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1 BIOO LIFE SCIENCE PRODUCTS FOR REFERENCE PURPOSES This manual is for Reference Purposes Only. DO NOT use this protocol to run your assays. Periodically, optimizations and revisions are made to the kit and protocol, so it is important to always use the protocol included with the kit. Alkaline Phosphatase (AP) ELISA Kit Manual Catalog #: 5505 BIOO Scientific Corp. 2010

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3 TABLE OF CONTENTS GENERAL INFORMATION... 1 Product Description... 1 Procedure Overview... 1 Kit Specificity... 2 Kit Contents, Storage and Shelf Life... 2 Required Materials Not Provided With the Kit... 2 Warnings and Precautions... 3 SAMPLE PREPARATION... 4 Serum... 4 Urine... 4 Cells... 4 Tissue... 4 ALKALINE PHOSPHATASE ELISA PROTOCOL... 5 Reagent Preparation... 5 ELISA Testing Protocol... 6 Alkaline Phosphatase Concentration Calculations... 7 TROUBLESHOOTING... 8 No Color Development or No Signals with Standards... 8 Low Optical Density (OD) Readings... 8 High Background or High Optical Density (OD) Readings... 8 High Intra-Plate Variance... 9 High Inter-Plate Variance... 9 MaxDiscovery Alkaline Phosphatase ELISA Kit is intended for laboratory use only, unless otherwise indicated. This product is NOT for clinical diagnostic use. MaxDiscovery is a Trademark of Bioo Scientific Corporation (BIOO).

4 MaxDiscovery Alkaline Phosphatase ELISA Kit Manual GENERAL INFORMATION GENERAL INFORMATION Product Description The MaxDiscovery Alkaline Phosphatase ELISA Kit is an enzyme immunoassay that analyzes the quantity of Alkaline Phosphatase in cells, tissues, serum or urine. Alkaline phosphatase is a well known hydrolase enzyme responsible for removing phosphate groups from molecules including nucleotides, proteins and alkaloids. In humans, alkaline phosphatase is present in all tissues throughout the body, but is concentrated in the liver, bile duct, kidney, bone and placenta. The normal range of alkaline phosphatase is IU/L. High alkaline phosphatase levels can show that the bile ducts are blocked. Levels are significantly higher in children and pregnant women. Also, elevated alkaline phosphatase indicates that there could be active bone deposition occurring as alkaline phosphatase is a by-product of osteoblast activity. Lowered levels of alkaline phosphatase exist in cases of disease including, hypophosphatasia, postmenopausal osteoporosis, women receiving estrogen therapy because of osteoporosis, Men with recent heart surgery, malnutrition, magnesium deficiency, hypothyroidism, or severe anemia, Children with achondroplasia and cretinism, children after a severe episode of enteritis, pernicious anemia, aplastic anema, and chronic myelogenous leukemia. Leukocyte alkaline phosphatase is found in white blood cells and is indicative of certain conditions. For example, higher levels are seen in polycythemia sera, essential thrombocytosis, primary myelofibrosis, and leukemoid reaction. Lower levels are found in chronic myelogenous leukemia and paroxysmal nocturnal hemoglobinuria. Like most ELISA assays, the MaxDiscovery Alkaline Phosphatase ELISA Kit relies on a Horseradish Perioxidase (HRP) conjugated antibody and the TMB (3,3,5,5 -tetramethylbenzidine) substrate. TMB is a chromogen that yields a blue color when oxidized with hydrogen peroxide (catalyzed by HRP) that has major absorbances at 370 nm and 652 nm. The color then changes to yellow with the addition of acid with maximum absorbance at 450 nm. The relative amount of alkaline phosphatase protein in the cells will be directly proportional to the amount of signal that is obtained at 450 nm. This kit contains materials for the extraction and quantitative detection of the alkaline phosphatase protein in cells, serum, tissues or urine. Procedure Overview This kit uses a sandwich ELISA format, in which samples are added to microtiter plate wells that have been coated with an antibody to alkaline phosphatase (the capture antibody ). After incubation, the sample is removed, the wells are washed and a second antibody to alkaline phosphatase, which is directly conjugated to HRP, is added (the detection antibody ). Signal is generated by reaction with the TMB substrate as described above. The intensity of the signal (measured at 450 nm) is directly proportional to the amount of alkaline phosphatase in the sample. Dilutions of the alkaline phosphatase standard, included in the kit, are used to construct a standard curve, from which the concentration of Alkaline Phosphatase in the samples are determined by extrapolation. This is described in more detail in Section, Alkaline Phosphatase Concentration Calculations. BIOO LIFE SCIENCE PRODUCTS 1

5 MaxDiscovery Alkaline Phosphatase ELISA Kit Manual Kit Specificity The alkaline phosphatase Antibody-HRP Conjugate included in this kit reacts with human alkaline phosphatase. Kit Contents, Storage and Shelf Life MaxDiscovery Alkaline Phosphatase ELISA Kit has the capacity for 96 determinations or testing of 42 samples in duplicate (assuming 6 wells for standards, in duplicate). Return any unused microwells to the foil bag and reseal them with the desiccant provided, in the original package. Store the kit at 4 C. The normal shelf life is 6 months from the date of manufacturing when the kit is properly stored. Check the bar code on the box for the specific expiration date. Kit Contents Amount Storage 1 Anti- Alk Phos Capture Antibody Coated Plate One 96-well plate (8 wells x 12 strips) 4 C Protein Extraction Buffer 25 ml 4 C Alk Phos Standard (10,000 U/L) 2 x 25 µl 4 C 2 Standard Diluent 15 ml 4 C Alk Phos Antibody-HRP Conjugate 30 µl -20 C 3 HRP Conjugate Diluent 15 ml 4 C 20X Wash Solution ** 28 ml 4 C Stop Buffer ** 20 ml 4 C TMB Substrate ** 12 ml 4 C IMPORTANT: 1 All reagents except for Alk Phos Standard) stock and Alk Phos Antibody-HRP Conjugate should be brought up to room temperature before use (30 min - 1 hour at C/68 77 F) 2 Do Not leave Alk Phos Standard stock at room temperature. Take out right before use and return the unused portion to 4 C 3 Do Not Leave ALK Phos Antibody-HRP Conjugate at room temperature, take out right before use and return unused portion to C. To avoid repeated freeze thaw cycles, aliquot HRP-conjugate and store at C ** Components with the same BIOO part numbers within their expiration dates are interchangeable among BIOO kits. Required Materials Not Provided With the Kit Microtiter plate reader (450 nm) 37 C Incubator 10, 20, 100 and 1000 µl pipettes and tips Multi-channel pipette: µl (Optional) 1.5 ml microcentrifuge tubes -80 C Freezer or dry ice/ethanol bath Micro-Homogenizer (Bioo Scientific cat. #: , Optional) MaxDiscovery Bradford Assay Kit (Bioo Scientific cat. #: , Optional) BIOO LIFE SCIENCE PRODUCTS 2

6 Warnings and Precautions MaxDiscovery Alkaline Phosphatase ELISA Kit Manual BIOO strongly recommends that you read the following warnings and precautions to ensure your full awareness of ELISA techniques and other details you should pay close attention to when running the assays. More information can also be found in Troubleshooting section. Periodically, optimizations and revisions are made to the kit and manual. Therefore, it is important to follow the protocol included with the kit. If you need further assistance, you may contact your local distributor or BIOO at Do not use the kit past the expiration date. Try to maintain a laboratory temperature of (20 25 C/68 77 F). Avoid running assays under or near air vents, as this may cause excessive cooling, heating and/or evaporation. Also, do not run assays in direct sunlight, as this may cause excessive heat and evaporation. Cold bench tops should be avoided by placing several layers of paper towel or some other insulation material under the assay plates during incubation. Make sure you are using only distilled deionized water since water quality is very important. When pipetting samples or reagents into an empty microtiter plate, place the pipette tips in the lower corner of the well, making contact with the plastic. BIOO makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made are of standard quality. There is no warranty of merchantability of this product, or of the fitness of the product for any purpose. BIOO shall not be liable for any damages, including special or consequential damage, or expense arising directly or indirectly from the use of this product. BIOO LIFE SCIENCE PRODUCTS 3

