Human Angiostatin K1-3 ELISA Kit User Manual
Contents
1. OR THERAPEUTIC PROCEDURES I INTRODUCTION Ang K1 3 is a single non glycosylated polypeptide chain containing 259 amino acids It represents a proteolytic fragment of plasminogen containing the first three kringle structures Ang K1 3 reduces endothelial cell proliferation and acts as a potent inhibitor of angiogenesis and tumor growth It displays increased inhibitory activity EDso 270 nM relative to kringles 1 4 EDso 135 nM FOR RESEARCH USE ONLY NOT FOR 0 OR THERAPEUTIC PROCEDURES Il ASSAY PRINCIPLES The Human Angiostatin K1 3 ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of Human Angiostatin K1 3 in Cell Culture Supernatants Serum Plasma This assay employs an antibody specific for Human Angiostatin K1 3 coated on a 96 well plate Standards and samples are pipetted into the wells and Angiostatin K1 3 present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti Human Angiostatin K1 3 antibody is added After washing away unbound biotinylated antibody HRP conjugated streptavidin is pipetted to the wells The wells are again washed a TMB substrate solution is added to the wells and color develops in proportion to the amount of Angiostatin K1 3 bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 FOR RESEARCH USE ONLY NO2. experiment The user will decide sample dilution fold by crude estimation of protein amount in samples 1 Add 100 ul of each standard and sample into appropriate wells 2 Cover well and incubate for 90 minutes at room temperature or over night at 4 C with gentle shaking 3 Remove the cover discard the solution and wash plate 3 times with Wash Buffer Working Solution and each time let Wash Buffer Working Solution stay in the wells for 1 2 minutes Blot the plate onto paper towels or other absorbent material Do NOT let the wells completely dry at any time 4 Add 100 ul of Biotin Labeled Detection Antibody Working Solution into each well and incubate the plate at 37 C for 60 minutes 5 Wash plate 3 times with Wash Buffer Working Solution and each time let Wash Buffer Working Solution stay in the wells for 1 2 minutes Discard the Wash Buffer Working Solution and blot the plate onto paper towels or other absorbent material 6 Add 100 ul of Streptavidin HRP Working Solution into each well and incubate the plate at 37 C for 45 minutes 7 Wash plate 5 times with Wash Buffer Working Solution and each time let wash buffer stay in the wells for 1 2 minutes Discard the wash buffer and blot the plate onto paper towels or other absorbent material 8 Add 100 ul of TMB Substrate Solution into each well and incubate plate at 37 C in dark for 30 minutes 9 Add 100 ul of Stop Solution into each well The color changes into yellow im
3. Human Angiostatin K1 3 ELISA Kit User Manual Catalog MBS824874 Sandwich Enzyme Linked Immunosorbent Assay for Quantitative Detection of Human Angiostatin K1 3 Concentrations in Cell Culture Supernatants Serum Plasma For research use only Not for diagnostic or therapeutic procedures FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Ie INTRODUCTION EE 2 1 ASSAY PRINCIPLES 3 Ih KIT COMPONENT S p 4 IV STORAGE 5 4 V MATERIALS REQUIRED BUT NOT 5 VI HEALTH AND SAFETY 5 nennen nennen 5 REAGENT PREPARATION eese nennen nennen le Seen 6 ASSAY PROCEDU RE 5 2 ni eorr enr rr tr tir e 9 IX ASSAY PROCEDURE 11 XD YPICAL iem ederet aeter MM o cocotte repe 12 XI SENSITIVITY iiiter urn as N e rere nnn 12 XII SPECIFICITY rete eres 12 XIII CROSS REACTIVITY 1 fierent eite 13 REFERENCES ia 13 XV TROUBLESHOOTING GUIDE eese een mnn nennen nennen enne 14 XVI TECHNICAL SUPPCOM INK iieri rere teer t egre n etate entree 15 XVII ej dcm me A oem EUER 15 FOR RESEARCH USE ONLY NOT FOR Qe
4. RP Incubation time too long Development time too long Reagent added in incorrect order or incorrectly prepared Standard has gone bad If there is a signal in the sample wells Assay was conducted from an incorrect starting point Insufficient washing unbound Streptavidin HRP remaining Too much Streptavidin HRP Plate sealer or reservoir reused resulting in presence of residual Streptavidin HRP Plate not developed long enough Improper calculation of standard curve dilution Sample matrix is masking detection Samples contain protein levels above assay range Uneven temperature around work surface Solution Increase number of washes Increase time of soaking between in wash Check dilution titration Reduce incubation time Decrease the incubation time before the stop solution is added Review protocol Check the condition of stored standard Reagents allows to come to 20 30 C before performing assay Increase number of washes Carefully Check dilution Use fresh plate sealer and reagent reservoir for each step Increase substrate solution incubation time Check dilution make new standard curve More diluted sample Recommended Dilute samples and run Again Avoid incubating plate in areas where environmental conditions vary Use plate sealer NOSTIC OR THERAPEUTIC PROCEDURES TECHNICAL SUPPORT For troubleshooting inform
