BioRad TeSeE™ (768 tests English) - TSE-LAB-NET
Contents
1. 2575 alcohol according to European legislation Reagents containing 0 1 ProClin 300 are classified as irritating preparations according to European legislation Xn Alcohol gt 25 0 1 ProClinTM 300 R 10 22 37 38 41 43 67 Flammable Harmful if swallowed Irritating to respiratory system and skin Risk of serious damage to eyes May cause sensitisation by skin contact Inhalation of vapour may cause drowsiness and dizziness S 7 9 13 26 28 37 39 46 Keep container tightly closed and in a well ventilated place Keep away from food drink and animal feed In case of contact with eyes rinse immediately with plenty of water and seek medical advice After contact with skin wash immediately with plenty of water Wear suitable protecting clothings gloves and eye face protection If swallowed seek medical advice immediately and show this container or label 7 REFERENCES 1 J GRASSI E COMOY S SIMON C CREMINON Y FROBERT S TRAPMANN H 5 5 HAWKINS J MOYNAGH JP DESLYS G A H WELLS 2001 Rapid Test for preclinical postmorten diagnosis of BSE in central nervous system tissue The Veterinary Record 149 577 582 2 JP DESLYS E COMOY S HAWKINS S SIMON H SCHIMMEL G WELLS J GRASSI 1 MOYNAGH 2001 Screening slaughtered cattle for BSE Nature 409 476 477 3 E COMOY 2000 Contribution au d veloppement d un test de diagnostic post mortem des bovins atteints2. User s manual 2 1 PRINCIPLE OF PURIFICATION OF PrP The reagents of the TeSeE Purification Kit allow purification concentration and solubilisation of PrP from samples of tissues obtained from infected animals Processing of the samples comprises the following steps Grinding of samples Treatment of samples with Proteinase K Concentration of PrP by precipitation Solubilisation of PrP for immunoenzymatic assay using the reagents of the TeSeE SAP Detection Kit Ref 355 1182 2 2 SAMPLES Bovine purification of PrP is performed on samples from Central Nervous System CNS The BSE extraction tool Ref 355 1130 can be used to collect brainstem Since distribution of PrP is heterogeneous in central nervous system obex area from brainstem must be preferably sampled for optimal detection Sampling syringe Ref 355 1175 allows easy and rapid sampling of obex area in a secure way Please refer to sampling protocol for detailed instructions on good sampling procedure Small ruminants and cervids purification of PrP is performed on samples from Central Nervous System CNS or peripheral tissues lymphoid nodes spleen The small ruminant extraction tool Ref 355 1184 can be used to collect both brainstem and cerebellum Since distribution of PrP is heterogeneous in central nervous system obex area from brainstem must be preferably sampled for optimal detection Samples are cut and weighed individually No
3. Store the kit at 2 C to 8 C before use all reagents are stable at this temperature until the expiry date indicated on the kit The shelflives of the reagents after preparation are as follows LABELLING REAGENT SHELF LIFE R1 Microplate in tightly closed sachet 1 month at 2 C to 8 C R2 Diluted wash solution 24 hours at room temperature 18 C to 30 C 2 weeks at 2 C to 8 C R4 Reconstituted positive control 2 hours at room temperature 18 C to 30 C 4 hours at 2 C to 8 C 6 months at 20 C is recommended to divide the reconstituted solution into 0 5 ml aliquots and to store them immediately at 20 C Can be submitted to 3 successive freezing thawing cycles R7 Reconstituted conjugate solution 8 hours at room temperature 18 C to 30 C with diluted wash solution R8 R9 Development solution 6 hours at room temperature 18 C to 30 C always protected from light 3 6 PREPARATION OF SAMPLES FOR PrP DETECTION BY EIA Purified samples chapter 2 6 must be diluted with 125 pl 10 of reagent R Diluted samples must be homogenized with vortex 5 sec 2 sec just before distribution into the plate R1 3 7 PROCEDURE TeSeE Detection Kit Short assay Protocol Ref 355 1182 Procedure Procedure for manual processing 12 13 14 Remove the microplate rack and the required number of rows R1 from the protective packaging Replace the u
4. E PURIFICATION KIT 768 tests Ref 355 1181 eSe DETECTION KIT 768 tests Short Assay Protocol Ref 355 1182 REAGENT KITS FOR IN VITRO PURIFICATION AND DETECTION OF PrP Within the European Union this test is approved as rapid test for the BSE and scrapie testing programmes on cattle sheep and goats which are set up in accordance with Annex lll chapter to Regulation EC No 999 2001 User s manual User s manual 1 TABLE CONTENTS GENERAL INFORMATION 2 TeSeE PURIFICATION KIT 7 Dx NO NO h2 h O h2 1 NOOR Principle of purification of PrP Samples Composition of the TeSeE Purification Kit Preparation of reagents Storage shelf life Procedure Limits of the purification protocol TeSeE SAP DETECTION KIT CO CO CO WWW CO CO Q R C Principle of PrP detection by EIA Samples Composition of the TeSeE SAP Detection Kit Preparation of reagents Storage shelf life Preparation of samples for PrP detection by EIA Procedure Calculation and interpretation of the results Limits of the test MATERIAL REQUIRED BUT NOT SUPPLIED PRECAUTIONS HYGIENE AND SAFETY INSTRUCTIONS REFERENCES 1 GENERAL INFORMATION Transmissible Spongiform Encephalopathies TSE s are slow degenerative diseases of the central nervous system induced by unconventional transmissible agents UTAs routinely called prions 5 are generally classified according to their et
