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FAO/IAEA Competitive Rinderpest ELISA Version 2.0, January 1999 Word 6.0/95 Filename: CORP20.doc

JOINT FAO/IAEA PROGRAMME

ANIMAL PRODUCTION AND HEALTH

Bench Protocol Version - RPV 2.0

January 1999

This kit has been prepared for the Animal Production and Health Subprogramme by the Institute for Animal Health, Pirbright, UK, in support of the projects of the FAO/IAEA Programme of Nuclear and Related Techniques in Food and Agriculture.

RINDERPEST ELISA KIT

COMPETITIVE ENZYME IMMUNOASSAY

FOR DETECTION OF ANTIBODY

TO RINDERPEST VIRUS


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NOTICE As part of the FAO/IAEA Programme for Quality Assurance, this manual is considered to be a Controlled Document. The manual may be photocopied for use by others, but responsibility for the contents can only be accepted by the FAO/IAEA Animal Production and Health Subprogramme when an original signature follows this notice. ______________

ENQUIRIES

Any enquiries relating to this kit should be addressed to either:

Your Technical Officer at the Agency Animal Production and Health Section

Joint FAO/IAEA Division International Atomic Energy Agency Wagramerstrasse 5, P.O.Box 100

A-1400 Vienna, Austria Tel: +43-1-2600-0 Fax: +43-1-2600-7

or

the Head of the Laboratory Unit Animal Production Unit

FAO/IAEA Agriculture and Biotechnology Laboratory Agency?s Laboratories Division

International Atomic Energy Agency Wagramerstrasse 5, P.O. Box 100

A-1400 Vienna, Austria Tel: +43-1-2600-28355 Fax: +43-1-2600-28222


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NOTE: With each individual kit, FACT SHEETS and PACKING LISTS are included. The FACT SHEETS contain the KIT IDENTIFICATION. With respect to problems with the kit, please include this KIT IDENTIFICATION (e.g. cRPV/98/10/01). The FACT SHEETS also contain information on preparation, aliquoting, storage and dilution procedures for the different biologicals, quality assurance (QA) data and other relevant information for use with this ELISA kit. This information may differ from batch to batch. The PACKING LISTS identify the reagents and quantities of each supplied in the kit. The FACT SHEETS and PACKING LIST should be kept with this protocol in Appendix II for easy access to the appropriate information.


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CONTENTS Sections page 1) Introduction 5

2) Equipment and Supplies Required 7

3) Reagents Supplied 10

4) Reagent and Sample Preparation 13

5) Assay Procedure 18

6) Assay Performance and Interpretation 24

7) Guide to problem solving 29

8) References 32

Appendices Appendix I. - Assay Flow chart - Plate layout - Work sheet - ELISA data sheet Appendix II. - Fact Sheets: * Reagent Information * QA Information - Packing Lists


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1. INTRODUCTION This kit has been developed for the detection of serum antibody to rinderpest virus. It has been specifically designed for use under the conditions prevailing in the majority of laboratories in countries participating in the Global Rinderpest Eradication Programme (GREP). The kit is based on a standard competitive enzyme-linked immunosorbent assay (ELISA) technique to determine the presence of anti-RPV antibody in serum as described by Anderson et al. (1991) and Libeau et al. (1991).The test is based upon the competition between the anti-RPV monoclonal antibody and antibodies in the serum sample for binding to the RPV antigen. The presence of antibodies to rinderpest virus in the serum sample will block reactivity of the monoclonal antibody (Mab) resulting in a reduction in expected colour following the addition of enzyme labelled anti-mouse conjugate and substrate/chromogen solution. As this is a solid phase assay, wash steps are required between each step to ensure the removal of unreacted reagents. The rinderpest antigen supplied is prepared from Madin-Darby Bovine Kidney (MDBK) cell cultures infected with the attenuated Kabete 'O' strain (RBOK) of rinderpest virus (RPV) (Anderson et al., 1982). The rinderpest infected cell culture supernatant has been concentrated by ammonium sulphate precipitation. The mouse monoclonal antibody (designated C1) supplied is directed against the hemagglutinin (HA) protein of the rinderpest virus (Anderson et al., 1991). The anti-species conjugate supplied is a polyclonal rabbit anti-mouse immunoglobulin conjugated to horseradish peroxidase. It has been absorbed against human and ox immunoglobulins to remove any cross reactivity. The kit is complete with control reagents and a protocol which


