ELISA/assay

Mouse IgE ELISA Kit

DEIA652

96T

Mouse IgE ELISA Kit

IgE

Mouse IgE ELISA Kit

sELISA

IgE

Mouse

Quantitative

Serum, Plasma

96T

Stored at 2-8°C for 6 months from date of receipt.

By immunoelectrophoresis and ELISA the antibodies in this kit react specifically with Mouse IgE, not with other mouse immunoglobulins or other mouse serum proteins. Cross-reactivity with other species has not been tested.

This enzyme linked immunosorbent assay (ELISA) is for the detection of Mouse IgE in serum or plasma. Due to idiotypic and allotypic variations, this product may not accurately quantitate hybridomas, monoclonals or myelomas.

There are five classes of mammalian immunoglobulins: IgA, IgD, IgE, IgG, and IgM. In mice, the IgG class is further divided into four subclasses: IgG1, IgG2a/ IgG2c (strain specific), IgG2b, and IgG3.The general immunoglobulin structure is described as two heavy and two light chain (H2L2) polypeptides linked together by disulfide bridges of cysteine residues. Mouse IgE, also known as reaginic antibody, exists in serum as a 4-chain monomer of 185 – 200 kDa. Like IgM, the IgE monomers contain a fourth constant region domain. Serum concentrations of IgE are reported to be > 10 ug/ml. IgE is cyolytic for mast cells, where it binds to the FcεRI receptor and is able to mediate the response of granular and lipid mediators of inflammation, including the PCA reaction. The IgE class switch is induced by IL-4 as part of the Th2 response. (Quimby & Luong Richard, 2007).

This kit is based on a sandwich ELISA. Mouse IgE present in the test sample is captured by anti-Mouse IgE antibody that has been pre-adsorbed on the surface of microtiter wells. After sample binding, unbound proteins and molecules are washed off, and a biotinylated detection antibody is added to the wells to bind to the captured IgE. A strepavidin-conjugated horseradish peroxidase (SA-HRP) is then added to catalyze a colorimetric reaction with the chromogenic substrate TMB (3,3',5,5'-tetramethylbenzidine). The colorimetric reaction produces a blue product, which turns yellow when the reaction is terminated by addition of dilute sulfuric acid. The absorbance of the yellow product at 450 nm is proportional to the amount of IgE analyte present in the sample and a fourparameter standard curve can be generated. The IgE concentrations in the test samples can then be quantified by interpolating their absorbance from the standard curve generated in parallel with the samples. After factoring sample dilutions, the IgE concentrations in the original sample can finally be calculated.

1. Mouse IgE Pre-Coated 96-well Strip Plate, 1 each 2. Mouse IgE Standard, 1000 ng/vial, 2 each 3. Mouse IgE Detection Antibody, 12 ml 4. 10× Dilution Buffer B, 25 ml 5. HRP Solution A, 12 ml 6. TMB Substrate, 12 ml 7. Stop Solution, 12 ml 8. 20× Wash Buffer, 50 ml 9. Sealing Tape, 6 sheets

1. Ultrapure water 2. Precision pipettors, with disposable plastic tips 3. Polypropylene or polyethylene tubes to prepare standard and samples. Do not use polystyrene, polycarbonate or glass tubes. 4. A container to prepare 1× Dilution Buffer B 5. A container to prepare 1× Wash Buffer 6. A 96-well plate washer or a wash bottle. An autoclavable plate washer is recommended, if available. 7. Disposable reagent reservoirs 8. A standard microtiter plate reader for measuring absorbance at 450 nm for end-point ELISA.

Sample Handling 1. This ELISA assay can be used for mouse serum or plasma. 2. All blood components and biological materials should be handled as potentially hazardous. Follow universal precautions when handling and disposing of infectious agents. 3. 100 μl of sample or standard is required per well. 4. Samples must be assayed in duplicate each time the assay is performed. 5. Store samples to be assayed within 24 hours at 2-8°C. For long-term storage, aliquot and freeze samples at -70°C. Avoid repeated freeze-thaw cycles when storing samples. 6. If particulates are present in samples, centrifuge prior to analysis. 7. If blood samples are clotted, grossly hemolyzed, lipemic, or the integrity of the sample is of concern, make a note on the Plate Template and interpret results with caution. Sample Preparation 1. The dilution schemes indicated below are only suggestions. Dilutions should be based on the expected concentration of the unknown sample such that the diluted sample falls within the dynamic range of the standard curve. Prepare one or more dilutions of the sample in 1× Dilution Buffer B using the same serial dilution technique described below until the desired concentration is obtained. 2. Serum and plasma – Recommended starting dilution is 1:20. Dilute by adding 20 μl of serum or plasma into 380 μl of 1× Dilution Buffer B to give a 1:20 dilution.