5. The assay comprises the following reactive steps 1 Distribution of negative R3 and positive controls R4 samples prepared with the reagents of the TeSeE Purification Kit Ref 355 1181 in the wells of the microplate coated with the first monoclonal antibody This distribution can be visually controlled as there is a marked colour difference between an empty well and a well containing a sample Incubation Washing then distribution of the peroxidase labelled antibody This distribution can also be visually controlled by the colour difference between an empty well and a well containing the conjugate solution Incubation Washing then revelation of enzymatic activity bound to the solid phase by addition of the substrate Stopping of the colour development determination of optical density at 450 nm 620 nm bichromatism mode and interpretation of the results 3 2 SAMPLES The assay can only be performed on samples obtained from collected tissues treated with the reagents and under the conditions of use of the TeSeE Purification Kit Ref 355 1181 Purified samples must be diluted with reagent R of the TeSeE SAP Detection Kit 3 3 COMPOSITION OF THE KIT LABELLING TYPE OF REAGENT PRESENTATION RI Microplate 12 strips of 8 wells coated with an anti PrP 8 plates monoclonal antibody Wash solution 10 fold concentrated Tris NaCl buffer R2 H 7 4 4 vials SEWA 250 ml Preservative Pro
6. d Encephalopathie Spongiforme Bovine Th se de doctorat v t rinaire Ecole Nationale V t rinaire d Alfort 4 EUROPEAN COMMISSION Directorate General DG XXIV 1999 Preliminary Report The evaluation of tests for the diagnosis of transmissible Spongiform Encephalopathy in bovines 5 JP DESLYS 1999 Prevention du risque d Encephalopathie Spongiforme Subaigu Trans missible La Revue du Praticien 49 966 970 6 R KNIGHT 1999 The relationship between new variant Creutzfeldt Jakob Disease and Bovine Spongiform Encephalopathy Vox sanguinis 76 203 208 7 D DORMONT 1997 Les Agents Transmissibles Non Conventionnels ou prions Virologie 1 11 22 8 F HILLA M DESBRULAIS S JOINER KCL SIDLE GOWLAND J COLLINGE lJ DOEY P LANTOS 1997 The same prion strain causes CJ disease and BSE Nature 389 448 450 9 CI LASMEZAS JP DESLYS O ROBAIN D DORMONT 1997 L agent secret des maladies prions La Recherche 46 53 10 AM HAYWOOD 1997 Transmissible Spongiform Encephalopathies The New England Journal of Medecine 337 25 1821 1828 11 J COLLINGE SIDLE J MEADS J IRONSIDE AF HILL 1996 Molecular analysis of prion strain variation and the aetiology of new variant CJD Nature 383 685 690 12 RG WILL J IRONSIDE M ZEIDLER SN COUSENS ESTIBEIRO ALPEROVITCH S POSER M POCCHIARI A HOFMAN PG SMITH 1996 A new variant of CreutzfeldtJakob disease in the U
7. positive greater than or equal to the cut off value The sample is considered to be negative according to the TeSeE SAP Detection Kit when these two values are less than the cutoff value Samples retested in duplicate and found to be negative according to the TeSeE SAP Detection Kit but for which one of the 2 values is close to the cutoff value cutoff value 10 must be interpreted cautiously 17 3 9 LIMITS OF THE TEST A negative result means that the test sample does not contain any PrP detectable by the TeSeE SAP Detection Kit However as very low levels of PrP may not be detected such a negative result does not exclude the possibility of infection Any sample with a reproducible positive result according to the test interpretation criteria must be confirmed in accordance with the countries national reference laboratory for TSEs or community reference laboratory in exceptional circumstances MATERIAL REQUIRED BUT NOT SUPPLIED Distilled or ultrapure water 20 000 ppm sodium hypochlorite final concentration and 1 M sodium hydroxide final concentration Absorbent paper Disposable gloves Protective glasses or mask with visor Purification step 2 ml polypropylene micro tesHubes with caps and appropriate tube rack Automatic or semiautomatic adjustable pipettes able to distribute volumes between 20 pl and 500 yl e Tissue homogenizer Ribolyser TeSeE PRECESS 24 or TeSeE PRECESS 48 e Centr
8. solution R10 grinding tubes reagent and reagent B Wash solution R2 sample diluent R6 peroxidase substrate buffer R8 chromogen R9 stop solution R10 and grinding tubes can be used with all kits from the TeSeE product line TeSeE and TeSeE sheep goat Allow the reagents to adjust to room temperature 18 to 30 C for 30 minutes before use Thoroughly reconstitute reagents avoiding any contamination Do not perform the test in the presence of reactive vapors acids alkalis aldehydes or dust which could alter the enzymatic activity of the conjugate Only use polypropylene tubes Use perfectly washed glassware rinsed in distilled water or preferably disposable material Do not let the microplate more than 5 minutes between the end of washing and distribution of the reagents The enzymatic reaction is very sensitive to all metals or metallic ions Consequently no metallic element must enter in contact with the various solutions containing the conjugate or the substrate The revelation solution substrate buffer chromogen must be colorless The appearance of a colour few minutes after reconstitution indicates that the reagent cannot be used and must be replaced The revelation solution should preferably be prepared with disposable plastic containers and distribution material or glassware previously washed in 1 N hydrochloric acid rinsed in distilled water and dried Store this solu