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contains a list of essential equipment required to conduct the assay. This protocol also describes reagent preparation, handling and use in the assay, as well as details of data acceptance and interpretation. A guide to problem solving has also been included. The bench protocol should be strictly followed in order to ensure a standard level of assay performance within and between laboratories. A software program (ELISA Data Interchange or EDI) is available through the FAO/IAEA Animal Production and Health Subprogramme, which is designed to facilitate data acquisition, processing, management, interpretation and reporting, as well as internal and external quality assurance. In addition, an external quality assurance programme (EQAP) with proficiency test panels is available through the Subprogramme. This programme is designed to facilitate international accreditation of laboratory competence using this kit or its equivalent.


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2. EQUIPMENT AND SUPPLIES REQUIRED 2.1 Photometer Flow laboratories, Titertek Multiskan Plus MKII microplate reader

(or equivalent) with an interference filter of 492 nm. 2.2 Orbital Shaker Flow Laboratories, Titertek Microplate Shaker (or equivalent). 2.3 Washer Flow Laboratories, Titertek Handiwash microplate washer (or

equivalent). Alternatively, plastic wash bottles or a wash fluid container connected to suitable tubing may be used.

2.4 Pipettes Multichannel Pipettor any brand (50-300 ul) Multichannel Pipettor any brand (5-50 ul) Single Channel Pipettors any brand (1-20 ul) Single Channel Pipettors any brand (20-200 ul) Single Channel Pipettors any brand (200-1000 ul) 2.5 Water Purification System Minimum; glass-distilled or deionized water, Optimum; Millipore, Milli-Q (or equivalent), Type I, 18 megaohm,

pyrogen-reduced water.


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2.6 Microplates NUNC Immuno I - Maxisorp (cat.# 439454), flat bottom, 96 well

microplates (substitution may alter diagnostic performance). According to manufacturer's recommendation, plates should be stored at temperatures between + 4EC and + 15EC.

2.7 Refrigerator Any type in the range of +2°C to +6°C. 2.8 Freezer Any type in the range of -15°C to -20°C. 2.9 Incubator Any type of radiant, warm wall incubator or hot room in the range

of +10°C to +37°C. 2.10 pH Meter Any type with an accuracy of 0.01 pH units. 2.11 Glassware/Plasticware A selection of beakers (20-4000 ml), flasks (50-1000 ml),

graduated cylinders (10-2000 ml), graduated pipettes (1-25 ml) with suitable safety bulbs, storage bottles with closures (1-100 ml), dilution tubes (2-4 ml) and suitable racks, wash fluid container with tap (5-10 l) and tubing for connection of this reservoir to plate washer or alternatively, wash bottles (250 ml).


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2.12 Cryopreservation Vials Recommended; polypropylene, conical bottom, screw cap with

internal thread and O-ring seal (1-5 ml) and appropriate storage racks.

2.13 Vortex mixer Any type with variable speed. 2.14 Timer Any type, preferably countdown-type with an audible alarm. 2.15 Absorbent towels Disposable or cloth, lint-free and non-abrasive. 2.16 Marker pens (water proof) and adhesive labels. 2.17 Hydrochloric Acid, A.C.S., F.W. 36.46.


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3. REAGENTS SUPPLIED WARNING: All of the biologicals listed herein are to be used in 'in vitro' test procedures only and are not to be introduced into laboratory, domesticated or wild animals (including birds and fish) by injection, instillation, ingestion or inhalation under any circumstances. All waste generated and containers used in the preparation and use of these biological reagents should be chemically disinfected and/or autoclaved prior to disposal or reuse. None of the biologicals or derivatives thereof are to be distributed to any other laboratory or persons without consultation with Agency staff.

3.1 Antigen Cell culture derived rinderpest virus antigen, freeze dried. Store at +4EC. 3.2 Control Sera (Bovine) C++, anti-RPV antibody positive (strong). C+, anti-RPV antibody positive (moderate). C- , anti-RPV antibody negative, also used in Blocking Buffer. All control sera are whole bovine sera, freeze dried. Store at +4EC.


