DRG EIA-2935 Insulin Elisa Lh Urine 96 Well Kit Instructions

DRG EIA-2935 Insulin Elisa Lh Urine 96 Well Kit

Specifications

• Product Name: Insulin ELISA EIA-2935
• Manufacturer: DRG International, Inc., USA
• Intended Use: Quantitative measurement of Insulin in serum and
  plasma (lithium heparin or EDTA plasma)

Product Usage Instructions:

Reagents
Ensure all reagents are at room temperature before use.

• Provided Reagents:
  • Microtiterwells coated with anti-Insulin antibody
  • Zero Standard ready for use
• Materials Required (not provided):
  • Microtiter plate reader (450 nm with reference wavelength at 620-630 nm)
  • Calibrated micropipettes
  • Absorbent paper, distilled water, timer
  • Graph paper or software for data reduction

Reagent Preparation
Dilute the concentrated Wash Solution with deionized water as per instructions. The diluted Wash Solution is stable for 1 week at room temperature.

Disposal of the Kit:
Dispose of the kit following national regulations. Refer to the Safety Data Sheet for specific disposal information.

Specimen Collection and Preparation:
Use serum or plasma (only lithium heparin or EDTA plasma) for the assay. Avoid samples containing sodium azide, haemolytic, icteric, or lipaemic specimens.

Frequently Asked Questions (FAQ)

• Q: Can I use samples containing sodium azide for the assay?
  A: No, samples containing sodium azide should not be used in the assay.

• Q: What should I do if the test kit or components are severely damaged?
  A: Inform DRG in writing within one week of receiving the kit. Do not use severely damaged components for testing and followofficial regulations for disposal.

Please use only the valid version of the Instructions for Use provided with the kit.

INTRODUCTION

Intended Use
The DRG Insulin ELISA is an enzyme immunoassay for the quantitative in vitro diagnostic measurement of Insulin in serum and plasma (lithium heparin or EDTA plasma).

Summary and Explanation
Insulin is the principal hormone responsible for the control of glucose metabolism. It is synthesized in the β-cells of the islets of Langerhans as the precursor, proinsulin, which is processed to form C-peptide and insulin. Both are secreted in equimolar amounts into the portal circulation. The mature insulin molecule comprises two polypeptide chains, the A chain and B chain (21 and 30 amino acids respectively). The two chains are linked together by two inter-chain disulphide bridges. There is also an intra-chain disulphide bridge in the A chain.
Secretion of insulin is mainly controlled by plasma glucose concentration, and the hormone has a number of important metabolic actions. Its principal function is to control the uptake and utilisation of glucose in peripheral tissues via the glucose transporter. This and other hypoglycaemic activities, such as the inhibition of hepatic gluconeogenesis and glycogenolysis are counteracted by the hyperglycaemic hormones including glucagon, epinephrine (adrenaline), growth hormone and cortisol.
Insulin concentrations are severely reduced in insulin-dependent diabetes mellitus (IDDM) and some other conditions such as hypopituitarism. Insulin levels are raised in non-insulin-dependent diabetes mellitus (NIDDM), obesity, insulinoma and some endocrine dysfunctions such as Cushing’s syndrome and acromegaly.

PRINCIPLE OF THE TEST

The DRG Insulin ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle.
The microtiter wells are coated with a monoclonal antibody directed towards a unique antigenic site on the Insulin molecule.
An aliquot of patient sample containing endogenous Insulin is incubated in the coated well with enzyme conjugate, which is an anti-Insulin antibody conjugated with Biotin. After incubation the unbound conjugate is washed off.
During the second incubation step Streptavidin Peroxidase Enzyme Complex binds to the biotin-anti-Insulin antibody. The amount of bound HRP complex is proportional to the concentration of Insulin in the sample.
Having added the substrate solution, the intensity of colour developed is proportional to the concentration of Insulin in the patient sample.