9. yl 50 13 52 yl 58 15 60 yl 66 17 68 yl 74 19 76 yl 82 21m 84 pl 90 23m 92 yl The volumes must be pipetted exactly The tip containing the PK has to be correctly rinsed by successive aspiration distribution cycles in reagent A After reconstitution homogenize the solution by successive inversions until you obtain a red homogeneous solution 2 5 STORAGE SHELF LIFE Store the TeSeE Purification Kit Ref 355 1181 at 2 C to 8 C All reagents are stable at this temperature until the expiry date indicated on the kit before and after opening of the vials After dilution the reconstituted proteinase K solution when stored at room temperature 18 C to 30 C must be used within 6 hours 2 6 PROCEDURE For the semi automatic processing of the purification protocol please refer to the TeSeE NSP operator s manual Procedure for manual processing 1 Sampling For peripheral tissues lymph nodes spleen insert one medium bead Ref 355 1171 in the grinding tube before to add the sample Take a mass of 350 mg 40 mg of nervous tissue preferably in the obex area or 200 mg 20 mg of peripheral tissue Deposit the samples in grinding tubes close firmly and proceed to the grinding step in the homogenizer Ribolyser TeSeE PRECESS 24 TeSeE PRECESS 48 systems 2 Sample grinding Place the tubes in the crown of the homogenizer Ribolyser TeSeE PRECESS 24 or TeSeE PRECESS 4
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12. 79 Take at least 400 yl 1000 pl with a 1000 pl tip and transfer in one well of the filter plate Ref 355 1179 exclude the first 6 positions from A1 to F1 Place a plastic sealing film on top the filter plate Set the vacuum gauge of the pump Ref 359 0350 to 25 4 Hg 2 5 Switch the pump on and check the gauge for correct vacuum then open manifold valve for 1 minute 6 seconds Close the valve switch off the pump and release the vacuum from the manifold Centrifugation technique Take at least 400 pl lt 1000 pl with a 1000 pl tip and transfer in one well of the filter plate Ref 355 1179 priorly fitted on a Deepwell plate Ref 359 0132 the master plate exclude the first 6 positions from A1 to F1 Place a plastic sealing film on top the filter plate Centrifuge the complete system filter plate and Deepwell plate for 1 min at 500 g Taking care to keep the filtration plate securely in position on the Deepwell plate Note Centrifuge must be equipped with Deepwell microplate rotor Ref 359 0136 for 5804R Eppendorf centrifuge Ref 359 1396 After either technique discard the filter plate and transfer 250 yl of filtered samples into another Deepwell purification plate for the manual protocol or directly place the master plate on board the NSP refer to the TeSeETM NSP operator s manual Note At this stage grinding tubes after homogenisation micro tesHubes and Deepwell plate after sampl
13. 8 systems Perform one agitation cycle with the following instrument parameters Ribolyser TeSeE PRECESS 24 or 48 Nervous tissues Peripheral tissues Nervous tissues Peripheral tissues Time 45 2 x 45 Speed 6 5 6 5 Program Program 1 Program 2 Wait a 5 minutes pause between the 2 agitation cycles When grinding is insufficient another 1 or 2 agitation cycles can be performed by ensuring that the temperature of the tube returns to room temperature 18 C to 30 C between each cycle using crushed ice for example 3 Sample transfer Remove the grinding tubes from the homogenizer resuspend the homogenate by inversion before opening the tubes Transfer the homogenate with one of the following methods Calibration syringe method Take 250 yl with the calibration syringe Ref 355 1174 taking care to immerse the needle in the pellet of beads to avoid sampling poorly homogenized tissue fragments Transfer each 250 pl sample into a 2 ml Eppendorf micro tesHtube or Deepwell Ref 359 0132 Filter plate method The transfer and the filtration are done separately using a filter plate 355 1179 and Deepwell plate Ref 359 0132 with either one of the two following filtration techniques Vacuum technique Fit the Deepwell plate Ref 359 0132 the master plate in the bottom of the vacuum manifold place the lead of the manifold and then the filter plate Ref 355 11
14. 8 MOI anon aw mon asy S00 Sas vid liqa 351 IWVN 0 L l go ala lasyWoLlon TPOEW 651 0 Sy 5 0 0 sz oyMd IM sz 008 e o sa pouow 8 2 2461 0104 6 61 07 4 81 1 7 4 07 10104 uw Wawa EWIN dv Wani SUN S 3WL 3WL MS lb 9NDVOS JON 108 108 MOH ainon AO IWMOA sv S080 SAAS PIAUOW avid uaa 351 3WVN s1949UiDaDd 49uspA 9p do bIW 16 3 8 CALCULATION AND INTERPRETATION OF THE RESULTS 1 Calculation of the mean optical density OD of the negative control OD R3 mean of the four OD of R3 wells 2 Calculation of the cut off value The cut off value is equal to OD R3 0 210 Example OD R3 0 020 Cutoff value 0 020 0 210 0 230 3 Condition of validation of the test Negative control R3 Validation of the individual negative control values The optical density of each individual negative control must be lower than 0 150 However a maximum of one individual aberrant value can be eliminated when its optical density is higher or equal to 0 150 The test must be repeated if more than one of the negative control lies outside of this limit b Homogeneity of the negative control values Calculate the mean of the negative contro
