S100B ELISA Kit

Catalog No. ABIN456467

Product Details S100B ELISA Kit

Target: S100B (S100 Calcium Binding Protein B (S100B))

Binding Specificity: Soluble

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 15.6-1000 pg/mL

Minimum Detection Limit: 15.6 pg/mL

Application: ELISA

Purpose: This immunoassay kit allows for the specific measurement of human Soluble protein-100B, S-100B concentrations in cell culture supernates, tissue homogenates, serum, and plasma.

Sample Type: Cell Culture Supernatant, Tissue Homogenate, Serum, Plasma

Analytical Method: Quantitative

Specificity: This assay recognizes recombinant and natural human S-100B.

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

Characteristics: Homo sapiens,Human,Protein S100-B,S-100 protein beta chain,S-100 protein subunit beta,S100 calcium-binding protein B,S100B

Components: Reagent (Quantity):

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

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

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol: This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for S-100B has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any S-100B present is bound by the immobilized antibody. An biotinylated polyclonal antibody specific for S-100B is added to the wells. Following a wash to remove any unbound reagent, an enzyme complex is added to the wells.After incubation and washing ,a substrate solution is added to the wells and color develops in proportion to the amount of S-100B 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. 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. The Sample Diluent serves as the zero standard (0 ng/ml).

Sample Collection: 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 human 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.

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 μL 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 μL 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 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 37 °C .
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 37 °C . 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 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 5 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 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 SAA 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 Assay Plate, Standard, Detection Reagent A and Detection Reagent B should be stored at -20°C upon being received. After receiving the kit , Substrate should be always stored at 4°C.

References for S100B ELISA Kit (ABIN456467)

Li, Xia, Zhang, Wu: "S100B protein, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor in human milk." in: PLoS ONE, Vol. 6, Issue 6, pp. e21663, (2011) (PubMed).

Target Details for S100B

Target: S100B (S100 Calcium Binding Protein B (S100B))

Alternative Name: S100B (S100B Products)

Synonyms: NEF ELISA Kit, S100 ELISA Kit, S100-B ELISA Kit, S100beta ELISA Kit, S100P ELISA Kit, AI850290 ELISA Kit, Bpb ELISA Kit, si:dkey-110p14.2 ELISA Kit, wu:fq18b10 ELISA Kit, zgc:92776 ELISA Kit, S100 calcium binding protein B ELISA Kit, S100 protein, beta polypeptide, neural ELISA Kit, S100 calcium binding protein, beta (neural) ELISA Kit, S100B ELISA Kit, S100b ELISA Kit, s100b ELISA Kit

Background: S-100B is a member of highly homologous Ca2+ binding proteins family that possess two EF-hand motifs. The family of S100 proteins consists of 19 members. Most S100 proteins exist as dimers (frequently homodimers) within cells. Exclusively expressed in vertebrates, S100 is implicated in various intracellular and extracellular regulatory activities. Studies indicate that S100 proteins are involved in the inhibition of protein phosphorylation, inhibition of cytosceletal constituent assembly, regulation of Ca2+ homeostasis, stimulation of enzyme activities, and interaction with transcription factors. S100B is abundant in the nervous system where it is predominantly expressed in astrocytes, oligodendrocytes and Schwann cells. When secreted by astrocytes, S100B has neurotrophic effects during development and nerve regeneration at physiologic (nanomolar) concentrations. However high (micromolar) concentrations of S100B have shown to be neurotoxic, participating in the physiology of neurodegenerative disorders. The clinical values have been demonstrated in stroke, cerebral complications association with cardiac arrest and in patients with severe as well as minor head injury.

Pathways: Regulation of Muscle Cell Differentiation, Positive Regulation of Immune Effector Process, Toll-Like Receptors Cascades, Regulation of long-term Neuronal Synaptic Plasticity, S100 Proteins