WARNINGS AND PRECAUTIONS

1. This kit is for in vitro diagnostic use only. For professional use only.
2. All reagents of this test kit which contain human serum or plasma have been tested and confirmed negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should be treated as potential biohazards in use and for disposal.
3. Before starting the assay, read the instructions completely and carefully. Use the valid version of instructions for use provided with the kit. Be sure that everything is understood.
4. The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil pouch and used in the frame provided.
5. Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step.
6. Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir for dispensing a substrate solution that had previously been used for the conjugate solution may turn solution coloured. Do not pour reagents back into vials as reagent contamination may occur.
7. Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse micro wells.
8. Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
9. Allow the reagents to reach room temperature (21 °C to 26 °C) before starting the test. Temperature will affect the absorbance readings of the assay. However, values for the patient samples will not be affected.
10. Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
11. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
12. Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or specimens may give false results.
13. Handling should be done in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation.
14. Do not use reagents beyond expiry date as shown on the kit labels.
15. All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes and microtiter plate readers.
16. Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells of different plates even of the same lot. The kits may have been shipped or stored under different conditions and the binding characteristics of the plates may result slightly different.
17. Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
18. Some reagents contain Proclin 300, BND and/or MIT as preservatives. In case of contact with eyes or skin, flush immediately with water.
19. TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with an abundant volume of water and skin with soap and abundant water. Wash contaminated objects before reusing them. If inhaled, take the person to open air.
20. Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national biohazard safety guideline or regulation.
21. For information on hazardous substances included in the kit please refer to Safety Data Sheets.
    Safety Data Sheets for this product are available upon request directly from DRG.

 REAGENTS

Reagents provided

1. Microtiterwells, 12 x 8 (break apart) strips, 96 wells;
   Wells coated with anti-Insulin antibody (monoclonal).

2. Zero Standard, 1 vial, 3 mL, ready to use 0 µIU/mL
   Contains non-mercury preservative.

3. Standard (Standard 1-5), 5 vials, 1 mL, ready to use;
   Concentrations: 6.25 – 12.5 – 25 – 50 and 100 µIU/mL,
   Conversion: µIU/mL x 0.0433 = ng/mL, ng/mL x 23.09 = µIU/mL
   The standards are calibrated against international WHO approved Reference material NIBSC 66/304.; Contain non-mercury preservative.

4. Enzyme Conjugate, 1 vial, 5 mL, ready to use, mouse monoclonal anti-Insulin conjugated to biotin;
   Contains non-mercury preservative.

5. Enzyme Complex, 1 vial, 7 mL, ready to use,
   Streptavidin-HRP Complex
   Contains non-mercury preservative.

6. Substrate Solution, 1 vial, 14 mL, ready to use,
   Tetramethylbenzidine (TMB).

7. Stop Solution, 1 vial, 14 mL, ready to use, contains 0.5 M H2SO4,
   Avoid contact with the stop solution. It may cause skin irritations and burns.

8. Wash Solution, 1 vial, 30 mL (40X concentrated), see „Preparation of Reagents“.

Note: Additional Zero Standard for sample dilution is available upon request.

Materials required but not provided

• A microtiter plate calibrated reader (450 nm, with reference wavelength at 620 nm – 630 nm) (e.g. the DRG Instruments Microtiter Plate Reader)
• Calibrated variable precision micropipettes
• Absorbent paper
• Distilled water
• Timer
• Graph paper or software for data reduction

Storage Conditions
When stored at 2 °C to 8 °C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date.
Opened reagents must be stored at 2 °C to 8 °C. Microtiter wells must be stored at 2 °C to 8 °C. Once the foil bag has been opened, care should be taken to close it tightly again.
Opened kits retain activity for 3 months if stored as described above.

Reagent Preparation
Bring all reagents and required number of strips to room temperature prior to use.

Wash Solution
Add deionized water to the 40X concentrated

Wash Solution.
Dilute 30 mL of concentrated Wash Solution with 1170 mL deionized water to a final volume of 1200 mL.
The diluted Wash Solution is stable for 1 week at room temperature.

Disposal of the Kit
The disposal of the kit must be made according to the national regulations. Special information for this product is given in the Safety Data Sheet.

Damaged Test Kits
In case of any severe damage to the test kit or components, DRG has to be informed in writing, at the latest, one week after receiving the kit. Severely damaged single components should not be used for a test run. They have to be stored until a final solution has been found. After this, they should be disposed according to the official regulations.

SPECIMEN COLLECTION AND PREPARATION

Serum or plasma (only lithium heparin or EDTA plasma) can be used in this assay.
Note: Samples containing sodium azide should not be used in the assay.
In general it should be avoided to use haemolytic, icteric or lipaemic specimens. For further information refer to chapter “Interfering Substances”.

Specimen Collection

Serum:
Collect blood by venipuncture (e.g. Sarstedt Monovette for serum), allow to clot, and separate serum by centrifugation at room temperature. Do not centrifuge before complete clotting has occurred. Patients receiving anticoagulant therapy may require increased clotting time.
Plasma:
Whole blood should be collected into centrifuge tubes containing anti- coagulant (e.g. Sarstedt Monovette with the appropriate plasma preparation) and centrifuged immediately after collection.