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16. Clin 300 0 01 Negative Control PBS buffer pH 7 2 supplemented with 4 vials R3 BSA 4 mi Preservative ProClin 300 0 1 ph Positive Control PBS buffer pH 7 4 supplemented with R4 non infectious synthetic peptide Lyophilized 4 vials Preservative ProClin 300 0 1 Sample diluent PBS buffer pH 7 2 supplemented with R6 BSA and phenol red ten Preservative ProClin 300 0 1 Conjugate 10 fold concentrated peroxidase labelled anti PrP monoclonal antibody in PBS buffer pH 7 1 4 vials R7 solution supplemented with bovine proteins and 2 8 ml coloured with phenol red i Preservative ProClin 300 0 1 Peroxidase Substrate Buffer Solution of citric acid and 4 vial R8 sodium acetate pH 4 0 containing 0 015 and 60 mi 4 dimethylsulfoxide DMSO m R9 Chromogen Tetramethylbenzidine 4 vials TMB solution 5 ml R10 Stop solution 1 N sulphuric acid 5 Adhesive films 1 The following reagents are generic components sample diluent R6 wash solution R2 peroxidase substrate buffer R8 chromogen R9 and stop solution R10 They can be used With all batches of the TeSeE SAP Detection Kits 3 4 PREPARATION OF REAGENTS Before use let the reagents of the TeSeE SAP Detection Kit adjust to room temperature 18 C to 30 C for 30 minutes 1 Ready to use reagents Microplates R1 Before opening the sealed bag with a desiccant let the microplate adjust t
17. K Lancet 347 911 925 13 SB PRUSINER amp AL 1993 Immunologic and molecular biologic studies of prion protein in Bovine Spongiform Encephalopathy The Journal of Infectious Diseases 167 602 613 21 Sample syringe 355 1175 SAMPLING METHOD FOR BIO RAD TSE SCREENING ASSAYS PLATELIA AND TeSeE SAP 24 TABLE OF CONTENTS GENERAL INFORMATION 1 1 Sample collection at the abattoir 1 2 Sample procedure at the laboratory BIO RAD SAMPLE SYRINGE SAMPLE MASS REQUIRED FOR THE TEST OPERATING PROCEDURE PRECAUTIONS ADVICE HEALTH AND SAFETY PROCEDURES 1 GENERAL INFORMATION The Bio Rad TSE screening assays are performed on a sample of 350 40 mg of central nervous tissues CNS The specific anatomical region for detecting PrP in infected animals is the brain stem more precisely in the area of the vagal nerve nucleus in the obex region This is the area of the brainstem where PrP is most concentrated Brain Sampling region right or left Obex Spinal cord Cross section of the brain stem at the level of the obex identifying the key target sites for diagnosis by histopathology and immunohistochemistry in BSE nucleus of the solitary tract 1 and the nucleus of the trigeminal tract V and scrapie dorsal nucleus of the vagus 3 Source OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 1 1 Sample collection at the abattoir The brain stem is easily and quickly coll
18. R8 R9 into each well and incubate the plate in darkness and at room temperature 18 C to 30 C for 30 mn 2 mn Do not use adhesive film during this incubation Add 100 pl 10 of stop solution R10 to each well according to the same sequence and same distribution rate as for the revelation solution Thoroughly wipe the bottom of the plate and determine the optical density at 450 nm 620 nm bichromatism mode within 30 minutes after stopping the reaction the rows must always be protected from light before reading 15 6 6 9 6 9 8 9 s lt Sel 0 Yl Pld 43385 43385 43385 43385 43385 MNMOQ Man 9945 43345 SOd hBA SOd DBA SOd BA SOd 34VHS ONDIVHS ONDIVHS 108 108 4610 45 TVOIBBA IVINOZOH 108 ONRELNI dsv 108 67611 20 IVI Md Ov Md 10 IVI 3WVN 11V14 0 L go amd fasvWOLIOG Zpouow 5 61 07 4 cr s 0 0 5 sz oyMd IM sz 008 0 sa 842 6251 0104 SZ 1 OYMd 81 LrMd 0rMd 10 03 uow Wawa WNN dv uojpunj Wani SIDE 3WL ML MOH 3WL MS poyrew IN JO N HW 9NDIVOS 108 WOllOd 10
19. aminated wastes Samples material and contaminated products must be eliminated after decontamination either by soaking in 1 M sodium hydroxide final concentration for 1 hour at room temperature 18 C to 30 C or by soaking in 20 000 ppm sodium hypochlorite solution for 1 hour at room temperature 18 C to 30 C or by autoclaving at 134 C minimum for at least 18 minutes under 3 bars of pressure Note never autoclave solutions containing sodium hypochlorite solution or reagent B All operations involved in Transmissible Spongiform Encephalopathy TSE screening tests are subject to regulations and must be performed in an isolated limited and controlled access laboratory devoted exclusively to this activity A laboratory coat overshoes gloves mask with visor or simple mask with safety glasses are required to ensure the operator s safety 19 20 Operators must receive specific training concerning the risks related to TSEs agents or prions and the validated modes of decontamination for unconventional agents Biosafety measures must be in agreement with recommendations of regular authorities of the country Avoid any contact of the substrate buffer chromogen and stopping solution with the skin and mucous membranes Neutralize and or autoclave all wash solutions or wash wastes or any liquid containing biological samples prior to their elimination Reagent B is a dangerous substance classified as nocive
