Home

Troubleshooting Aspects - The Tulip Group, India

image

Contents

1. 2 ELISA Troubleshooting Aspects TECHNICAL SERIES P Setting trends O p Mi Soa eS SS P Other Technical Series published by TULIP Group Quality Assurance for routine Haemostasis Laboratory Monitoring oral anticoagulant therapy Concepts amp Practice Lupus Anticoagulants Basic concepts and Laboratory Diagnosis Mycobacterium Tuberculosis AFB staining Culture and sensitivity Anti Human Globulin Reagent Basic Concepts and Practice Syphilis Daignosis Human Immunodeficiency Virus Perspectives Malaria and its Diagnosis Rapid Diagnostic Tests for Malaria CK and its isoenzymes The time tested biomarker for diagnosis and monitoring of MI Hepatitis C Virus Perspectives Agar Agar and dehydrated culture media Foreword Qualpro Diagnostics is a part of the innovative TULIP Group of companies based at Goa India The group s commitment in building products of international standards through indigenous R amp D has accorded the company virtual leadership in most product segments in the Indian marketplace Its state of art manufacturing facility conforms to the strictest FDA India and GMP regulations In its efforts to build world class Quality products the group has recently received the ISO 9001 2000 certification from TUV It is this commitment to Quality which has given the group international acclaim The products are now exported to over 45 co
2. Use freshly prepared substrate A and substrate B in 2 reagent substrate systems e Do not hold substrate solution longer then 1 hour e Follow procedure of working substrate solution e The temperature of solution is important because it effect s rate of colour reaction e Do not add fresh substrate to reagent bottle containing old substrate e Clean old substrate solution bottle with H SO and thoroughly rinse with distilled water Conjugates e Store at recommended temperature e Never store excessively diluted conjugate for use at some later time e Always make up the working dilution of conjugate just before you need it e Never leave conjugate on the bench for excessive time Addition of Samples Problems caused by e Failure to put sample into buffer in well leaving it on the side of the plate Stopping Reagents Stopping reagents are added to prevent further enzyme reaction in ELISA The stopping is usually made at a time when the relationship among the enzyme substrate product is in the linear phase Molar concentration of strong acids or strong bases stops enzyme activity by quickly denaturing enzymes Some stopping reagents are enzyme specific Sodium azide is a potent inhibitor of HRPO whereas EDTA inhibits Alkaline phosphatase by the chelation of metal ion cofactors Since addition of stopping agents may alter the absorption spectrum of the product the absorption peak must be known 11 FLSA Troubleshoo
3. solution so that no air bubbles are trapped in the well or a thumb is not placed over corner wells Strip Plate Washers e Various washing cycles can be programmed e Careful maintenance is essential since they are prone to machine errors such as having a particular nozzle being blocked Washing Tips e Follow procedure for preparation of wash buffer e Check washer alignment daily as part of routine instrument start up procedures e Ensure that the plate is leveled e Make certain will is completely filled when washing to ensure residual conjugate is removed ue ELISA Troubleshooting aspects Examine the fill volume a slight dome should be observed at the top of the well When washing do not allow wells to overflow Reduce pressure in wash system Check washers before use to determine they are working properly Perform routine maintenance Be certain to wash the specified number of times Allow approximately 20 seconds soak time between the addition of wash solution and subsequent aspiration if soak time is not indicated in the assay pack insert Examine the wells for complete aspiration of contents Upon completion of wash cycle blot to remove residual fluid Pipetting Tips Calibrate pipettes regularly according to manufacture s instructions Avoid touching side wall of well with tips Avoid splashing of sample and reagents Avoid blowing out tip contents Use a new tip for each sample control r
4. action and stabilizes final colour reaction e Insufficient conjugate concentrate added in e Prepare conjugate accurately preparing working stock e Follow working reagent preparation as described by the manufacture FSA Troubleshooting aspects Problem Entire plate gives positive OD or colour all over plate Possible Causes Corrective Action e Inadequate wash volume or contamination e When washing fill the wells to the rim of substrate by residual conjugate left in and ensure no overflow well e Too strong conjugate e Check dilution e Antispecies antibodies react e Check suitable controls with absorbed antigen e Serum factors in heated sera e Do not heat sera e Substrate solution contaminated by e Check pipette barrels for residual fluids or conjugate dried material remove if present e Pipette tips should be long enough to provide air space between top of tip and pipette barrel e For automated system make sure regent lines are in proper position Do not switch lines e Substrate solution is not fresh e Do not hold substrate solution longer than Manufacturer claims e Failure to stop reaction e Check bottle before use e Acid not added e Check assay procedure e Plate sat idle too long before reading e Read within 30 minutes of adding stop solution e Chromogen may not be working e Use fresh chromogen e Substrate solution container is dirty e Do not add fresh substrate t
