Human Lactoferrin
Contents
1. enssaYeRO Human Lactoferrin ELISA Kit CE n Assaypro LLC 3400 Harry S Truman Blvd St Charles MO USA 63301 T 1 636 447 9175 F 1 636 395 7419 WWW assaypro com Emergo Europe Molenstraat 15 2513 BH The Hague The Netherlands 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 IVD For In Vitro Diagnostic Use Expiration Date Temperature Requirements Consult Instructions for Use Catalog Number Lot Number E e Sn Manufacturer Authorized Representative For any questions regarding troubleshooting or performing the assay please contact our support team at support assaypro com Thank you for choosing Assaypro Assay Template AssayMax Human Lactoferrin ELISA Kit EL2011 4 gt lt 30 APR 2017 Sample protocol for reference use only Read package protocol completely before using product Follow instructions carefully when performing tests failure to follow protocol may result in inaccurate results Introduction Lactoferrin is an 80 kDa iron binding glycoprotein produced by many exocrine glands with a major constituent in the2. 10 minutes and assay If necessary dilute samples within the range of 1 2 to 1 10 into MIX Diluent and assay The undiluted samples can be Explore more at Assaypro com stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e Saliva Collect saliva to sample tube Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 1000 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 Milk Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 100000 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 CSF Collect cerebrospinal fluid CSF using sample pot Centrifuge samples at 3000 x g for 10 minutes Dilute samples 1 10 into MIX Diluent and assay 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 protocol for specific dilution suggested 1 100 1 10000 A 4ulsample 396 ul buffer 100x A 4ulsample 396 ul buffer 100x 100 fold dilution B 4ulofA 396 ul buffer 100x 10000 fold dilution Assuming the needed volume is less than Assuming the needed volume is less than or equal to 400 ul or equal to 400 ul 1 1000 1
3. 100000 4 ul sample 396 ul buffer 100x 4 ul sample 396 ul buffer 100x 24 ul of A 216 ul buffer 10x 4 ul of A 396 ul buffer 100x 1000 fold dilution 24 ul of B 216 ul buffer 10x 100000 fold dilution Assuming the needed volume is less than Assuming the needed volume is less than or equal to 240 ul 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 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 280 ng of Human Lactoferrin Standard with 3 5 ml of MIX Diluent to generate an 80 ng 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 Explore more at Assaypro com diluting the standard stock solution 80 ng ml 1 2 using equal volume of MIX Diluent to produce 40 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 solution should be frozen at 20 C and used within 30 days Standard Lactoferrin pom Dimon O O gra 40 00 20 00 1 part P2 1 part MIX Diluent 10 00 2 500 1 250 0 625 0 000 P1 i P2 i P3 i P4 5 000 P5 i P7 i e
4. of lactoferrin to be used in conjunction with clinical evaluation and patient risk assessment Lactoferrin can be used as an aid in predicting risk for inflammation This assay is intended for in vitro diagnostic use Explore more at Assaypro com Principle of the Assay The AssayMax Human Lactoferrin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human lactoferrin in plasma serum urine saliva milk CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique which measures lactoferrin in less than 4 hours A polyclonal antibody specific for lactoferrin has been pre coated onto a 96 well microplate with removable strips Lactoferrin in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for lactoferrin which is recognized by a streptavidin peroxidase conjugate All unbound material is then 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 Prepare all reagents working diluent wash buffer standard biotinylated antibody and SP conjugate as instructed prior to running the assay e Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents e Prepare all samples prior to running the assay The dilution factors for samples are suggested in this protocol However
5. Biotinylated Human Lactoferrin 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 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 Lactoferrin 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 absorben
6. Supplies Required e Microplate reader capable of measuring absorbance at 450 nm e Program with statistical calculator to perform linear regression analysis e Device for delivery of wash buffer solution e g automatic wash system e Containers necessary for preparation of reagents e Pipettes 1 20 ul 20 200 ul 200 1000 ul and multiple channel pipette e Disposable tips and a multichannel micropipette reservoir 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 50 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 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 50 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 the remaining samples at 20 C or below Avoid repeated freeze thaw cycles e Urine Collect urine using sample pot Centrifuge samples at 800 x g for
7. determine the optimal dilution factor for samples Contamination of e A new tip must be used for each addition of different samples or reagents during the assay procedure Contents of wells e Verify that the sealing film is firmly in place before placing e Pipette properly in a controlled and careful manner Improper pipetting e Check pipette calibration e Check pipette for proper performance e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Deficient Standard Curve Fit Insufficient mixing of reagent dilutions References 1 Naot D etal 2005 Clinical Medicine amp Research Vol 3 No 2 93 101 2 Brink W October 2000 LE Magazine 3 Yamauchi K et al 1993 Infection and Immunity Vol 61 No 2 p 719 728 4 Conneely O M 2001 J of the Am Col of Nutrition Vol 20 No 5 389S 395S Version 1 0 Explore more at Assaypro com Page 11