5. T FOR Q OR THERAPEUTIC PROCEDURES KIT COMPONENTS 96 well Plate Coated With Anti Human Angiostatin K1 3 12 x 8 Strips Antibody IV STORAGE AND STABILITY All kit components are stable at 2 to 8 C Standard recombinant protein should be stored at 20 C or 80 C recommended at 80 C after reconstitution Opened Microplate Wells or reagents may be store for up to 1 month at 2 to 8 C Return unused wells to the pouch containing desiccant pack reseal along entire edge Note the kit can be used within one year if the whole kit is stored at 20 C Avoid repeated freeze thaw cycles FOR RESEARCH USE ONLY NOT FOR 0 OR THERAPEUTIC PROCEDURES V MATERIALS REQUIRED BUT NOT PROVIDED 1 Microplate reader capable of measuring absorbance at 450 nm 2 Adjustable pipettes and pipette tips to deliver 2 ul to 1 ml volumes 3 Adjustable 1 25 ml pipettes for reagent preparation 4 100 ml and 1 liter graduated cylinders 5 Absorbent paper 6 Distilled or deionized water 7 Computer and software for ELISA data analysis 8 Tubes to prepare standard or sample dilutions VI HEALTH AND SAFETY PRECAUTIONS 1 Reagents provided in this kit may harmful if ingested inhaled or absorbed through the skin Please carefully review the MSDS for each reagent before conducting the experiment 2 Stop Solution contains 2 N Sulfuric Acid 504 and is an extremely corrosive agent Please wear proper eye hand and face pro
6. ation or assistance please go online XVII NOTES FOR RESEARCH USE ONLY NOT FOR USE I NOSTIC OR THERAPEUTIC PROCEDURES
7. eces and homogenize with a tissue homogenizer in PBS or in lysate solution lysate solution tissue net weight 10ml 1g i e Add 10ml lysate solution to 1g tissue Centrifuge at approximately 5000 X g for 5 minutes Assay immediately or aliquot and store homogenates at 20 C Avoid repeated freeze thaw cycles Urine Urinary samples should be cleared by centrifugation and then can be used directly without dilution Storage at 20 C 2 Human Angiostatin K1 3 Standard Preparation Reconstitute the lyophilized Human Angiostatin K1 3 Standard by adding 1 ml of Standard Sample Diluent to make the 10000 pg ml standard stock solution Allow FOR RESEARCH USE ONLY NOT FOR OR THERAPEUTIC PROCEDURES solution to sit at room temperature for 5 minutes then gently vortex to mix completely Use within one hour of reconstituting Two tubes of the standard 10 ng per tube are included in each kit Use one tube for each experiment Perform 2 fold serial dilutions of the top standards to make the standard curve within the range of this assay 156 pg ml 10000 pg ml as below Standard Sample Dilution Buffer serves as the zero standard 0 pg ml Standard Add Into 10000 pg ml 5000 pg ml 500 ul of the Standard 10000 pg ml 500 ul of the Standard Sample Diluent 2500 pg ml 500 ul of the Standard 5000 pg ml 1250 pg ml 500 ul of the Standard 2500 pg ml 625 pg ml 500 ul of the Standard 1250 pg ml 312 5 pg ml 500 ul of the Standa
8. mediately FOR RESEARCH USE ONLY NOT FOR OR THERAPEUTIC PROCEDURES 10 Read the O D absorbance at 450nm in a microplate reader within 30 minutes after adding the Stop Solution For calculation the relative O D 450 the O D 450 of each well the O D 450 of Zero well The standard curve can be plotted as the relative O D 450 of each standard solution Y vs the respective concentration of the standard solution X The concentration of the samples can be interpolated from the standard curve Note If the samples measured were diluted multiply the dilution factor to the concentrations from interpolation to obtain the concentration before dilution FOR RESEARCH USE ONLY NOT FOR USE I NOSTIC OR THERAPEUTIC PROCEDURES IX ASSAY PROCEDURE SUMMARY Prepare all reagents samples and standards Add 100 ul Standard or Sample Wash plate 3 times with Wash Buffer Working Solution Wash plate 3 times with Wash Buffer Working Solution m v hd Add 100 ul Streptavidin HRP Wa g Solution b Wash plate 5 times Wash Buffer Working Solution Add 100 ul TN y ubstrate Solution f z Add 100 ul Stop Solution A Readthe plate at 450nm FOR RESEARCH USE ONLY NOT FOR Q OR THERAPEUTIC PROCEDURES X TYPICAL DATA The standard curve is for demonstration only A standard curve must be run with each assay 10 amp e 2 E M 8 2 l
