C-peptide ELISA kit instructions

C- peptide ELISA kit instructions

  (Germany DRG : EIA-1293 )

1, the clinical application of DRG company C- peptide ELISA kit

1)   Functional evaluation of residual β - cells in diabetic patients receiving insulin therapy .

2)   Remission in patients with type I diabetes detection and monitoring phase.

3)   As Type I diabetes (insulin-dependent) and Type II diabetes aids (non-insulin dependent) differential diagnosis.

4)   Diagnosis of pseudohypoglycemia caused by insulin.

5)   Diagnosis of helper insulinoma. (Insulin inhibition test)

6)   A prognostic index for detecting pregnancy success in pregnant women with diabetes.

7)   Evaluation of insulin secretion in patients with liver disease.

8)   Monitoring of pancreatectomy.

2 , the principle of experiment

DRG company C- peptide ELISA kit is based on the principle of competition. An unknown amount of C- peptide present in the sample and a known concentration of C- peptidase linkage compete with each other for binding to the binding site of the mouse anti- C- peptide antiserum. It is then bound to an anti-mouse antibody coated on the detection well. Unbound enzyme complexes are washed down. After the addition of the substrate solution, the C- peptide concentration in the sample is inversely proportional to the measured optical density.

3 , kit composition

1)        Microwell plate, 96 (well), coated with anti-mouse antibody.

2)        Enzyme linked, 14ml . The ready-to-use type contains a C- peptide biotin label and a stabilizing buffer.

3)        Enzyme complex, 14ml , ready-to-use, containing horseradish peroxidase

4)        Standard series ( 0-5 ), 0-16ng/ml ( see the reagent bottle label for specific concentration ) . Freeze-dried powder, 6 pieces, add 0.75mL of distilled water before use .

5)        Sample dilution, 3 ml , ready-to-use.

6)        Antiserum: mouse anti- C- peptide monoclonal antibody, 1 branch, 7 mL .

7)        Substrate solution, TMB , 14ml

8)        Stop solution, 0.5M The H ₂ SO _4, 14ml.

9)        Concentrated sputum wash solution, 40 times concentrated, 30 ml .

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

1)   Microplate reader

2)   Pipettes, 100 ; 200 and 1000 μl

3)   Standard refrigerator

4)   Absorbent paper

5)   Deionized water

5 , storage conditions

When stored in 2‐ 8 °C The unopened reagents are valid for the period of validity. Do not use expired reagents. After the semi-standard is prepared, all remaining standards must be refrigerated at 2‐ 8 °C The environment. For longer storage, reconstituted standards can be frozen in -20 °C The environment. C- peptide biotin linkages, enzyme complexes, substrate solutions, washes, and zero standards must be refrigerated in 2‐ 8 °C The environment. Microporous reaction plates must also be refrigerated in 2‐ 8 °C The environment. Once the bag is taken apart, it must be resealed.

6 , sample collection and preparation

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. Sample in 2~ 8 °C Can store for 24 hours, if you need to store longer, you need to - 20 °C Frozen down, and can only freeze once

Plasma: Whole blood was collected into a centrifuge tube containing an anticoagulant and centrifuged immediately after collection. Sample in 2~ 8 °C Can store for 24 hours, if you need to store longer, you need to - 20 °C Frozen down, and can only freeze once

Urine:

1)        The total amount of urine excreted within 24 hours should be collected and mixed in a separate container.

2)        Record the total volume of urine.

3)        Mix the sample thoroughly. Centrifuge the sample to clarify, at 2~ 8 °C Can store for 36 hours; if you want to save longer, you need to - 20 °C Freeze and freeze only once.

4)        Experimental samples according to sample dilution 1: 20 dilution.

7 , reagent preparation

Standard: The lyophilized standard was dissolved in 0.75 ml of distilled water. It can be left still until it is completely melted, then mix thoroughly by gently inverting. Restandard standard in 2~ 8 °C Can be saved for 3 days, if you need to save longer, you need to place it - 20 °C Freezing

Washing solution: 30ml concentrated washing liquid is diluted with deionized water to 1170ml 1200ml. The diluted washing solution can be stored stably for 2 weeks at room temperature .

8 , experimental steps

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

2)        Add 100 μl of C -peptide standard and sample to the appropriate detection wells.

3)        Added to each well 50 μ l of antiserum.

4)        Each well was added 100 μ l of enzyme was linked.

5)        Shake well for 10 seconds.

6)        Incubate for 60 min at room temperature .

7)        Discard the reaction solution in the well.

8)        Washed three times with a washing solution (per well of 400 μ l). The reaction plate was patted on the absorbent paper for the last time.

9)        Add 100 μ l each of an enzyme complex in the micropores.

10)    Incubate for 30 minutes at room temperature .

11)    Discard the reaction solution in the well. Washed three times with a washing solution (per well of 400 μ l). The last time the reaction plate was patted on absorbent paper.

12)    At the same time intervals was added 100 μ l substrate solution in each microwell.

13)    Allow to stand at room temperature for 20 minutes.

14)    At the same time intervals was added 100 μ l in each microwell to stop the enzyme reaction stop solution.

15)    The OD value was measured at 450 ± 10 nm within 10 minutes after the addition of the stop solution .

9 , the result of calculation

Any microplate reader that can measure the absorbance at 450 ± 10 nm can be used . The C -peptide content value of each sample serum can be determined as follows .

1)        On a linear or semi-logarithmic coordinate paper, a standard curve is made for their respective concentration values ​​( X , ng/ml ) with the average absorbance value ( Y ) of each reference standard . We recommend that you use four parameters as the standard curve.

2)        Using the average absorbance value of each serum sample, the corresponding concentration values ​​can be obtained by reading the corresponding C- peptide values from the standard curve and , if necessary, multiplying the dilution factor of the initial sample. Values can be read using DRG 's ELIZAMAT 3000 and regression programs, and computer-aided interpretations can be obtained by using four-parameter logic functions or stereo regression.

Expected value:

The normal range of C -peptide in serum and urine .

    It is recommended that each laboratory establish its own range of normal human C -peptide levels. The normal range values ​​obtained by using DRG 's C -peptide enzyme-linked kit to detect normal males and females are listed below:

     In addition, glucose was measured in 6 normal adults after fasting for 12 hours. After 12 hours on an empty stomach , serum was drawn. The person taking the test follows 50 -60 grams Glucose was taken from the serum samples after 30-45 minutes and found to increase the C- peptide level by 200-600% .

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

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