5. binds unexpectedly to any reagent E Grou Ip Problem Low positive control value or low absorbance Possible Causes Corrective Action e Reagent not at room temperature e Make certain all kit components are at RT 22 28 C e Test volume low e Ensure pipette tips are fitted correctly I tightly e Check pipette barrels for obstructions e Check calibration of pipettes e Substrate A amp B not freshly combined or e Prepare substrate immediately before use incorrectly prepared in case of 2 reagent e Follow working reagent preparation substrate system e Contamination of substrate with or e Re run the assay with fresh reagents bacterial contamination of positive control e Incubation time too short e Check calibration of timers e Record time of incubation e Moisture in pouches e Check whether desiccant in pouch is in working condition e Seal unused wells in pouches e Date pouches when first opened e Improper incubation temperature e Check incubator temperature Room Temperature 22 28 C e Room temperature too low for substrate e Check temperature of the working area incubation e Washing step too vigorous e Reduce pressure in wash system e Reagent not mixed before using e Mix the reagents before use e Wells allowed to dry after assay e Complete all assay steps without has started interruption e Failure to add stop solution e Addition of stop solution increases intensity of colour re
6. eagent addition New tips should be used on the multichannel pipettes for each reagent to be added Reverse pipette when using the multichannel pipettes to add conjugate and substrate solution Forward pipette when using the multichannel pipettes to add stop solution Check pipette tips are long enough to provide air space between top of tip and pipette barrel Check pipette barrel for residual fluid of dried material remove if present Ensure pipettes tips are fitted tightly Microplates Bring microplate pouches to room temperature before opening Level microwells evenly in microplate frame as the individual breakaway wells have very flexible plate frames leading to bowing off wells and yield poor washes Place plates in dark immediately after addition of substrate solution provided the substrate is sensitive to light Grasp holder on grip marks when tapping to avoid strips slipping from holder Rotate strips 180 and re insert or use correct holder if strips do not fit in holder 10 Ul P 8 e Seal unused wells in purchase along with the desiccant e Date the pouches when first opened e Clean bottom surface of plates with wash buffer to remove fingerprints e Make sure microwells are at level during washing reagent addition and plate strip reading e Wipe the bottom the plate with a lint free cloth towel before reading e Do not allow microwells to become dry once the assay has begun Substrate Preparation e
7. eagents has occurred e Toimprove accuracy itis recommended that samples and standards be run in duplicate In this technical series we present various problems commonly encountered in ELISAs and the necessary corrective action E ELSA Troubleshooting aspects Problem High negative control value or high background Possible Causes e Contamination of negative control wells by positive control e Contamination of negative control vial e Insufficient washing or contamination of negative control by conjugate e Non specific attachment of antibodies e Antispecies conjugate reacts with reagent coated on plate Corrective Action e When washing do not allow wells to overflow e Check pipette barrel for residual fluid of dried material Remove if present e Always format negative control wells before positive control e Use new pipette tip for each sample e Check that pipette tips are long enough to provide air space between top of tip and the barrel e Re run with fresh reagents e Make sure wells are completely filled While washing ensure residual conjugate is removed from well e Pipette all specimen and reagents in the center bottom of the micro well Avoid contact with inner wall and rim e Re wash e Unsuitable blocking buffer or omission of blocking buffer e Wells not pre processed to prevent non specific attachment of antibodies e Set up controls to assess weather any reagent
8. g Reagents Temperature Rotation of plates while incubating reagents Laboratory conditions References amp suggested reading Page No 1 oo CO o O O N N N NNNNOO OO OO RA W N 10 10 11 11 11 11 12 12 12 zdi INTRODUCTION ELISAs have emerged as the mainstay for diagnosis of various human diseases in a modern clinical laboratory Despite being an extremely sensitive amp specific assay format several pre analytical and analytical factors may affect the performance of ELISAs Thus it is imperative that laboratory professionals be aware of the problems so that he she has more control over the final assay results Many errors can be avoided if the protocol is read and fully understood before starting the assay e On identifying assay failure check the expiration dates of the individual reagents and ensure that all the reagents have been stored as indicated on the product label e Once this has been established check for signs of instability or deterioration inreagent solutions e g precipitation or discoloration e All substrate solutions should be colorless e Use clean plastic disposable pipettes tips and containers for reagent preparation and storage e Avoid cross contamination of kit reagents by changing pipette tips between addition of each calibrator sample and reagent e Ensure that specified incubation times and temperatures have been adhered to and that no substitution of kit r
