Chapter: Enzyme Linked Immunosorbent Assay (ELISA - ASHI-U
Contents
1. 4 Verify calibration of micropipets refer to notebook for pipetman Hamilton calibration Micropipets will be designated for ELISA use only Calibration to be performed bi annually 5 Check that microplate reader is enabled and communication with computer is available Turn power switch on rear of reader to on position and start up computer Open software program on computer hard disk Under Control on the menu bar select Close Drawer then select Open Drawer Verify that the microplate reader performs a self calibration 6 Run Linear Validation Plate LVP once a month to check photo diode linearity of the reader Results of the LVP are to be stored in a notebook labeled Photo Diode Linearity Notebook 7 Reagents should be examined for expiration dates Do not use reagents which have exceeded the manufacturer s expiration date All reagents should be visually examined for turbidity or contamination Discard any reagents found to be turbid or contaminated 8 Internal quality control measures in the form of positive and negative controls have been incorporated into each assay Verify that Positive and Negative Control sera are added to each microplate to be tested and results are within acceptable limits Corrective Action Deviation from an acceptable range or specifications in any of the quality control standards for ELISA Class or II screening or identification assays must be resolved immediately Resolution m2. Negative Serum Control to designated wells according to position on the recording sheet 9 Add 50 ul of diluted Positive Serum Control to designated wells according to position on recording sheet 10 Add 50 ul of each diluted patient serum to designated wells according to position on recording sheet 11 Cover plate with plate sealer and incubate in dry incubator at 37 C for 45 minutes 12 Decant contents of each well by flipping contents out inverting tray onto absorbent material and tapping against a solid surface making sure to blot any residual fluid Add 200 300 ul of Working Wash Solution to each well Repeat wash step for a total of four washes Keep trays inverted on moist paper towel 13 Dilute anti human IgG 1 1000 in Specimen Diluent Mix well 14 Add 50 ul diluted IgG to all wells except those identified as Blanks Cover plate with plate sealer and incubate in dry incubator at 37 C for 45 minutes 15 Decant contents of each well by flipping contents out inverting tray onto absorbent material and tapping against a solid surface making sure to blot any residual fluid Add 200 300 ul of Working Wash Solution to each well Repeat wash step for a total of four washes Keep trays inverted on moist paper towel Proceed quickly through the next three steps 16 Dissolve one vial of PNPP Substrate with 500 ul of deionized or distilled water 17 Dilute PNPP Substrate 1 1000 in Enzyme Substrate Buffer Mix well avoiding direct l
3. antibodies as appropriate for clinical applications D 4 6 18 12 Ensure that there is a procedure to monitor and adjust for non specific binding of antibody D 4 6 18 13 Use an HLA antibody screening method that is at least as sensitive or equivalent to and predictive of the routine crossmatch method and is consistent with clinical transplant protocols E3 100 Test sera at concentrations determined to be optimal for detection of antibody to HLA antigens and document the dilution s in the test records E3 200 Use a human serum demonstrated to be non reactive as a negative control in each assay E3 200 Use a human serum of an appropriate isotype and specificity as a positive control in each assay E3 200 Use HLA molecules that are bound to solid supports e g beads plates or expressed on human cells e g peripheral blood lymph node spleen cell lines as targets E4 10 Use a panel of antigens sufficient in number and phenotypic distribution with respect to individual antigens and or crossreactive groups CREGs for the intended use of the test results and for the population served E4 200 When applicable use a method that detects antibodies to HLA class II antigens and distinguishes them from antibodies to HLA class antigens E4 200 Document the HLA class and or class II phenotypes of the panel for assays intended to provide information on HLA antibody specificity D 4 11 20 Laboratories performing ELISA assays must use positive negative
4. assay is not quantitative It simply identifies the presence or absence of anti MHC antibodies There is data that suggests that the positivity of a reaction in units of ocular density may correlate with a quantitative value but to date this has not been established 13 STANDARDS D 4 6 13 Laboratories performing ELISA must ensure that components of the test system are prepared according to the manufacturer s directions and verify their performance If the test system is modified the laboratory must establish and verify the performance specifications D 4 3 Laboratories performing antibody screening and identification must have protocols that are appropriate for the clinical application D 4 6 18 1 Use a technique s that detects HLA specific antibody with sensitivity equivalent or superior to that of the basic complement dependent microlympho cytotoxicity assay D 4 6 18 2 Use a panel of antigens sufficient in number and phenotypic distribution with respect to individual antigens and or crossreactive groups CREGs for the intended use of the test results and for the population served D 4 6 18 10 Document that the pooled cells or antigens used for a present not present detection of antibody include the major antigen specificities or CREGs or be derived from a population of sufficient size to ensure representation of major antigen specificities D 4 6 18 11 Have a process for distinguishing HLA class and class II antibodies from non HLA
5. other HLA antibodies IgM antibodies or any other class of immunoglobulin or non HLA antibodies Some non cytotoxic HLA antibodies may be detected that are not observed in the standard lymphocytotoxic assay 2 This assay is not intended to identify an antibody but rather to detect the presence or absence of an antibody Antibodies with low titer or low avidity to HLA Class or II may not be detectable 3 Antibody to low frequency HLA Class or Il antigens may not be detected by this assay 4 Immune complexes or aggregates may cause non specific binding leading to false positive reactions 5 Anti coagulated samples and samples treated with immunoglobulin therapies such as IVIG and ATG may have false positive or false negative results 6 The ocular density value for the positive control must be equal to or greater than 1 500 and the mean of the negative controls must be between 0 100 and 0 250 Precautions This assay involves the handling of potentially infectious serum and appropriate precautions should be taken when handling patient and control serum Some reagents contained in the kits contain sodium azide as a preservative Sodium azide reacts with lead and copper plumbing forming highly explosive metal azides If flushed down a sink reagents containing sodium azide should be flushed with large amounts of water to prevent azide build up This procedure is a modification of the manufacturer s product insert guidelines QUAL
6. Chapter Enzyme Linked Immunosorbent Assay ELISA Section Application module Module Antibody Detection Author s Pat Adams Ohio State University adams 5 medctr osu edu Dean Sylvaria Beth Israel Deaconess Medical Center dsylvari bidmc harvard edu Kathryn Tinckam University Health Network Toronto Kathryn tinckam uhn on ca Date Prepared 10 08 2005 OBJECTIVES gt Understand and identify the components of the ELISA antibody identification assay including the target antigens secondary antibodies enzyme substrates and their by products gt Identify a positive or negative reaction understand how this determination was obtained and how it applies to the clinical assessment of the transplant candidate gt Understand and identify the ASHI standards that pertain to the compliance of the assay the plate reader and automated washer and the quality control measures incorporated in the test system INTRODUCTION The ELISA enzyme linked immunosorbent assay assay is designed to detect antibodies that bind to antigens coated onto solid surfaces One application of this assay is the qualitative screening for the presence or absence of anti MHC Class or Class II antibodies Enzyme conjugated antibodies are used to detect the binding of IgG to antigens coupled to the plastic surface of a microwell The antigens coated to the plastic surface of the microwell have been obtained from EBV transformed B cell lines from among a lar
7. ITY CONTROL ASSURANCE Procedure Quality Control for ELISA Class II Antibody Screen and Identification Purpose To describe the standards involved in maintaining appropriate quality control measures to ensure the validity and accuracy of the ELISA Class II screen and identification methodology The quality control of these assays is dependent on both mechanical performance and biological reagents Some of the quality control standards are accounted for in daily laboratory QC Some standards are performed at longer intervals i e pipet calibration and others are needed every time the assay is run Daily quality control records are documented on the ELISA Class II Quality Control Checklist and stored in the ELISA Class II Daily Quality Control Log Materials Refrigerator 0 10 C Dry Incubators 22 C and 37 C Microcentrifuge Micropipets 10 100 ul amp 100 1000 ul Microplate reader Commercial ELISA kits Standards 1 Ensure refrigerator holding screen and identification kits are within acceptable temperature range Monitored daily with results written in Daily QC book 2 Ensure 22 C and 37 C incubators are within acceptable temperature range Monitored daily with results written in Daily QC book 3 Check documentation of microcentrifuge for verification of performance standards Calibration of microcentrifuge performed by Wilkinson Associates Inc Documentation can be found on sticker on the side of the microcentrifuge
8. and reagent controls that are appropriate for the intended use of the assay and test results The dilution of reagents and test specimens must be documented D 4 7 9 Laboratories performing ELISA techniques must follow the manufacturer s recommendations for verifying calibration and setting positive and negative cutoff values D 4 9 4 1 1 Demonstrate that the light source and filter produce the intensity and wavelength of light required for the test system D 4 9 4 1 2 Perform and document calibration verification of plate alignment and instrument linearity according to the manufacturer s instructions or at least once every six months D 4 9 4 2 Verify and record precise movement of the plate D 4 9 4 3 Microplate Washer Check and document microplate washer performance during each month of use 14 LITERATURE CITED RECOMMENDED READING References GTI B Screen product insert 2001 Dombrausky Osowski L Quality assurance of information data in the laboratory New test validation patient test management computerization and laboratory data maintenance ASHI Laboratory Manual 4 Edition Volume 1 2000 Dombrausky Osowski L Gutierrez M Muth B HLA antibody screening and identification by ELISA methodology ASHI Laboratory Manual 4 Edition Volume 1 2000 Zachary AA Solid Phase Immunoassays tests for HLA specific antibodies ASHI NE Regional Ed 1998 Molecular Devices Corporation Emax Precision Microplate Reader User s M
9. anual 1989 Recommended Reading Lucas DP Paparounis ML Myers L Hart JM Zachary AA Detection of HLA class I specific antibodies by the QuikScreen enzyme linked immunosorbant assay Clin Diag Lab Immunolol 4 252 257 1997 Zaer F Metz S Scornik JC Antibody screening by enzyme linked immunosorbant assay using pooled soluble HLA in renal transplant candidates Transplantation 63 48 51 1997 Bryan CF Baier KA Flora Ginter G Shield CF Warady BA et al Detection of HLA IgG antibodies by two enzyme linked immunoassays solubilized HLA class and PRA STAT Comparison with the AHG PRA Transplantation 60 1588 1594 1995 Buelow R Chiang T R Monteiro F Cornejo MC Ellingston L et al Soluble HLA antigens and ELISA a new technology for crossmatch testing Transplantation 60 1594 99 1995 15
10. ay include but is not limited to discarding reagents mechanical repairs performed by authorized service representatives repeating assays or performing confirmatory testing of the assay using well characterized anti sera Any corrective action must be documented 10 Technologist Date PASS FAIL Corrective Action if necessary ELISA Class II Quality Control Checklist GTI B Screen amp Quik ID Class II OBJECTIVE Emax precision microplate reader communicating with computer Emax precision microplate reader operates self calibration MedTec Linear Validation Plate LVP run to check photo diode linearity Results documented if applicable monthly Reagents examined for expiration dates documented on worksheets Reagents examined for turbidity or contamination Positive and Negative Control sera plated and results within acceptable limits B Screen O D for Positive Control must be 1 500 Mean O D of Negative Controls must be 0 100 0 250 Quik ID Mean O D of Positive Contols must be 8X the mean of the Negative Controls 11 FREQUENTLY ASKED QUESTIONS Question Can this assay detect antibodies other than those classified as IgG Answer Yes if patient serum is suspected to have anti HLA antibodies other than of the Class IgG replacing the IgG secondary antibody with an IgM class antibody will allow for detection of IgM anti HLA class antibodies The secondary anti human IgM antibody will atta
11. ay is required by the laboratory prior to clinical use regardless of the quality control provided by the manufacturer Alternately the detection of anti MHC Class I and Class II antibodies by ELISA can be developed in the laboratory however this requires extensive quality control and expertise PROTOCOLS Procedure Detection of Class or II lgG HLA Antibodies by Enzyme Linked Immunosorbent Assay ELISA Purpose To describe the steps involved in testing patient sera for IgG antibodies directed against HLA Class or Il antigens Controls and patient sera are added to separate wells containing previously bound affinity purified HLA Class or Il glycoproteins obtained from EBV transformed B lymphocytes Unbound antibody is washed away and an anti human IgG antibody is allowed to attach to the HLA antibody present Any unattached IgG is washed away and a chromogenic substrate added The intensity of color in each well is read by an ELISA reader A negative cut off is established using two times the mean of the negative control wells Positive results are identified as an ocular density value greater than the negative cut off The information obtained from this assay will be used to identify potential recipients with antibody to HLA Class or II antigens to identify potential recipients who may require further identification of the Class or Il antibody detected and to establish crossmatch requirements for some potential recipients Mate
12. ch to the HLA antibody present in the assay A chromogenic substrate is added and the color change measured and quantified A secondary IgM Class antibody is not included in commercially available kits Question Is the established negative cut off an absolute criteria for identification of a positive or negative determination Answer No the negative cut off is a quantitative measurement that establishes a threshold for what can be defined as either positive or negative but is not absolute There is a range above and below this negative cut off which must be determined by each laboratory individually that should indicate repeat or reflex testing by the same or a more sensitive method Question Why are blank wells included in the assay and what function do they serve Answer Blank wells are included as part of the assays internal control system Plastics have an inherent ability to absorb light at different wavelengths The absorbance measured in units of ocular density detected in these blank wells is subtracted from the ocular density values obtained in the patient and control wells In this way the absorbance used to determine the positivity of a serum sample does not include any inconsequential absorbance due to the nature of the plastic material used to make the microwell 12 FREQUENTLY ASKED QUESTIONS Question Can a percent reactive antibody or PRA be determined by the ELISA antibody identification assay Answer No this
