INHBA Kit ELISA

N° du produit ABIN578861

Détail du produit INHBA Kit ELISA

Antigène: INHBA (Inhibin, beta A (INHBA))

Reactivité: Rat

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Gamme de detection: 15.6-1000 pg/mL

Seuil minimal de détection: 15.6 pg/mL

Application: ELISA

Fonction: This immunoassay kit allows for the in vitro quantitative determination of rat inhibin B, INH-B concentrations in cell culture supernates, serum, plasma and other biological fluids.

Type d'échantillon: Cell Culture Supernatant, Plasma, Serum

Analytical Method: Quantitative

Specificité: This assay recognizes recombinant and natural rat INH-B.

Réactivité croisée (Details): No significant cross-reactivity or interference was observed.

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

Attributs du produit: Rattus norvegicus,Rat,Inhibin beta A chain,Activin beta-A chain,Inhba

Ingrédients: Reagent (Quantity ): Assay plate (1×20ml), Standard (2), Sample Diluent (1 × 20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1 × 120μl), Detection Reagent B (1 × 120μl), Wash Buffer (25 x concentrate) (1 × 30ml), Substrate (1x10ml), Stop Solution (1x10ml), Plate sealer for 96 wells (5), Instructions (1)

Matériel non inclus: Luminometer. Pipettes and pipette tips. EP tube Deionized or distilled water.

Information d'application

Volume d'échantillon: 100 μL

Plaque: Pre-coated

Protocole: The microtiter plate provided in this kit has been pre-coated with an antibody specific to INH-B. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for INH-B 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 INH-B, 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 INH-B in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Préparation des réactifs:

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 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 80 U/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (80 U/mL). The Sample Diluent serves as the zero standard (0 U/mL). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.

Prélèvement de l'échantillon: Cell culture supernates - Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, heart and lung tissue from one rabbit was rinsed with 1X PBS to remove excess blood, homogenized in 20 mL of 1X PBS and stored overnight at ≤ -20 °C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 x g. Remove the supernate and assay immediately or aliquot and store at ≤ -20 °C. Note: Citrate plasma has not been validated for use in this assay.

Procédure de l'essai:

Allow all reagents to reach room temperature. Arrange and label required number of strips.
1. Prepare all reagents, working standards and samples as directed in the previous sections.
2. Add 100 uL of Standard, Control, or sample* per well. Cover with the adhesive strip. Incubate for 2 hours at 37 °C.
3. Remove the liquid of each well, don’t wash.
4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) 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.
6. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37 °C.
7. Repeat the aspiration/wash as in step
5. 8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Important Note:1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
2. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
3. Duplication of all standards and specimens, although not required, is recommended.
4. When mixing or reconstituting protein solutions, always avoid foaming.
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. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.

Calcul des résultats:

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 y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the iNOS concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. 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

Stockage

Conseil sur la manipulation: 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.
3.

Stock: 4 °C/-20 °C

Stockage commentaire: 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.

Détail du antigène

Antigène: INHBA (Inhibin, beta A (INHBA))

Autre désignation: Inhba (INHBA Produits)

Synonymes: EDF Kit ELISA, FRP Kit ELISA, actba Kit ELISA, inhba Kit ELISA, inhbaa-001 Kit ELISA, edf Kit ELISA, frp Kit ELISA, INHB Kit ELISA, inhbal Kit ELISA, zgc:158348 Kit ELISA, inhibin beta A subunit Kit ELISA, inhibin beta-A Kit ELISA, inhibin, beta Aa Kit ELISA, inhibin beta A Kit ELISA, inhibin, beta Ab Kit ELISA, INHBA Kit ELISA, Inhba Kit ELISA, inhbaa Kit ELISA, inhba Kit ELISA, inhbab Kit ELISA

Sujet: Inhibin is a peptide that is an inhibitor of FSH synthesis and secretion, and participates in the regulation of the menstrual cycle. Inhibin contains an alpha and beta subunit linked by disulfide bonds. Two forms of inhibin differ in their beta subunits (A or B), while their alpha subunits are identical. Inhibin belongs to the transforming growth factor- beta (TGF- beta ) superfamily. In women, FSH stimulates the secretion of inhibin from the granulosa cells of the ovarian follicles in ovary. In turn, inhibin suppresses FSH. Inhibin secretion is diminished by GnRH, and enhanced by insulin-like growth factor-1 (IGF-1). Inhibin B reaches a peak in the early- to mid-follicular phase, and a second peak at ovulation. Inhibin A reaches its peak in the mid-luteal phase. Inhibin is produced in the gonads, pituitary gland, placenta and other organs. In men, it is a hormone that inhibits FSH production. It is secreted from the Sertoli cells, located in the seminiferous tubule inside the testes.

ID gène: 3074

Pathways: Hormone Transport, Peptide Hormone Metabolism, Hormone Activity, Negative Regulation of Hormone Secretion, Autophagy