Mouse IAA (Insulin Autoantibodies) ELISA Kit - Information
The Assay Genie Mouse IAA (Insulin Autoantibodies) ELISA Kit can assay for Mouse IAA (Insulin Autoantibodies) in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.How our Mouse IAA (Insulin Autoantibodies) ELISA Kits Work?
The Assay Genie Mouse IAA (Insulin Autoantibodies) ELISA Kit is a highly sensitive assay for the quantitative measurement of a specific analyte in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.
This kit was based on indirect enzyme-linked immune-sorbent assay technology. Antigen was pre-coated onto 96-well plates. And the biotin conjugated antibody was used as detection antibodies. The standards, test samples and biotin conjugated detection antibody were added to the wells subsequently, and washed with wash buffer. HRP-Streptavidin was added and unbound conjugates were washed away with wash buffer. TMB substrates were used to visualize HRP enzymatic reaction. TMB was catalysed by HRP to produce a blue colour product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the target amount of sample captured in plate. Read the O.D. absorbance at 450nm in a microplate reader, and then the concentration of target can be calculated.
Mouse IAA (Insulin Autoantibodies) ELISA Kit Data
|Product Code|| |
IAA ELISA Kit, Insulin Autoantibodies ELISA Kit
|Detection method|| |
Indirect ELISA - Biotin
This immunoassay kit allows for the in vitro quantitative determination of Mouse IAA (Insulin Autoantibodies) concentrations in serum plasma and other biological fluids.
4'C for 6 months
Matrices listed below were spiked with certain level of Mouse IAA (Insulin Autoantibodies) and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse IAA (Insulin Autoantibodies) in samples.
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse IAA (Insulin Autoantibodies) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
For Research Use Only
Mouse IAA (Insulin Autoantibodies) ELISA Kit Protocol
The below protocol is the sample protocol for a Mouse IAA (Insulin Autoantibodies) ELISA Kit. This indirect ELISA kit allow for the detection of an analyte in a sample. Before adding to the wells, equilibrate the TMB substrate for at least 30 mins at 37C. When diluting samples and reagents, ensure they are mixed completely and evenly. It is recommended to plot a standard curve for each test.
|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 2 times.|
|6.||Add 100µl of Biotin-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 60 mins.|
|8.||Wash: Remove the cover, and wash plate 3 times with Wash buffer.|
|9.||HRP-Streptavidin Conjugate (SABC): Add 100?l of SABC Working Solution into each well, cover the plate and incubate at 37°C for 30 minutes.|
|10.||Wash: Remove the cover and wash plate 5 times with Wash Buffer.|
|11.||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.|
|12.||Add 50µl of Stop solution into each well and mix thoroughly. The colour changes into yellow immediately.|
|13.||Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.|
Mouse IAA (Insulin Autoantibodies) ELISA Kit components
|ELISA Microplate(Dismountable)||8×12 strips||4°C for 6 months|
|Sample/Standard Dilution Buffer||20ml||4°C|
|Biotin-labeled Antibody (Concentrated)||120ul||4°C (Protect from light)|
|Antibody Dilution Buffer||10ml||4°C|
|HRP-Streptavidin Conjugate(SABC)||120ul||4°C (Protect from light)|
|SABC Dilution Buffer||10ml||4°C|
|TMB Substrate||10ml||4°C (Protect from light)|
Other materials and equipment required:
The Assay Genie Mouse IAA (Insulin Autoantibodies) ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
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.
If 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.
Collect 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 Fluid
Collect 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 supernatant
Collect 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.
Solubilize 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.
The 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.
Rinse 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.
Collect 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.