IgM ELISA Kit

Catalog No. ABIN455413

Product Details IgM ELISA Kit

Target: IgM

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 7.8-500 ng/mL

Minimum Detection Limit: 7.8 ng/mL

Application: ELISA

Purpose: This immunoassay kit allows for the in vitro quantitative determination of human IgM concentrations in cell culture supernates, serum, plasma and other biological fluids.

Sample Type: Cell Culture Supernatant, Plasma, Serum

Analytical Method: Quantitative

Specificity: This assay recognizes recombinant and natural human IgM.

Cross-Reactivity (Details): No significant cross-reactivity or interference was observed.

Sensitivity: < 0.06 μg/mL
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.

Characteristics: Immunoglobulin M,IgM

Components: Reagent (Quantity): Assay plate (1×20ml), Standard (2), Sample Diluent (1×20ml), Assay Diluent A (1×10ml), Assay Diluent B (1×10ml), Detection Reagent A (1×120 μl), Detection Reagent B (1×120μl), Wash Buffer(25 x concentrate) (1×30ml), Substrate (1×10ml), Stop Solution (1×10ml), Plate sealer for 96 wells (5), Instruction (1)

Material not included: Luminometer. Pipettes and pipette tips. EP tube Deionized or distilled water.

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol: The microtiter plate provided in this kit has been pre-coated with an antibody specific to IgM. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for IgM and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain IgM, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution 2 and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of IgM in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reagent Preparation:

Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 15 ug/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the high standard (15 ug/mL). The Sample Diluent serves as the zero standard (0 ug/mL). ug/mL 15 7.5 3.75 1.88 0.94 0.47 0.24 0 Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A and B (1:100), respectively.

Sample Collection: Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 × g. Remove serum and assay immediately or aliquot and store samples at -20C or -80C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g at 2 - 8C within 30 minutes of collection. Store samples at -20C or -80C. Avoid repeated freeze-thaw cycles. Cell culture supernates and other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20C or -80C. Avoid repeated freeze-thaw cycles. Sample preparation - Serum/plasma samples require about 100 fold dilution. Note: Serum, plasma, and cell culture supernatant samples to be used within 7 days may be stored at 2-8 C, otherwise samples must stored at -20C (≤ 1 months) or -80C (≤ 2 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature.

Assay Procedure:

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37C directly.). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at 4C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
1. Add 100 μl of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37C. 4
2. Remove the liquid of each well, don’t wash.
3. Add 100 μl of Detection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37C. Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (approximately 400 μl) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
5. Add 100 μl of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hours at 37C.
6. Repeat the aspiration/wash as in step
4. 7. Add 90 μl of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 30 minutes at 37C. Protect from light.
8. Add 50 μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well at once, using a microplate reader set to 450 nm.
Important Note:
1. Absorbance is a function of the incubation time. Therefore, prior to starting the assay it is recommended that all reagents should be freshly prepared prior to use and all required strip-wells are secured in the microtiter frame. This will ensure equal elapsed time for each pipetting step, without interruption.
2. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards can be used only once. This assay requires pipetting of small volumes. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10μl for once pipetting.
3. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay.
4. For each step in the procedure, total dispensing time for addition of reagents to the assay plate should not exceed 10 minutes.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
7. Duplication of all standards and specimens, although not required, is recommended. 5
8. Substrate Solution is easily contaminated. Please protect it from light.

Calculation of Results:

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the IgM concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 13.0. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Restrictions: For Research Use only

Handling

Handling Advice: 1. The kit should not be used beyond the expiration date on the kit label. 3
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

Storage: 4 °C/-20 °C

Storage Comment: The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.

Target Details for IgM

Target: IgM

Alternative Name: Immunoglobulin M (IgM Products)

Synonyms: IgM constant region ELISA Kit, IGM ELISA Kit

Target Type: Antibody

Background: Immunoglobulins belong to a large group of related glyco-proteins that make up approximately 20% of the serum proteins. The serum immunoglobulins react with antigens and confer immunity to individuals. All immunoglobulins share the basic structure of: 2 identical heavy chains joined by disulfide bonds to 2 identical light chains. Both the heavy (H) chains and the light (L) chains are divided into constant and variable regions. The constant regions have similar amino acid composition between all the immunoglobulin classes while the variable regions encompasses about 110 amino acids characterized by a high degree or sequence variability. Its H-chain type, based on the amino acid sequence, determines the classes of an immunoglobulin. There are 5 types of H-chains that correspond to the following immunoglobulin classes: IgG, IgA, IgM, IgD, and IgE. IgG is further subdivided into 4 subclasses with ~95% homology. There are 2 subclasses of IgA. IgG and IgA exists in serum as a monomer consisting of a single 4-polypeptide unit. IgM exists in serum as a pen tamer. IgA may also polymerize to form polymers containing 2-5 structural units. It is important to measure the level of immunoglobulins in serum for Antibody deficiency conditions, such as Primary hypogammagobulinaemia, or other immune deficiency diseases such as AIDS. When evaluating patients with recurrent infections, suspected immunodeficiency, allergic disease and many other conditions, it may be necessary to quantify the levels of immunoglobulins.