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3.3 Monoclonal Antibody Mouse anti-RPV monoclonal antibody supplied as freeze dried

hybridoma cell culture supernatant. Store at +4EC. 3.4 Anti-Species Conjugate Horseradish peroxidase (HRP) conjugated, rabbit anti-mouse

immunoglobulin, liquid. Store at +4EC. 3.5 Coating Buffer Phosphate buffered saline (PBS) powder (Sigma Chemicals; catalog no. 1000-3). Store dry at room temperature. 3.6 Wash and Blocking Buffer Base Phosphate buffered saline (PBS) powder (Sigma Chemicals; catalog no. 1000-3). Store dry at room temperature. 3.7 Blocking Detergent Tween 20, liquid. Store at room temperature.


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3.8 Substrate

WARNING: It is the responsibility of the user to acquaint themselves with any hazards and disposal requirements associated with the chemicals used in this kit. Material safety data sheets (MSDS) should be consulted, especially for ABTS. These MSDS's may be obtained free of charge from the Animal Production Unit.

Hydrogen peroxide tablets. Store dry at +4EC. 3.9 Chromogen Ortho-phenylenediamine (OPD) tablets. Store blister pack containing tablets in bottle at +4EC. 3.10 Reconstitution Diluent Water. Pyrogen-free, deionized water and bottle have been autoclaved

to ensure sterility. Store at +4EC.


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4. REAGENT AND SAMPLE PREPARATION NOTE: The quality of the distilled/deionized water produced in your laboratory may be affected by the efficiency of the water purification system and subsequent storage conditions. Care must be taken to ensure that the purity of the locally produced distilled/deionized water used for the preparation of your stocks, buffers and stopping solution is of an acceptable standard.

NOTE: It is good laboratory practice to plan in advance the amounts of buffers required in order to avoid running out of buffers during an assay or to avoid excessive buffer storage periods.

4.1 RPV Antigen Stock

IMPORTANT: Consult the FACT SHEETS for specific information on the preparation, aliquoting and storage procedures for the different biologicals. Because individual batches of different reagents may differ in their final working dilution, a greater dilution factor may be required for the preparation of 'Working Stock'. If necessary, this will be indicated on the accompanying FACT SHEET under 'Working Stock Preparation'.

Reconstitute the freeze dried contents of a vial with precisely 1

ml of the sterile water (reconstitution diluent) supplied with the kit and mix gently until completely dissolved. The stock may be stored in its original vial at -20EC as it can be thawed and refrozen several times without a significant loss of activity. Use one vial at a time until depleted and then reconstitute the freeze dried contents of a second vial.


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4.2 Anti-Rinderpest Monoclonal Antibody Stock Reconstitute the freeze dried contents of a vial with precisely 1

ml of the sterile water (reconstitution diluent) supplied with the kit and mix gently until completely dissolved. The stock may be stored in its original vial at -20EC as it can be thawed and refrozen several times without a significant loss of activity. Use one vial at a time until depleted and then reconstitute the freeze dried contents of a second vial.

4.3 Anti-Species Conjugate Stock The rabbit anti-mouse immunoglobulin HRPO conjugate stock

supplied should be further subdivided into 500 :l aliquots in 1 ml cryopreservation vials supplied, labelled and stored at +4EC. Use one vial at a time until depleted.

4.4 Control Serum Stocks Reconstitute the freeze dried contents of a vial of each control

serum (C++, C+ and C-) with precisely 1 ml of sterile water (reconstitution diluent) supplied with the kit and agitate gently until completely dissolved. These control serum stocks may be stored in their original vials at -20EC as they can be thawed and refrozen several times without a significant loss of activity. Use one vial at a time until depleted and then reconstitute the freeze dried contents of a second vial.

4.5 Chromogen Stock 3.7 mM OPD Dissolve one OPD tablet in 75 ml of locally produced

distilled/deionized water just before the substrate/ chromogen incubation step and store in the dark at 4EC.


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This stock solution will provide enough reagents for 15 plates. If the whole volume is not required for a days' test, prepare aliquots of 6 and/or 12 ml and store at - 20EC in the dark.

4.6 Substrate Stock 3% (w/v) H2O2 (882 mM) Place one hydrogen peroxide tablet in the brown bottle supplied

and dissolve with 10 ml of locally produced distilled/deionized. This will give a 3% solution. Store at +4EC until depleted at which point a fresh stock should be prepared in the same manner.

