozen. -20 °C In the environment, and can only be frozen once, can not be frozen repeatedly. Thawed samples must be turned upside down several times before the experiment.

7. Dilution of the sample:

In the first experiment, if the concentration of the sample was higher than the highest standard, the sample application was diluted with a zero standard as described in the experimental procedure and retested. And you need to multiply this dilution factor when calculating the concentration.

example:

a)   By 1:10 dilution: 10 μ L of serum +90 μ L zero standard (fully shake).

b)   1: 100 diluted Release: 10 μ L of diluted by a) +90 μ L of serum zero standard (Shake)

8 , experimental steps

All standards, controls, and samples must be double tested to ensure all test conditions are the same. There is a standard curve for each experiment.

1 ) Place the required number of slats on the pallet. .

2) The disposable nozzle ﹑ standards controls and samples were added to 15 μ L corresponding to the test wells.

3) the assay buffer added to each well of 100 μ L.

4 ) Mix well for 10 seconds, and it is very important to mix well in this step.

5 ) Incubate for 120 minutes at room temperature .

6) Rapid discarded contents of the test wells, each well was washed with 300 μ L plate was washed three times, shoot dry on absorbent paper to remove residual liquid. Note: The correct operation of the washing step will significantly affect the sensitivity and accuracy of the experiment.

7) 100 μ L per well of anti-serum.

8 ) Incubate for 30 minutes at room temperature .

9) Quick discarded contents of the test wells, each well was washed with 300 μ L of the plate was washed three times, and pat dry on absorbent paper to remove residual liquid.

10) Add 100 μ L per well enzyme conjugate.

11 ) Incubate for 30 minutes at room temperature .

12) Fast discarded contents of the test wells, each well was washed with 300 μ L of the plate was washed three times, and pat dry on absorbent paper to remove residual liquid.

13) Add 100 μ L per well of substrate solution.

14 ) Incubate for 15 minutes at room temperature .

15) 50 μ L were added to each well to terminate the enzyme reaction stop solution.

16 ) Read the OD value at 450 ± 10 nm within 10 minutes after the addition of the stop solution .

9 , the result of calculation

1)        Calculate the average absorbance value for each standard, control, and patient sample.

2)        Using the absorbance value as the ordinate ( y ) and the concentration value as the abscissa ( x ), the corresponding concentration values ​​of each standard are plotted to make a standard curve.

3)        The corresponding concentration was determined on the standard curve using the average absorbance value of each sample.

4)        Automatic calculation method: On the IFU , it automatically calculates the result using a four-parameter curve. Other inductive methods may yield slightly different results.

5)        The concentration of the sample can be read directly from the standard curve. Further dilution of the HPL concentration in the sample above the highest standard must be used. This dilution factor should be taken into account when calculating the sample concentration.

10 , reference value

We strongly recommend that each experimental city establish its own normal and outliers. The following results are based on a certain number of blood samples from the normal population:

This translation is for reference only, please refer to the original for details.