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Proinsulin EIA 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 a Proinsulin molecule. An aliquot of patient sample containing endogenous Proinsulin is incubated in the coated wells. After washing off the samples in a second step an enzyme conjugate, which is an anti-Proinsulin antibody conjugated with horseradish peroxidase is incubated in the wells. After incubation the unbound conjugate is washed off with wash solution. Having added the substrate solution, the intensity of colour developed is proportional to the concentration of Proinsulin in the patient sample.
| Product Name | Human Proinsulin ELISA Kit |
|---|---|
| SKU/Catalog Number | EK7001 |
| Description | Sandwich High Sensitivity ELISA kit for Quantitative Detection of Human Proinsulin. 96wells/kit, with removable strips. |
| Cite This Product | Human Proinsulin ELISA Kit (Boster Biological Technology, Pleasanton CA, USA, Catalog # EK7001) |
| Validated Species | Human |
| Application | ELISA *Our Boster Guarantee covers the use of this product in the above tested applications. |
| Cross Reactivity | There is no detectable cross-reactivity. |
| Pack Size | 96wells/kit, with removable strips. |
| Sensitivity | 2.6 pmol/L *Sensitivity, or Lower Limit of Detection (LLD), is the minimum level of target protein the ELISA assay can detect. We measure 20 blank wells and if the O.D. value is 2 standard deviations higher than the blanks' average O.D. the sample can be deemed positive. |
|---|---|
| Assay Range | 2.6-66 pmol/L *This assay range is determined using common samples. For samples with low target protein concentrations, users can adjust the standard curve to extend the lower limit of assay range. |
| Sample Type | Serum *The above listed samples are the ones valided with the assay. If you do not see your sample of interest listed, as long as there is enough level of target protein present in the sample, this Picokine? ELISA kit should detect it. **For protocol and tips regarding preparing your sample of interest, please check our ELISA sample preparation guide. |
| Storage | Store the kit at 2°C to 8°C. Keep microwells sealed in a dry bag with desiccants. The reagents are stable until expiration of the kit. Do not expose reagent to heat, sun, or strong light. Avoid multiple freeze-thaw cycles(Shipped with wet ice.) |
| Description | Quantity |
| Microwell coated with anti Pro-insulin Antibody | 12x8x1 Microwells |
| Proinsulin Standards | 6 vials (ready to use) 1ml |
| Pro-Insulin Enzyme Conjugate 11X | 1 vial 1.2 ml |
| TMB Substrate | 1 bottle (ready to use) 14ml |
| Stop Solution | 1 bottle (ready to use) 14ml |
| Wash concentrate 40X | 1 bottle (ready to use) 30ml |
| Sample Diluent | 1 vial (ready to use) 2 mL |
| Conjugate Diluent | 1 bottle (ready to use) 12 mL |
| control (low & high) | 2 ml |
| Assay Buffer | 1 bottle (ready to use) 12 mL |
1. Distilled or deionized water
2. Precision pipettes
3. Disposable pipette tips. Multichannel pipettes are recommended in the condition of large amount of samples in the detection.
4. ELISA reader capable of reading absorbance at 450nm
5. Absorbance paper or paper towel
6. Graph paper
Proinsulin is a 9390 MW polypeptide of 86 amino acids, that is synthesized in the ? cells of the pancreas and is the precursor molecule for insulin. Most proinsulin is converted to insulin and C-Peptide, which are secreted in equimolar amounts into the blood. About 15 % is not converted and is released as proinsulin. The biological activity of proinsulin is only about 10% of Insulin, but the half life of proinsulin is three times as long as insulin. The level of proinsulin in serum can be a reflection of ? cell status. Both IDDM and NIDDM are characterized by dysfunction of the pancreatic ? cells. Elevated proinsulin levels have been noted at the onset of IDDM and in healthy siblings of IDDM patients. Proinsulin levels may also be increased in patients with established NIDDM. Increased levels of circulating proinsulin are found in older patients, pregnant or obese diabetics, patients with insulinomas, functional hypoglycemia and hyperinsulinemia, a rare syndrome. Because the structure of proinsulin is similar to insulin, proinsulin may be detected as immunoreactive insulin in the insulin assay. Immunoreactive insulin levels are generally determined in conventional RIA's, which overestimate the insulin level because the methods use antibodies which crossreact with proinsulin. By calculating the molar ration of proinsulin to true insulin (P/I), a better assessment of ? cell function can be made.
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1. For Research Use Only. Not for use in diagnostic procedures.
2. Potential biohazardous materials: The calibrator and controls contain human source components, which have been tested and found non-reactive for hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. However, there is no test method that can offer complete assurance that HIV, Hepatitis B virus or other infectious agents are absent. These reagents should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Control/National Institutes of Health manual, "Biosafety in Microbiological and Biomedical Laboratories" 1984.
3. Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which specimens or kit reagents are handled.
4. The components in this kit are intended for use as an integral unit. The components of different lots should not be mixed.
5. It is recommended that standards, control and serum samples be run in duplicate.
6. Optimal results will be obtained by strict adherence to this protocol. Accurate and precise pipetting, as well as following the exact time and temperature requirements prescribed are essential. Any deviation from this may yield invalid data.
1. Collect blood specimens and separate the serum immediately.
2. Typically, specimens may be stored refrigerated at (2°C to 8°C) for 5 days. If storage time exceeds 5 days, store frozen at (-20°C) for up to one month.
3. Avoid multiple freeze-thaw cycles.
4. Prior to assay, frozen sera should be completely thawed and mixed well.
5. Do not use grossly lipemic specimens.
Prepare 1X Wash buffer by adding the contents of the bottle (30 mL, 40X) to 475 mL of distilled or deionized water. Store at RT.
Dilute the concentrated Enzyme Conjugate in the Conjugate Diluent (100 ?l Enzyme Conjugate + 1000 ?l Conjugate Diluent). For every well you need 100 ?l diluted Enzyme Conjugate. The diluted Enzyme Conjugate is stable for 24 h at room temperature.
Prior to assay, allow reagents to stand at room temperature. Gently mix all reagents before use.
1. Secure the desired number of coated Microtiter wells in the holder.
2. Dispense 100 ?l of Proinsulin Standards control and samples into appropriate wells.
3. Dispense 100 ?l of Assay buffer into each well.
4. Mix thoroughly for 10 seconds. It is important to achieve a complete mixing in this step.
5. Cover the plate with a plate sealer and incubate overnight (16-24 hours) at 4° C in a humidity chamber.
6. Briskly shake out the contents of the wells. Rinse the wells 3 times with diluted Wash Solution (350 ?l per well). Strike the Wells sharply on absorbance paper to remove residual droplets.
7. Dispense 100 ?l of diluted Enzyme-Conjugate into each well.
8. Mix thoroughly for 10 seconds. It is important to achieve a complete mixing in this step.
9. Incubate for 60 minutes at room temperature without agitation.
10. Briskly shake out the contents of the wells. Rinse the wells 5 times with diluted Wash Solution (350 ?l per well). Strike the wells sharply on absorbent paper to remove residual droplets.
11. Add 100 ?l of Substrate Solution to each well at timed intervals.
12. Incubate for 30 minutes at room temperature.
13. Stop the enzymatic reaction by adding 50 ?l of Stop Solution to each well.
14. Read the OD at 450±10 nm within 15 minutes after adding the stop solution.
The standard curve is constructed as follows:
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