4.7 Coating Buffer 0.01 M Phosphate Buffered Saline, pH 7.4 +/- 0.20 Dissolve the contents of one bottle per 1 litre of locally produced

distilled/deionized water. Label and store at +4EC for no longer than two weeks. If not being used immediately, store in aliquots of 50 ml at -20EC.

4.8 Blocking Buffer 0.01 M Phosphate Buffered Saline, pH 7.4 +/- 0.20 plus 0.1% (v/v) Tween 20 plus 0.3% (v/v) normal bovine serum (C- control serum) NOTE: Because Tween 20 is very viscous, care must be taken to ensure that the pipette or tip used to transfer this detergent is properly rinsed with the Phosphate Buffered Saline to which it is being added and that all of the Tween is dispersed in the buffer. Cutting approximately 4 mm off the end of the pipette tip will greatly facilitate transfer of the Tween 20.

Dissolve the contents of one bottle per 1 litre of locally produced


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distilled/deionized water. Label and store at +4EC for no longer than two weeks.

4.9 Wash Buffer 0.002 M Phosphate Buffered Saline, pH 7.4 +/- 0.20 In 1 liter of locally produced distilled/deionized water dissolve the

contents of one bottle. Transfer to a wash fluid container with a tap to which tubing may be attached and further dilute with the addition of 4 litres of distilled/deionized water, mix well and store at room temperature for no longer than two weeks.

4.10 Stopping Solution 1 M Sulphuric Acid Slowly add 55 ml of concentrated sulphuric acid to 945 ml of

locally produced distilled/deionized water. Label and store at room temperature.

WARNING: Always add concentrated acid to water, not water to concentrated acid.


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NOTE: It is important that the buffers and stocks described above be within the pH ranges indicated. Therefore the pH of each solution should be tested. If the pH should not fall within the specified ranges, then steps should be taken to correct the problem. Poor water quality and unclean glassware are most frequently implicated. Should any buffer pH stock solution show signs of deterioration (ie. formation of a precipitate) or contamination (ie. evidence of bacterial or algal growth), discard immediately and prepare a fresh batch in clean glassware. Wash bottles, wash fluid containers, tubing and washer should be thoroughly rinsed with distilled water prior to the addition of freshly prepared washing buffer.

4.11 Test Sera Whole blood samples should be allowed to clot in the tubes into

which they were collected and the serum may be sampled directly off the clot if no suspended cells are apparent. If suspended cells are apparent, the sample should be centrifuged at 1000-2000 G for 10 minutes, after which the serum may be sampled.

If not tested immediately, test sera should be collected from the

clot of the blood samples and stored at -20EC in appropriately labelled cryopreservation vials.


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5. ASSAY PROCEDURE NOTE: It is good laboratory practice to plan your tests in advance. Check that all equipment is available and working, and that all the reagents and control sera and/or test sera are prepared and thawed if necessary. For optimal performance, those buffers which will be used at temperatures greater than + 4°C should be equilibrated to room temperature before use. This reduces the effect of thermal gradient formation as microplates tend to warm from the outer edges towards the centre. Thermal gradients may become even more apparent if microplates are stacked in the incubator. Biological reactivity is temperature dependent. Therefore, if thermal gradient formation occurs, warmer wells around the outer edges of the microplate will tend to develop higher background and/or specific colour than the cooler wells towards the inner areas of the plate, Prepare work sheets showing the position of the Controls and test sera according to the proposed plate layout (Appendix I). When using a single or multichannel pipette, it is important to ensure that all the pipette tips are seated tightly and functioning properly (i.e. tips should fill equally with no air bubbles). For each pipetting operation, new and clean disposable tips must be used. Only NUNC - Immuno I Maxisorp (cat.# 439454) ELISA microplates may be used for the test and they are not reusable. Any change of plate type may alter the diagnostic performance.

NOTE: Please consult the FACT SHEET which has accompanied the kit to determine the appropriate working dilution of the antigen. This is necessary because different batches of antigen may vary in their optimal working dilutions.


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5.1 Coating of Microplates Gently mix an aliquot of reconstituted RPV antigen stock to

ensure uniform dispersion. Immediately prepare a working dilution of RPV antigen in coating

buffer in a volume sufficient for the number of plates required (6 ml of working dilution per microplate).