20. e by closing the top of the syringe barrel and pushing the green piston until the air gaps have been eliminated At the same time ensure that the tissue nearest the opening of the syringe barrel is retained Holding the top of the syringe barrel still move the green piston to the nearest symbol Check that the sample core covers at least one zone corresponding to m as described in the previous section of this document sample mass required for the test Take a grinding tube and remove the lid with the sample syringe carefully depress the green piston to the next identical symbol to ensure that the correct mass of tissue m is dispensed in the grinding tube Remember that you must move the piston to the corresponding position of the next symbol as indicated in Sample mass required for the test Cut the sample core by gripping the top of the sample syringe against the inner edge of the grinding tube Samples of extremely bad quality should be either disected or if very autolysed pipetted up The unused part of the sample core can be stored by placing the sample syringe in the original container with the remaining piece of brain stem 27 5 PRECAUTIONS ADVICE As for any pipetting device Bio Rad recommends that operators using the sample syringe should be periodically monitored for a representative statistical population of samples taken so ensuring that sample weights are within range The sample syringes are to be used on
21. e incubator and the homogenization Homogenization is performed under the same conditions as in step 4 6 Concentration of the PrP centrifugation Within 30 minutes after reagent B distribution and mixing centrifuge the micro test tubes or purification plate as follows Centrifugation Micro tesHubes Deepwell plate Speed g 20 000 15 000 2 000 Time mm 5 7 10 Temperature C 20 20 4 Note For Deepwell plate allow a 5 minute delay at 37 C or a 10 minute delay at room temperature 18 C to 30 C before centrifugation 7 Sample clarifying Discard the supernatant by inverting the micro test tubes over a waste container Dry the micro testtubes by inverting onto absorbent paper for 5 minutes Or load the Deepwell plate on DW40 unit Ref 359 0137 Select TSE DW program and select number of strips to be performed Deepwell plate wells must be dried at the end of the DW40 process by inverting the plate on absorbent paper for 5 minutes Distribute 25 pl 10 of reagent into all micro testtubes or Deepwell wells Do not exceed an interval of 10 minutes between the end of the drying operation and distribution of buffer C Incubate immediately for 5 1 minute at 100 C 5 C Do not exceed 2 minutes between the reagent C distribution and the beginning of the incubation Do not seal the Deepwell plate during incubation Note If using Deepwell heating block must be equipped with a Deepw
22. e of the following methods by soaking in 1 M sodium hydroxide final concentration for 1 hour at room temperature 18 C to 30 C by soaking in 20 000 ppm sodium hypochlorite solution for 1 hour at room temperature 18 C to 30 C by autoclaving at a temperature of at least 134 C for a minimum of 18 minutes at 3 bar pressure Note never autoclave solutions containing bleach All operations involved in Transmissible Spongiform Encephalopathy TSE screening tests are subject to local safety guidelines and must be performed in an isolated limited and controlled access laboratory devoted exclusively to this activity A laboratory coat or boiler suit overshoes gloves two pairs mask with visor or simple mask with safety glasses are required to ensure the Operator s safety Operators must receive specific training concerning the risks related to TSE agents or prions and the validated methods of decontamination for unconventional agents Bio safety measures must comply with the Guidelines of the regulatory authorities of the country concerned Group Headquarters Bio Rad Laboratories 2000 Alfred Nobel Drive Hercules California 94547 Phone 510 741 1000 Toll Free Phone 14800 4246723 Fax 510 741 5800 Subsidiaries of Bio Rad Laboratories Australia Bio Rad Laboratories Pty Ltd PO Box 210 Regents Park Block Y Unit 1 Regents Park Industrial Estate 393 Park Road Regents Park New South Wales 21
23. e transfer can be stored closed At room temperature At 2 C to 8 C S At 20 C 18 C to 30 C in ice or refrigerator for 1 P for 8 hours for 15 hours Ra u Grinding tubes and Yes Yes Yes micro testtubes Deepwell plate Yes Yes No Frozen samples must be thawed at room temperature 18 C to 30 C Samples can be submitted to a maximum of 3 freezing thawing cycles Samples must always be homogenized by inversion before use 4 PK treatment Distribute 250 yl 10 of reconstituted proteinase K solution see paragraph 2 4 into each micro tube or Purification plate well Do not exceed intervals of 5 minutes for distribution of reconstituted proteinase K between the first and the last sample Immediately homogenise closed tubes or Deepwell sealed with aluminium film 10 times by inversion Do not exceed 2 minutes between the homogenization and the incubation at 37 C Incubate at 37 C 2 C in a heating block incubator for 10 1 minute Note If using Deepwell heating block must be equipped with Deepwell rack adaptor for heating block Ref 359 0134 Precipitation of PrP with reagent B Remove the micro testtubes or Deepwell plate from the heating block incubator Open the tubes and distribute 250 yl x 10 of reagent B into all micro tesHubes or Deepwell wells Observe the same order of distribution as described in step 4 Do not exceed intervals of 2 minutes between the exit of th