9. rd 625 pg ml 156 25 pg ml 500 ul of the Standard 312 5 pg ml 0 ng ml 1 ml of the Standard Sample Diluent Note The standard solutions are best used within 2 hours The 10000 pg ml standard solution should be stored at 4 C for up to 12 hours or at 20 C for up to 48 hours Avoid repeated freeze thaw cycles 3 Biotin Labeled Detection Antibody Working Solution Preparation The Biotin Labeled Detection Antibody should be diluted in 1 100 with the Detection Antibody Diluent and mixed thoroughly The solution should be prepared no more than 2 hours prior to the experiment 4 Streptavidin HRP Working Solution Preparation The Streptavidin HRP should be diluted in 1 100 with the Streptavidin HRP Diluent and mixed thoroughly The solution should be prepared no more than 1 hour prior to the experiment FOR RESEARCH USE ONLY NOT FOR e OR THERAPEUTIC PROCEDURES 5 Wash Buffer Working Solution Preparation Pour entire contents 30 ml of the Wash Buffer Concentrate into a clean 1 000 ml graduated cylinder Bring final volume to 600 ml with glass distilled or deionized water 1 20 FOR RESEARCH USE ONLY NOT FOR USE I NOSTIC OR THERAPEUTIC PROCEDURES Vill ASSAY PROCEDURE The Streptavidin HRP Working Solution and TMB Substrate Solution must be kept warm at 37 C for 30 minutes before use When diluting samples and reagents they must be mixed completely and evenly Standard detection curve should be prepared for each
10. t lt 100 1000 10000 Human Angiostatin K1 3 Concentration pg ml Xl SENSITIVITY The minimum detectable dose of Human Angiostatin K1 3 is typically less than 10 pg ml XII SPECIFICITY The Human Angiostatin K1 3 ELISA Kit allows for the detection and quantification of endogenous levels of natural and or recombinant Human Angiostatin K1 3 proteins within the range of 156 pg ml 10000 pg ml FOR RESEARCH USE ONLY NOT FOR OR THERAPEUTIC PROCEDURES XIII CROSS REACTIVITY No detectable cross reactivity with other relevant proteins XIV REFERENCES 1 O Relly MS Holmgren L Shin Y Chen C Rosenthal RA Moses M et al Angiostatin a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma Cell 1994 79 315 328 2 Cao Y Ji RW Davidson D Schaller J Marti D Sohndel S et Kringle domains of human angiostatin JBC 1996 271 29461 29467 FOR RESEARCH USE ONLY NOT FOR O OR THERAPEUTIC PROCEDURES XV TROUBLESHOOTING GUIDE Problem High signal and background in all wells No signal Too much signal whole plate turned uniformly blue Standard curve achieved but poor discrimination between point No signal when a signal is expected but standard curve looks fine Samples are reading too high but standard curve is fine Edge effect FOR RESEARCH USE ONLY NOT FOR USE I Possible Cause Insufficient washing Too much Streptavidin H
11. tection when handling this material When the experiment is finished be sure to rinse the plate with copious amounts of running water to dilute the Stop Solution prior to disposing the plate FOR RESEARCH USE ONLY NOT FOR OR THERAPEUTIC PROCEDURES Vil REAGENT PREPARATION 1 Sample Preparation Store samples to be assayed within 24 hours at 2 8 C For long term storage aliquot and freeze samples at 20 C Avoid repeated freeze thaw cycles Cell culture supernates Remove particulates by centrifugation assay immediately or aliquot and store samples at 20 C Serum Allow the serum to clot in a serum separator tube about 4 hours at room temperature Centrifuge at approximately 1000 X g for 15 minutes Analyze the serum immediately or aliquot and store samples at 20 C Plasma Collect plasma using heparin or EDTA as an anticoagulant Centrifuge for 15 minutes at 1500 X g within 30 minutes of collection Assay immediately or aliquot and store samples at 20 C Cell Lysates Collect cells and rinse cells with PBS Homogenize and lyse cells throughly in lysate solution Centrifuge celllysates at approximately 10000 X g for 5 minutes to remove debris Aliquots of the cell lysates were removed and assayed Bone Tissue Extract demineralized bone samples in 4 M Guanidine HCl and protease inhibitors Dissolve the final sample in 2 M Guanidine HCl Tissue Homogenates Rinse tissue with PBS to remove excess blood chopped into 1 2 mm pi
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