AssayMaxTM Human Complement C5 ELISA Kit
Contents
1. 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 32 ng of Human Complement C5 Standard with 1 6 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 Prepare duplicate or triplicate standard points by serially diluting the standard stock solution 20 ng ml 1 2 with MIX Diluent to produce 10 5 2 5 1 25 0 625 and 0 313 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 Point Dilution C5 ng ml P1 1 part Standard 20 ng ml 20 00 1 part P1 1 part MIX Diluent 10 00 1 part P2 1 part MIX Diluent 5 000 a ae 1 part P3 1 part MIX Diluent 2 500 Ps 1partPS 1partMiXDiluent 0625 IP gt gt MxDlere 0 000 e Biotinylated Human Complement C5 Antibody 50x Spin down the2. Biotinylated Human Complement C5 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 develops 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 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 four parameter or log log logistic curve fit e Determine the unknown sample con
3. 24 1682 1688 6 Patel SN et al 2008 J Exp Med 205 1133 1143 7 Copland DA et al 2010 Clin Exp Immunol 159 3 303 314 Version 2 3R Related Products EC1102 1 AssayMax Human Complement Cir ELISA Kit Plasma Serum Urine Milk Saliva CSF and Cell Culture samples e EC1101 1 AssayMax Human Complement C1q ELISA Kit Plasma Serum Urine Milk Saliva CSF and Cell Culture samples EC1111 1 AssayMax Human Complement C1 ELISA Kit Plasma Serum Urine Milk Saliva CSF and Cell Culture samples e EC2001 1 AssayMax Human Complement C2 ELISA Kit Plasma and Serum samples e EC2101 1 AssayMax Human Complement C3 ELISA Kit Plasma and Serum samples EC3201 1 AssayMax Human Complement C3 ELISA Kit Urine Milk Saliva CSF and Cell Culture samples EC3301 1 AssayMax Human Complement C3b ELISA Kit Plasma Serum Urine Milk Saliva CSF and Cell Culture samples e EC2102 1 AssayMax Human Complement C4 ELISA Kit Plasma and Serum samples EC3202 1 AssayMax Human Complement C4 ELISA Kit Urine Milk Saliva CSF and Cell Culture samples EC2202 1 AssayMax Human Complement C4BP ELISA Kit Plasma Serum Urine Milk Saliva CSF and Cell Culture samples EC6101 1 AssayMax Human Complement C6 ELISA Kit Plasma Serum Urine Milk Saliva CSF and Cell Culture samples EC7101 1 AssayMax Human Complement C7 ELISA Kit Plasma Serum Urine Milk Saliva CSF and Cell Culture samples EC8101 1 AssayMax Hu
4. 3 The C5a mediated early pro inflammatory responses include sepsis systemic lupus erythamatosis and cerebral malaria 4 6 The larger C5b interact with complement components C6 C7 C8 and C9 to form a membrane attack complex C5b 9 which is involved in cell apoptosis cell activation and production of proinflammatory mediators 7 Principle of the Assay The AssayMax Human Complement C5 ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of C5 in human plasma serum saliva milk CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures C5 in less than 4 hours A polyclonal antibody specific for C5 has been pre coated onto a 96 well microplate with removable strips C5 in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for C5 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 suggeste
5. 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 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 Complement C5 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
6. centration 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 20 00 P2 10 00 P3 5 000 P4 2 500 P5 1 250 P6 0 625 P7 0 313 P8 0 000 Sample Pool Normal Sodium Citrate Plasma 20000x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Human Complement C5 Standard Curve OD 450 nm 10 100 10 hC5 ng ml Reference Value e Normal human complement C5 plasma levels range from 45 to 115 ug ml e Human plasma and serum samples from healthy adults were tested n 40 On average complement C5 level was 65 ug ml Sample Average Value ug ml Human Pool Normal Plasma 59 Human Normal Plasma 56 Human Pool Normal Serum 72 Performance Characteristics e The minimum detectable dose of human complement C5 as calculated by 2SD from the mean of a zero standard was established to be 0 1 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 Sa