1. Preparation of 1× Dilution Buffer B Prepare 1× Dilution Buffer B by diluting 25 ml of 10× Dilution Buffer B into 225 ml of ultra pure water. Mix well. Store reconstituted 1× Dilution Buffer B at 2-8°C for up to six (6) months. Do not use 1× Dilution Buffer B if it becomes visibly contaminated during storage. 2. Preparation of Standard Solutions Standard solutions should be treated as a biological material and universal precautions should be followed. 1) Reconstitute the 1000-ng IgE standard vial with 1.0 ml of 1× Dilution Buffer B to achieve a final concentration of 1000 ng/ml. Mix well. 2) Label seven (7) tubes, one for each standard curve point: 333, 111, 37, 12.3, 4.1, 1.37, and 0 ng/ml. The original vial of re-suspended standard solution represents the top standard at 1000 ng/ml. 3) Add 300 μl of 1× Dilution Buffer B into each of the seven tubes. 4) Serially dilute 1:3 by adding 150 μl of the 1000 ng/ml standard into the first tube containing 300 μl of 1× Dilution Buffer B. Mix well. Continue the dilution by adding 150 μl of the previous standard into 300 μl of 1× Dilution Buffer B in the next tube until the sixth tube (1.37 ng/ml). 5) The seventh tube containing 300 μl of 1× Dilution Buffer B serves as the zero standard value or blank. Using this dilution scheme, only 350 μl of the re-suspended standard solution should be used. The remaining standard solution can be used for another assay within the same day. Otherwise, use the second vial of lyophilized standard. 3. Preparation of 1× Wash Buffer Prepare 1× Wash Buffer by diluting 20× Wash Buffer in ultrapure water. For example, if preparing 1 L of 1× Wash Buffer, dilute 50 ml of 20× Wash Buffer into 950 ml of ultrapure water. Mix well. Store reconstituted 1× Wash Buffer at 2-8°C for up to six (6) months. Do not use 1× Wash Buffer if it becomes visibly contaminated during storage.

Sample Incubation Determine the number of strips required. Leave these strips in the plate frame. Place unused strips in the foil pouch with desiccant and seal tightly. Store unused strips at 2-8°C. After completing assay, keep the plate frame for additional assays. Use a Plate Template to record the locations of the standards and unknown samples within the wells. 1. Add 100 μl of appropriately diluted standards or samples to each well. Run each standard, sample, or blank in duplicate. Note: Serum or plasma samples must be diluted prior to testing (see Specimen Collection And Preparation section above). 2. Carefully cover the wells with a new adhesive plate cover and incubate for one (1) hour at room temperature, 20-25°C. 3. Carefully remove the adhesive plate cover and wash FOUR times with 1× Wash Buffer, as described in the Plate Washing section below. Plate Washing Using a manual or automated plate washer 1. Rinse the tips of the plate washer by dispensing the 1× Wash Buffer into the wash trough and aspirating the solution. Repeat this step five times. For automated plate washers, program the rinse step accordingly. Note: This initial rinse step is necessary especially if the plate washer has been idle for several days or longer. Automated plate washers are susceptible to microbial growth in the fluid lines and cavities. 2. Aspirate the solutions from the wells. Fill the wells to about 90% full with 1× Wash Buffer and then aspirate the wash solution. Repeat this wash step three more times. For automated plate washers, program 4 washes at 300 μl per wash, according to the manufacturer's instructions. Using a squirt bottle 1. Gently squeeze the long sides of plate frame before washing to ensure all strips remain securely in the frame. 2. Empty the plate contents by quickly dumping the contents of the wells into the sink using a quick, flipping motion. 3. Use a squirt wash bottle to fill each well completely with 1× Wash Buffer, and then empty the plate contents. Repeat procedure three more times for a total of FOUR washes. Blot the plate onto paper towels or other absorbent material. Incubation with Detection Antibody Only remove the required amount of Detection Antibody reagent for the number of strips being used. 1. Add 100 μl of Detection Antibody to each well containing standard, sample or blank. Mix well by gently tapping the plate several times. 2. Carefully attach a new adhesive plate cover and incubate the plate for one hour at room temperature, 20-25°C. 3. Carefully remove the adhesive plate cover, discard plate contents and wash FOUR times with 1× Wash Buffer (see Plate Washing section above). Incubation with HRP Solution A Remove only the required amount of HRP Solution A for the number of strips being used. 1. Add 100 μl of HRP Solution A to each well containing standard, sample or blank. 2. Carefully attach a new adhesive plate cover and incubate plate for 30 minutes at room temperature, 20-25°C. 3. Carefully remove the adhesive plate cover, discard plate contents and wash FOUR times with 1× Wash Buffer (see Plate Washing section). Blot off any residual liquid at the bottom of the wells that might interfere later with the absorbance readings. Incubation with TMB Substrate and Stopping the Reaction Remove only the required amount of TMB Substrate Solution and Stop Solution for the number of strips being used. Do NOT use glass pipette to measure the TMB Substrate Solution. Do NOT cover the plate with aluminum foil or metalized mylar. Do NOT return leftover TMB Substrate to bottle. Do NOT contaminate the unused TMB Substrate Solution. If the solution is blue before use, DO NOT USE IT! 1. Add 100 μl of TMB Substrate Solution into each well and allow the enzymatic reaction to develop a blue color at room temperature (20-25°C) in the dark for 30 minutes. Do NOT cover plate with a plate sealer. 2. Stop the reaction by adding 100 μl of Stop Solution to each well. Tap plate gently to mix. The solution in the wells should immediately change in color from blue to yellow. Absorbance Measurement Note: Wipe the underside of the wells with a lint-free tissue. 1. For end-point ELISA, measure the absorbance on an ELISA plate reader set at 450 nm within 30 minutes after the addition of the Stop Solution.

