Human Serum Amyloid P Component ELISA Kit
Contents
1. Cross Reactivity Species Cross Reactivity Canine None Bovine None Monkey lt 5 Mouse None Rat None Swine None Rabbit None Human 100 e 10 FBS in culture media will not affect the assay Troubleshooting Causes Course of Action Low Precision Use of expired components e Check the expiration date listed before use e Do not interchange components from different lots Improper wash step e Check that the correct wash buffer is being used e Check that all wells are dry after aspiration e Check that the microplate washer is dispensing properly e If washing by pipette check for proper pipetting technique Splashing of reagents while loading wells e Pipette properly in a controlled and careful manner Inconsistent volumes loaded into wells e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance Insufficient mixing of reagent dilutions e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Improperly sealed microplate e Check the microplate pouch for proper sealing e Check that the microplate pouch has no punctures e Check that three desiccants are inside the microplate pouch prior to sealing a c oo n lt E e wn 2 a YU E mo w E o w Q x c gt Microplate was left unattended bet2. can be cut to fit the format of the individual assay Human Serum Amyloid P Standard Human SAP in a buffered protein base 60 ng lyophilized Biotinylated Human Serum Amyloid P Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against SAP 140 pul MIX Diluent Concentrate 10x A 10 fold concentrated buffered protein base 30 ml Wash Buffer Concentrate 20x A 20 fold concentrated buffered surfactant 30 ml 2 bottles Streptavidin Peroxidase Conjugate SP Conjugate A 100 fold concentrate 80 ul Chromogen Substrate A ready to use stabilized peroxidase chromogen substrate tetramethylbenzidine 8 ml Stop Solution A 0 5 N hydrochloric acid to stop the chromogen substrate reaction 12 ml Storage Condition Upon arrival immediately store components of the kit at recommended temperatures up to the expiration date Store SP Conjugate and Biotinylated Antibody at 20 C Store Microplate Diluent Concentrate 10x Wash Buffer Stop Solution and Chromogen Substrate at 2 8 C Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed May be stored for up to 30 days in a vacuum desiccator Diluent 1x may be stored for up to 30 days at 2 8 C Store Standard at 2 8 C before reconstituting with Diluent and at 20 C after reconstituting with Diluent Other Supplies Required Microplate reader capable of measuring absorbance at 450 nm Pipettes 1 20 ul 20
3. months Avoid repeated freeze thaw cycles Refer to Sample Dilution Guidelines below for further instruction Guidelines for Dilutions of 1 100 or Greater for reference only please follow the insert for specific dilution suggested 1 100 1 10000 4 ul sample 396 ul buffer 100x A 4ulsample 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 1 1000 1 100000 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 1 10 with reagent grade water Store for up to 30 days at 2 8 C e Standard Curve Reconstitute the 60 ng of Human Serum Amyloid P Standard with 3 ml of MIX Diluent to generate a 20 ng ml standard stock solution Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions P
4. 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 e Calculate the mean value of the duplicate or triplicate readings for each standard and sample e To generate a standard curve plot the graph using the standard concentrations on the x axis and the corresponding mean 450 nm absorbance on the y axis The best fit line can be determined by regression analysis using log log or four parameter logistic curve fit e Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor Typical Data e 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 10 00 P2 5 000 P3 2 500 P4 1 250 PS 0 625 P6 0 313 P7 0 156 P8 0 000 Sample Pool Normal Sodium Citrate Plasma 20000x Standard Curve e The curve is used for illustration only A standard curve should be generated each time the assay is performed Human Serum Amyloid P Standard Curve 1 0F OD 450 nm 0 1 1 1 107 10 fi 10 hSAP ng ml Reference Va
5. 200 ul 200 1000 ul and multiple channel 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 Dilute samples 1 20000 into MIX Diluent and assay 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 Dilute samples 1 20000 into MIX Diluent and assay The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e Cell Culture Supernatants Collect cell culture media and centrifuge at 3000 x g for 10 minutes at 4 C to remove debris The samples can be stored at 20 C or below Avoid repeated freeze thaw cycles e Urine Collect urine using sample pot Centrifuge samples at 800 x g for 10 minutes Dilute urine samples 1 2 into MIX Diluent and assay 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 Dilute samples 1 20 into MIX Diluent and assay The undiluted samples can be stored at 80 C for up to 3
6. lue e Normal human SAP plasma levels range from 20 to 50 ug ml e Human plasma and serum samples from healthy adults were tested n 40 On average SAP level was 37 ug ml Sample Average Value ug ml Human Pool Normal Plasma 30 Human Normal Plasma 39 Human Pool Normal Serum Performance Characteristics e The minimum detectable dose of SAP as calculated by 25D from the 43 mean of a zero standard was established to be 0 06 ng ml e Intra assay precision was determined by testing replicates of three plasma samples 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 20 CV Average CV Spiking Recovery e _ Recovery was determined by spiking two plasma samples with different serum AP concentrations Unspiked Sample ng ml Spike Recovery ng ml 1 5 2 8 2 9 104 1 3 3 0 4 3 4 4 102 7 5 8 8 7 6 86 1 5 5 0 4 8 96 3 0 6 5 6 7 103 7 5 11 0 10 4 95 Average Recovery 98 Expected Observed Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 10000 102 89 1 20000 99 97 1 40000 105 106