Immediately dispense 50 :l volumes of the working dilution of

RPV antigen into all 96 wells of the microplates. Tap the sides of the microplates to ensure that the antigen is evenly distributed over the bottom of each well.

Cover or seal the microplates and place on an orbital plate

shaker housed in a 37EC warm air incubator or hot room and incubate for 1 hour with continuous shaking.

Return the remainder of the reconstituted RPV antigen stock to -

20EC. 5.2 Preparation of Blocking Buffer 0.01 M Phosphate Buffered Saline, pH 7.4 +/- 0.20 plus 0.1%

(v/v) Tween 20 plus 0.3% (v/v) normal bovine serum (C- control serum).

On the day of testing, calculate the amount of blocking buffer

required and add Tween 20 to a final concentration of 0.1% (v/v) and normal bovine serum (C- control serum) to a final concentration of 0.3% (v/v) to the 0.01 M phosphate buffered saline (PBS).

For example: to prepare 100 ml blocking buffer add 100 :l

Tween 20 and 300 :l normal bovine serum (C- control serum) to 100 ml of PBS.

5.3 Addition of Test Sera and Control Sera, and


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Monoclonal Antibody Agitate the test sera and all 3 control sera (C++, C+ and C-)

gently to ensure homogeneity. By inverting the microplate and using an abrupt downward hand

motion, discharge the contents of all antigen coated microplates into a sink or other reservoir and slap the inverted microplates onto a lint-free absorbent to remove all residual contents. Using the Handiwash or equivalent, fill all 96 wells of all microplates with wash buffer. After filling, again discharge the contents of the microplates and slap the inverted microplates onto a lint-free absorbent to remove all residual contents. Repeat with two more wash cycles of filling and emptying.

After 3 complete wash cycles and ensuring that no residual

contents are left in the microplates, immediately dispense 40 :l volumes of blocking buffer to all 96 wells of all the microplates.

According to the plate layout (Appendix I), add 10 :l volumes of

test and control sera to the appropriate wells. This will result in the initial serum dilution of 1/5. Add 10 :l of blocking buffer to the monoclonal antibody control (Cm) wells and 60 :l of blocking buffer to the conjugate control (Cc) wells.

Immediately prepare a working dilution of the monoclonal

antibody in blocking buffer for all the plates (6 ml of working dilution per plate). Both the monoclonal antibody stock and its working dilution should be handled with care, mixing should be gentle but thorough.

Add 50 :l volumes of the working dilution of the monoclonal

antibody to all the wells of the microplates except the conjugate control (Cc) wells following the plate layout. This will result in the final serum dilution of 1/10.

Cover or seal the microplates and place on an orbital plate

shaker housed in a +37EC warm air incubator or hot room and


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incubate for 1 hour with continuous shaking. Return the test sera and the remainder of the control sera and

monoclonal antibody stocks to -20EC. 5.4 Addition of Conjugate Immediately before the end of the serum/monoclonal antibody

incubation, prepare a working dilution of the conjugate in blocking buffer in a volume sufficient for all the microplates (6 ml of working dilution per microplate). Both the conjugate stock and its working dilution should be handled with care; agitation should be gentle but thorough.

Return the conjugate stock to +4EC. After 1 hour of serum incubation, remove the microplates from

the incubator and wash the microplates with wash buffer as described previously (see Section 5.3).

Immediately after washing, add 50 :l volumes of the working

dilution of conjugate to all 96 wells of the microplates. Again, it is important not to let wells of the microplates become dry. Tap the sides of the microplates to ensure that the conjugate working dilution is evenly distributed over the bottom of each well.

Cover or seal the microplates and incubate for 1 hour at +37EC

with continuous shaking.


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5.5 Addition of Substrate/Chromogen and Stopping Solutions

Immediately before the end of the conjugate incubation, prepare

a working dilution of the substrate/chromogen solution in a volume sufficient for the number of microplates [e.g. for 1 plate, dilute 24 :l of substrate stock (H2O2) in 6 ml of chromogen stock (OPD) solution]. This represents 3.7 mM OPD and 3.5 mM H2O2. This final substrate/chromogen solution should be colourless and stored in the dark. If coloured, it should be discarded.