9. kes sure temperature of substrate is solution correct PD ee Grou Ip TECHNICAL TIPS Washing The purpose of washing is to separate bound and unbound free unwanted reagents serum components This involves the emptying of microwells of reagents followed by the addition of liquid into the wells Such a process is performed at least 3 6 times for every well The liquid used to wash wells is usually buffered PBS in order to maintain isotonicity since most Ag Ab reactions are optimal under such conditions Tap water is not recommended since tap water varies greatly in composition pH molarity and so on Generally the mechanical action of flooding wells with a solution is enough to wash wells of unbound reagents Some workers leave washing solution for a short time soak time after each addition 1 5 minutes Sometimes detergents notably Tween 20 0 05 are added to washing buffers This can cause problems where excessive frothing takes place producing poor washing conditions since air is trapped and prevents the washing solution from contacting the well surface For most cases this addition does not contribute significantly to the washing procedure When using detergents care has to be taken that they do not affect reagents adversely denature Ag and greater care is needed to prevent frothing in the wells Normal Washing In washing plate manually the most important factor is that each well receives the washing
10. mixing during rotation Stationary incubation relies on the diffusion of molecules amp thus is dependent on temperature Laboratory conditions The laboratory should be devoid of any acid fumes Group p REFERENCES amp SUGGESTED READING 1 James T Wu Quantitative Immunoassay A practical Guide for Assay Establishment Troubleshooting and Clinical Application AACC press 2000 Lothar Thomas Clinical Laboratory Diagnostics Use and assessment of Clinical Laboratory Results TH Books 1998 Varley s Practical Clinical Chemistry 6th edition 1996 John R Crowther ELISA Theory and Practice Volume 42 Humana Press 1995 Teitz Textbook of Clinical Chemistry Second Edition WB Saunders publishing 1994 ee 1 SA Troubleshooting aspects NOTES For the use of Registered Medical Practioners and Laboratories only DIAGNOSTICS QUALPRO DIAGNOSTICS Gitanjali Tulip Block Dr Rego Bagh Alto Santacruz Bambolim Complex Post office Goa 403 202 India Te l 91 832 2458546 51 Fax 91 832 2458544 E mail tulip sancharnet in Website www tulipgroup com
11. ntinuously throughout manufacturer entire assay procedure e Plates being held too long after first incubation before further processing Problem High absorbance of calibrator Possible Causes Corrective Action e Plates being held too long after first incuba e Process plate continuously throughout tion at a higher temperature than what is entire assay procedure recommended by the manufacturer before further processing e Insufficient sample volume added e Check calibration of pipettes PDD ee Grou Ip Problem Specimen absorbance out of range of calibrators Possible Causes Corrective Action e Concentration in specimen is too high e Dilute with 0 calibrator amp re assay Problem Overall low absorbance Possible Causes Corrective Action e Temperature of room lt 20 C e Increase time of reaction between enzyme substrate Check with manufacturer e It is recommended to maintain 22 28 C ambient temperature in the laboratory Problem Controls out of range Possible Causes Corrective Action e Contamination of controls e Re run assay with new controls e Contamination of calibrators e Re run assay with new calibrators Problem Strips slip from holder Possible Causes Corrective Action e Improper handling e Grasp holder on grip marks when tapping Problem Strips do not fit in holder Possible Causes Corrective Action e Improper alignment or incorrect holder e Rotate
12. o reagent bottle containing old substrate e Clean old solution bottle with acid and thoroughly rinse with distilled water e Plate exposed to light during substrate e Place plates in dark immediately after incubation addition of substrate solution Check Product Insert 1 RR Grou Ip Problem False positive reactions Possible Causes Corrective Action e Inadequate washing e Check washer before use to determine they are working properly e Clogged cannulas in washer e Perform routine maintenance e Contamination of wells by conjugate e Carefully add conjugate to wells Pipette reagent to center bottom of microwell e Splashing of conjugate on rims of wells e Avoid contact with sides and rims of wells during conjugate addition e Check alignment and delivery of automated systems e Check pipette barrels for residual fluid or dried material Remove if present e Check pipette tips are long enough to provide air space between top of tip amp pipette barrel e RBCs in test sample e Centrifuge before use e Evaporation of sample of conjugate during e Place the covered test plate in a the 37 C incubation if 37 C isa must not pre warmed 37 C moist incubation box applicable in RT incubation inside the incubator dry or humidified e Contamination of substrate solution by conjugate e Mold in incubation box and or wash buffer e Visually check incubation boxes amp wash bottle buffer bo