7 MaxDiscovery Alkaline Phosphatase ELISA Kit Manual SAMPLE PREPARATION SAMPLE PREPARATION Be sure samples are properly stored. Samples should be refrigerated at 2-4 C for no more than 1-2 days. If samples need to be stored for longer than 2 days, freeze them to -20 C or colder. Frozen samples can be thawed at room temperature (20 25 C/68 77 F) or in a refrigerator before use. Serum 1. Blood should be collected without anticoagulant and left at room temperature for 3 hours or at 4 C overnight, to clot. 2. Centrifuge at 3,000 x g for 10 minutes at 4 C. 3. Take the serum from the upper layer of the blood sample. 4. Use 100 µl of the serum directly or dilute the sample as per instructions under Reagent Preparation and use 100 µl of that in the MaxDiscovery Alkaline Phosphatase ELISA Kit. Urine Use 100 µl directly or dilute the sample as per instructions under Reagent Preparation and use 100 µl. Cells 1. For suspension cells, collect cells and centrifuge at 4 C for 10 minutes at 3,000 x g. For adherent cells skip to step Remove the media. 3. Add 50 µl of Protein Extraction Buffer (Cat. #: ) per 20,000-50,000 cells (1 x 10 6 cells = 1 ml of Protein Extraction Buffer) and vortex or pipet up and down about 10 times to lyse the cells. 4. Freeze the sample for 30 minutes in a -80 C freezer or a dry ice/ethanol bath. 5. Thaw sample at room temperature (20 25 C/68 77 F). 6. Dilute cell lysate with Bradford Diluent provided with the MaxDiscovery Bradford Assay Kit (Cat. #: ). The recommended starting dilutions are 1:20 and 1: Determine the total concentration of protein using the MaxDiscovery Bradford Assay. 8. The total concentration of protein in the sample should be normalized using Secondary Standard Diluent to mg/ml. 9. Use 100 µl of the normalized sample or dilute the sample as per instructions under Reagent Preparation and use 100 µl of that in the assay. Tissue 1. Add 500 µl of Protein Extraction Buffer (Cat. #: ) to 500 mg or less of tissue samples. 2. Grind the sample using a Micro-Homogenizer (Cat. #: ) or a pestle. 3. Freeze the sample for 30 minutes in a -80 C freezer or a dry ice/ethanol bath. 4. Thaw sample at room temperature (20 25 C/68 77 F) and spin at 14,000g for 10 minutes. 5. Take out the supernatant to a new tube and dispose of the pellet. 6. Dilute the sample in the Bradford Diluent provided with the MaxDiscovery Bradford Assay Kit (Cat. #: ). The recommended starting dilutions are 1:50 and 1: Quantify the total protein using the MaxDiscovery Bradford Assay Kit. 8. The total concentration of protein in the sample should be normalized using Secondary Standard Diluent to mg/ml. 9. Use 100 µl of the normalized sample or dilute the sample as per instructions under Reagent Preparation and use 100 µl of that in the MaxDiscovery Alkaline Phosphatase ELISA Kit. BIOO LIFE SCIENCE PRODUCTS 4

8 MaxDiscovery Alkaline Phosphatase ELISA Kit Manual ALKALINE PHOSPHATASE ALKALINE ELISA PROTOCOL PHOSPHATASE Reagent Preparation IMPORTANT: Make sure you read Warnings and Precautions section on page 3. All reagents, except for Alk Phos Standard and Alkaline Phos Antibody-HRP Conjugate, should be brought to room temperature before use (30 min - 1 hour at C/68 77 F). Take the Standard Stock and Antibody-HRP Conjugate out of the freezer right before use, return the unused portions to C immediately, or tests may fail. Solutions should be prepared just prior to performing the ELISA test. All reagents should be thoroughly mixed