C-Peptide Kit ELISA

N° du produit ABIN997070

Ce kit ELISA Colorimetric est destiné à la mesure quantitative de Humain C-Peptide.

Aperçu rapide pour C-Peptide Kit ELISA (ABIN997070)

Antigène: C-Peptide

Reactivité: Humain

Méthode de détection: Colorimetric

Type de méthode: Competition ELISA

Application: ELISA

Détail du produit C-Peptide Kit ELISA

Fonction: The essential reagents required for an immunoenzymometric assay include high affinity and specificity antibodies (Ab). (enzume conjugated and immobilized), with different and distinct epitope recognition, in excess, and native antigen (Ag). In this procedure, the immobilization takes place during the assay at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated monoclonal C-peptide antibody.

Analytical Method: Quantitative

Specificité: The DAI C-PEPTIDE (ng/mL) quantitative was tested for cross-reactivity with the following:
Human pro-insulin (32-33 split): 63 % .
Human pro-insulin (64-65 split): 87 %
Human insulin: 0 %
Human C-Peptide of Insulin: 100 %
Fasting concentrations of intact and split pro-insulin are typically only 1-2 % of C-PEPTIDE concentrations. Cross-reactivity with these molecules is not clinically significant.

Sensibilité: 0.020ng/mL

Information d'application

Commentaires:

Quality Control:
Each laboratory should assay controls at levels in the low, normal and elevated ranges for monitoring assay performance. These controls should be treated as unknowns and values determined in every test procedure performed. Quality control charts should be maintained to follow the performance of the supplied reagents. Pertinent statistical methods should be employed to ascertain trends. Significant deviation from established performance can indicate unnoticed change in experimental conditions or degradation of kit reagents. Fresh reagents should be used to determine the reason for the variations.
Limitations of procedure:
1. It is important that the time of reaction in each well is held constant for reproducible results.
2. Pipetting of samples should not extend beyond ten (10) minutes to avoid assay drift.
3. If more than one (1) plate is used, it is recommended to repeat the dose response curve.
4. Addition of the substrate solution initiates a kinetic reaction, which is terminated by the addition of the stop solution. Therefore, the addition of the substrate and the stopping solution should be added in the same sequence to eliminate any time deviation during reaction.
5. Plate readers measure vertically. Do not touch the bottom of the wells.
6. Failure to remove adhering solution adequately in the aspiration or decantation wash step(s) may result in poor replication and spurious results.
7. Highly lipemeic, hemolysed or grossly contaminated specimen(s) should not be used. 8. Patient samples with C-Peptide concentrations above 10 ng/mL may be diluted with the zero calibrator and re-assayed. Multiply the value obtained by the dilution factor to obtain the corrected value. Use components from the same lot. No intermixing of reagents from different batches.

Durée du test: 2 - 3 h

Plaque: Pre-coated

Préparation des réactifs:

1. Wash Buffer Dilute contents of Wash concentrate to 1000 mL with distilled or deionized water in a suitable storage container. Store at room temperature (2-30 °C) for up to 60 days.
   2. Working Substrate Solution Pour the contents of the vial labeled Solution 'A' into the clear vial labeled Solution 'B'. Place the yellow cap on the clear vial for easy identification. Mix and label accordingly. Store at 2-8 °C.
   Note: Do not use the working substrate if it looks blue. Do not use reagents that are contaminated or have bacteria growth.

Préparation de l'échantillon:

The specimens shall be blood serum in type and the usual precautions in the collection of venipuncture samples should be observed. For accurate comparison to established normal values, a fasting morning serum sample should be obtained. The blood should be collected in a plain red-top venipuncture tube without additives. Allow the blood to clot. Centrifuge the specimen to separate the serum from the cells. Samples may be refrigerated at 2-8 °C for a maximum period of two (2) days. If the specimen(s) cannot be assayed within this time, the sample(s) may be stored at temperatures of -20 °C for up to 30 days. Avoid repetitive freezing and thawing. When assayed in duplicate, 0.100 mL of the specimen is required.

Procédure de l'essai:

Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20 °C). Test procedure should be performed by a skilled individual or trained professional.
1. Format the microplates' wells for calibrator, control and patient specimen to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and store at 2-8 °C.
2. Pipette 0.050 mL (50 μL) of the appropriate calibrators, controls and samples into the assigned wells.
3. Add 0.100 mL (100 μL) of the C-Peptide Enzyme Reagent to each well. It is very important to dispense all reagents close to the bottom of the microwell.
4. Swirl the microplate gently for 20-30 seconds to mix. Cover with a plastic wrap.
5. Incubate for 120 minutes at room temperature (20-25 °C).
6. Discard the contents of the microplate by decantation or aspiration. If decanting, tap and blot the plate dry with absorbent paper.
7. Add 350 μL of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two (2) additional times for a total of three (3) washes. An automatic or manual plate washer can be used. Follow the manufacturer's instruction for proper usage. If a squeeze bottle is used, fill each well to the top by squeezing the container. Avoiding air bubbles. Decant the wash and repeat two (2) additional times.
8. Add 0.100 mL (100 μL) of working substrate solution to all wells (see Reagent Preparation Section). DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION
9. Incubate at room temperature for fifteen (15) minutes.
10. Add 0.050 mL (50 μL of stop solution to each well and mix gently for 15-20 seconds. Read the absorbance in each well at 450 nm (using a reference wavelength of 620-630 nm to minimize well imperfections) in a microplate reader. The results should be read within thirty (30) minutes of adding the stop solution.

NOTE: Always add reagents in the same order to minimize reaction time differences between wells.

Calcul des résultats:

A dose response curve is used to ascertain the concentration of C-Peptide in unknown specimens.
1. Record the absorbance obtained from the printout of the microplate reader as outlined in Example 1

2. Plot the absorbance for each duplicate serum reference versus the corresponding C-Peptide concentration in ng/mL on linear graph paper (do not average the duplicates of the serum references before plotting).

3. Draw the best-fit curve through the plotted points.
4. To determine the concentration of C-Peptide for an unknown, locate the average absorbance of the duplicates for each unknown on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration (in ng/mL) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated). In the following example, the average absorbance (0.433) intersects the dose response curve at
1.03 ng/mL for the C-Peptide concentration (See Figure 1).
Note: Computer data reduction software designed for DAI (ELISA) assays may also be used for the data reduction. * The data presented in Example 1 and Figure 1 is for illustration only and should not be used in lieu of a dose response curve prepared with each assay.
EXAMPLE 1
Sample I.D. Well Number Abs (A) Mean Abs (B) g l l e Cal A A1 0.022 0.022 0 B1 0.023 Cal B C1 0.097 0.103 0.2 D1 0.107 Cal C E1 0.421 0.429 1 F1 0.439 Cal D G1 0.889 0.901 2 H1 0.910 Cal E A2
1.976
1.971 5 B2
1.966 Cal F C2
2.717
2.643 10 D2
2.570 Ctrl 1 E2 0.429 0.433
1.03 F2 0.437 Ctrl 2 G2
1.861
1.887
4.64 H2
1.913 Patient 1 A3 0.388 0.405 0.82 B3 0.421 The data presented in Example 1 and Figure 1 is for illustration only and should not be used in lieu of a dose response curve prepared with each assay. QC PARAMETERS In order for the assay results to be considered valid the following criteria should be met:
1. The absorbance (Od) of calibrator 'A' should be < 0.
1.

2. The absorbance (OD) of calibrators 'F' should be >
1.
3.

3. Four out of six quality control pools should be within the established ranges. RISK ANALYSIS A. Assay Performance It is important that the time of reaction in each well is held constant for reproducible results.

1.
2.
3.4.5.6. Pipetting of samples should not extend beyond ten (10) minutes to avoid assay drift. Highly lipemeic, hemolysed or grossly contaminated specimen(s) should not be used. If more than one (1) plate is used, it is recommended to repeat the dose response curve. The addition of the substrate solution initiates a kinetic reaction, which is terminated by the addition of the stop solution. Therefore, the addition of the substrate and the stopping solution should be added in the same sequence to eliminate any time deviation during reaction. Plate readers measure vertically. Do not touch the bottom of the wells.
7. Failure to remove adhering solution adequately in the aspiration or decantation wash step(s) may result in poor replication and spurious results.
8. Use components from the same lot. No intermixing of reagents from different batches.
9. Accurate and precise pipetting, as well as following the exact time and temperature requirements prescribed are essential. Any deviation from DAI IFU may yield inaccurate results.
1. All applicable national standards, regulations and laws, including, but not limited to, good laboratory procedures, must be strictly followed to ensure compliance and proper device usage.
11. It is important to calibrate all the equipment e.g. Pipettes, Readers, Washers and/or the automated instruments used with this device, and to perform routine preventative maintenance.
12. Risk Analysis as required by CE Mark IVD Directive 98/79/EC - for this and other devices, made by DAI can be requested via email from www.rapidtest.com. B. Interpretations
1. Laboratory results alone are only one aspect for determining patient care and should not be the sole basis for therapy, particularly if the results conflict with other determinanats.

2. For valid test results, adequate controls and other parameters must be within the listed rages and assay requirements.