Specimen Storage and Preparation
Specimens should be capped and may be stored for up to 7 days at 2 °C to 8 °C prior to assaying.
Specimens held for a longer time (up to 18 months) should be frozen only once at -20 °C prior to assay. Thawed samples should be inverted several times prior to testing.

Specimen Dilution
If in an initial assay, a specimen is found to contain more than the highest standard, the specimens can be diluted with Zero Standard and re-assayed as described in Assay Procedure.
For the calculation of the concentrations this dilution factor has to be taken into account.

Example:

• dilution 1:10: 10 μL sample + 90 μL Zero Standard (mix thoroughly)
• dilution 1:100: 10 μL dilution a) 1:10 + 90 μL Zero Standard (mix thoroughly).

ASSAY PROCEDURE

General Remarks

• All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed without foaming.
• Once the test has been started, all steps should be completed without interruption.
• Use new disposal plastic pipette tips for each standard, control or sample in order to avoid cross contamination.
• Absorbance is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption.
• As a general rule the enzymatic reaction is linearly proportional to time and temperature.

Test Procedure
Each run must include a standard curve.

1. Secure the desired number of Microtiter wells in the frame holder.

2. Dispense 25 µL of each Standard, control and samples with new disposable tips into appropriate wells.

3. Dispense 25 µL Enzyme Conjugate into each well.
   Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.

4. Incubate for 30 minutes at room temperature.

5. Briskly shake out the contents of the wells.
   Rinse the wells 3 times with 400 µL diluted Wash Solution per well, if a plate washer is used – or – rinse the wells 3 times with 300 µL diluted Wash Solution per well for manual washing.
   Strike the wells sharply on absorbent paper to remove residual droplets.
   Important note:
   The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure!

6. Add 50 µL of Enzyme Complex to each well.

7. Incubate for 30 minutes at room temperature.

8. Briskly shake out the contents of the wells.
   Rinse the wells 3 times with 400 µL diluted Wash Solution per well. , if a plate washer is used – or – rinse the wells 3 times with 300 µL diluted Wash Solution per well for manual washing.
   Strike the wells sharply on absorbent paper to remove residual droplets.

9. Add 50 µL of Substrate Solution to each well.

10. Incubate for 15 minutes at room temperature.

11. Stop the enzymatic reaction by adding 50 µL of Stop Solution to each well.

12. Determine the absorbance (OD) of the solution in each well at 450 nm (reading) and at 620 – 630 nm (background subtraction, recommended) with a microtiter plate reader.
    It is recommended that the wells be read within 10 minutes after adding the Stop Solution.

Calculation of Results

1. Calculate the average absorbance values for each set of standards, controls and patient samples.

2. Using linear graph paper, construct a standard curve by plotting the mean absorbance obtained from each standard against its concentration with absorbance value on the vertical (Y) axis and concentration on the horizontal (X) axis.

3. Using the mean absorbance value for each sample determine the corresponding concentration from the standard curve.

4. Automated method: The results in the Instructions for Use have been calculated automatically using a 4-Parameter curve fit. (4 Parameter Rodbard or 4 Parameter Marquardt are the preferred methods.)
   Other data reduction functions may give slightly different results.

5. The concentration of the samples can be read directly from this standard curve. Samples with concentrations higher than that of the highest standard have to be further diluted or reported as > 100 µIU/mL. For the calculation of the concentrations this dilution factor has to be taken into account.

Example of Typical Standard Curve
The following data is for demonstration only and cannot be used in place of data generations at the time of assay.

EXPECTED NORMAL VALUES
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
In a study conducted with apparently normal healthy adults, using the DRG Insulin ELISA the following values are observed:

2 µIU/mL to 25 µIU/mL

The results alone should not be the only reason for any therapeutic consequences. The results should be correlated to other clinical observations and diagnostic tests.

QUALITY CONTROL
Good laboratory practice requires that controls be run with each calibration curve. A statistically significant number of controls should be assayed to establish mean values and acceptable ranges to assure proper performance.
It is recommended to use control samples according to state and federal regulations. The use of control samples is advised to assure the day to day validity of results. Use controls at both normal and pathological levels.
The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to the kit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for direct comparison of the results.
It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy of the results.
Employ appropriate statistical methods for analysing control values and trends. If the results of the assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid.
In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods.
After checking the above mentioned items without finding any error contact your distributor or DRG directly.

PERFORMANCE CHARACTERISTICS

Assay Dynamic Range
The range of the assay is between 1.76 – 100 µIU/mL.