by gently inverting or swirling prior to use. Prepare volumes that are needed for the number of wells being run. Do not return the reagents to the original stock tubes/bottles. BIOO recommends using disposable reservoirs when handling reagents to minimize the risk of contamination. Preparation of Sample: If the sample contains high alkaline phosphatase levels, then an initial dilution of the sample is required. The recommended starting dilutions are 1:2, 1:5 or 1:10 of the sample into the Standard Diluent solution, provided in the kit, to identify the right dilution factor. Preparation of 1X Wash Solution Mix 1 volume of the 20X Wash Solution with 19 volumes of distilled water. Preparation of Standards (Only Prepare immediately before use, return the unused portion to 4 C) IMPORTANT: Quickly spin (10 seconds) the Alk Phos Standard (10,000 U/L) tube to ensure the standard solution is at the bottom of the tube. Prepare the standards in sterile 1.5 ml centrifuge tubes as per the table below. Standards Preparation Negative Control (0 U/L) Use 100 µl of Standard Diluent per well 300 U/L Add 15 µl of 10,000 U/L to 500 µl of Standard Diluent 150 U/L Add 250 µl of 300 U/L to 250 µl of Standard Diluent 125 U/L Add 3 µl of 10,000 U/L to 237 µl of Standard Diluent 100 U/L Add 3 µl of 10,000 U/L to 297 µl of Standard Diluent 75 U/L Add 250 µl of 150 U/L to 250 µl of Standard diluent Preparation of 1:500 Alk Phos Antibody-HRP Conjugate (Prepare immediately before use, return the unused portion to -20 C) IMPORTANT: Quickly spin (10 seconds) the tube to ensure the antibody solution is at the bottom of the tube. Mix the Alk Phos Antibody-HRP Conjugate into the room temperature HRP Conjugate Diluent minutes before adding to wells. For every 8 well strip used, 1 ml of Alk Phos Antibody-HRP Conjugate is needed. To prepare 1 ml of Alk Phos Antibody-HRP Conjugate, add 2 µl of Alk Phos Antibody-HRP Conjugate to 1 ml of HRP Conjugate Diluent. Mix thoroughly. BIOO LIFE SCIENCE PRODUCTS 5

9 ELISA Testing Protocol MaxDiscovery Alkaline Phosphatase ELISA Kit Manual Label the individual strips that will be used and aliquot reagents as shown below: Component Volume per Reaction 24 Reactions Alk Phos Antibody-HRP Conjugate 100 µl 2.4 ml 1X Wash Solution 1.5 ml 36 ml Stop Buffer 100 µl 2.4 ml TMB Substrate 100 µl 2.4 ml 1. Dilute the Wash Buffer, Standards, and Alk Phos Antibody-HRP Conjugate as indicated in the reagent preparation section above. 2. Add 100 µl of each Alk Phos Standard, in duplicate, into appropriate wells ( Add standards to plate only in the order from low concentration to high concentration in the following order: Negative, Negative, 75 U/L, 75 U/L, 100 U/L, 125 U/L, 150 U/L, 300 U/L). 3. Add 100 µl of each sample, in duplicate, into appropriate sample wells. 4. Incubate the plate for 2 hours at 37 C ( Covering the microtiter plate with foil while incubating is recommended). (Optional: After adding the standard & samples, gently tap the plate for ~one minute and thrity seconds to ensure good mixing) 5. Aspirate all fluid from each well and wash the plate 3 times with 250 µl/well of 1X Wash Solution (Optional: Allow ~ one minute for soaking during each wash step). 6. After the last wash, invert the plate and gently tap the plate on paper towels to remove the residual liquid ( Perform the next step immediately after plate washings. Do not allow the plate to air dry between working steps). 7. Add 100 µl of 1:500 diluted Alk. Phos. Antibody-HRP Conjugate. Incubate the plate for 1 hour at 37 C. ( Covering the microtiter plate with foil while incubating is recommended). (Optional: After adding the HRP Conjugate, gently tap the plate for ~one minute and thirty seconds to ensure good mixing) 8. Aspirate all fluid from each well and wash the plate 3 times with 250 µl/well of 1X Wash Solution (Optional: Allow ~ one minute for soaking during each wash step). 