Duplicate absorbance values should be within 10% of each other. Care should be taken when interpreting data with differences in absorbance values greater than 10%. 1. Prepare a standard curve to determine the amount of IgE in an unknown sample. Plot the average absorbance obtained for each standard concentration on the vertical (Y) axis versus the corresponding IgE concentration on the horizontal (X) axis using curve-fitting software. 2. Calculate the IgE concentration in unknown samples using the prepared standard curve. Determine the amount of IgE in each unknown sample by noting the IgE concentration (X axis) that correlates with the absorbance value (Y axis) obtained for the unknown sample. 3. If the sample was diluted, multiply the observed IgE concentration by the dilution factor to determine the concentration of IgE in the original, undiluted sample. Note: Most plate readers come with appropriate templates and curve-fitting software. The standard curve of this assay can be fitted into a 4-parameter curve fitting equation that can be programmed to calculate and display a table (or tables) consisting of the raw absorbance readings, net absorbance readings, the analyte concentration in the assay solution, dilution factors, and analyte concentration in the original unknown sample.

This typical standard curve was generated using the Mouse IgE ELISA Kit Protocol. This standard curve is for demonstration only. A standard curve must be generated for each assay. *Suggested standard curve points are 1000 ng/ml, 333, 111, 37, 12.3, 4.1 1.37, and 0 ng/ml. Assay Range: 1.37 – 1000 ng/ml

1. Store all reagents at 2-8°C. Do not freeze reagents. 2. All reagents must be at room temperature (20-25°C) before use. 3. Vigorous plate washing is essential. 4. Use new disposable pipette tips for each transfer to avoid cross-contamination. 5. Use a new adhesive plate cover for each incubation step. 6. Minimize lag time between wash steps to ensure the plate does not become completely dry during the assay. 7. Avoid microbial contamination of reagents and equipment. Automated plate washers can easily become contaminated and result in erroneous data. 8. Do not contaminate the TMB Solution. Do not expose TMB Substrate solution to glass, foil, or metal. If the solution is blue before use, DO NOT USE IT. 9. Individual components may contain preservatives. Wear gloves while performing the assay and follow proper disposal procedures.

This enzyme linked immunosorbent assay (ELISA) is for the detection of Mouse IgE in serum or plasma. Due to idiotypic and allotypic variations, this product may not accurately quantitate hybridomas, monoclonals or myelomas.

1. Mouse IgE Pre-Coated 96-well Strip Plate, 1 each 2. Mouse IgE Standard, 1000 ng/vial, 2 each 3. Mouse IgE Detection Antibody, 12 ml 4. 10× Dilution Buffer B, 25 ml 5. HRP Solution A, 12 ml 6. TMB Substrate, 12 ml 7. Stop Solution, 12 ml 8. 20× Wash Buffer, 50 ml 9. Sealing Tape, 6 sheets

This typical standard curve was generated using the Mouse IgE ELISA Kit Protocol. This standard curve is for demonstration only. A standard curve must be generated for each assay. *Suggested standard curve points are 1000 ng/ml, 333, 111, 37, 12.3, 4.1 1.37, and 0 ng/ml. Assay Range: 1.37 – 1000 ng/ml

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet