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Human α-Fetoprotein ELISA Kit

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1. e assavPRo AssayMax Human alpha Fetoprotein 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 8 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 alpha Fetoprotein ELISA Kit Catalog No EF6011 1 Sample insert for reference use only Introduction Alpha fetoprotein AFP alpha fetoglobulin is a fetal specific glycoprotein with a molecular weight of approximately 70 kDa It is expressed in the embryonic liver by cells of the vitelline sac and by the fetal intestinal tract in the first trimester of pregnancy 1 After birth the synthesis of alpha fetoprotein decreases rapidly In adults AFP level is low but detectable 2 Alpha fetoprotein has no known function in healthy adults High l
2. for proper performance e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Low Precision Inconsistent volumes loaded into wells Insufficient mixing of reagent 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 amount of reagents added to wells e Check pipette calibration e Check pipette for proper performance Wash step was skipped Improper wash buffer e Consult the provided procedure for all wash steps 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 Deficient Standard Curve Fit Non optimal sample dilution e Sandwich ELISA If samples generate OD values higher than the highest st
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4. al to completely remove the liquid e Add 50 ul of Biotinylated Human alpha Fetoprotein Antibody to each well Gently tap plate to thoroughly coat the wells Break any bubbles that may have formed Cover wells with a sealing tape and incubate for 1 hour e Wash the microplate as described above e Add 50 ul of SP Conjugate to each well Gently tap plate to thoroughly coat the wells Break any bubbles that may have formed Cover wells with a sealing tape 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 to each well Gently tap plate to thoroughly coat the wells Break any bubbles that may have formed Incubate for 8 minutes or until the optimal blue color density develops e Add 50 ul of Stop Solution to each well The color will change from blue to yellow Gently tap plate to ensure thorough mixing Break any bubbles that may have formed 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 tri
5. andard point P1 dilute samples further and repeat the assay e Competitive ELISA If samples generate OD values lower 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 Anew 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 Improper pipetting e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance Insufficient mixing of reagent dilutions e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions 10 References 1 2 3 4 5 6 7 8 9 Asmaa Gomaa et al 2009 World J Gastroenterol 15 11 1301 1314 Blohm ME et al 1998 Pediatr Hematol Oncol 15 2 135 42 Stewart Sell 2008 Tumour Biol 29 3 161 180 Alberto Maringhini et al 1988 Digestive Diseases and Sciences 33 1 0163 2116 Chen L et al 2010 Ann Surg Oncol DOI 10 1245 s10434 010 1038 8 Hiroshima K et al 2002 Pathol Int 52 46 53 Chen J et al 2003 Dig Dis 21 357 362 J Kelleher et al 1974 Gut 15 401 403 Pauniaho SL et al 2010 Tumour Biol DOI 10 1007 s13277 010 0026 8
6. blished to be 0 55 ng ml e Intra assay precision was determined by testing three plasma samples twenty times 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 96 Average CV 96 Recovery Standard Added Value 2 5 20 ng ml Recovery 96 89 11496 Average Recovery Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1x 9596 9396 2x 10296 9796 4x 10696 10696 Cross Reactivity Species Cross Reactivity Bovine 3096 Mouse Rat Swine Rabbit Canine Monkey Troubleshooting Issue Causes Course of Action Use of improper 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 empty after aspiration Improper wash step e Check that the microplate washer is dispensing properly e If washing by pipette check for proper pipetting technique Splashing of reagents e Pipette properly in a controlled and careful manner while loading wells e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette
7. evels of alpha fetoprotein in an adult individual may be associated with a hepatocellular carcinoma HCC malignant tumor of the liver 1 3 Thus the concentration of alpha fetoprotein in serum can be measured as a first step in HCC diagnosis 4 5 Moreover the elevated level of alpha fetoprotein has been observed in lung cancer 6 gastric cancer 7 8 yolk sac tumor and adenocarcinoma 9 Principle of the Assay The AssayMax Human alpha Fetoprotein ELISA Enzyme Linked Immunosorbent Assay Kit is designed for detection of alpha fetoprotein in human plasma serum and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures human alpha fetoprotein in less than 4 hours A polyclonal antibody specific for human alpha fetoprotein has been pre coated onto a 96 well microplate with removable strips Alpha fetoprotein in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for human alpha fetoprotein which is recognized by a streptavidin peroxidase SP 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 intended for use in diagnostic procedures Prepare all reagents diluent buffer wash buffer standard biotinylated antibody and SP conjuga