9. After the last wash, invert the plate and gently tap the plate on paper towels to remove the residual liquid ( Perform the next step immediately after plate washings. Do not allow the plate to air dry between working steps). 10. Add 100 µl of TMB substrate and incubate at room temperature (20 25 C/68 77 F) for 15 minutes. ( Avoid direct sunlight and cold bench tops during the incubation.) Start timing the reaction immediately after adding the substrate to the last well. Mix the solution by gently rocking the plate manually for 1 minute while incubating ( Covering the microtiter plate with foil while incubating is recommended. Do not put any substrate back to the original container to avoid any potential contamination. Any substrate solution exhibiting coloration is indicative of deterioration and should be discarded). 11. Add 100 µl of Stop Buffer to stop the enzyme reaction. 12. Read the plate as soon as possible following the addition of Stop Buffer on a plate reader with 450 nm wavelength. BIOO LIFE SCIENCE PRODUCTS 6

10 MaxDiscovery Alkaline Phosphatase ELISA Kit Manual Alkaline Phosphatase Concentration Calculations A standard curve is constructed by plotting the absorbance obtained from each reference standard against its concentration in ng/ml, as shown below. The following figure is a typical alkaline phosphatase standard curve. Using the equation generated by the standard curve, the alkaline phosphatase level (U/L) of the sample can be determined. To determine the total amount of alkaline phosphatase protein in the sample, use the following equation and solve for the x value: x=(y-b)/m Where: b=the y-axis intercept of your standard curve m=the slope of your standard curve x=the alkaline phosphatase concentration y=the Absorbance of the sample at 450 nm For example: If the OD reading of a 1:10 diluted sample is: , using the equation and the Standard Curve shown above, the alkaline phosphatase concentration would be: U/L. BIOO LIFE SCIENCE PRODUCTS 7

11 MaxDiscovery Alkaline Phosphatase ELISA Kit Manual TROUBLESHOOTING TROUBLESHOOTING No Color Development or No Signals with Standards Possible Causes Reagents were used in the wrong order or a step was skipped. HRP Conjugate was prepared incorrectly or has deteriorated. TMB substrate has deteriorated. Recommended Action Follow the protocol carefully and repeat the assay. Make sure that the HRP Conjugate used is the one that comes with the kit. The HRP Conjugate is kit- and lot-specific. Make sure that the correct HRP Conjugate and diluent are mixed in correct volumes. Use a new set of BIOO TMB substrate. Low Optical Density (OD) Readings Possible Causes Reagents were expired or mixed with a different lot number or were prepared incorrectly. Wash solution was prepared incorrectly. Too many wash cycles were used. Incubation times were too short. Lab temperature was too low. Reagents and plates were too cold. Reader was at wrong wavelength, or reader was malfunctioning. Excessive kit stress has occurred. Assay plates were compromised Protein expression is low Recommended Action Verify the expiration dates and lot numbers and that the reagents were prepared correctly. Use the wash solution for the kit and verify that it is prepared correctly. Make sure to use the number of washes per the protocol instruction. Time each plate separately to ensure accurate incubation times, follow protocol. Maintain the lab room temperature within (20 25 C/68 77 F). Do not run assays under air conditioning vents or near cold windows. Make sure plates and reagents are brought up to room temperature. Keep the kit components out of the kit box for at least 1 hour before starting the assay. Make sure the wavelength is 450 nm for the assay and read the plate again. Verify reader calibration and lamp alignment. Check records to see how many times the kit has cycled from the refrigerator. Check to see if the kit was left at extreme temperatures for too long. Always refrigerate plates in sealed bags with the desiccant provided to maintain stability. Prevent condensation