Specificity of Antibodies (Cross Reactivity)
The cross reactivities were determinated by addition of different analytes to serum containing 4 ng/mL ( 100 µIU/mL) Insulin and measuring the apparent Insulin concentration.

Added analyte to a high value serum (4 ng/mL)| Observed Insulin value (ng/mL)| Cross reaction (%)
---|---|---
Porcine Insulin          8 ng/mL

Bovine Insulin          8 ng/mL

Dog Insulin              16 ng/mL

Rabbit Insulin           16 ng/mL

Rat Insulin               16 ng/mL Human Proinsulin 32 ng/mL Porcine Proinsulin 16 ng/mL Bovine Proinsulin      16 ng/mL

| 17

17.8

17.2

14.1

4.0

4.1

4.0

4.1

| > 100

100

82

63

0

0

0

0

Sensitivity
The analytical sensitivity of the DRG ELISA was calculated by adding 2 standard deviations to the mean of 20 replicate analyses of the Zero Standard and was found to be 1.76 µIU/mL.

Reproducibility

Intra-Assay
The within assay variability is shown below:

Inter-Assay
The between assay variability is shown below:

Recovery
Samples have been spiked by adding Insulin solutions with known concentrations in a 1:1 ratio.
The expected values were calculated by addition of half of the values determined for the undiluted samples and half of the values of the known solutions. The % Recovery has been calculated by multiplication of the ratio of the measurements and the expected values with 100.

| Sample 1| Sample 2
---|---|---
Concentration (µIU/mL)| 21.2| 69.0
Average Recovery (%)| 106.5| 95.5

Range of Recovery (%)

| from| 101.1| 91.8
to| 109.6| 100.1

Linearity
Samples were measured undiluted and in serial dilutions with zero standard. The recovery (%) was calculated by multiplying the ratio of expected and measured values with 100.

| Sample 1| Sample 2
---|---|---
Concentration (µIU/mL)| 21.2| 69.0
Average Recovery (%)| 100.8| 100.5

Range of Recovery (%)

| from| 88.5| 88.4
to| 110.3| 110.4

 LIMITATIONS OF USE

Reliable and reproducible results will be obtained when the assay procedure is performed with a complete understanding of the package insert instruction and with adherence to good laboratory practice.
Any improper handling of samples or modification of this test might influence the results.

Interfering Substances
Haemoglobin (up to 4 mg/mL), bilirubin (up to 0.5 mg/mL) and triglyceride (up to 30 mg/mL) have no influence on the assay results.
A biotin concentration of up to 1200 ng/mL in a sample has no influence on the assay results.

Drug Interferences
Until today no substances (drugs) are known to us, which have an influence to the measurement of Insulin in a sample.

High-Dose-Hook Effect
No hook effect was observed in this test up to 1600 µIU/mL of Insulin.

LEGAL ASPECTS

Reliability of Results
The test must be performed exactly as per the manufacturer’s instructions for use. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable national standards and/or laws. This is especially relevant for the use of control reagents. It is important to always include, within the test procedure, a sufficient number of controls for validating the accuracy and precision of the test.
The test results are valid only if all controls are within the specified ranges and if all other test parameters are also within the given assay specifications. In case of any doubt or concern please contact DRG.
Therapeutic Consequences
Therapeutic consequences should never be based on laboratory results alone even if all test results are in agreement with the items as stated under point 11.1. Any laboratory result is only a part of the total clinical picture of a patient.
Only in cases where the laboratory results are in acceptable agreement with the overall clinical picture of the patient should therapeutic consequences be derived.
The test result itself should never be the sole determinant for deriving any therapeutic consequences.

Liability
Any modification of the test kit and/or exchange or mixture of any components of different lots from one test kit to another could negatively affect the intended results and validity of the overall test. Such modification and/or exchanges invalidate any claim for replacement.
Claims submitted due to customer misinterpretation of laboratory results subject to point 11.2 are also invalid. Regardless, in the event of any claim, the manufacturer’s liability is not to exceed the value of the test kit. Any damage caused to the test kit during transportation is not subject to the liability of the manufacturer.

SYMBOLS USED

DRG Instruments GmbH, Germany Frauenbergstraße. 18, D-35039 Marburg
Phone: +49 (0)6421-1700 0, Fax: +49 (0)6421-1700 50 Website: www.drg- diagnostics.de
E-mail: drg@drg-diagnostics.de

DRG International, Inc., USA
841 Mountain Ave., Springfield, NJ 07081 Phone: 973-564-7555, Fax: 973-564-7556 Website: www.drg- international.com
E-mail: corp@drg-international.com

References

• Analyticon® Biotechnologies GmbH

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