24. ected with an appropriate tool or sample collection spoon via the occipital foramen without opening the cranial cavity Sample collection with the Bio Rad collection spoon 25 26 1 2 Sampling procedure at the laboratory The whole brain stem sample is sent to the testing laboratory ensuring that appropriate bio safety measures recommended by the regulatory authorities of the particular country are followed In the laboratory the appropriate amount of cerebral material is cut scalpel blade from the obex region or collected with the Bio Rad sample syringe Ref 355 1175 which makes it possible to sample the required amount of the appropriate area quickly and safely without any risk of sharps injuries The following describes the procedure to effectively collect the sample from the obex region using the Bio Rad sample syringe without damaging the tissue BIO RAD SAMPLE SYRINGE The Bio Rad sample syringe consists of a green piston and a transparent syringe barrel The syringe barrel is labelled with a series of geometric shapes 0 gt Marks black Cutting wing Bio Rad gt 01 Barrel of the syringe transparent Piston green SAMPLE MASS REQUIRED FOR THE TEST The sample mass should occupy the space between two symbols of the same shape which corresponds to a mass m of 350 40 mg m m OPERATING PROCEDURE Take a sample syringe and pull out the green pi
25. ell rack adaptor for heating block Ref 359 0134 Remove the micro tesHubes or the Deepwell from the incubator and homogenate the tubes with a vortex 5 2 seconds Samples in micro test tubes or Deepwell can be stored for 5 hours at 2 C to 8 C or frozen for 72 hours at 20 C Frozen samples must be thawed at room temperature 18 to 30 C and homogenized with a vortex 5 2 seconds Please refer to information on TeSeE SAP Detection package insert Ref 355 1182 for detailed detection assay protocol 2 7 LIMITS OF THE PURIFICATION PROTOCOL Difficulties can be encountered during the grinding step when using dehydrated samples or peripheral tissues If necessary the grinding step step No 2 of the procedure may need to be repeated several times for this type of sample TeSeE DETECTION KIT Short Assay Protocol 768 TESTS 355 1182 REAGENTS FOR IN VITRO DETECTION OF PrP AFTER PURIFICATION User s manual 3 1 PRINCIPLE OF PrP DETECTION BY EIA The TeSeE SAP Detection Kit is an immuno enzymatic technique sandwich format using 2 monoclonal antibodies for the detection of the abnormal prion protein resistant to proteinase tissues collected from infected animals The kit contains sufficient reagents to assay 768 tests including controls The solid phase is composed of 12 strips of 8 polystyrene wells coated with the first monoclonal antibody The second monoclonal antibody is bound to peroxidase
26. ifuge adapted to micro testtubes e One micro testtube heating block thermostated at 37 C 2 C and one micro testtube heating block thermostated at 100 C 5 C For the semi automatic purification of the sample TeSeE NSP System Detection step Automatic or semiautomatic adjustable or fixed pipettes able to distribute 50 pl 100 yl 200 yl and 1000 pl 10 ml 20 ml 100 ml graduated test tubes Contaminated waste containers e Microplate incubator thermostated at 37 C 2 C Refrigerated chamber at 2 C to 8 C Automatic or semiautomatic microplate washing system Microplate reading apparatus equipped with 450 nm and 620 nm filters Microplate system for the automation of the assay protocol stages The performances of the system must conform with the requirements of the test protocol Contact BioRad for the list of available instruments PRECAUTIONS The quality of the results depends on compliance with the following good laboratory practices Reagents must be stored at 2 C to 8 C Do not use reagents whose shelflife has expired Do not use the reconstituted and stored at room temperature 18 C to 30 C proteinase K over 6 hours Do not mix reagents derived from different batches of the TeSeE SAP kits during the same manipulation with the exception of generic reagents wash solution R2 sample diluent R6 peroxidase substrate buffer R8 chromogen R9 stop
27. iology as iatrogenic familial and or sporadic Sheep scrapie has been reported in the 18 century and transmission demonstrated including to goats However the modes of contamination within flocks remain obscure TSEs were also described in deer and elk chronic wasting disease CWD and in cow Bovine Spongiform Encephalopathy BSE Humans are also susceptible to certain forms of TSE There is compelling evidence supporting that Bovine Spongiform Encephalopathy 5 has passed from cattle to human probably through consumption of contaminated meat In addition to this variant form of CreutzfeldtJakob disease vCJD other forms in humans include the Kuru and the iatrogenic CreutzfeldtJakob disease Pure hereditary forms such as the Gerstmann Str ussler Scheinker syndrome GSS and or sporadic CJD have been demonstrated in humans but their incidences are low We do not know if similar sporadic TSE cases exist in animals The main characteristics of these diseases are a slowly progressive but always fatal course absence of conventional infectious agents progressive accumulation in the central nervous system of an abnormal isoform of the natural prion protein PrP called PrP This isoform is characterized by particular biochemical properties and especially by an increased resistance to proteases The strikingly long incubation period that precedes the neurological symptoms suggests that important events of TSE pathogenese