8. e calibration e Check pipette for proper performance e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions e Check the microplate pouch for proper sealing Improperly sealed e Check that the microplate pouch has no punctures microplate e Check that three desiccants are inside the microplate pouch prior to sealing Inconsistent volumes loaded into wells Insufficient mixing of reagent dilutions Microplate was left e Each step of the procedure should be performed unattended between uninterrupted steps Omission of step e Consult the provided procedure for complete list of steps Steps performed in e Consult the provided procedure for the correct order incorrect order Insufficient amount of e Check pipette calibration reagents added to e Check pipette for proper performance wells Improper reagent e Consult reagent preparation section for the correct Insufficient or e Consult the provided procedure for correct incubation prolonged incubation time periods Intensity c 20 w 08 Ir San rs gt l So D e Q Q 2 x o c 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
9. e mean of a zero standard was established to be 0 35 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 20 20 20 20 __ st 20 aa E Spiking Recovery e Recovery was determined by spiking two plasma samples with different lactoferrin concentrations Explore more at Assaypro com napike Spike Recover Sample Sample faecal Expected Observed y o ng ml 4 104 1 50 5 0 0 8 6 107 O ae 103 Average Recovery 102 Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Cross Reactivity Species Cross Reactivity Troubleshooting issue Causes Course of Action 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 Explore more at Assaypro com Page 10 c 2 Ba O cD Sen a gt e aa e Pipette properly in a controlled and careful manner e Check pipett
10. 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 e Itis suggested to use a control sample of known concentration with each assay If the values obtained are not within the expected range of the control the assay results may be invalid Each testing facility should establish its own acceptable range 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 Explore more at Assaypro com Standard Point ng ml OD AverageOD 0 370 0 202 0 128 0 049 Sample Pool Normal 0 741 0 739 Sodium Citrate Plasma 50x 0 737 Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed H Lactoferrin Standard Curve OD 450 nm 1 10 100 Lactoferrin ng ml Explore more at Assaypro com Reference Value e Normal human lactoferrin plasma levels range from 150 to 850 ng ml e Human plasma and serum samples from healthy adults were tested n 40 On average lactoferrin level was 330 ng ml Pp Sample Average Value ng ml Human Pooled Normal Plasma Human Normal Plasma Human Pooled Normal Serum Performance Characteristics e The minimum detectable dose of lactoferrin as calculated by 2SD from th
11. say e Human Lactoferrin Standard Human lactoferrin in a buffered protein base 280 ng lyophilized e Biotinylated Human Lactoferrin Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against human lactoferrin 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 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 unopened components of the kit at recommended temperatures Component y Before opening or y After opening or reconstituting reconstituting Wash Buffer 1x Chromogen Substrate Stop Solution MIX Diluent Concentrate 2 8 C 2 8 C 10x Wash Buffer Concentrate 2 8 C 2 8 C 20x Explore more at Assaypro com e Microplate Unused wells may be returned to the foil pouch with the desiccant packs and resealed May also be stored for up to 30 days ina vacuum desiccator Expiration Date e Kit should not be used beyond the expiration date Before opening or z After opening or reconstituting reconstituting All Reagents 30 APR 2017 Use within 30 days Other
12. 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 involved 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 Intended Use The AssayMax Lactoferrin ELISA Kit is an enzyme immunoassay for the quantitative determination
13. t material to completely remove the liquid Explore more at Assaypro com e Add 50 ul of Biotinylated Human Lactoferrin 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 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 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
14. the user should determine the optimal dilution factor e Intended for in vitro diagnostic use and should not be used as the sole testing method in therapeutic procedures e The Stop Solution is an acidic solution e All reagents should be considered potentially hazardous It is recommended that this product is handled only by professionals trained in laboratory techniques and is used in accordance with the principles of good laboratory practice e The device contains materials of human and animal origin and should be handled as a potential transmitter of disease All human source material have tested negative for antibodies to HIV HbsAg and HCV However no test method can offer complete assurance that infectious agents are not present PPE should be used when handling any patient sera or serum based products e Any material in contact with potentially infectious material must be disinfected or discarded into a biohazard container Dispose of reagents and specimens as clinical waste Any unused reagents should be flushed away with copious amounts of water Disposal must be performed in accordance with local legislation Explore more at Assaypro com Reagents e Human Lactoferrin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human lactoferrin e Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes that can be cut to fit the format of the individual as
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