7. d 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 Complement C5 Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human C5 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 Complement C5 Standard Human C5 in a buffered protein base 32 ng lyophilized e Biotinylated Human Complement C5 Antibody 50x A 50 fold biotinylated polyclonal antibody against human C5 120 ul 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 Streptavidin Peroxidase Conjugate SP Conjugate A 100 fold concentrate 80 ul e Chromogen Substrate A ready to use stabilized peroxidase chromogen substrate tetramethylbenzidine 8 ml e Stop Solution A 0 5 N hydrochloric acid to stop the chromogen substrate reaction 12 ml 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 Sto
8. 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 Low Precision 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 Microplate was left unattended between 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 Insuf
9. ficient or prolonged incubation periods Unexpectedly Low or High Signal Intensity e Consult the provided procedure for correct incubation time 10 Deficient Standard Curve Fit further and repeat the assay further and repeat the assay e Sandwich ELISA If samples generate OD values higher than the highest standard point P1 dilute samples Non optimal sample e Competitive ELISA If samples generate OD values lower dilution than the highest standard point P1 dilute samples e User should determine the optimal dilution factor for samples Contamination of e A new tip must be used for each addition of different reagents samples or reagents during the assay procedure Contents of wells e Verify that the sealing film is firmly in place before placing evaporate the assay in the incubator or at room temperature Improper pipetting e Check pipette calibration e Check pipette for proper performance e Pipette properly in a controlled and careful manner Insufficient mixing of at a reconstitution reagent dilutions e Thoroughly mix dilutions e Thoroughly agitate the lyophilized components after References 1 Sandoval A et al 2000 J Immunol 165 2 1066 1073 2 Ward PA 2009 J Mol Med 87 375 378 3 Gerard NP and Gerard C 1991 Nature 349 6310 614 617 4 Chenoweth DE and Hugli TE 1978 Proc Natl Acad Sci U S A 75 8 3943 3947 5 Jacob A et al 2010 FASEB J
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11. mple 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 complement C5 concentrations Unspiked Sample ng ml Spike Recovery ng ml 1 0 4 5 5 1 113 3 5 4 0 7 5 77 103 8 0 11 5 11 4 99 1 0 8 4 8 6 102 4 0 11 4 10 7 94 8 0 15 4 14 6 95 Average Recovery 101 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 105 98 1 20000 97 97 1 40000 101 104 Cross Reactivity Species Cross Reactivity Monkey lt 50 Mouse None Rat None Swine None Canine None Bovine None Rabbit None Human 100 Proteins Cross Reactivity Complement C1 None Complement C3 None Complement C4 None Complement C5 100 Complement C6 None Complement C7 None Complement C8 None Complement C9 None Troubleshooting Issue Causes Course of Action Use of expired e Check the expiration date listed before use components 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
12. re 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 Diluent 1x may be stored for up to 30 days at 2 8 C 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 Dilute samples 1 20000 into MIX Diluent and assay The undiluted samples at 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 Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris Collect s
13. upernatants and assay Store the remaining samples at 20 C or below Avoid repeated freeze thaw cycles e Milk Collect milk using sample tube Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 40 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 Saliva Collect saliva using sample tube Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 8 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 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 A 4ulsample 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 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
14. yssarpro AssayMax Human Complement C5 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 Complement C5 ELISA Kit Catalog No EC5101 1 Sample insert for reference use only Introduction Human complement component 5 C5 is the fifth component of the complement system C5 has a molecular weight of about 195 kDa and matures into heterodimers with alpha and beta chains of 120 kDa and 75 kDa respectively 1 Upon complement activation C5 is cleaved into a small C5a and larger C5b polypeptides by C5 convertase The potent pro inflammatory anaphylatoxin C5a binds to receptors C5aR and C5L2 to initiate acute inflammatory responses 2
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