13. date should have a two week post transfusion sample tested and subsequently be tested for three consecutive months if no reactivity is detected ELISA testing is sensitive versatile and most importantly specific for the detection of antibody binding to purified MHC proteins Simply put alloantibody testing by ELISA replaces the 3 complex and dynamic cell surface used in cell panel based testing with a solid matrix plastic well coated with a single class of protein MHC Class or MHC Class II for the purpose of detecting alloantibody While the simplification of the ligand target plastic surface v cell surface is undoubtedly the priority advantage ELISA testing offers several additional advantages to cell surface based testing There are no cell viability issues The reaction score optical density is objective and not dependent on subjective technologist interpretation ELISA is also an ideal approach to testing serial samples and can be very useful in monitoring patients undergoing antibody reduction protocols Similarly it is an easy way to monitor alloantibody production during the post transplant period METHODOLOGIES Commercial kits are available and come supplied with all required reagents as well as having undergone extensive quality control measures to determine the appropriate working conditions of the assay The manufacturer provides training for their product and continuous technical support Validation of the ass
14. ge pool of donors These antigens have been tested against known anti sera to ensure reactivity against a broad range of known HLA specificities Secondary antibodies e g goat anti human IgG are often linked to enzymes such as horse radish peroxidase or alkaline phosphatase These enzymes are coupled to the Fc region of the IgG molecule leaving the Fab regions free to bind to their specific antigen human IgG Acolorless enzyme substrate is used to detect the binding of the secondary antibodies which if present convert the substrate into a colored enzyme by product Thus the production of a colored by product greater than two times the average of the negative controls in the ELISA antibody identification assay indicates the presence of MHC reactive alloantibodies in the serum of a transplant candidate The presence of MHC reactive alloantibodies indicates the need for further testing of the transplant candidate s antibody profile Once it has been determined that an anti HLA IgG antibody is present the next step is to attempt to determine the specificity of the antibody detected If no antibody is detected the transplant candidate is considered unsensitized and no further antibody testing is required at this time Candidates who are considered sensitized should be tested monthly while unsensitized candidates may be tested on a quarterly basis unless a sensitizing event such as a blood transfusion has been documented In such a case the candi
15. ight 18 Add 100 ul of diluted PNPP solution to all wells except those identified as Blanks Incubate in the dark at 22 25 C for exactly 30 minutes do not seal plate 19 Add 100 ul of ELISA Stopping Solution to all wells Add an additional 100 ul of ELISA Stopping Solution to wells designated as Blanks 20 Read the absorbance of each well at 405 nm within 15 minutes of adding the stop solution Allow at least five minutes before reading plate keep in dark until read and analyzed Be sure to align the plate with the Blank wells in the lower left corner when loading into the ELISA reader 21 Print ocular density readings of each well and transcribe the results to appropriate wells on the recording sheet Calculate the average of the two readings for each well 22 Calculate the mean value of negative control wells The mean value must be between 0 100 and 0 250 to be valid The positive control must have an ocular density greater than or equal to 1 500 23 Establish a negative cut off value which is equal to two times the mean of the negative controls Record this value on the B Screen Recording Sheet 24 Identify sera which have an average ocular density reading greater than or equal to the negative cut off These sera are positive for the detection of antibody to HLA Class II antigens Limitations 1 Each assay detects only the Class of IgG HLA antibodies identified by the manufacturer I or Il and therefore will not detect
16. rials GTI Class l Screen or B Screen Kit 12 ELISA Microwell Strips 1 X 8 Wells with Holder 1 X 60 ml Concentrated Wash Solution 1 X 15 ml Specimen Diluent Solution 1 X 80 ul Anti Human IgG Reagent 6 X 500 ul PNPP p nitrophenyl phosphate Reagent 1 X 15 ml Enzyme Substrate Buffer 1 X 15 ml ELISA Stopping Solution 3M Sodium Hydroxide Solution 1 X 450 ul Positive Serum Control 1 X 0 7 ml Negative Serum Control 12 Plate Sealers Microcentrifuge tubes Micro Centifuge Deionized or distilled water Transfer pipets Micropipets 10 100 ul amp 100 1000 ul Pipet tips Microplate reader Procedure 1 Bring all reagents to room temperature 2 Make working wash solution using a 1 10 dilution of the Concentrated Wash Solution with deionized or distilled water 3 Assign and record each patient sample two wells on the recording sheet 4 If using removable strips remove required strips and reseal unused strips in pouch provided Strips placed in frame must have yellow line at the bottom of the strip If a complete tray is to be used remove it now 5 Dilute positive and negative controls and patient sera 1 2 using Specimen Diluent Solution Mix well 6 Add 250 ul of Working Wash Solution to all wells Let stand for 5 10 minutes at room temperature 7 Decant all wells by flipping contents out inverting tray onto absorbent material and tapping against a solid surface making sure to blot any residual fluid 8 Add 50 ul of diluted
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