A clean microplate (not coated with antigen) will be used as the

'blanking plate' for the photometric reading. After 1 hour of conjugate incubation, wash the microplates as

described previously (see Section 5.3).

NOTE: It is important not to let the wells of the microplate become dry.

Immediately after washing, add 50 :l volumes of the substrate/

chromogen solution to the wells of the microplates, starting with the first column of the 'blanking plate' followed by all 96 wells of the microplates in the test run. Immediately begin timing the substrate/chromogen development and incubate at room temperature for 10 minutes without plate shaking.

After 10 minutes of substrate/chromogen incubation,

immediately add 50 :l volumes of the stopping solution (1 M sulphuric acid) to the wells of the microplates, starting with the first column of the 'blanking plate', followed by all 96 wells of the microplates in the test run. Briefly shake the microplates using the orbital shaker to ensure thorough mixing. All wells should now contain 50 :l of substrate/ chromogen solution plus 50 :l of stopping solution.


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5.6 Measurement of Substrate Development Place the 'blanking plate' in the carriage of the microplate reader

and initiate the blanking sequence. Place the microplate of the test run in the carriage of the blanked

reader and initiate reading sequence. Repeat for each microplate.


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6. ASSAY PERFORMANCE AND INTERPRETATION 6.1 Data Expression Microplate readings will be used in two types of data analysis:

A) Percent Inhibition (PI) values which are used for Quality Assurance (QA) acceptance and are calculated as follows:

x100)Control Cm of Value OD Median

Control each of Value OD Replicate(100 −

and

B) Percent Inhibition (PI) values which are used for acceptance of replicate values for test sera and diagnostic interpretation and are calculated as follows:

x100)Control Cm of Value OD Median

Serum Test of Value OD Replicate(100 −

6.2 Calculation and Acceptance of Control Data The data expressed in OD values and PI values for the

monoclonal antibody control (Cm) and the data expressed in PI values for the four other Controls (C++, C+, Cc and C-) are used to determine whether or not the test has (been) performed within acceptable limits of variability and therefore, whether or not the test sera data may be accepted for any given microplate.

The Controls included with the kit have been tested extensively

at the Seibersdorf Laboratory. Given that the variation in OD values and PI values should be normally distributed, Upper (UCL) and Lower Control Limits (LCL) for these Controls have been established.


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NOTE: The Upper Control Limits (UCL) and Lower Control Limits (LCL) for the Controls included with any individual kit may be found on the FACT SHEET on 'Quality Assurance (QA) Information' accompanying that kit. For those laboratories using FAO/IAEA ELISA Software, the UCL and LCL values should be updated by the user according to program instructions.

The Controls should be examined in the following order: 6.2.1. First Level of Microplate Acceptance Monoclonal Antibody Control (Cm)

Before calculating PI values, compare the OD values of the Cm replicates to the Lower and Upper Control Limits indicated on the QA Fact Sheet. At least three out of four of these replicates must fall within these limits. If not, the plate must be rejected (“Outside Limits”).

The OD values of the three or four Cm replicates that fall within

the Lower and Upper Control Limits should be be used for the calculation of the median Cm OD value and used in subsequent PI calculations.

6.2.2. Second Level of Microplate Acceptance Monoclonal Antibody (Cm), Strong Positive (C++),

Moderate Positive (C+), Negative (C-) and Conjugate (Cc) Controls

For the monoclonal antibody control (Cm) and for each control

serum (C++, C+, C- and Cc), the Replicate PI values should be calculated and recorded on the ELISA DATA SHEET.


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NOTE: Use the Tables 6A and 6B only if the individual microplate is accepted according to the 'First Level of Microplate Acceptance', Section 6.2.1. (i.e., at least three of the OD values for the Cm replicates must fall within the UCL and LCL indicated on the QA Fact Sheet).

i. Monoclonal Antibody (Cm), Strong Positive (C++) and

Moderate Positive (C+) Controls Compare the Replicate PI values (as calculated in Section

6.1 A) for the Cm, C++ and C+ controls to the UCL's and LCL's indicated on the QA Fact Sheet and use the criteria in Table 6A to accept or reject each individual microplate.

