AssayMaxTM Human Complement C8 ELISA Kit
Contents
1. 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 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 v A e Pipette properly in a controlled and careful manner a Inconsistent volumes 3 ARE A e Check pipette calibration 3 loaded into wells X o e Check pipette for proper performance l 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 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 amount2. minutes and assay Store samples at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e Milk Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 20 into EIA Diluent and assay The undiluted samples can be stored 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 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 EIA Diluent Concentrate 10x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute EIA Diluent Concentrate 1 10 with
3. 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 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 mixi
4. 0 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 C8 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 C8 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 bubb
5. MassaPro AssayMax Human Complement C8 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 C8 ELISA Kit Catalog No EC8101 1 Sample insert for reference use only Introduction Complement component 8 C8 is a 150 kDa complex composed of three genetically distinct subunits C8 alpha 64 kDa C8 beta 64 kDa and C8 gamma 22 kDa C8 alpha and C8 beta are highly homologous to each other and to C6 C7 and C9 It also contains a common membrane attack complex perforin MACPF domain C8 gamma has a lipocalin fold and shares no homology with any other complement protein 1 C8 plays a central role in membrane attack comp
6. aliva 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
7. les 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 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 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 40 00 P2 20 00 P3 10 00 P4 5 000 PS 2 500 P6 1 250 P7 0 625 P8 0 000 Sample Normal Sodium Citrate Plasma 10000x Standard Cu
8. lex MAC assembly by coordinating the interaction with complement proteins C5b 7 and the pore forming protein C9 on pathogen membranes It is also the first component to penetrate the lipid bilayer 2 3 Principal of the Assay The AssayMax Human Complement C8 ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of C8 in human plasma serum saliva milk and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures C8 in less than 4 hours A polyclonal antibody specific for C8 has been pre coated onto a 96 well microplate with removable strips C8 in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for C8 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 Spin down the SP conjugate vial and the biotinylated antib
9. ng of reagent dilutions References 1 Bubeck D et al 2011 J Mol Biol 405 2 325 330 2 Hadders MA et al 2007 Science 14 317 5844 1552 1554 3 Lovelace LL et al 2011 J Biol Chem 286 20 17585 17592 Version 1 2R1 10 Related products e EC1101 1 AssayMax Human Complement C1q ELISA Kit Plasma Serum Saliva Urine Milk and Cell Culture samples EC1102 1 AssayMax Human Complement Cir ELISA Kit Plasma Serum Urine Saliva Milk and Cell Culture samples EC1111 1 AssayMax Human Complement C1 ELISA Kit Plasma Serum 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 Milk Saliva 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 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 Milk Saliva and Cell Culture samples EC7101 1 AssayMax Human Complement C7 ELISA Kit Plasma Serum Urine Milk S
10. 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 C8 Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against C8 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 C8 Standard Human complement C8 in a buffered protein base 40 ng lyophilized Biotinylated Human Complement C8 Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against C8 140 ul EIA 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 be returned to the foil pouch
11. reagent grade water Store for up to 30 days at 2 8 C e Standard Curve Reconstitute the 40 ng of Human Complement C8 Standard with 1 ml of EIA Diluent to generate a 40 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 1 2 with equal volume of EIA Diluent to produce 20 10 5 2 5 1 25 and 0 625 ng ml solutions EIA Diluent serves as the zero standard 0 ng ml Any remaining solution should be frozen at 20 C and used within the next 30 days Standard Point Dilution C8 ng ml P1 1 part Standard 40 ng ml 40 00 1 part P1 1 part EIA Diluent 20 00 1 part P2 1 part EIA Diluent 10 00 Pa 1partP3 1 part EIA Diluent 5 000 P6 1partP5 1 part EIA Diluent 1250 LPs O FlaDiluvent f 000 e Biotinylated Human Complement C8 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 EIA Diluent Any remaining solution should be frozen at 2
12. rve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Human Complement C8 Standard Curve OD 450 nm 1 1 1 1 0 1 1 10 100 hC8 ng ml Performance Characteristics e The minimum detectable dose of C8 as calculated by 25D from the mean ofa zero standard was established to be 0 4 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 Sample 1 2 3 1 2 3 n 20 20 20 20 20 20 CV Average CV Recovery Standard Added Value 1 5 15 ng ml Recovery 88 111 Average Recovery 103 Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 5000 96 94 1 10000 100 99 1 20000 106 107 Cross Reactivity Species Cross Reactivity Canine None Monkey lt 20 Mouse None Rat 1 Swine None Rabbit None Bovine None Proteins Cross Reactivity Complement C1 None Complement C3 None Complement C4 None Complement C5 None Complement C6 None Complement C7 1 Complement C8 100 Complement C9 1
13. with the desiccant packs and resealed May be stored for upto 30 daysina 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 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 10000 into EIA 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 10000 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 Saliva Collect saliva using sample tube Centrifuge samples at 800 x g for 10
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