13. strip 180 amp re insert or use correct holder Problem Substrate A is blue Possible Causes Corrective Action e Contaminated e Obtain fresh Substrate A Problem Substrates A and B turn blue when mixed Possible Causes Corrective Action e Contaminated e Obtain Fresh substrate A amp B FLSA Troubleshooting aspects Problem Stop solution yellow Possible Causes Corrective Action e Contamination e Obtain fresh stop solution Problem Waited over 30 minutes before measuring plate Possible Causes Corrective Action e End product of enzyme reaction may e Re run the essay precipitate and cause error Problem No colour even after 30 minutes incubation with substrate Possible Causes Corrective Action e mproper mixing of Substrate A amp B e Re mix the substrates e Substrate not working e Contact manufacturer Problem Colour develops very quickly Possible Causes Corrective Action e Contaminated enzymes e Common in wells pre treatment may be necessary e Make sure all reservoirs are clean Problem Colour develops too slowly Possible Causes Corrective Action e Sample not at room temperature e Bring samples to room temperature before assay run e Conjugate too weak e Check dilutions amp time when diluted e Contamination inhibits activity of enzyme e Avoid wrong preservatives e g sodium azide on peroxidase e Low temperature of laboratory or substrate e Ma
14. ting aspects Temperature e Bring test reagents to room temperature 22 28 C approximately 30 minutes prior to use e Maintain proper incubation temperature Lower temperature can decrease OD values Higher temperatures can increase OD values Evaporation in wells can cause edging effect e The optimal temperature for incubation is 22 28 C e Check temperature against calibrated thermometer e Strict adherence to time must be maintained e Check calibration of timers e Record time of incubation e Read plate with specified time limits of adding stop solution Rotation of plates while incubating reagents In certain ELISA systems the plates are rotated during incubation for better antigen antibody reaction The effect of rotating plates is to mix the reactants completely during the incubation step Since the solid phase limits the surface area of the absorbed reactant the mixing ensures that potentially reactive molecules are continuously coming into contact with the solid phase During stationary incubation mixing only takes place because of diffusion of reagents Thus to allow maximum reaction from reagents in stationary conditions greater times of incubation may be required than if they are rotated Rotation also allow ELISA to be performed independent of temperature conditions The interaction of antigen amp antibodies relies on their closeness and the kinetic energy provided to the system which is encouraged with the
15. ttles e Clean any moldy containers e Be sure all containers are free of cleaning agents before using e Set up routine cleaning schedule e T00 much conjugate concentrate used in e Prepare fresh working conjugate preparing working stock e Follow conjugate preparation e Incubation temperature too high e Check room temperature whether at 22 28 C I Check AC Zz E ELISA Troubleshooting aspects Problem Poor reproducibility or bad duplication Possible Causes Corrective Action e Bubbles in wells e Use pin or needle to burst Use separate pin for each well e Finger tips on plates e Clean bottom surface of plate with wash buffer blot to dry e Mis aligned wells in plate e Re align wells e Improper washing technique e Be certain to wash the specified no of times Fill each well to the rim with wash buffer Do not allow well to overflow Blot plate dry at end of wash Problem Poor sensitivity Possible Causes Corrective Action e Usage of non human serum based e Re run with human serum based calibrators for quantitative assays calibrators Primary Standards e Insufficient conjugate concentration e Prepare fresh working conjugate follow added in preparing working stock working procedure e Error in pipetting working conjugate e Check calibration of pipettes e First incubation time insufficient e Repeat run using proper incubation time e Temperature more than suggested by e Process plate co
16. untries globally with an ever increasing user base With decades of experience in in vitro diagnostics IVD TULIP has created a strong knowledge base TULIP believes that in the knowledge based society of the 21 century regular upgradation of knowledge is essential not only for better diagnosis and patient care but also to improve the overall quality of life Publishing of Technical Series is one such initiative to make available to the Laboratory professionals and clinicians updated knowledge that is vital for them to set trends in their day to day practice es 1 ISA Troubleshooting aspects CONTENTS Introduction Troubleshooting aspects High negative control value or high background Low positive control value or low absorbance Entire plate gives positive OD or colour all over plate False positive reaction Poor reproducibility or bad duplication Poor sensitivity High absorbance of calibrator Specimens give absorbance out of range of calibrators Overall low absorbance Controls out of range Strips slip from holder Strips do not fit in holder Substrates A is blue Substrates A and B turn blue when mixed Stop solution yellow Waited over 30 minutes before measuring plate No colour even after 30 minutes incubation with substrate Colour develops very quickly Colour develops too slowly Technical tips Washing Pipetting Tips Microplates Substrate Preparation Conjugates Addition of Samples Stoppin

Download Pdf Manuals

image

Related Search

Related Contents

PENDOPAD 10.1”  HP 995CK User's Manual  HS DAMPER 製品保証書  T- トップ - ヘビー  Using - Burco  UFSconnect 906  Kelly Motor Controller  6303478 ORTH XG 3D Inst es.book  EV-X01  LOGICTECH  

Copyright © All rights reserved.
Failed to retrieve file