from forming on plates by allowing them equilibrate to room temperature (20 25 C/68 77 F) while in the packaging. If the Alk Phos protein level in the diluted sample is low use a more concentrated sample to obtain a higher reading. High Background or High Optical Density (OD) Readings Possible Causes Poor quality water was used in wash solution. Substrate solution has deteriorated. There was insufficient washing or poor washer performance. Reader was malfunctioning or not blanked properly. This is a high possibility if the OD readings were high and the color was light. Lab temperature was too high Reagents were intermixed, contaminated or prepared incorrectly. Recommended Action If water quality is questionable, try substituting an alternate distilled water source to prepare the wash solution. Make sure the substrate is colorless prior to addition to the plate. Use the number of washes per the protocol instruction. Make sure that at least 250 µl of wash solution is dispensed per well per wash. Verify the performance of the washer system; have the system repaired if any ports drip, dispense or aspirate poorly. Verify the reader s performance using a calibration plate and check the lamp alignment. Verify the blanking procedure, if applicable, and reblank. Maintain the room temperature within (20 25 C/68 77 F). Avoid running assays near heat sources or in direct sunlight. Ensure that the correct reagents were used, that working solutions were prepared correctly and that contamination has not occurred. BIOO LIFE SCIENCE PRODUCTS 8

12 High Intra-Plate Variance MaxDiscovery Alkaline Phosphatase ELISA Kit Manual Possible Causes Inconsistent time was taken when adding standards, reagents or samples to the assay plate. Multichannel pipette was not functioning properly. There was inconsistent washing or washer system malfunctioning. Recommended Action Make sure all materials are set up and ready to use. Use a multichannel pipette to add reagents to multiple wells whenever possible. Do not interrupt while adding standards, reagents and samples. Verify pipette calibration and check that tips are on tight. Be sure all channels of the pipette draw and dispense equal volumes. Check performance of the wash system. Have the system repaired if any ports drip or dispense/aspirate poorly. High Inter-Plate Variance Possible Causes Inconsistent incubation times occurred from plate to plate. Inconsistent washing occurred from plate to plate. Pipette was working improperly. Kit plates, reagents, standards and samples were at different temperatures. Reagents used were intermixed from different kit lots, or the kits were of different expiration dates. Recommended Action Time each plate separately to ensure consistent incubation times. Make sure to use the number of washes per the protocol instruction. Verify performance of the wash system and have the system repaired if any ports drip or dispense/ aspirate poorly. Check the pipette calibration. Verify that pipette tips are on tight before use and that all channels draw and dispense equal volumes. Make sure to allow sufficient time for kit plates, reagents, standards and samples come to room temperature (20 25 C/68 77 F). Larger volumes will require longer equilibration time. If using a water bath to hasten equilibration, make sure it is maintained at room temperature; do not use a warm water bath to warm reagents, samples and kit standards. Carefully label each reagent to make sure the reagents are not intermixed. Kits with different expiration dates might generate different range of OD readings, however, the relative absorbance values may very well be comparable. In general, a value of less than 0.6 in zero standard reading may indicate certain degrees of deterioration of reagents. Bioo Scientific Corporation 3913 Todd Lane Suite 312 Austin, TX USA Tel: Fax: (512) Made in USA BIOO Research Products Group info@biooscientific.com techsupport3@biooscientific.com BIOO LIFE SCIENCE PRODUCTS 9