Anti-Lactoferrin IgG
Contents
1. Account for this further dilution factor when calculating the value of the sample Dilution Multiplication Factor For Factor Calculating Values Example Serum with normal level of anti Lactoferrin IgG Serum with elevated level of anti Lactoferrin IgG Serum with elevated level of anti Lactoferrin IgG 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 100 0 P2 50 00 P3 25 00 P4 12 50 P5 6 250 P6 3 125 P7 1 563 P8 0 000 Normal Level Sample 40x Serum with normal level of anti Lactoferrin IgG Elevated Level Sample 80x Serum with elevated level of anti Lactoferrin IgG Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Human Lactoferrin Autoantibody Standard Curve 1 0F OD 450 nm 0 1p 1 10 100 hLactoferrin IgG AU ml Reference Value e Human plasma and serum samples from healthy adults were tested n 20 Moreover patient serum samples containing high levels of anti lactoferrin IgG were tested n 11 The following ranges have been established Sample Anti Lactoferrin IgG AU ml Normal Level lt 15 0 Elevated Level gt 15 0 It is recommended2. 00 1 part P2 1 part MIX Diluent 25 00 Pa 1partP3 1 part MIX Diluent 1250 Ps A partPS ipartmixDiluent_ 3125 Pf miDilen 0 000 e HRP Conjugate 50x Spin down the HRP Conjugate briefly and dilute the desired amount of the conjugate 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 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 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
3. ay technique that measures autoantibodies anti Lactoferrin IgG in less than 4 hours A lactoferrin antigen has been pre coated onto a 96 well microplate with removable strips Autoantibody specific for lactoferrin in standards and samples is sandwiched by the immobilized antigen and an antibody HRP 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 working diluent buffer wash buffer standard HRP 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 HRP conjugate 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 Reagents e Human Lactoferrin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with lactoferrin 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 Lactoferrin Standard Plasma standard 100 AU lyophilized e HRP Conjugate 50x A 50 fold concentrated HRP antibody conju
4. d in the up regulation of natural killer NK cell activity 2 Lactoferrin is present in maternal milk saliva tears vaginal secretions semen bronchoalveolar lavage fluid and specific granules of polymorphonuclear leukocytes PMNs 3 Lactoferrin is found mainly in the oral cavity where it can come into direct contact with pathogens such as viruses bacteria etc Lactoferrin directly inhibits viruses by binding to viral receptor sites thus preventing the virus from infecting healthy cells Lactoferrin has a direct bactericidal function to certain bacteria such as Streptococcus mutans Vibrio cholerae Escherichia coli Actinobacillus actinomycetemcomitans and Legionella pneumophila 2 4 Also it has a bacteriostatic effect that deprives iron requiring bacteria of this essential growth nutrient 4 Lactoferrin is also considered an antioxidant that scavenges free iron helping to prevent uncontrolled iron based free radical reactions thus protecting certain cells from peroxidation 2 Autoantibodies against lactoferrin belong to the pANCA class Perinuclear Anti Neutrophil Cytoplasmic Antibodies Principle of the Assay The AssayMax Human Lactoferrin Autoantibody ELISA Enzyme Linked Immunosorbent Assay kit is designed for the quantitative determination of autoimmune response IgG to a target antigen lactoferrin The kit detects autoantibodies in plasma and serum samples This assay employs a quantitative sandwich enzyme immunoass
5. eck 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 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 Deficient Standard Curve Fit April 2015 Non optimal sample dilution e Sandwich ELISA If samples generate OD values higher than the highest standa
6. gate 120 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 1 bottle 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 HRP Conjugate 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 40 into MIX Diluent and assay The undiluted sa
7. mples 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 40 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 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 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 Standard 100 AU with 1 ml of MIX Diluent to generate a 100 AU ml standard stock solution Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions Prepare duplicate or triplicate points by serially diluting the standard stock solution 100 AU ml 1 2 using equal volume of MIX Diluent to produce 50 25 12 5 6 25 3 125 and 1 563 AU ml solutions MIX Diluent serves as the zero standard 0 AU ml Any remaining solution should be frozen at 20 C and used within 30 days Standard Dilution Lactoferrin Point AU ml P1 1 part Standard 100 AU ml 100 0 1 part P1 1 part MIX Diluent 50
8. rd 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 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 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 References 1 2 3 4 Naot D etal 2005 Clinical Medicine amp Research Vol 3 No 2 93 101 Brink W October 2000 LE Magazine Yamauchi K et al 1993 Infection and Immunity Vol 61 No 2 p 719 728 Conneely O M 2001 J of the Am Col of Nutrition Vol 20 No 5 389S 395S Version 1 1R Related Products EL2011 1 AssayMax Lactoferrin ELISA Kit Plasma Serum Urine Saliva Milk CSF and Cell Culture samples www assaypro com e e mail Support assaypro com April 2015
9. that each laboratory establishes its own normal and pathological ranges of antibodies Performance Characteristics e The minimum detectable dose of autoantibodies as calculated by 25D from the mean of a zero standard was established to be 1 AU 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 Linearity e Serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Serum 1 20 90 1 40 98 1 80 105 Troubleshooting 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 technique c 2 2 Q D i 3 e Splashing of reagents e Pipette properly in a controlled and careful manner while loading wells Inconsistent volumes loaded into wells e Pipette properly in a controlled and careful manner e Ch
10. ul of HRP Conjugate to each well and incubate for 1 hour e Wash the microplate as described above e Add 50 ul of Chromogen Substrate per well and incubate for 30 minutes or till the optimal blue color density develops Gently tap the 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 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 e Although normal samples have been diluted 1 40 do not multiply the value by the dilution factor Samples with elevated level of autoantibodies can be diluted further for example 1 80
11. yssaypro AssayMax Human Lactoferrin Autoantibody ELISA Kit Anti Lactoferrin IgG 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 HRP Conjugate per well Incubate 1 hour Step 3 Wash then add 50 ul of Chromogen Substrate per well Incubate 30 minutes Step 4 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 Lactoferrin Autoantibody ELISA Kit Anti Lactoferrin IgG Catalog No EL7011 1 Sample insert for reference use only Introduction Lactoferrin is an 80 kDa iron binding glycoprotein produced by many exocrine glands with a major constituent in the secondary granules of neutrophilic leukocytes Serum lactoferrin concentration is much higher during inflammation 1 Lactoferrin is known to be an immune modulator or enhancer due to specific receptors for lactoferrin that are found on many key immune cells such as lymphocytes monocytes and macrophages Lactoferrin is known to be directly involve
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