3. If test kits are altered, such as by mixing part of different ktis, which could produce false test results, or if results are incorrectly interpreted, DAI shall have no liability.
4. If computer controlled data reduction is used to interpret the results of the test, it is imperative that the predicted values for calibrators fall within 10 % of the assigned concentrations.
A. Precision The within and between assay precision of the C-Peptide DAI ELISA test system were determined by analyses on three different levels of pool control sera. The number (N) mean value (X), standard deviation (sigma) and coefficient of variation (C.V.) for each of these control sera are presented in Table1 and Table

2. TABLE 1 Within Assay Precision (Values in ng/mL)
SAMPLE
N X sigma C.V. Pool 1 20
1.43 0.11
7.7 % Pool 2 20
5.07 0.46
9.0 % Pool 3 20
7.81 0.73
9.3 % TABLE 2 Between Assay Precision (Values in ng/mL) SAMPLE N X sigma C.V. Pool 1 20
1.27 0.12
9.7 % Pool 2 20
5.40 0.54
9.9 % Pool 3 20
8.18 0.50
6.1 % As measured in ten experiments in duplicate over ten days. B. Accuracy The C-Peptide ELISA test system was compared with a predicate radioimmunoassay assay. Biological specimens from population (symptomatic and asymptomatic) were used. (The values ranged from 0.2 ng/mL -
11.8 ng/mL). The total number of such specimens was 124. The data obtained is displayed in Table
4. TABLE 3 Method Mean (x)
1.068
1.066 This Method (y) Reference (x) Least Square Regression Correlation Coefficient 0.962 Analysis y = 0.2079 + 0.8036(x) Only slight amounts of bias between the C-Peptide DAI ELISA test system and the reference method are indicated by the closeness of the mean values. The least square regression equation and correlation coefficient indicates excellent method agreement. C.
C-Peptide values are consistently higher in plasma than in serum, DAI advises that a serum sample be used for accurate determination. Compared with fasting values in non-obese non-diabetic individuals, C-Peptide levels are higher in obese non-diabetic subjects and lower in trained athletes. Each laboratory is advised to establish its own ranges for normal and abnormal populations. These ranges are always dependent upon locale, population, laboratory, technique and specificity of the method. It is important to keep in mind that any normal range establishment is dependent upon a multiplicity of factors like the specificity of the method, the locale, the population tested and the precision of the method in the hands of technicians. For these reasons each laboratory should depend upon the range of expected values established by the manufacturer only until an in-house range can be determined by the technicians using the method with a population indigenous to the area in which the laboratory is located. Based on the clinical data gathered by Diagnostic Automation, Inc. in concordance with the published literature the following ranges have been assigned. These ranges should be used as guidelines only: Adult (Normal) 0.7 -
1.9 ng/mL

Restrictions: For Research Use only

Stockage

Stock: 4 °C

Stockage commentaire: Store at 2-8 °C

Date de péremption: 12-14 months

Détail du antigène

Antigène: C-Peptide

Sujet: Human insulin and C-PEPTIDE originate as a single polypeptide chain known as proinsulin(M.Wt 9000) in the pancreatic cell. Proinsulin is cleaved proteolytically to form equimolar amounts of mature insulin and C-PEPTIDE that are released into the portal vein. So called because it connects the A and B chains of insulin in the proinsulin molecule. C-PEPTIDE is a single chain of 31 amino acid (Mol. Wt 30200). Unlike insulin has no known physiological function. Because C-PEPTIDE has a longer half-life than insulin (2-5 times longer), high concentrations of C-PEPTIDE persist in the peripheral circulation and these level fluctuate less insulin. For these reasons, in plasma C-PEPTIDEe concentrations may reflect pancreatic insulin secretion more reliable than the level of insulin itself 1. C-PEPTIDE is cleaved from the body by the kidney. Urine concentrations of C-PEPTIDE are 20-50 times higher than in plasma, unlike plasma insulin levels, which fluctuate in response to meals, measurement of the 24 hour urinary excretion of C-PEPTIDE provides a useful monitor of average cell insulin secretion 2. C- PEPTIDE measurements are useful in insulinoma diagnosis, especially in patients treated with insulin. Elevated C-PEPTIDE levels are indicative of insulinoma. C-PEPTIDE measurements are useful in the need for progression to insulin therapy in non-insulin dependent diabetics (NIDDM). C-PEPTIDE measurements are useful as a marker for residual pancreatic tissue after pancreatectomy. It may also be used to monitor the progress of pancreas or islet cell transplantation. C-PEPTIDE measurements are useful in the diagnosis of hypoglycemia brought on by surreptitious insulin administration.