Hemoglobin Subunit beta ELISA Kit

Catalog No. ABIN579148

Product Details Hemoglobin Subunit beta ELISA Kit

Target: Hemoglobin Subunit beta (HBB) (Hemoglobin beta (HBB))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.78-50 ng/mL

Minimum Detection Limit: 0.78 ng/mL

Application: ELISA

Purpose: This immunoassay kit allows for the specific measurement of human HB-beta concentrations in serum and plasma.

Sample Type: Serum, Plasma

Analytical Method: Quantitative

Specificity: This assay recognizes recombinant and natural human HB-beta.

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

Characteristics: Homo sapiens,Human,Hemoglobin subunit beta,Beta-globin,Hemoglobin beta chain,HBB

Components: Reagent (Quantity ): Assay plate (1), Standard 2 Sample Diluent (1 × 20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A 1×120μl Detection Reagent B 1×120μl 2 Wash Buffer (25 x concentrate) (1 × 30ml), Substrate (1x10ml), Stop Solution (1 x 10ml)

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol: This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for HB-beta has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any HB-beta present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for HB-beta is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of HB-beta bound in the initial step. The color development is stopped and the intensity of the color is measured.

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 1,000 pg/mL. Allow the standard to sit for about 10 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 highest standard (1,000 pg/mL). The Sample Diluent serves as the zero standard (0 pg/mL). pg/mL 1,000 500 250 125 62.5 31.2 15.6 0 Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A or B (1:100), respectively.

Sample Collection: Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 20 minutes at approximately 1000 g. Remove serum and assay immediately or aliquot and store samples at -20 or -80 . Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 g at 2 - 8 within 30 minutes of collection. Store samples at -20 or -80 . Avoid repeated freeze-thaw cycles. Other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20 or -80 . Avoid repeated freeze-thaw cycles. Note: Serum and plasma to be used within 7 days may be stored at 2-8, otherwise samples must stored at -20 ( 1 month) or -80 ( 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 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 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 of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for two hours at 37 .
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 . 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, 4 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 one hour at 37 .
6. Repeat the aspiration/wash process for five times as conducted in step
4. 7. Add 90 μ l of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 30 minutes at 37 . Protect from light.
8. Add 50 μ l 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 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 samples 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 PCT 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 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.
5. Limited by the current condition and scientific technology, we can't completely conduct 3 the comprehensive identification and analysis on the raw material provided by suppliers. So there might be some qualitative and technical risks to use the kit.

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 Hemoglobin Subunit beta

Target: Hemoglobin Subunit beta (HBB) (Hemoglobin beta (HBB))

Alternative Name: HBB (HBB Products)

Synonyms: HBB ELISA Kit, HBB1 ELISA Kit, HBBA ELISA Kit, HBBB ELISA Kit, CD113t-C ELISA Kit, beta-globin ELISA Kit, hbb ELISA Kit, hemoglobin, beta ELISA Kit, hemoglobin subunit beta ELISA Kit, hemoglobin subunit beta-like ELISA Kit, beta globin ELISA Kit, hemoglobin beta chain complex ELISA Kit, hemoglobin subunit beta-1 ELISA Kit, HBB ELISA Kit, HBB2 ELISA Kit, LOC100720944 ELISA Kit, LOC480784 ELISA Kit, Hbb ELISA Kit, LOC100136576 ELISA Kit, LOC101098159 ELISA Kit, LOC100976465 ELISA Kit, LOC100410611 ELISA Kit, LOC105468624 ELISA Kit

Target Type: Viral Protein

Background: In adult humans, the most common hemoglobin type is a tetramer (which contains 4 subunit proteins) called hemoglobin A, consisting of two alpha and two beta subunits non-covalently bound, each made of 141 and 146 amino acid residues, respectively. This is denoted as alpha2beta2. The subunits are structurally similar and about the same size. Each subunit has a molecular weight of about 17,000 daltons, for a total molecular weight of the tetramer of about 68,000 daltons. Hemoglobin A is the most intensively studied of the hemoglobin molecules. The four polypeptide chains are bound to each other by salt bridges, hydrogen bonds, and hydrophobic interactions. There are two kinds of contacts between the alpha and beta chains: alpha1beta1 and alpha1beta2.

Gene ID: 3859