AssayMaxTM Human IgA ELISA Kit
Contents
1. 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 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 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 Reagent Preparation 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 Freshly dilute all reagents and bring all reagents to room temperature before use 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 Standard Curve Reconstitute the 200 ng 24 mU ml of Human IgA Standard with 2 ml of MIX Diluent to generate a 100 ng ml 12 mU ml standard stock solution Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions Prep2. 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 Calculate the mean value of the duplicate or triplicate readings for each standard and sample 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 Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor Typical Data 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 50 00 P2 25 00 P3 12 50 P4 6 250 PS 3 125 P6 1 563 P7 0 781 P8 0 000 Sample Pool Normal Sodium Citrate Plasma 80000x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Human IgA Standard Curve 2 1 0b Q Q O 0 1 1 1 1 ait J 1 10 100 hIgA ng ml Performance Characteristics respectively The m
3. repeated freeze thaw cycles e Urine Collect urine using sample pot Centrifuge samples at 800 x g for 10 minutes Dilute urine 1 20 with MIX Diluent or within the range of 1 10 to 1 100 and assay Depending on application needs user should determine proper dilutions 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 Dilute saliva 1 2000 with MIX Diluent or within the range of 1 1000 to 1 10000 and assay Depending on application needs user should determine proper dilutions The undiluted samples can be stored 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 milk 1 10000 with MIX Diluent or within the range of 1 2000 to 1 40000 and assay Depending on application needs user should determine proper dilutions 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 1 500 into MIX Diluent or within the range of 1 200 to 1 2000 and assay Depending on application needs user should determine proper dilutions The undiluted samples can be stored at 80 C for up to 3 months Avoid repeated freeze thaw cycles
4. 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 80000 into MIX Diluent or within the range of 1 20000 to 1 200000 and assay Depending on application needs user should determine proper dilutions 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 80000 into MIX Diluent and assay or within the range of 1 20000 to 1 200000 and assay Depending on application needs user should determine proper dilutions 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
5. A assarPro AssayMax Human IgA 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 20 minutes Step 5 Add 50 ul of Stop Solution per well Read at 450 nm immediately Symbol Key ci Consult instructions for use Assay Template 12 11 10 Human Immunoglobulin A IgA ELISA Kit Catalog No El7001 1 Sample insert for reference use only Introduction Human Immunoglobulin A IgA is the most abundant antibody isotype in mucosal secretions and exists in two subclasses IgA1 and IgA2 1 While circulating serum IgA1 occurs mainly in the monomeric 160 kDa form 2 mucosal secretary IgA2 is in dimeric form and serves as the first line of defense against microorganisms through immune exclusion 3 Selective IgA deficiency is the most common primary immunodeficiency observed by a maturation defect
6. are duplicate or triplicate standard points by serially diluting the standard stock solution 100 ng ml 1 2 with MIX Diluent to produce 50 25 12 5 6 25 3 125 1 563 and 0 781 ng ml solutions MIX Diluent serves as the zero standard 0 ng ml Any remaining solution should be frozen at 20 C and use within 30 days Standard Sa IgA Point Dilution ng ml 1 part Standard 100 ng ml Bl 1 part MIX Diluent 50 90 1 part P1 1 part MIX Diluent 25 00 3 000 1 part P2 1 part MIX Diluent 12 50 1 500 Pa 1partP3 1 part MIX Diluent 6 250 0 750 Ps 1partP5 1 part MIX Diluent 1563 0 188 MIX Diluent 0 000 o 000 e Biotinylated Human IgA Antibody 60x Spin down the antibody briefly and dilute the desired amount of the antibody 1 60 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 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
7. dure for correct incubation time 10 Deficient Standard Curve Fit 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 dilution lower than the highest standard point P1 dilute samples further and repeat the assay e User should determine the optimal dilution factor for samples e A new tip must be used for each addition of different samples or reagents during the assay procedure Contamination of reagents e Verify that the sealing film is firmly in place before placing the assay in the incubator or at room temperature Contents of wells evaporate e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance Improper pipetting e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Insufficient mixing of reagent dilutions References 1 2 3 4 Delacroix DL et al 1982 Immunology 47 383 385 Kerr MA 1990 Biochem J 271 285 296 Corth sy B 2007 J Immunol 178 1 27 32 Yel L 2010 J Clin Immunol 30 1 10 16 Version 1 8R Related Products EI7200 1 AssayMax Human IgG ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples El7201 1 AssayMax Human IgG3 ELISA Kit Plasma Serum Urine M
8. 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 IgA 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 IgA Antibody to each well and incubate for 1 hour e Wash the microplate as described above 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 Wash the microplate as described above Add 50 ul of Chromogen Substrate per well and incubate for 20 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 Add 50 ul of Stop Solution to each well The color will change from blue to yellow Read the absorbance on a microplate reader at a wavelength of 450 nm immediately If wavelength correction is available subtract readings at 570
9. gents e Human IgA Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human IgA 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 IgA Standard Human IgA in a buffered protein base 200 ng lyophilized e Biotinylated Human IgA Antibody 60x A 60 fold concentrated biotinylated polyclonal antibody against IgA 100 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 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 days in a vacuum desiccator e Diluent 1x may be stored for up to 30 days at
10. ilk Saliva and Cell Culture samples El7301 1 AssayMax Human IgM ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples El7800 1 AssayMax Human IgD ELISA Kit Plasma Serum Urine Milk Saliva and Cell Culture samples www assaypro com e e mail Support assaypro com
11. in B cells to produce IgA 4 Principle of the Assay The AssayMax Human IgA ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human IgA in plasma serum urine saliva milk CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures human IgA in less than 4 hours A polyclonal antibody specific for human IgA has been pre coated onto a 96 well microplate with removable strips IgA in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for IgA 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 Solution is an acidic solution e The kit should not be used beyond the expiration date Rea
12. inimum detectable dose of IgA is typically 0 7 ng ml Intra assay and inter assay coefficients of variation were 5 0 and 7 2 e Kit standard has been calibrated against WHO International Reference Recovery Standard Added Value 3 13 25 ng ml Recovery 85 113 Average Recovery Linearity 98 e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 40000 92 91 1 80000 99 100 1 160000 Cross Reactivity Species 105 103 Cross Reactivity Canine None Bovine None Monkey lt 5 Mouse None Rat None Swine None Rabbit None Immunoglobulins Cross Reactivity IgM lt 5 IgA1 100 IgA2 100 IgG1 lt 1 1862 None 1863 None 1864 None IgD lt 1 IgE lt 1 Troubleshooting Issue Causes Course of Action e Check the expiration date listed before use Use of expired i y e Do not interchange components from different components lots e Check that the correct wash buffer is being used e Check that all wells are dry after aspiration e Check that the microplate washer is dispensing Improper wash step properly e If washing by pipette check for proper pipetting technique c Splashing of e Pi
13. pette properly in a controlled and careful 2 reagents while manner 3 loading wells a E e Pipette properly in a controlled and careful 2 Inconsistent manner 3 volumes loaded into wells 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 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 e Check pipette calibration e Check pipette for proper performance to wells Wash step was e Consult the provided procedure for all wash steps skipped Improper wash e Check that the correct wash buffer is being used buffer Improper reagent preparation e Consult reagent preparation section for the correct dilutions of all reagents Insufficient or prolonged incubation periods e Consult the provided proce
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