AssayMax Human Complement C1 ELISA Kit
Contents
1. 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 12 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 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 t2. 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 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 EC3301 1 Human Complement C3b ELISA Kit Plasma Serum Urine Milk Saliva CSF and Cell Culture samples e EC2102 1 Human Complement C4 ELISA Kit Plasma and Serum 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 e 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 e EC9101 1 Human Complement C9 ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples www assaypro com e e mail Support assaypro com
3. DA 15SAYPRO AssayMax Human Complement C1 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 12 minutes Step 5 Add 50 ul of Stop Solution per well Read at 450 nm immediately Symbol Key BE Consult instructions for use Assay Template 12 11 10 Human Complement C1 ELISA Kit Catalog No EC1111 1 Sample insert for reference use only Introduction Complement component C1 C1 is a calcium dependent serine protease complex with an approximate mass of 790 kDa and acts as the first component of the classical complement pathway C1 is formed from the association of a recognition protein C1q and two catalytic subunits C1r and Cis respectively 1 2 The globular heads of the C1q bind to the Fc fragment of IgM or IgG on the surface of a pathogen resulting in the activation of C1r The activa
4. 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 40000 into MIX 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 Dilute samples 1 40000 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 supernatants and assay Store the remaining samples at 20 C or below Avoid repeated freeze thaw cycles e Urine Collect urine using sample tube Centrifuge samples at 800 x g for 10 minutes and as
5. e 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 256 ng of Human Complement C1 Standard with 4 ml of MIX Diluent to generate a 64 ng ml standard stock solution Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions The standard stock solution 64 ng ml should be further diluted 1 8 with MIX Diluent to produce an 8 ng ml standard working solution Prepare duplicate or triplicate standard points by serially diluting the standard working solution 8 ng ml 1 2 with MIX Diluent to produce 4 2 1 0 5 0 25 and 0 125 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 C1 Point ng ml 1 part Standard 64 ng ml 7 parts MIX Diluent 8 000 1 part P1 1 part MIX Diluent 4 000 1 part P2 1 part MIX Diluent 2 000 Pa 1partP3 1 part MIX Diluent 1000 Ps 1partP5 1 part MIX Diluent 0 250 la MiX Diluent 0000 e Biotinylated Human Complement C1 Antibody 100x Spin down the antibody briefly and dilute the desired amount of the antibody 1 100 with MIX Diluent Any remaining solution should be frozen at 20 C e Wash Buffer Concentrate 20x If crystals have formed i
6. he assay is performed Human Complement C1 Standard Curve OD 450 nm 0 1 1 1 1 107 10 10 hC1 ng ml Performance Characteristics e The minimum detectable dose of C1 is typically 0 1 ng ml e Intra assay and inter assay coefficients of variation were 4 7 and 7 0 respectively Linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 20000 88 93 1 40000 99 98 1 80000 104 105 Recovery Standard Added Value 0 3 5 ng ml Recovery 84 115 Average Recovery 98 Cross Reactivity Species Cross Reactivity Monkey lt 5 Mouse None Rat None Swine None Canine lt 5 Bovine None Human 100 Proteins Cross Reactivity Complement C1 100 Complement C2 None Complement C3 None Complement C4 None Complement C5 None Complement C6 None Complement C7 None Complement C8 None Complement C9 None Reference Value e On average normal human complement C1 plasma level is 75 ug ml References 1 Arlaud GJ et al 2002 Mol Immunol 39 383 394 2 Fabian DG et al 2006 J Immunol 176 2950 2957 3 Duncan AR and Winter G 1988 Nature 332 6166 738 740 4 Volanakis JE 2002 Curr Top Microbiol Immunol 266 41 56 Version 1 1R3 Related Products EC1111 1 Human Complement C1 ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples EC1102 1 Human
7. lution is an acidic solution e The kit should not be used beyond the expiration date Reagents e Human Complement C1 Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human C1 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 C1 Standard Human Complement C1 in a buffered protein base 256 ng lyophilized e Biotinylated Human Complement C1 Antibody 100x A 100 fold biotinylated polyclonal antibody against human complement C1 80 pul 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 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
8. n 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 C1 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 Complement C1 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
9. say Store samples 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 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 and assay Store samples at 20 C or below 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 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 befor
10. ted Cir is able to activate C1s which in turn activates C2 and C4 leading to the production of the C4b C2a form of C3 convertase 3 4 Principle of the Assay The AssayMax Human Complement C1 ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of C1 in human plasma serum saliva urine milk and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures C1 in less than 4 hours A polyclonal antibody specific for C1 has been pre coated onto a 96 well microplate with removable strips C1 in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for C1 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 So
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