AssayMax Human Prekallikrein ELISA Kit
Contents
1. Storage Condition e Upon arrival immediately store components of the kit at recommended temperatures up to the expiration date e Store SP Conjugate and Biotinylated Antibody at 20 C e _ Store Microplate Diluent Concentrate 10x Wash Buffer Stop Solution and Chromogen Substrate at 2 8 C e Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed May be stored for up to 30 daysina vacuum desiccator e Store Standard at 2 8 C before reconstituting with Diluent and at 20 C after reconstituting with Diluent Other Supplies Required e Microplate reader capable of measuring absorbance at 450 nm Pipettes 1 20 ul 20 200 ul 200 1000 ul and multiple channel e Deionized or distilled reagent grade water Sample Collection Preparation and Storage e Plasma Collect plasma using one tenth volume of 0 1 M sodium citrate as an anticoagulant Centrifuge samples at 3000 x g for 10 minutes and collect plasma A 4000 fold sample dilution is suggested into MIX Diluent however user should determine optimal dilution factor depending on application needs The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles EDTA or Heparin can also be used as an anticoagulant e Serum Samples should be collected into a serum separator tube After clot formation centrifuge samples at 3000 x g for 10 minutes and remove serum A 4000 fold sample dilut2. 4 a Recovery Standard Added Value 5 40 ng ml Recovery 87 114 Average Recovery Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 2000x 95 93 4000x 101 98 8000x 107 104 Cross Reactivity Species Cross Reactivity Canine None Bovine None Monkey 20 Mouse None Rat None Swine None Rabbit None Protein Cross Reactivity 10 FBS in culture media will not affect the assay Troubleshooting Causes Course of Action Use of improper e Check the expiration date listed before use components e Do not interchange components from different lots e Check that the correct wash buffer is being used e Check that all wells are empty after aspiration Improper wash step e Check that the microplate washer is dispensing properly e If washing by pipette check for proper pipetting technique Splashing of reagents e Pipette properly in a controlled and careful manner while loading wells e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions e Check the microplate pouch for proper sealing Improperly sealed e Check that the microplate pouch has no pu
3. samples e A new tip must be used for each addition of different samples or reagents during the assay procedure Contents of wells evaporate e Verify that the sealing film is firmly in place before placing the assay in the incubator or at room temperature Improper pipetting Deficient Standard Curve Fit Insufficient mixing of reagent dilutions References e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions 1 Girolami A et al 2010 Expert Rev Hematol 3 6 685 695 2 Chung DW et al 1986 Biochemistry 25 9 2410 2417 3 Tait JF Fujikawa K 1986 J Biol Chem 261 33 15396 15401 4 Wynne J et al 2004 Brit J Haemat 127 220 223 Version 2 1 www assaypro com e mail support assaypro com 10
4. cleaved by factor Xlla PK is converted into kallikrein with an N terminal heavy chain 371 amino acids and a catalytic light chain 248 amino acids held together by a disulfide bond 2 Plasma kallikrein liberates kinins bradykinin and kallidin from the kininogens to regulate vasodilation and inflammation 3 PK deficient patients have prolonged activated partial thromboplastin time without having any bleeding disorder 4 Principle of the Assay The AssayMax Human Prekallikrein ELISA Enzyme Linked Immunosorbent Assay Kit is designed for detection of prekallikrein in human plasma serum saliva milk CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures human prekallikrein in approximately 4 hours A polyclonal antibody specific for human prekallikrein has been pre coated onto a 96 well microplate with removable strips Prekallikrein in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for human prekallikrein which is recognized by a streptavidin peroxidase SP conjugate All unbound material is washed away and a peroxidase enzyme substrate is added The color development is stopped and the intensity of the color is measured Caution and Warning e This product is for Research Use Only and is not intended for use in diagnostic procedures Prepare all reagents diluent buffer wash buffer standard biotin
5. may also include biofluids cell culture and tissue homogenates If necessary user should determine optimal dilution factor depending on application needs Refer to Dilution Guidelines for further instruction Guidelines for Dilutions of 100 fold or Greater for reference only please follow the insert for specific dilution suggested 100x 10000x A 4plsample 396 ul buffer 100x 4 ul sample 396 ul buffer 100x 100 fold dilution 4 ul of A 396 ul buffer 100x 10000 fold dilution Assuming the needed volume is less than Assuming the needed volume is less than or equal to 400 ul or equal to 400 ul 1000x 100000x 4 ul sample 396 ul buffer 100x 4 ul sample 396 ul buffer 100x 24 ul of A 216 ul buffer 10x 4 ul of A 396 ul buffer 100x 1000 fold dilution 24 ul of B 216 ul buffer 10x 100000 fold dilution Assuming the needed volume is less than Assuming the needed volume is less than or equal to 240 ul or equal to 240 ul Reagent Preparation e Freshly dilute all reagents and bring all reagents to room temperature before use e MIX Diluent Concentrate 10x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute the MIX Diluent Concentrate 10 fold with reagent grade water to produce a 1x solution Store for up to 30 days at 2 8 C e Human Prekallikrein Standard Reconstitute the Human Prekallikrein Standard 96 ng with 1 2 ml of MIX Diluen
