Amyloid beta 1-42 (Abeta 1-42) ELISA Kit

Antigen: Amyloid beta 1-42 (Abeta 1-42)

Reactivity: Human

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN365017

Additional Information

Characteristics: This immunoassay kit allows for the in vitro quantitative determination of human A1-42 concentrations in cell culture supernates, serum, plasma and other biological fluids.

Alternative name: Amyloid beta peptide 1-42 (Abeta1-42)

Sample Type: Serum, Plasma

Description: One of the hallmarks of Alzheimer's disease is the self-aggregation of the amyloid peptide (A) in extracellular amyloid fibrils. A is a peptide composed of 40 to 42 (43) amino acids, and is said to be cleaved out of the precursor protein APP (a protein composed of 695, 751, or 770 amino acids) by the action of - or ?-secretase.In addition, the presence of numerous variant A molecules has been demonstrated in the culture fluid of mouse neuroblastoma cells transfected with cDNA coding human amyloid precursor protein (APP). Among the different forms of A, the 42-residue fragment (A1-42) readily self-associates and forms nucleation centers from where fibrils can quickly grow. The strong tendency of A1-42 to aggregate is one of the reasons for the scarcity of data on its fibril formation process. A1-40 is the most common form secreted from cultured cells and found in cerebro-spinal fluid (CSF). A1-42 is the major component of senile plaques and considered as the most crucial factor in AD pathogenesis.

Specificity: This assay recognizes recombinant and natural human A1-42. No significant cross-reactivity or interference was observed.

Application Details

Principle: The microtiter plate provided in this kit has been pre-coated with an antibody specific to A1-42. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for A1-42 and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3'5, 5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain A1-42, 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 A1-42 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protocol: Preparation of Reagents: 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 500 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 350 pg/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 (350 pg/mL). The Sample Diluent serves as the zero standard (0 pg/mL). Biotin-antibody - Dilute to the working concentration specified on the vial label using Biotin-antibody Diluent(1:100), respectively. HRP-avidin - Dilute to the working concentration specified on the vial label using HRP-avidin Diluent(1:100), respectively. Sample Collection and Storage: 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 1000 x g. Remove serum and assay immediately or aliquot and store samples at _ -20 C. Avoid repeated freeze-thaw cycles. Plasma - Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at _ -20 C. Avoid repeated freeze-thaw cycles. Assay Procedure: 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 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 A1-42 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. Limitations of the Procedure: The kit should not be used beyond the expiration date on the kit label. Do not mix or substitute reagents with those from other lots or sources. It is important that the Calibrator Diluent selected for the standard curve be consistent with the samples being assayed. If samples generate values higher than the highest standard, dilute the samples with the appropriate Calibrator 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. This assay is designed to eliminate interference by soluble receptors, 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.

Assay Procedure: Assay Time: 1-3h
Sample Volume: 50-100ul

Application Notes: Detection Wavelength: 450 nm

Components: Reagent Quantity Assay plate 1 Standard 2 Sample Diluent 1 x 20ml Biotin-antibody Diluent 1 x 10ml HRP-avidin Diluent 1 x 10ml Biotin-antibody 1 x 120ul HRP-avidin 1 x 120ul Wash Buffer 1 x 30ml (25 x concentrate) TMB Substrate 1 x 10ml Stop Solution 1 x 10ml STORAGE Unopened test kits should be stored at 2-8°Cupon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit . Refer to the package label for the expiration date. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Material not included: Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. Pipettes and pipette tips. Deionized or distilled water. Squirt bottle, manifold dispenser, or automated microplate washer.

Storage: Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit . Refer to the package label for the expiration date. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Research Area: Alzheimer's Disease

Restrictions: For Research Use only

Publications

Publications: Liang, Yang, Yin et al.: "Estrogen stimulates degradation of beta-amyloid peptide by up-regulating neprilysin." in: The Journal of biological chemistry, Vol. 285, Issue 2, pp. 935-42, 2010 (PubMed).

Liu, Li, Qiu et al.: "The inhibitory effects of different curcuminoids on β-amyloid protein, β-amyloid precursor protein and β-site amyloid precursor protein cleaving enzyme 1 in swAPP HEK293 cells." in: Neuroscience letters, 2010 (PubMed).