AssayMax Human Complement C3b ELISA Kit
Contents
1. Monkey None Mouse None Rat None Swine None Canine None Bovine None Human 100 Proteins Cross Reactivity Complement C1 None Complement C1q None Complement Cir None Complement C1s None Complement C3 80 Complement C3b 100 Complement C4 None Complement C5 None Complement C6 None Complement C7 None Complement C8 None Complement C9 Troubleshooting None 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 e Check that the correct wash buffer is being used e Check that all wells are dry after aspiration Improper wash step e Check that the microplate washer is dispensing properly e If washing by pipette check for proper pipetting E technique o Splashing of reagents e Pipette properly in a controlled and careful manner 8 while loading wells L a e Pipette properly in a controlled and careful manner a Inconsistent volumes RES A e Check pipette calibration 3 loaded into wells R o 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 proper2. 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 e Each step of the procedure should be performed T unattended between uninterrupted E steps a Omission of step e Consult the provided procedure for complete list of steps Steps performed in e Consult the provided procedure for the correct order zZ incorrect order 5 z Insufficient amount of e Check pipette calibration 2 3 reagents added to e Check pipette for proper performance 3 g wells zs Wash step was skipped e Consult the provided procedure for all wash steps 3 Improper wash buffer e Check that the correct wash buffer is being used E Improper reagent e Consult reagent preparation section for the correct Q preparation dilutions of all reagents a Insufficient or e Consult the provided procedure for correct incubation gt prolonged incubation time periods e Sandwich ELISA If samples generate OD values higher than the highest standard point P1 dilute samples further and repeat the assay Non optimal sample e Competitive ELISA If samples generate OD values lower dilution 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 e A new tip must be used for each addition of different reagents samples or reagents du
3. ed within 30 days C3b ng ml P1 1 part Standard 10 ng ml 10 00 1 part P1 1 part EIA Diluent 5 000 1 part P2 1 part EIA Diluent 2 500 pa 1 part P3 1 part EIA Diluent 1 250 Ps ipartPS ipartFlADiluent 0 513 ls iluent_ 0 000 e Biotinylated Complement C3b Antibody 50x Spin down the antibody briefly and dilute the desired amount of the antibody 1 50 with EIA 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 ElA 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 C3b Standard or sample per well Cover wells with a sealing tape and incubate for 2 hours Start the timer after the last sample addition e Wash five times with 200 ul of Was
4. ess than or equal to 400 ul 4 ul sample 396 ul buffer 100x 4 ul of A 396 ul buffer 100x 10000 fold dilution Assuming the needed volume is less than or equal to 400 ul 1 1000 1 100000 4 ul sample 396 ul buffer 100x 24 ul of A 216 ul buffer 10x 1000 fold dilution Assuming the needed volume is less than or equal to 240 ul 4 ul sample 396 ul buffer 100x 4 ul of A 396 ul buffer 100x 24 ul of B 216 ul buffer 10x 100000 fold dilution Assuming the needed volume is less than or equal to 240 ul Reagent Preparation e Freshly dilute all reagents and bring all reagents to room temperature before use e EIA Diluent Concentrate 10x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute the EIA Diluent Concentrate 1 10 with reagent grade water Store for up to 30 days at 2 8 C e Standard Curve Reconstitute the 20 ng of Human Complement C3b Standard with 2 ml of EIA Diluent to generate a 10 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 10 ng ml 1 2 with EIA Diluent to produce 5 2 5 1 25 0 625 0 313 and 0 156 ng ml solutions EIA Diluent serves as the zero standard 0 ng ml Any remaining solution Standard Point Dilution should be frozen at 20 C and us
5. h 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 Complement C3b Antibody to each well and incubate for 1 hour e Wash the microplate as described above e Add 50 ul of Streptavidin Peroxidase Conjugate to each 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 10 minutes or till the optimal blue color density develops Gently tap 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 the 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 readi
6. ngs 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 OD 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 Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed H C3b Standard Curve 1 0F OD4so nm 0 1 oorl AN 107 10 10 H C3b ng ml Reference Value e Normal human C3b plasma levels range from 0 3 to 1 6 mg ml Performance Characteristics e The minimum detectable dose of complement C3b is typically 0 1 ng ml e Intra assay and inter assay coefficients of variation were 5 1 and 7 2 respectively Recovery Standard Added Value 0 25 2 5 ng ml Recovery 83 112 Average Recovery 97 5 Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 200000 91 92 1 400000 97 98 1 800000 106 108 Average Percentage of Expected Value Sample Dilution Milk 1 2000 96 1 4000 99 1 8000 103 Cross Reactivity Species Cross Reactivity