6. d at 20 C Assay Procedure e Prepare all reagents standard solutions and samples as instructed Bring all reagents to room temperature before use The assay is performed at room temperature 20 25 C e Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccants inside Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator e Add 50 ul of Human Prekallikrein Standard or sample to each well Gently tap plate to thoroughly coat the wells Break any bubbles that may have formed Cover wells with a sealing tape and incubate for 2 hours Start the timer after the last addition e Wash the microplate manually or automatically using a microplate washer Invert the plate and decant the contents hit 4 5 times on absorbent material to completely remove the liquid If washing manually wash five times with 200 ul of Wash Buffer per well Invert the plate each time and decant the contents hit 4 5 times on absorbent material to completely remove the liquid If using a microplate washer wash six times with 300 ul of Wash Buffer per well invert the plate and hit 4 5 times on absorbent material to completely remove the liquid Add 50 ul of Biotinylated Human Prekallikrein Antibody to each well Gently tap plate to thoroughly coat the wells Break any bubbles that may have formed Cover wells with a sealing tape and incubate for 1 hour Wash the microplat
7. e as described above Add 50 ul of SP Conjugate to each well Gently tap plate to thoroughly coat the wells Break any bubbles that may have formed Cover wells with a sealing tape and incubate for 30 minutes Turn on the microplate reader and set up the program in advance Wash the microplate as described above Add 50 ul of Chromogen Substrate to each well Gently tap plate to thoroughly coat the wells Break any bubbles that may have formed Incubate for 20 minutes or until the optimal blue color density develops Add 50 ul of Stop Solution to each well The color will change from blue to yellow Gently tap plate to ensure thorough mixing Break any bubbles that may have formed Read the absorbance on a microplate reader at a wavelength of 450 nm immediately If wavelength correction is available subtract readings at 570 nm from those at 450 nm to correct optical imperfections Otherwise read the plate at 450 nm only Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes which will reduce the readings Data Analysis Calculate the mean value of the duplicate or triplicate readings for each standard and sample To generate a standard curve plot the graph using the standard concentrations on the x axis and the corresponding mean 450 nm absorbance OD on the y axis The best fit line can be determined by regression analysis using log log or four parame
8. ion is suggested into MIX Diluent however user should determine optimal dilution factor depending on application needs The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e Saliva Collect saliva using sample tube Centrifuge samples at 800 x g for 10 minutes The sample is suggested for use at 1x however user should determine optimal dilution factor depending on application needs The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e Milk Collect milk using sample tube Centrifuge samples at 800 x g for 10 minutes A 10 fold sample dilution is suggested into MIX Diluent however user should determine optimal dilution factor depending on application needs The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e CSF Collect cerebrospinal fluid CSF using sample pot Centrifuge samples at 3000 x g for 10 minutes A 10 fold sample dilution is suggested into MIX Diluent however user should determine optimal dilution factor depending on application needs The undiluted samples can be stored at 80 C for up to 3 months Avoid repeated freeze thaw cycles e Cell Culture Supernatant Centrifuge cell culture media at 1500 rpm for 10 minutes at 4 C to remove debris and collect supernatant Samples can be stored at 80 C Avoid repeated freeze thaw cycles Applicable samples
9. nctures microplate e Check that three desiccants are inside the microplate pouch prior to sealing Inconsistent volumes loaded into wells Low Precision Insufficient mixing of reagent dilutions Microplate was left e Each step of the procedure should be performed op unattended between uninterrupted steps 5 gt Omission of step e Consult the provided procedure for complete list of steps 3 a Steps performed in e Consult the provided procedure for the correct order So incorrect order gt s Insufficient amount of e Check pipette calibration E 5 reagents added to e Check pipette for proper performance o S wells oo a a Wash step was skipped e Consult the provided procedure for all wash steps a Improper wash buffer e Check that the correct wash buffer is being used 5 Improper reagent e Consult reagent preparation section for the correct preparation dilutions of all reagents Insufficient or prolonged incubation periods e Consult the provided procedure for correct incubation time Non optimal sample dilution Contamination of reagents e Sandwich ELISA If samples generate OD values higher than the highest standard point P1 dilute samples further and repeat the assay e Competitive ELISA If samples generate OD values lower than the highest standard point P1 dilute samples further and repeat the assay e User should determine the optimal dilution factor for
