Mouse Ovalbumin Specific IgE / OVA sIgE ELISA Kit

SKU:
MOFI01017
€599

Description

ELISA Kit Technical ManualMSDS

Mouse OVA sIgE (Ovalbumin Specific IgE) ELISA Kit - Information

The Assay Genie Mouse OVA sIgE (Ovalbumin Specific IgE) ELISA Kit can assay for Mouse OVA sIgE in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How do our Mouse OVA sIgE (Ovalbumin Specific IgE) ELISA Kits Work?

The Assay Genie Mouse OVA sIgE (Ovalbumin Specific IgE) 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. HRP conjugated antibody is used as detection antibodies. The standards, test samples and HRP conjugated detection antibody were added to the wells subsequently, and washed 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 OVA sIgE (Ovalbumin Specific IgE) ELISA Kit Data

Product Code

MOFI01017

Alias

OVA sIgE

Detection method

Indirect ELISA - HRP

Application

This immunoassay kit allows for the in vitro quantitative determination of Mouse OVA sIgE concentrations in serum plasma and other biological fluids.

Size

96T

Range

1.563-100ng/ml

Sensitivity

0.938ng/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of Mouse OVA sIgE and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse OVA sIgE in samples.

MatrixRecovery range(%)Average(%)
serum(n=5)89-10398
EDTA plasma(n=5)91-10498
UFH plasma(n=5)92-10096
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse OVA sIgE and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample1:21:41:8
serum(n=5)89-103%89-103%89-97%
EDTA plasma(n=5)87-98%82-101%88-100%
UFH plasma(n=5)86-99%81-96%82-98%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Mouse OVA sIgE (Ovalbumin Specific IgE) ELISA Kit Protocol

The below protocol is the sample protocol for a Mouse OVA sIgE (Ovalbumin Specific IgE) 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 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.

Assay Procedure:

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.

Mouse OVA sIgE (Ovalbumin Specific IgE) ELISA Kit components

96 Assays

Storage

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:

The Assay Genie Mouse OVA sIgE (Ovalbumin Specific IgE) ELISA Kit will require other equipment and materials tocarry 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
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir

Sample Preparation

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

Sample TypeProtocol

Serum

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

If serum separator tubes are not being used, allow samples to clotovernight 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.

Plasma

Collect plasma using EDTA or heparin as an anticoagulant. Centrifugesamples at 4°C for 15 mins at 1000 × g within 30 mins ofcollection. Collect the plasma fraction and assay promptly oraliquot and store the samples at -80°C. Avoid multiplefreeze-thaw cycles.Note: Over haemolysed samples are not suitable foruse with this kit.

Urine & Cerebrospinal Fluid

Collect the urine (mid-stream) in a sterile container, centrifugefor 20 mins at 2000-3000 rpm. Remove supernatant and assayimmediately. If any precipitation is detected, repeat thecentrifugation step. A similar protocol can be used forcerebrospinal fluid.

Cell culture supernatant

Collect the cell culture media by pipette, followed bycentrifugation at 4°C for 20 mins at 1500 rpm. Collect the clearsupernatant and assay immediately.

Cell lysates

Solubilize cells in lysis buffer and allow to sit on ice for 30minutes. Centrifuge tubes at 14,000 x g for 5 minutes to removeinsoluble material. Aliquot the supernatant into a new tube anddiscard the remaining whole cell extract. Quantify total proteinconcentration using a total protein assay. Assay immediately oraliquot and store at ≤ -20 °C.

Tissue homogenates

The preparation of tissue homogenates will vary depending upontissue type. Rinse tissue with 1X PBS to remove excess blood &homogenize in 20ml of 1X PBS (including protease inhibitors) andstore overnight at ≤ -20°C. Two freeze-thaw cycles arerequired to break the cell membranes. To further disrupt the cellmembranes you can sonicate the samples. Centrifuge homogenates for 5mins at 5000xg. Remove the supernatant and assay immediately oraliquot and store at -20°C or -80°C.

Tissue lysates

Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with atissue homogenizer in PBS. Add an equal volume of RIPA buffercontaining protease inhibitors and lyse tissues at room temperaturefor 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 Milk

Collect milk samples and centrifuge at 10,000 x g for 60 min at4°C. Aliquot the supernatant and assay. For long term use, storesamples at -80°C. Minimize freeze/thaw cycles.

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Additional Information

Product type:
ELISA
Reactivity:
Mouse
ELISA Type:
Sandwich
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