Alkaline Phosphatase, Bone ELISA Kit

Antigen: Alkaline Phosphatase, Bone

Synonyms: HOPS, TNAP, TNSALP, AP-TNAP, FLJ40094, FLJ93059, MGC161443, MGC167935, Akp2, Akp-2, PHOA, MGC93545, ALPL, TNS-AP, MGC139479, alpl, MGC52917, MGC130818, ALP, iap

Reactivity: Human

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN366901

Additional Information

Characteristics: This immunoassay kit allows for the in vitro quantitative determination of human BALP concentrations in serum, plasma and other biological fluids.

Alternative name: bone alkaline phosphatase (BALP)

Sample Type: Serum, Plasma

Description: Alkaline phosphatase (ALP) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. ALP is made in the liver, bone, and the placenta and normally present in high concentrations in growing bone and in bile. The enzyme is termed alkaline phosphatase because it works under alkaline (non-acidic) conditions, as opposed to acid phosphatase. ALP is released into the blood during injury and during such normal activities as bone growth and pregnancy. It is measured in a routine blood test. Abnormally high blood levels of ALP may indicate disease in bone or liver, bile duct obstruction, or certain malignancies. The enzyme is often elevated in the leukemic cells in chronic myelogenous leukemia. Abnormally, low levels of ALP is a genetic condition called hypophosphatasia which results in bone deformities. The major sources of the ALP in the plasma are bone and liver and the total concentration represents the sum of the activity. The measurement of the serum bone specific alkaline phosphatase (BALP) level may be a more accurate index of osteoblastic activity. 3

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

Sensitivity: The minimum detectable dose of human BALP is typically less than 0 .78 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. Detection Range: The minimum detectable dose of human BALP is typically less than 0 .78 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.

Application Details

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

Protocol: Reagent Preparation:
Bring all reagents to room temperature before use. 1. Wash Buffer If crystals have formed in the concentrate, warm up 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. 5 2. Standard Centrifuge the standard vial at 6000-10000rpm for 30s. Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 200 ng/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 (200 ng/ml). The Sample Diluent serves as the zero standard (0 ng/ml). Prepare fresh for each assay. Use within 4 hours and discard after use. 3. Biotin-antibody Centrifuge the vial before opening. Dilute to the working concentration using Biotin-antibody Diluent(1:100), respectively. 4. HRP-avidin Centrifuge the vial before opening. Dilute to the working concentration using HRP-avidin Diluent(1:100), respectively. Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
Assay Procedure:
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate. All the reagents should be added directly to the liquid level in the well. The pipette should avoid contacting the inner wall of the well. 1. Add 100μl of Standard, Blank, or Sample per well. Cover with the adhesive strip. Incubate for 2 hours at 37°C. 2. Remove the liquid of ea ch well, don’t wash. 3. Add 100μl of Biotin-antibody working solution to each well. Incubate for 1 hour at 37°C. Biotin-antibody working solution may appear cloudy. Warm up 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: Fill each well with Wash Buffer (200μl) and let it stand for 2 minutes, then remove the liquid by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel. Complete removal of liquid at each step is essential to good performance. 8 5. Add 100μl of HRP-avidin working solution to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 hour at 37°C. 6. Repeat the aspiration and wash five times as step 4. 7. Add 90μl of TMB Substrate to each well. Incubate for 10-30 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark. 8. Add 50μl of Stop Solution to each well when the first four wells containing the highest concentration of standards develop obvious blue color. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 9. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Calculation of Results:
Using the professional soft ",Curve Exert 1.3", to make a standard curve is recommended, which can be downloaded from our web. 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) 9 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 BALP 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 Standard 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 Standard Diluent and repeat the assay. 10Any 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.
Sample collection and storage:
Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum and assay immediately or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles. Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles. Note: Grossly hemolyzed samples are not suitable for use in this assay.

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 (1x20ml), Biotin-antibody Diluent (1x10ml), HRP-avidin Diluent (1x10ml), Biotin-antibody (1x120μl), HRP-avidin (1x120μl), Wash Buffer (25×concentrate) (1x20ml), TMB Substrate (1x10ml), Stop Solution (1x10ml)

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. An incubator which can provide stable incubation conditions up to 37°C±0.5°C.

Storage: 1. Unopened test kits should be stored at 2-8 C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit, provided it is stored as prescribed above. Refer to the package label for the expiration date. 2. Opened test plate should be stored at 2-8 C in the aluminum foil bag with desiccants to minimize exposure to damp air. The kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Restrictions: For Research Use only

Publications

Publications: Roghani, Torkaman, Movasseghe et al.: "Effects of short-term aerobic exercise with and without external loading on bone metabolism and balance in postmenopausal women with osteoporosis." in: Rheumatology international, 2012 (PubMed).