10. t to generate an 80 ng ml standard stock solution Allow the vial to sit for 10 minutes with gentle agitation prior to making dilutions Prepare duplicate or triplicate standard points by serially diluting from the standard stock solution 80 ng ml 2 fold with equal volume of MIX Diluent to produce 40 20 10 5 2 5 and 1 25 ng ml solutions MIX Diluent serves as the zero standard 0 ng ml Any remaining stock solution should be stored at 20 C and used within 30 days Avoid repeated freeze thaw cycles Standard un Prekallikrein x Dilution Point ng ml P1 1 part Standard 80 ng ml 80 1 part P1 1 part MIX Diluent 1 part P2 1 part MIX Diluent 1 part P3 1 part MIX Diluent 1 part P4 1 part MIX Diluent 1 part P5 1 part MIX Diluent 1 part P6 1 part MIX Diluent MIX Diluent e Biotinylated Human Prekallikrein Antibody 50x Spin down the antibody briefly and dilute the desired amount of the antibody 50 fold with MIX Diluent to produce a 1x solution The undiluted antibody should be stored at 20 C e Wash Buffer Concentrate 20x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute the Wash Buffer Concentrate 20 fold with reagent grade water to produce a 1x solution e SP Conjugate 100x Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100 fold with MIX Diluent to produce a 1x solution The undiluted conjugate should be store
11. ter logistic curve fit Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor Typical Data The typical data is provided for reference only Individual laboratory means may vary from the values listed Variations between laboratories may be caused by technique differences Standard Point Average OD P1 2 246 of as P3 20 fae 1 122 P4 10 Gp 0 660 P5 5 0 a 0 404 P6 25 none 0 259 P7 1 25 ate 0 189 Sample Pooled Normal EDTA Plasma 4000x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed nm OD 450 Human Prekallikrein Standard Curve 0 1 eet 1 and cad 1 10 100 H Prekallikrein ng ml Reference Value e Plasma and serum samples from healthy adults were tested n 40 On average human prekallikrein level was 49 ug ml Performance Characteristics e The minimum detectable dose of human prekallikrein as calculated by 2SD from the mean of a zero standard was established to be 0 65 ng ml e Intra assay precision was determined by testing three plasma samples twenty times in one assay e Inter assay precision was determined by testing three plasma samples in twenty assays Intra Assay Precision Inter Assay Precision Sample 1 2 3 1 2 3 n 20 20 20 20 20 2 CV 4 4 4 9 4 5 9 7 9 5 10 1 Average 4 69 9 89 CV
12. ylated antibody and SP conjugate as instructed prior to running the assay e Prepare all samples prior to running the assay The dilution factors for the samples are suggested in this insert However the user should determine the optimal dilution factor e Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents e The Stop Solution is an acidic solution e The kit should not be used beyond the expiration date Reagents e Human Prekallikrein Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human prekallikrein e Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes that can be cut to fit the format of the individual assay e Human Prekallikrein Standard Human prekallikrein in a buffered protein base 96 ng lyophilized e Biotinylated Human Prekallikrein Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against human prekallikrein 120 pl e _ MIX Diluent Concentrate 10x A 10 fold concentrated buffered protein base 30 ml e Wash Buffer Concentrate 20x A 20 fold concentrated buffered surfactant 30 ml 2 bottles e SP Conjugate 100x A 100 fold concentrate 80 ul e Chromogen Substrate 1x A stabilized peroxidase chromogen substrate tetramethylbenzidine 8 ml e Stop Solution 1x A 0 5 N hydrochloric acid solution to stop the chromogen substrate reaction 12 ml
13. yssaypro AssayMax Human Prekallikrein ELISA Kit Assaypro LLC 3400 Harry S Truman Blvd St Charles MO 63301 T 636 447 9175 F 636 395 7419 www assaypro com For any questions regarding troubleshooting or performing the assay please contact our support team at support assaypro com Thank you for choosing Assaypro Assay Summary Step 1 Add 50 ul of Standard or Sample per well Incubate 2 hours Step 2 Wash then add 50 ul of Biotinylated Antibody per well Incubate 1 hour Step 3 Wash then add 50 ul of SP Conjugate per well Incubate 30 minutes Step 4 Wash then add 50 ul of Chromogen Substrate per well Incubate 20 minutes Step 5 Add 50 ul of Stop Solution per well Read at 450 nm immediately Symbol Key ci Consult instructions for use Assay Template 12 11 10 Human Prekallikrein PK ELISA Kit Catalog No EK2111 1 Sample insert for reference use only Introduction Prekallikrein PK also known as Fletcher factor is an 88 kDa serine protease that mostly circulates as a complex with high molecular weight kininogen 1 Human plasma PK is synthesized as a precursor with a signal peptide of 19 amino acids and the mature circulating protein is a single chain polypeptide of 619 amino acids It participates in the surface dependent activation of blood coagulation fibrinolysis kinin generation and inflammation When
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