28. ls with the individual remaining values Values higher than the mean of the negative controls 40 OD R3 40 must be eliminated If one individual value is eliminated in one additional value can be eliminated lf no negative control value is eliminated in two values maximum can be eliminated in The test must be repeated if more than two values of the negative control are eliminated criteria a b Positive control R4 The mean of the positive control optical densities R4 ODs must be higher or equal than 1 000 The test must be repeated if the mean of the positive control optical densities R4 ODs is strictly lower than this limit 4 Interpretation of the results Samples with an optical density lower than the cutoff value are considered to be negative according to the TeSeE SAP Detection Kit However results situated just below the cutoff value cutoff value 10 must be interpreted cautiously and the corresponding samples should be retested in duplicate starting from the original homogenate Samples with an optical density greater than or equal to the cutoff value are considered to be initially reactive according to the TeSeE SAP Detection Kit and should be retested in duplicate starting from the original homogenate before the final interpretation After repeating the test the sample is considered to be positive according to the TeSeE SAP Detection Kit when at least one of the 2 measurements is
29. ly once and then discarded in order to prevent any cross sample contamination The sample must be taken with all due precautions in order to ensure that risk of contamination for operators is minimized The syringes used are to be discarded after being decontaminated see Health amp Safety instructions IF the sample core does not fill the entire syringe barrel despite carrying out the procedure correctly it is advisable to weigh the sample 6 HEALTH amp SAFETY PROCEDURES 28 The hygiene conditions bio safety measures and good laboratory practices must comply with the guidelines of the regulatory authorities in the country The sample syringe is intended for use in in vitro diagnostic procedures only Wear disposable gloves when handling reagents and samples and wash your hands thoroughly after handling them Any equipment that has come into direct contact with the samples must be considered to have been contaminated Contaminated surfaces must be cleaned with 20 000 ppm sodium hypochlorite solution When the contaminating liquid is an acid contaminated surfaces must first be neutralized with sodium hydroxide before using sodium hypochlorite Surfaces must be rinsed with distilled water dried with ethanol and wiped with absorbent paper The material used for cleaning must be discarded in a specific container for contaminated waste Samples equipment and contaminated products must be discarded after decontamination using on
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31. nused rows with the desiccated bag in the microplate sachet and hermetically close it Prepare the positive control R4 as described in chapter 3 4 2 For each series of tests and every single plate distribute 100 pl 10 of control sample into wells in the following order Wells AT B1 C1 D1 negative control R3 Wells E1 F1 positive control RA Wells G1 H1 etc sample diluted with reagent 86 Samples are performed in singulate Cover with adhesive film and incubate for 30 mn 2 mn at 37 C 2 C Prepare wash solution R2 Prepare conjugate solution R7 Remove the adhesive film perform 3 wash cycles Optimal washing conditions are obtained with PWAO PW41 or 1575 BioRad plate washers with program TSE 3 Do not let the microplate stand for more than 5 minutes after the last wash cycle Dry by inversion on absorbent paper before the following step Distribute 100 yl 10 of conjugate solution R7 into each well Cover with adhesive film and incubate 30 mn 2 mn at 2 C to 8 C Prepare the enzymatic revelation solution R8 R9 Remove the adhesive film perform 5 wash cycles Optimal washing conditions are obtained with PWAO PW41 or 1575 BioRad plate washers with program TSE 5 Do not let the microplate stand for more than 5 minutes after the last wash cycle Dry by inversion on absorbent paper before the following step Distribute 100 pl 10 of revelation solution
32. o room temperature 18 C to 30 C in its protective packaging to avoid any water condensation in the wells Open at the solder point and immediately return the unused rows to the sachet Tightly close the bag after expelling any air then store at 2 C to 8 C The negative control R3 sample dilution solution 86 and stop solution R10 are ready to use 13 2 Reqgents to reconstitute Wash solution R2 Dilute wash solution R2 to 1 10 in distilled or ultrapure water example 100 ml of reagent R2 in 900 ml of distilled water Positive control R4 Gently tap the vial of positive control R4 on the laboratory bench to detach any substance adherent to the rubber stopper Open the vial and dissolve the content in 4 ml of diluent R Reseal the vial and let stand for approximately 1 minute homogenizing gently and occasionally to facilitate dissolution Conjugate R7 Dilute reagent R7 to 1 10 in the freshly reconstituted wash solution example O 1 ml of reagent R7 in 0 9 ml of reconstituted wash solution bearing in mind that 1 ml of reqdy for use conjugate is sufficient for 1 row Homogenize gently Avoid using a vortex agitator Enzymatic development solution R8 R9 Dilute reagent R9 to 1 11 in reagent R8 example 0 1 ml of reagent R9 in 1 ml of reagent R8 bearing in mind that 1 1 ml of enzymatic revelation solution is sufficient for 1 row Homogenize gently Avoid using a vortex agitator 3 5 STORAGE SHELF LIFE