7. r at room temperature Improper pipetting e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance 10 e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Insufficient mixing of reagent dilutions References 1 2 3 Cathcart ES Shirahama T Cohen AS 1967 Biochim Biophy Acta 147 392 393 Jenny NS et al 2007 Arteriosclerosis Thrombosis and Vascular Biology 2007 27 352 Koenig W 2007 Arterioscler Thromb Vasc Biol 2007 27 698 700 Version 2 6R www assaypro com e mail Support assaypro com
8. rame 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 Serum Amyloid P Standard or sample per well Cover wells with a sealing tape and incubate for 2 hours Start the timer after the last addition e Wash five times with 200 ul of Wash Buffer manually Invert the plate each time and decant the contents hit 4 5 times on absorbent material to completely remove the liquid If using a machine wash six times with 300 ul of Wash Buffer and then invert the plate decanting the contents hit 4 5 times on absorbent material to completely remove the liquid e Add 50 ul of Biotinylated Human Serum Amyloid P Antibody to each well and incubate for 1 hour e Wash the microplate as described above e Add 50 ul of Streptavidin Peroxidase Conjugate per well and incubate for 30 minutes Turn on the microplate reader and set up the program in advance e Wash the microplate as described above e Add 50 ul of Chromogen Substrate per well and incubate for 15 minutes or till the optimal blue color density develop Gently tap the plate to ensure thorough mixing and break the bubbles in the well with pipette tip e Add 50 ul of Stop Solution to each well The color will change from blue to yellow e Read the absorbance on a microplate reader at a wavelength of 450 nm immediately If wavelength correction is available subtract
9. repare duplicate or triplicate standard points by serially diluting the standard stock solution 20 ng ml twofold with equal volume of MIX Diluent to produce 10 5 2 5 1 25 0 625 0 313 and 0 156 ng ml solutions MIX Diluent serves as the zero standard 0 ng ml Any remaining solution should be frozen at 20 C and used within 30 days Standard madden SAP Point ng ml P1 1 part Standard 20 ng ml 1 part MIX Diluent 10 00 1 part P1 1 part MIX Diluent 5 000 1 part P2 1 part MIX Diluent 2 500 Pa tpartP3 1 part MIX Diluent 1250 Ps 1partP5 1 part MIX Diluent 0313 prs MixDiuent o000 e Biotinylated Human Serum Amyloid P Antibody 50x Spin down the antibody briefly and dilute the desired amount of the antibody 1 50 with MIX Diluent Any remaining solution should be frozen 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 1 20 with reagent grade water e SP Conjugate 100x Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1 100 with MIX Diluent Any remaining solution should be frozen 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 f
10. ssaypro AssayMax Human Serum Amyloid P Component 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 15 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 Serum Amyloid P Component ELISA Kit Catalog No EA8201 1 Sample insert for reference use only Introduction Serum amyloid P component SAP Serum AP APCS a 25kDa pentameric protein is a normal plasma protein and a universal non fibrillar constituent of amyloid deposits 1 SAP is a pentraxin similar to C reactive protein 2 3 Principle of the Assay The AssayMax Human Serum Amyloid P Component ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human SAP in plasma serum urine CSF and cell cul
11. ture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures SAP in less than 4 hours A polyclonal antibody specific for SAP has been pre coated onto a 96 well microplate with removable strips SAP in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for SAP which is recognized by a streptavidin peroxidase 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 For Use In Diagnostic Procedures Prepare all reagents working diluent buffer wash buffer standard biotinylated 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 Human Serum Amyloid P Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human SAP Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes that
12. ween steps e Each step of the procedure should be performed uninterrupted Omission of step e Consult the provided procedure for complete list of steps Steps performed in incorrect order e Consult the provided procedure for the correct order Insufficient amount of reagents added to wells e Check pipette calibration e Check pipette for proper performance Wash step was skipped e Consult the provided procedure for all wash steps Improper wash buffer e Check that the correct wash buffer is being used Improper reagent preparation e Consult reagent preparation section for the correct dilutions of all reagents Insufficient or prolonged incubation periods e Consult the provided procedure for correct incubation time Deficient Standard Curve Fit Non optimal sample dilution 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 samples Contamination of reagents 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 o
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