IgD ELISA Kit

Catalog No. ABIN1440250

Product Details IgD ELISA Kit

Target: IgD

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Minimum Detection Limit: ~ 0.06 ng/mL

Application: ELISA

Purpose: The AssayMax Human IgD ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human IgD in plasma, serum, urine, saliva, milk, and cell culture supernatants. This assay employs a quantitative sandwich enzyme immunoassay technique that measures human IgD in less than 4 hours. A polyclonal antibody specific for human IgD has been pre-coated onto a 96-well microplate with removable strips. IgD in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for IgD, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Brand: AssayMax

Sample Type: Serum, Milk, Saliva, Urine, Plasma, Cell Culture Supernatant

Analytical Method: Quantitative

Cross-Reactivity (Details): Cross-Reactivity: Monkey <2%, Mouse <2%

Characteristics: Standard Added Value: 0.06- 1 ng/mL

Components: Human IgD Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human IgD.
Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay.
Human IgD Standard: Human IgD in a buffered protein base (8 ng, lyophilized).
Biotinylated IgD Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against IgD (140 µL).
EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 mL).
Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 mL, 2 bottles).
Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80 µL).
Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 mL).
Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 mL).

Material not included: Microplate reader capable of measuring absorbance at 450 nm.
Pipettes (1-20 µL, 20-200 µL, 200-1000 µL and multiple channel).
Deionized or distilled reagent grade water.

Application Details

Sample Volume: 50 μL

Assay Time: < 4 h

Plate: Pre-coated

Protocol: Add 50 µL of standard/samples per well. Incubate 2 hours. Wash, then add 50 µL of biotinylated antibody per well. Incubate 1 hour. Wash, then add 50 µL of SP per well. Incubate 30 minutes. Wash, then add 50 µL of Chromogen Substrate per well. Incubate 15 minutes. Add 50 µL of Stop Solution per well. Read at 450 nm immediately.

Reagent Preparation:

Freshly dilute all reagents and bring all reagents to room temperature before use.
EIA Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the EIA Diluent Concentrate 1:10 with reagent grade water. Store for up to 1 month at 2-8°C.
Standard Curve: Reconstitute the 8 ng of IgD Standard with 2 mL of EIA Diluent to generate a solution of 4 ng/mL. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting the standard solution (4 ng/mL) 1:2 with EIA Diluent to produce 2, 1, 0.5, 0.25, 0.125, and 0.063ng/mL solutions. EIA Diluent serves as the zero standard (0 4 ng/mL). Any remaining solution should be frozen at -20°C and use within 30 days.
Biotinylated IgD Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:50 with EIA Diluent. Any remaining solution should be frozen at -20°C.
Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 1:20 with reagent grade water.
SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with EIA Diluent. Any remaining solution should be frozen at -20°C.

Sample Preparation:

Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 3000 x g for 10 minutes. Dilute samples 1:100000 into EIA Diluent and assay. If necessary dilute samples within the range of 1:20000 to 1:200000. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. (EDTA or Heparin can also be used as an anticoagulant.)
Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 3000 x g for 10 minutes and remove serum. Dilute samples 1:100000 into EIA Diluent and assay. If necessary dilute samples within the range of 1:20000 to 1:200000. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles.
Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store the remaining samples at -20°C or below. Avoid repeated freeze-thaw cycles.
Urine: Collect urine using sample pot. Centrifuge samples at 800 x g for 10 minutes. Dilute samples 1:10 with EIA Diluent and assay. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze- thaw cycles.
Saliva: Collect saliva using sample tube. Centrifuge samples at 800 x g for 10 minutes. Dilute samples 1:100 with EIA Diluent and assay. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze- thaw cycles.
Milk: Collect milk using sample tube. Centrifuge samples at 800 x g for 10 minutes. Dilute samples 1:2000 with EIA Diluent and assay. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze- thaw cycles.

Assay Procedure:

Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20-30°C).
Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccants inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator.
Add 50 µL of IgD Standard or sample per well. Cover wells with a sealing tape and incubate for 2 hours. Start the timer after the last sample addition.
Wash five times with 200 µL of Wash Buffer manually. Invert the plate each time and decant the contents, hit 4-5 times on absorbent material to completely remove the liquid. If using a machine, wash six times with 300 µL of Wash Buffer and then invert the plate, decanting the contents, hit 4-5 times on absorbent material to completely remove the liquid.
Add 50 µL of Biotinylated IgD Antibody to each well and incubate for 1 hour.
Wash the microplate as described above.
Add 50 µL of Streptavidin-Peroxidase Conjugate to each well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance.
Wash the microplate as described above.
Add 50 µL of Chromogen Substrate per well and incubate for about 15 minutes or till the optimal blue color density develops. Gently tap plate to ensure thorough mixing and break the bubbles in the well with pipette tip.
Add 50 µL of Stop Solution to each well. The color will change from blue to yellow.
Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings.

Calculation of Results:

Calculate the mean value of the duplicate or triplicate readings for each standard and sample.
To generate a standard curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. The best-fit line can be determined by regression analysis using four-parameter or log-log logistic curve-fit.
Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor.

Assay Precision: Intra-assay and inter-assay coefficients of variation were 4.8% and 7.1% respectively.

Restrictions: For Research Use only

Handling

Handling Advice: Prepare all reagents (working diluent buffer, wash buffer, standards, biotinylated antibody, and SP conjugate) as instructed, prior to running the assay.
Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this protocol. However, the user should determine the optimal dilution factor.
Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents.
The kit should not be used beyond the expiration date.
The Stop Solution is an acidic solution.

Storage: 4 °C/-20 °C

Storage Comment: Store components of the kit at 2-8°C or -20°C upon arrival up to the expiration date.
Store SP Conjugate and biotinylated antibody at -20°C.
Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C.
Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed. May be stored for up to 1 month in a vacuum desiccator.
Diluent (1x) may be stored for up to 1 month at 2-8°C.
Store standard at 2-8°C before reconstituting with diluent and at -20°C after reconstituting with diluent.

Target Details for IgD

Target: IgD

Abstract: IgD Products

Synonyms: IgD ELISA Kit, Igh-5 ELISA Kit, immunoglobulin heavy constant delta ELISA Kit, immunoglobulin delta heavy chain constant region ELISA Kit, Ighd ELISA Kit, Igd ELISA Kit, IGHD ELISA Kit

Target Type: Antibody

Background: Immunoglobulin D (IgD) is an antibody of the immunoglobulin isotype that is a monomer made up two identical heavy and light chains organized into variable and constant domains similar to IgG. It has soluble and transmembrane forms and is found as an antigen receptor on the surface of B cells. IgD is an important immunomodulatory molecule that promotes immune defense. Overactivation of this pathway can cause inflammation and tissue damage. Serum IgD was increased in patients with AIDS, tuberculosis, infectious hepatitis, and respiratory diseases.