Human Complement C4BP ELISA Kit
Contents
1. 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 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 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 the assay is performed Human C4BP Standard Curve OD 450 nm 0 1F 1 10 1002. hC4BP ng ml Performance Characteristics e The minimum detectable dose of complement C4BP is typically 0 9 ng ml Intra assay and inter assay coefficients of variation were 5 0 and 7 1 respectively Linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 20000 90 91 1 40000 98 98 1 80000 107 106 Average Percentage of Expected Value Sample Dilution Milk 1 400 92 1 800 99 1 1600 Recovery Standard Added Value 105 1 87 30 ng ml Recovery 85 111 97 Average Recovery Cross Reactivity Species Cross Reactivity Monkey None Mouse None Rat None Swine None Canine None Bovine None Human 100 Proteins Cross Reactivity Complement C1 None Complement C3 None Complement C4BP 100 Complement C4 None Complement C5 None Complement C6 None Complement C7 None Complement C8 None Complement C9 Reference Value None e On average normal human C4BP plasma level is 200 ug ml References 1 2 3 4 5 6 Gigli et al 1979 Proc Natl Acad Sci USA 76 6596 6600 Hillarp A and Dahlb ck B 1990 Proc Natl Acad Sci USA 87 3 1183 1187 S nchez Pernaute O et al 2006 Ann Rheum Dis 65 10 1279 1285 Trouw LA et al 2
3. 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 C4BP 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 C4BP 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
4. 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 Complement C4BP Standard with 1 ml of MIX Diluent to generate a 60 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 60 ng ml 1 2 with MIX Diluent to produce 30 15 7 5 3 75 1 875 and 0 938 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 Dilution C4BP Point ng ml P1 1 part Standard 60 ng ml 60 00 1 part P1 1 part MIX Diluent 30 00 1 part P2 1 part MIX Diluent 15 00 Pa 1partP3 1 part MIX Diluent 7500 PS 1partP5 1 part MIX Diluent 1875 Ps 5 MxDlee 0 000 e Biotinylated Human Complement C4BP 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
5. 008 Mol Immunol 45 13 3649 3660 Holmberg MT et al 2001 J Immunol 167 2 935 939 Okroj M et al 2008 Mol Immunol 45 1 169 179 Version 1 0 Related products EC1102 1 AssayMax Human Complement Cir ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples EC1101 1 AssayMax Human Complement C1q ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples EC1111 1 AssayMax Human Complement C1 ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples EC2001 1 AssayMax Human Complement C2 ELISA Kit Plasma Serum Saliva and Cell Culture samples EC2101 1 AssayMax Human Complement C3 ELISA Kit Plasma and Serum samples EC3201 1 AssayMax Human Complement C3 ELISA Kit Urine Milk Saliva and Cell Culture samples EC3301 1 AssayMax Human Complement C3b ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples EC2102 1 AssayMax Human Complement C4 ELISA Kit Plasma and Serum samples EC3202 1 AssayMax Human Complement C4 ELISA Kit Urine Milk Saliva and Cell Culture samples EC5101 1 AssayMax Human Complement C5 ELISA Kit Plasma Serum Milk Saliva and Cell Culture samples EC6101 1 AssayMax Human Complement C6 ELISA Kit Plasma Serum Urine Saliva Milk and Cell Culture samples EC7101 1 AssayMax Human Complement C7 ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples EC8101 1 AssayMax Human Complement C8 ELISA Kit Plasma Serum Urine M
6. ent 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 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 Saliva Collect saliva using sample pot Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 80 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 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 1 800 into MIX 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 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 Reagent Preparation e Freshly dilute all reagents and bring
7. entical 70 kDa alpha chains each containing a binding site for the complement protein C4b and a unique 45 kDa beta chain which contains a binding site for the vitamin K dependent protein S 2 C4BP was overexpressed in the synovial membranes of patients with rheumatoid arthritis 3 It was detected in amyloid beta plaques and on apoptotic cells in Alzheimer s disease brain 4 C4BP could directly bind to ovarian tumor cell lines and was associated with non small cell lung cancer 5 6 Principle of the Assay The AssayMax Human Complement C4BP ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human complement C4BP in plasma serum urine milk saliva CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures human complement C4BP in less than 4 hours A polyclonal antibody specific for human complement C4BP has been pre coated onto a 96 well microplate with removable strips Complement C4BP in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for complement C4BP 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 Prepare all reagents working diluent buffer wash buffer standard biotinylated antibody and SP con
8. ilk Saliva and Cell Culture samples EC9101 1 AssayMax Human Complement C9 ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples www assaypro com e e mail Support assaypro com
9. jugate 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 protocol 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 This kit is for research use only e The kit should not be used beyond the expiration date Reagents e Human Complement C4BP Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human complement C4BP 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 C4BP Standard Human complement C4BP ina buffered protein base 60 ng lyophilized e Biotinylated Human Complement C4BP Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against complement C4BP 140 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
10. 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 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 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 40000 into MIX Dilu
11. yssaypro AssayMax Human Complement C4BP 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 BE Consult instructions for use Assay Template 12 11 10 Human Complement C4b Binding Protein C4BP ELISA Kit Catalog No EC2202 1 Sample protocol for reference use only Introduction Complement component C4b binding protein C4BP regulates the complement system by accelerating the decay of the complement component C3 convertase and by acting as a cofactor to the serine protease factor in the degradation of C4b 1 C4BP is a high molecular mass 570 kDa glycoprotein and is present in plasma in various isoforms with different alpha beta composition The major form of C4BP is composed of seven id
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