7. nts e The Stop Solution is an acidic solution e The kit should not be used beyond the expiration date Reagents e Human Complement C3b Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human complement C3b 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 C3b Standard Human complement C3b in a buffered protein base 20 ng lyophilized e Biotinylated Complement C3b Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against complement C3b 120 pul e EIA 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 _ Store Microplate Diluent Concentrate 10x Wash Buffer Stop Solution and Chromogen Substrate at 2 8 C e Unused microplate wells may be
8. returned to the foil pouch with the desiccants and resealed May be stored for up to 30 days in a 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 400000 into EIA Diluent and assay The undiluted samples can be stored at 20 C or below for upto 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 400000 into ElA 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 Urine Collect urine using sample pot Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 4 into ElA Diluent and assay If necessary dilute samples within the range of 1 2 to 1 8 Depending on application needs user should determine optimal dilu
9. ring 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 e Pipette properly in a controlled and careful manner Improper pipetting e Check pipette calibration e Check pipette for proper performance Deficient Standard Curve Fit e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Insufficient mixing of reagent dilutions References 1 Morris KM et al 1982 Science 215 399 400 2 de Bruijn MH and Fey GH 1985 Proc Natl Acad Sci U S A 82 3 708 712 3 Hugli TE 1975 J Biol Chem 250 21 8293 8301 4 Wiesmann C et al 2006 Nature 444 7116 217 220 5 Janssen BJ et al 2006 Nature 444 7116 213 216 Version 1 2R Related products EC1102 1 Human Complement Cir ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples EC1101 1 Human Complement C1q ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples EC1111 1 Human Complement C1 ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples EC2001 1 Human Complement C2 ELISA Kit Plasma Serum Saliva and Cell Culture samples e EC2101 1 Human Complement C3 ELISA Kit Plasma and Serum samples EC3201 1 Human Complement C3 ELISA Kit Urine Milk Saliva and Cell Culture samples e EC2102 1 Human Complement C4 ELISA Kit Plasma and Se
10. rum samples EC3202 1 Human Complement C4 ELISA Kit Urine Milk Saliva and Cell Culture samples EC2202 1 Human Complement C4BP ELISA Kit Plasma Serum Urine Milk Saliva CSF and Cell Culture samples EC5101 1 Human Complement C5 ELISA Kit Plasma Serum Milk Saliva and Cell Culture samples EC6101 1 Human Complement C6 ELISA Kit Plasma Serum Urine Saliva Milk and Cell Culture samples EC7101 1 Human Complement C7 ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples EC8101 1 Human Complement C8 ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples EC9101 1 Human Complement C9 ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples www assaypro com e mail Support assaypro com
11. tions 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 pot Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 100 into ElA 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 supernatants and assay Samples can be stored 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 and assay Milk dilution is suggested at 1 4000 into ElA Diluent however the user should determine the optimal dilution factor 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 1000 into ElA 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 4 ul sample 396 ul buffer 100x 100 fold dilution Assuming the needed volume is l
12. tor can bind covalently to pathogen surfaces to promote phagocytosis 4 5 Principle of the Assay The AssayMax Human Complement C3b ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human complement C3b in plasma serum urine milk saliva CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures human complement C3b in less than 4 hours A polyclonal antibody specific for human complement C3b has been pre coated onto a 96 well microplate with removable strips Complement C3b in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for complement C3b 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 conte
13. yssarpro AssayMax Human Complement C3b 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 10 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 C3b ELISA Kit Catalog No EC3301 1 Sample insert for reference use only Introduction Complement component 3 C3 plays a central role in all three complement activation pathways The C3 precursor contains 1663 amino acids and has a molecular weight of about 180 kDa 1 Human C3 has 77 identity to mouse C3 at the amino acid level 2 C3 is cleaved by C3 convertase into two activated fragments C3a and C3b The anaphylatoxin C3a is a vasoactive peptide and a mediator of local inflammatory process 3 The C3b in complex with recep
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