Human IAA / Insulin Autoantibodies ELISA Kit

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SKU:
HUFI01166

Description

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Product Name:Human IAA / Insulin Autoantibodies ELISA Kit
Product Code:HUFI01166
Size:96 Assays
Alias:IAA, Insulin Autoantibodies
Detection method:Indirect ELISA - HRP
Application:This immunoassay kit allows for the in vitro quantitative determination of Human IAA concentrations in serum plasma and other biological fluids.
Sensitivity:0.469ng/ml
Range:0.781-50ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human IAA and the recovery rates were calculated by comparing the measured value to the expected amount of Human IAA in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)89-10596
EDTA plasma(n=5)85-10595
UFH plasma(n=5)85-10393
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human IAA and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)91-105%91-103%88-103%
EDTA plasma(n=5)84-100%84-95%83-97%
UFH plasma(n=5)83-89%90-100%83-92%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate(Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
HRP-labeled Antibody (Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

Other materials and equipment required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to the wells, equilibrate the TMB substrate for at least 30 mins at 37°C. When diluting samples and reagents, ensure they are mixed completely and evenly. It is recommended to plot a standard curve for each test.

StepProtocol
1. Set standard, test sample (diluted at least ½ with Sample Dilution Buffer) and control (blank) wells on the pre-coated plate and record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (blank) wells!
2.Aliquot 100µl of standard solutions into the standard wells.
3.Add 100µl of properly diluted Sample into the sample wells.
4.Seal the plate with a cover and incubate at 37 °C for 90 mins.
5.Wash: Repeat the aspiration/wash process for 3 times.
6. HRP-labelled antibody: Add 100µl of HRP-labelled antibody working solution into the bottom of each well (standard, test sample & zero wells) without touching the side walls.
7.Seal the plate with a cover and incubate at 37°C for 30 mins.
8. Wash: Remove the cover, and wash plate 5 times with Wash buffer.
9. TMB Substrate: Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark for 10-20 mins. (Note: This incubation time is for reference only, the optimal time should be determined by the end-user.) As soon as a blue colour develops in the first 3-4 wells (with most concentrated standards) and the other wells show no obvious colour, terminate the reaction.
10. Add 50µl of Stop solution into each well and mix thoroughly. The colour changes into yellow immediately.
11. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample TypeProtocol
SerumIf using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

PlasmaCollect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal FluidCollect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatantCollect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysatesSolubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenatesThe preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysatesRinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast MilkCollect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

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Additional Information

Product Type:
ELISA Kit
Size:
96 Assays
Sensitivity:
0.469ng/ml
Range:
0.781-50ng/ml
ELISA Type:
Indirect
Synonyms:
IAA, Insulin Autoantibodies
Reactivity:
Human
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