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34. s might take place in extra nervous sites and especially in peripheral lymphoid tissues In spite of many unknown and or incertitudes the detection of abnormal PrP is now established as the method to confirm TSE diagnosis This detection is mainly achieved from post mortal collected nervous tissues Abnormal PrP has also been detected in a number of lymphoid tissue and organs in the germinal centres of spleen lymph nodes tonsils and or mucosa associated lymphoid tissue but at the research area in animal models or in scrapie sheeps CWD deers and elks and vCJD patients Reagent designed by the Commissariat l nergie Atomique CEA French Atomic Energy Commission developed produced and marketed by Bio Rad allow PrP detection on samples of nervous tissues taken from animals This determination comprises the following reaction steps Purification of PrP 768 tests Step performed with the following reagents and accessories TeSeE Purification Kit 768 tests Calibration syringe and needle x 200 or Filter plates x 50 Deepwell microplates x 50 Medium beads x 2000 o PrP detection 768 tests Step performed with the following reagents TeSeE Detection Kit 768 tests Short Assay Protocol Ref Ref Ref Ref Ref Ref 355 1181 355 1174 355 1179 359 0132 355 1171 355 1182 TeSeE PURIFICATION KIT 768 TESTS 355 1181 REAGENT KIT FOR IN VITRO PURIFICATION OF PrP
35. ston to approximately 1 cm from its home position then push home again Firmly grasp the brain stem in one hand using a disposable wrapper plastic bag glove etc in order to avoid possible cross sample contamination The end of the brain stem should remain accessible If the brainstem received has a cord too long the user should trim it Samplers should received proper training regarding the pr cise location of the targetted area Use the other hand to position the open end of the sampling syringe on the right or left side of the caudal end of the brain stem Note a complete hemi section of brain stem with an intact obex region must remain available after sample collection for confirmatory testing Insert the syringe barrel gradually into the brain stem whilst holding the green piston stationary relative to the brain stem Note While collecting the sample from the obex region take care that the syringe barrel remains within the selected side of the brain stem Stop this movement when the top of the syringe barrel has reached the upper limit of the sampling zone Cut the sample core by twisting the syringe barrel through one complete turn Slowly remove the sample syringe from the brain stem taking care not to damage surrounding tissues The remaining brain stem can be placed in its original sample container Check whether there are any air gaps in the core sample collected If needed compress the sample cor
36. te other tissues tonsils ileum eyelid can be used for research purposes only Samples are stored at 2 C to 8 C when purification is performed within 24 hours or can be stored frozen for several months They can only be submitted to 3 freezing thawing cycles If these samples must be transported they should be packaged in accordance with current local regulations 2 3 COMPOSITION OF THE TeSeE PURIFICATION KIT LABELLING TYPE OF REAGENTS PRESENTATION STORAGE eem Grinding tube containing ceramic beads nang in a buffer solution 8 bm 2 C to 25 C Preservative ProClinTM 300 0 1 Reagent A Denaturing solution ae 2 C to 8 C Clarifying solution 4 vials E Reagent B Colouring bromophenol blue 55 ml Resolving buffer vials 6 Reagent Colouring malachite green Z ml La Proteinase K Vials Colouring phenol red 0 5 ml sada Reagent reagent B and grinding tubes are generic components They can be used with all batches of the TeSeE Purification Kits 2 4 PREPARATION OF REAGENTS All reagents of the TeSeE Purification Kit except proteinase are ready for use Reagent is the dilution buffer for proteinase K The solution must be prepared in the following way 4 pl of proteinase K in 1 ml of reagent A NUMBER REAGENT A PROTEINASE K OF SAMPLES 2 1 ml 4 yl 10 3 ml 12 pl 18 5 ml 20 pl 26 7 ml 28 yl 34 9 ml 36 yl 42 11m 44
37. tion protected from light Use a new pipette tip for each sample Washing of the wells is an essential step of the procedure respect the recommended number of washing cycles and ensure that all wells are completely filled then completely emptied Inadequate washing can give incorrect results Never use the same container and pipette to distribute the conjugate and the revelation solution HYGIENE AND SAFETY INSTRUCTIONS Generally hygiene conditions biosafety mesures and good laboratory practices must be in agreement with recommendation of regular authorities of the country All reagents of the kit are intended for use in in vitro diagnosis Wear disposable gloves when handling reagents and samples and wash your hands thoroughly after handling them Do not pipette with the mouth Use polypropylene containers to avoid any wounds with broken glass All the materials directly in contact with the samples and the wash solutions must be considered as contaminated Avoid splashing samples or solutions containing samples Contaminated surfaces must be cleaned with 20 000 ppm sodium hypochlorite solution bleach When the contaminating liquid is an acid contaminated surfaces must be first neutralized with sodium hydroxide before using bleach Surfaces must be rinsed with distilled water dried with ethanol and wiped with absorbent paper The material used for cleaning must be discarded in a special container for cont
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