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AssayMaxTM Human Complement C4 ELISA Kit

1. Recovery Standard Added Value 0 3 3 0 ng ml Recovery 88 109 Average Recovery 98 Linearity Average Percentage of Expected Value Sample Dilution Milk 1 4000 98 1 8000 99 1 16000 104 Cross Reactivity Species Cross Reactivity Canine lt 5 Bovine None Monkey lt 10 Mouse None Rat None Swine None Rabbit 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 c technique 2 Splashing of reagents e Pipette properly in a controlled and careful manner 8 while loading wells L e Pipette properly in a controlled and careful manner a Inconsistent volumes a TN e Check pipette calibration 3 loaded into wells E 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 proper sealing e Check that the microplate pouch has no punctures
2. e Check that three desiccants are inside the microplate pouch prior to sealing Unexpectedly Low or High Signal Intensity 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 Insufficient or prolonged incubation periods e Consult the provided procedure for correct incubation time 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 during the
3. and Cell Culture samples e EC2102 1 AssayMax Human Complement C4 ELISA Kit Plasma and Serum samples EC2202 1 AssayMax Human Complement C4BP ELISA Kit Plasma Serum Urine Saliva Milk CSF 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 Milk Saliva Urine and Cell Culture samples EC7101 1 AssayMax Human Complement C7 ELISA Kit Plasma Serum Milk Saliva Urine and Cell Culture samples EC8101 1 AssayMax Human Complement C8 ELISA Kit Plasma Serum Urine Milk 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
4. 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 20 200 ul 200 1000 ul and multiple channel e Deionized or distilled reagent grade water Sample Collection Preparation and Storage 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 1 200 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 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 Milk dilution is suggested at 1 8000 into MIX Diluent however the user should determine the optimal dilution factor The undiluted samples can be
5. contraction 4 C4b the major activation product is an essential subunit of the C3 and C5 convertases of the classical complement pathway Principle of the Assay The AssayMax Human Complement C4 ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human complement C4 in urine milk saliva CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures human complement C4 in less than 4 hours A polyclonal antibody specific for human complement C4 has been pre coated onto a 96 well microplate with removable strips Complement C4 in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for complement C4 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 e 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 antib
6. 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 1 500 into MIX Diluent and assay If necessary further dilute samples 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 4 ul sample 396 ul buffer 100x 100 fold dilution Assuming the needed volume is less 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 _ MIX Diluent Concentrate 10x If crystals have formed in the concentrate mix gently until the crystals have comp
7. MA assarpro AssayMax Human Complement C4 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 3 Consult instructions for use Assay Template 12 11 10 Human Complement C4 ELISA Kit Catalog No EC3202 1 Sample insert for reference use only Introduction Complement component 4 C4 plays a key role in the activation of the classical complement pathway C4 is synthesized as a single chain precursor molecule 200 kDa but processed to the three chain disulphide linked structure with alpha 93 kDa beta 78 kDa and gamma 33 kDa chains prior to secretion 1 3 After activation by C1s C4 is processed to C4a and C4b C4a anaphylatoxin is a mediator of local inflammation and induces smooth muscle
8. 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 Deficient Standard Curve Fit e Check pipette for proper performance e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Insufficient mixing of reagent dilutions References 1 Roos MH etal 1982 Nature 298 5877 854 856 2 Miura N etal 1987 J Biol Chem 262 15 7298 7305 3 Belt KT etal 1984 Cell 36 4 907 914 4 Moon KE etal 1981 J Biol Chem 256 16 8685 8692 Version 2 4R Related Products e EC1101 1 AssayMax Human Complement C1q ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples EC1102 1 AssayMax Human Complement C1r 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 e EC2101 1 AssayMax Human Complement C3 ELISA Kit Plasma and Serum samples EC3201 1 AssayMax Human Complement C3 ELISA Kit Urine Saliva Milk and Cell Culture samples EC3301 1 AssayMax Human Complement C3b ELISA Kit Plasma Serum Urine Milk Saliva CSF
9. ith 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 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 Human Complement C4 Standard Curve 10 0 OD 450 nm 0 1 0 01 0 10 1 00 10 00 1100 00 hC4 ng ml Performance Characteristics e The minimum detectable dose of C4 is typically 0 07 ng ml e Intra assay and inter assay coefficients of variation were 4 8 and 7 1 respectively
10. letely 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 90 ng of Human Complement C4 Standard with 4 5 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 4 with MIX Diluent to produce 5 1 25 0 3125 and 0 0781 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 Complement C4 Point ng ml P1 1 part Standard 20 ng ml 20 000 1 part P1 3 parts MIX Diluent 5 0000 Pa 1partP3 3 parts MIX Diluent 0315 LPs MWXDilent 0000 e Biotinylated Human Complement C4 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 Pr
11. ocedure 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 C4 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 C4 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 w
12. ody vial before opening and using contents The Stop Solution is an acidic solution The kit should not be used beyond the expiration date Reagents Human Complement C4 Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human complement C4 Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes that can be cut to fit the format of the individual assay Human Complement C4 Standard Human complement C4 in a buffered protein base 90 ng lyophilized Biotinylated Human Complement C4 Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against complement C4 120 ul 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

AssayMaxTM Human Complement C4 ELISA Kit

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