Table 6A - Cm, C++ and C+ Control Data

Replicate PI Values Status In Out ____________________________________________________ 4 0 Accept 3 1 Accept 2 2 Reject 1 3 Reject 0 4 Reject ____________________________________________________

In: Within UCL and LCL range

Out: Outside UCL and LCL range Microplate rejected if any of the Controls (Cm, C++ or C+) fails

to meet PI acceptance criteria. ii. Negative (C-) and Conjugate (Cc) Controls


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Compare the Replicate PI values (as calculated in Section 6.1 A)

for the C- and Cc controls to the UCL's and LCL's indicated on the QA Fact Sheet and use the criteria in Table 6B to accept or reject each individual microplate.

Table 6B - C- and Cc Control Data

Replicate PI Values Status In Out ____________________________________________________ 2 0 Accept 1 1 Reject 0 2 Reject

In: Within UCL and LCL range

Out: Outside UCL and LCL range Microplate rejected if any one of the Controls (C- or Cc) fails to

meet PI acceptance criteria. 6.3 Acceptance of Individual Test Sera Data The diagnostic threshold for this assay has been set at 50%

inhibition (50 PI) of the monoclonal antibody control (Cm) for seromonitoring purposes. If the assay is used for any purpose other than monitoring the efficacy of a vaccination programme, the threshold should be established for that purpose by each individual laboratory.


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To accept individual test sera, both replicate PI values of a test serum must fall either above or below the established threshold.

Test sera should be retested if their replicates PI values lie on

either side of the threshold. 6.4 Diagnostic Interpretation of Test Sera Data If accepted according to the Criteria in 6.3, the mean PI value for

the two replicates should be calculated. Test sera demonstrating mean PI values equal to or greater than the established threshold (50 PI for seromonitoring) are considered to be positive. Test sera demonstrating mean PI values less than the established threshold are considered to be negative.


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7. GUIDE TO PROBLEM SOLVING If the results of a test are not as expected, it is vital to retrace the

test procedure and ascertain if all the correct steps were taken. In particular try to recall if the correct dilution of all reagents was prepared.

It is useful to keep a sample of the conjugate working dilution and

substrate/chromogen solution used in the test until the end. If there is no colour development or the colour is slow to develop, the enzyme system may be initially checked as outlined in Table 7A, using the check procedure shown below. A fresh working dilution of conjugate and a fresh solution of substrate/chromogen should be prepared from the stocks and buffers used in the preceding test. The results of the different combinations of the old and freshly prepared conjugate working dilutions and old and freshly prepared substrate/ chromogen solutions may identify errors in the preparation of either the old working dilution of conjugate and/or the old substrate/chromogen solution used in the preceding test.

If the initial preparation check procedure does not identify an

error, new conjugate working stock, chromogen and substrate stocks, and substrate and diluent buffers should be prepared (see Section 4).

Conjugate working dilutions and substrate/chromogen solutions

should be prepared using the new stocks and buffers and old stocks and buffers (see Section 5). Test in the combinations outlined in Table 1b. according to the check procedure shown below.

Check procedure - in a clean microplate - to 100 µl of the substrate/chromogen solution - add 50 µl of the conjugate working dilution


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Table 7A - INITIAL PREPARATION CHECK PROCEDURE

Old Working Fresh Working Preparation Solution Dilution Solution Dilution Error (A+B) (C+D) (A+B) (C+D) in old

Step dilution/solution 1) 100 (µl) 50 - - - 2) 100 - - 50 (C+D)

3) - 50 100 - (A+B)

4) - - 100 50 See Tab. 1b

*) Should be freshly prepared from the stocks and buffers A-D used in the preceding test. **) If colour develops within 30 sec's, then error has occurred in preparation of old Dilution/solution. If no colour develops within 30 sec's, proceed to next sequential step.

Table 7B - INITIAL REAGENT CHECK PROCEDURE Functional Old New Detorioration

A B C D A B C D Of old Step Stock/buffer

1) A - C D - B - - B

2) - B C D A - - - A

3) - - C D A B - - A,B

4) A B C - - - - D D

5) A B - D - - C - C

6) A B - - - -B C D C,D

7) - - - - A C D ?**

*) If colour develops within 30 sec's, then functional deterioration has occurred in old Stock/buffer; use new stock/buffer. If no or slow colour develops, proceed to next Sequential step **) Contact Agency staff members. A = chromogen stock C = conjugate working stock B = substrate stock D = diluent buffer


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If a problem with the enzyme system is identified and resolved, run a test of internal quality controls (Cm, C++, C+ , C- and Cc) with the appropriate changes to the enzyme system. If again there is no colour development or the colour is slow to develop, contact Agency staff members (see Enquiries) and include all internal quality control data with the correspondence.

