IgA ELISA Kit

Catalog No. ABIN455399

Product Details IgA ELISA Kit

Target: IgA

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.312-20 μg/mL

Minimum Detection Limit: 0.312 μg/mL

Application: ELISA

Purpose: This immunoassay kit allows for the in vitro quantitative determination of human IgA 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 IgA.

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

Sensitivity: < 0.078 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 A,IgA

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 (1×120 μl), Wash Buffer(25 x concentrate) (1×30ml), Substrate (1×10ml), Stop Solution (1×10ml), Plate sealer for 96 wells (5), 3 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 IgA. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for IgA 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 IgA, 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 and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of IgA 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 20 ug/mL. Allow the standard to sit for a minimum of 15 4 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 (20 ug/mL). The Sample Diluent serves as the zero standard (0 ug/mL). ug/mL 20 10 5 2.5 1.25 0.625 0.312 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 -20 C or -80 C . Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g at 2 - 8 C within 30 minutes of collection. Store samples at -20 C or -80 C . 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 -20 C or -80 C . 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 -20 C ( ≤ 1 months) or -80 C ( ≤ 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 37 C 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 4 C 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 μ l of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37 C .
2. Remove the liquid of each well, don ’ t wash.
3. Add 100 μ l of Detection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37 C . 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 μ l) 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 μ l of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hours at 37 C .
6. Repeat the aspiration/wash as in step
4. 7. Add 90 μ l of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 30 minutes at 37 C . Protect from light.
8. Add 50 μ l of Stop Solution to each well. If color change does not appear uniform, 5 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 10 μ l 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.
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 6 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 IgA 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.
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 IgA

Target: IgA

Alternative Name: Immunoglobulin A (IgA Products)

Synonyms: MB-1 ELISA Kit, IG-alpha ELISA Kit, IGA ELISA Kit, IgA ELISA Kit, Igh-2 ELISA Kit, CD79a molecule ELISA Kit, immunoglobulin heavy constant alpha ELISA Kit, CD79A ELISA Kit, Igha ELISA Kit

Target Type: Antibody

Background: Immunoglobulin A (IgA) is an antibody playing a critical role in mucosal immunity. More IgA is produced in mucosal linings than all other types of antibody combined. However, sources are correct when they indicate immunoglobulin G as the most common form of immunoglobulin in the human body. In its secretory form, is the main immunoglobulin found in mucous secretions, including tears, saliva, colostrum, intestinal juice, vaginal fluid and secretions from the prostate and respiratory epithelium. It is also found in small amounts in blood. Because it is resistant to degradation by enzymes, secretory IgA can survive in harsh environments such as the digestive and respiratory tracts, to provide protection against microbes that multiply in body secretions. IgA is a poor activator of the complement system, and opsonises only weakly. Its heavy chains are of the type alpha . IgA is found in secretions in a specific form called secretory IgA', polymers of 2-4 IgA monomers linked by two additional chains. One of these is the J chain (joining chain), which is a polypeptide of molecular mass 15kD, rich with cysteine and structurally completely different from other immunoglobulin chains. This chain is formed in the IgA-secreting cells. The oligomeric forms of IgA in the external (mucosal) secretions also contain a polypeptide of a much larger molecular mass (70 kD) called the secretory component that is produced by epithelial cells. This molecule originates from the poly-Ig receptor (130 kD) that is responsible for the uptake and transcellular transport of oligomeric (but not monomeric) IgA across the epithelial cells and into secretions such as tears, saliva, sweat and gut fluid. The high prevalence of IgA in mucosal areas is a result of a cooperation between plasma cells that produce polymeric IgA (pIgA), and mucosal epithelial cells that express an immunoglobulin receptor called the polymeric Ig receptor (pIgR). pIgA is released from the nearby activated plasma cells and binds to pIgR. This results in transportation of IgA 2 across mucosal epithelial cells and its cleavage from pIgR for release into external secretions. In the blood, IgA interacts with an Fc receptor called Fc alpha RI (or CD89), which is expressed on immune effector cells, to initiate inflammatory reactions. Ligation of Fc alpha RI by IgA containing immune complexes causes antibody-dependent cell-mediated cytotoxicity (ADCC), degranulation of eosinophils and basophils, phagocytosis by monocytes, macrophages, neutrophils and eosinophils, and triggering of respiratory burst activity by polymorphonuclear leukocytes. Polymeric IgA (mainly the secretory dimer) is produced by plasma cells in the lamina propria adjacent to mucosal surfaces. It binds to the polymeric immunoglobulin receptor on the basolateral surface of epithelial cells and is taken up into the cell via endocytosis. The receptor-IgA complex passes through the cellular compartments before being secreted on the luminal surface of the epithelial cells, still attached to the receptor. Proteolysis of the receptor occurs and the dimeric IgA molecule, along with a portion of the receptor known as the secretory component, are free to diffuse throughout the lumen. In the gut, it can bind to the mucus layer on top of the epithelial cells to form a barrier capable of neutralizing threats before they reach the cells.