8. ing 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 10 fold with reagent grade water to produce a 1x solution Store for up to 30 days at 2 8 C e Human alpha Fetoprotein Standard Reconstitute the Human alpha Fetoprotein Standard 44 ng with 1 1 ml of MIX Diluent to generate a 40 ng ml standard stock solution Allow the vial to sit for 10 minutes with gentle agitation prior to making dilutions Prepare duplicate or triplicate standard points by serially diluting from the standard stock solution 40 ng ml 2 fold with equal volume of MIX Diluent to produce 20 10 5 2 5 1 25 and 0 625 ng ml solutions MIX Diluent serves as the zero standard 0 ng ml Any remaining stock solution should be stored at 20 C and used within 30 days Avoid repeated freeze thaw cycles Standard Bilton AFP Point ng ml P1 1 part Standard 40 ng ml 40 1 part P1 1 part MIX Diluent 1 part P2 1 part MIX Diluent 1 part P3 1 part MIX Diluent 1 part P4 1 part MIX Diluent 1 part P6 1 part MIX Diluent 0 625 MIX Diluent 00 P4 P6 1 part P5 1 part MIX Diluent 1 25 P8 e Biotinylated Human alpha Fetoprotein Antibody 50x Spin down the antibody briefly and dilute the desired amount of the antibody 50 fold with MIX Diluent to produce a 1x solution The undiluted an
9. ival 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 with the desiccant packs and resealed May be stored for up to 30 daysina vacuum desiccator 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 Deionized or distilled reagent grade water Sample Collection Preparation and Storage 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 and collect plasma A 2 fold sample dilution is suggested into MIX Diluent however user should determine optimal dilution factor depending on application needs 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 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 A 2 fold sample dilution is suggested into MIX Diluent however user should determi
10. ne optimal dilution factor depending on application needs The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles Cell Culture Supernatant Centrifuge cell culture media at 1500 rpm for 10 minutes at 4 C to remove debris and collect supernatant Samples can be stored at 80 C Avoid repeated freeze thaw cycles Applicable samples may also include biofluids cell culture and tissue homogenates If necessary user should determine optimal dilution factor depending on application needs Refer to Dilution Guidelines for further instruction Guidelines for Dilutions of 100 fold or Greater for reference only please follow the insert for specific dilution suggested 100x 10000x A 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 1000x 100000x 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 br
11. plicate 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 2 042 P2 1 460 P3 0 987 P4 3 0 638 P5 0 430 P6 0 336 P7 0 298 P8 0 0 Sample Pooled Normal Sodium Citrate Plasma 2x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Human alpha Feto Standard Curve nm OD 450 p soss s ss os saa 1 10 100 H alpha Feto ng ml Reference Value e Normal human alpha fetoprotein plasma levels range from O to 6 ng ml e Plasma and serum samples from healthy adults were tested n 30 On average human alpha fetoprotein level was 3 9 ng ml Performance Characteristics e The minimum detectable dose of human alpha fetoprotein as calculated by 2SD from the mean of a zero standard was esta
12. te 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 antibody 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 alpha Fetoprotein Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human alpha fetoprotein Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes that can be cut to fit the format of the individual assay Human alpha Fetoprotein Standard Human alpha fetoprotein in a buffered protein base 44 ng lyophilized Biotinylated Human alpha Fetoprotein Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against human alpha fetoprotein 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 SP Conjugate 100x A 100 fold concentrate 80 pl Chromogen Substrate 1x A stabilized peroxidase chromogen substrate tetramethylbenzidine 8 ml Stop Solution 1x A 0 5 N hydrochloric acid solution to stop the chromogen substrate reaction 12 ml Storage Condition Upon arr
13. tibody should be stored 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 20 fold with reagent grade water to produce a 1x solution e SP Conjugate 100x Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100 fold with MIX Diluent to produce a 1x solution The undiluted conjugate should be stored 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 alpha Fetoprotein Standard or sample to each well Gently tap plate to thoroughly coat the wells Break any bubbles that may have formed 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 materi

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