If a problem with excessive colour development occurs, run a test

of the internal quality controls (Cm, C++, C+, C- and Cc) using fresh reagents and buffers. Pay strict attention to buffer pH, reagent dilutions and incubation times and temperatures. If the problem persists, contact Agency staff members (see Enquiries) and include all internal quality control data with the correspondence.


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8. REFERENCES - Anderson J, McKay JA and Butcher RN (1991). The use of

monoclonal antibodies in competitive ELISA for the detection of antibodies to rinderpest and peste des petits ruminants viruses. IAEA-SM-318, International Symposium on Nuclear and Related Techniques in Animal Production and Health, Vienna, Austria.

- Libeau G, Diallo A, Calvez D and Lefevre PC (1991). A

competitive ELISA using anti-N monoclonal antibodies for specific detection of rinderpest antibodies in cattle and small ruminants. IAEA-SM318, International Symposium on Nuclear and Related Techniques in Animal Production and Health, Vienna, Austria.

- Anderson J, Rowe LW, Taylor WP and Crowther JR (1982). An

enzyme-linked immunosorbent assay for the detection of IgG, IgA and IgM antibodies to rinderpest virus in experimentally infected cattle. Res Vet Sci 32: 242-257.


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APPENDIX I. - ASSAY FLOW CHART - PLATE LAYOUT - WORK SHEET - ELISA DATA SHEET

Competitive Rinderpest ELISA Version 2.0, January 1999 Word 6.0/95 Filename: CORP20.doc

Competitive Rinderpest ELISA

Assay Conditions

Assay Steps Incubation Time

Incubation Temperature

Plate Shaking

Wash Step

1) Coat RPV Ag 1 hour + 37°C Yes 3 Times 2) Add Test Sera/Mab 1 hour + 37°C Yes 3 Times 3) Add Conjugate 1 hour + 37°C Yes 3 Times 4) Add Substrate &

Chromogen 10 Min + 37°C No -

5) Add Stop Solution None Room Temp. Brief - 6) Read Reaction (492 nm filter must be in plate reader)


Competitive ELISA Plate Layout Controls Serum samples (in duplicate)

1 2 3 4 5 6 7 8 9 10 11 12

A Cc Cc 1 5 9 13 17 21 25 29 33 37 B C++ C++ 1 5 9 13 17 21 25 29 33 37

C C++ C++ 2

D C+ C+ 2

E C+ C+ 3

F Cm Cm 3

G Cm Cm 4 40

H C- C- 4 40 Note: Cc: Conjugate Control (no serum) C++ Strong Positive Serum Control C+ Moderate Positive Serum Control Cm Monoclonal Control C- Negative Serum Control


Competitive ELISA Work Sheet

Plate no.: Date: Run ID.: Operator: Controls Serum samples (in duplicate)

1 2 3 4 5 6 7 8 9 10 11 12

A Cc Cc B C++ C++

C C++ C++

D C+ C+

E C+ C+

F Cm Cm

G Cm Cm

H C- C- Note:


ELISA DATA SHEET – Competitive Rinderpest ELISA

Disease: Plate No.: Date: Run ID: Operator: Filter:

Plate Reader Blank Value: Comment:

IQC Data OD1 OD2 OD3 OD4 UCL LCL Median OD Cm

PI IQC Data OD1 OD2 OD3 OD4 PI1 PI2 PI3 PI4 UCL LCL

Cc

C++

C+

Cm

C-

TEST SAMPLE DATA PI threshold = % (See Section 6.3)

ID OD1 / OD2 PI1/PI2 Mean PI ID OD1 / OD2 PI1/PI2 Mean PI 1 21

2 22

3 23

4 24

5 25 6 26

7 27

8 28

9 29

10 30

11 31

12 32

13 33

14 34

15 35

16 36

17 37

18 38

19 39

20 40

APPENDIX II.


- FACT SHEETS: * Reagent Information * QA Information - PACKING LISTS

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