BioRad TeSeE™ Combi Kit - TSE-LAB-NET
Contents
1. 8 C Preservative ProClin 300 0 1 Conjugate 10 fold concentrated peroxidase labelled anti PrP monoclonal antibody in PBS 1 vial 2 vials 4 vials R7 buffer pH 7 1 solution supplemented with 2 8 ml 2 8 ml 2 8 ml 2 to 8 C bovine proteins and coloured with phenol red i i Preservative ProClin 300 0 1 Peroxidase Substrate Buffer Solution of citric R8 acid and sodium acetate pH 4 0 containing 1 vial 1 vial 2 vials 42 C to 48 C 0 015 H O and 4 dimethylsulfoxide 60 ml 120 ml 120 ml DMSO Chromogen Tetramethylbenzidine TMB 1 vial 1 vial 1 vial s R9 solution 6m tom Cte 8 TE 1 vial 1 vial 1 vial a R10 Stop solution 1 N sulphuric acid 28 ml 56 ml 112 2 to 8 Adhesive films 8 12 16 The following reagents are generic components Reagent A reagent B 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 Kits 2 4 Preparation of Reagents Before use let the reagents of the TeSeE SAP Combi Kits adjust to room temperature 18 to 30 for 30 minutes 1 Ready to use reagents Reagent A B C the negative control R3 sample dilution solution R6 and stop solution R10 are ready to use Microplates R1 Before opening the sealed bag with a desiccant let the microplate adjust to room temperature 18 t2. Bb LO WH H3GWNN SIGNI dSV HILNI SUM S310A40 Ivu awu HSVM MOH Inu MS pouen apow JOAN I3A SNDVOS 108 WoLio8 108 MO fanon faao dsv aaow SdiHLS alvi 1103 S ASL E 3 0 s s 5 E 0 s sed fdSvWOLLOS gt Z POUR 9 81 09 sr E 0 Lr 0 z 9 0 IM sz 008 eo SA Geld HSYM E aor 68 96 Lyme 8 2 core Jejurejed E E fear usyorMd g L LHMdOrMd HASANN MEIN dv uogoun SUM HAN S31049 dsv awu HSvM Mond MS pou 134 108 WOLLOS 108 aano dsv aaow PIQUE 1103 ASL 3INVN 11 2 7 Calculation and Interpretation of the Results 1 2 Calculation of the mean optical density OD of the negative control OD R3 mean of the four OD of R3 wells Calculation of the cut off value The cut off value is equal to OD R3 0 210 Example OD R3 0 020 Cut off value 0 020 0 210 0 230 3 4 12 Condition of validation of the test Negative con
3. PK treatment Distribute 250 ul 10 of reconstituted proteinase 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 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 a Deepwell rack adaptor for heating block Ref 359 0134 5 Precipitation of PrP with reagent Remove the micro test tubes or Deepwell plate from the heating block incubator Open the tubes and distribute 250 ul 10 of reagent B into all micro test tubes 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 the 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 test tubes 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
4. The performances of the System must conform with the requirements of the test protocol Contact Bio Rad for the list of available instruments 4 PRECAUTIONS The quality of the results depends compliance with the following good laboratory practices Reagents must be stored at 2 C to 8 C Do not use reagents whose shelf life has expired Do not use the reconstituted and stored at room temperature 18 C to 30 C proteinase 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 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 TeSeE SAP and TeSeE sheep goat assays Allow the reagents to adjust to room temperature 18 to 30 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 no
5. 1997 The same prion strain causes CJ disease and BSE Nature 389 448 450 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 KC 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 K ESTIBEIRO A ALPEROVITCH S POSER M POCCHIARI A HOFMAN PG SMITH 1996 A new variant of Creutzfeldt Jakob disease in the U K Lancet 347 911 925 13 SB PRUSINER amp AL 1993 16 Immunologic and molecular biologic studies of prion protein in Bovine Spongiform Encephalopathy The Journal of Infectious Diseases 167 602 613 Sample Syringe Ref 355 1175 SAMPLING METHOD FOR Bio Rad TSE SCREENING ASSAYS 18 TABLE CONTENTS 1 GENERAL INFORMATION 1 1 Sample Collection at the Abattoir 1 2 Sample Procedure at the Laboratory 2 Bio Rad SAMPLE SYRINGE 3 SAMPLE MASS REQUIRED FOR THE TEST 4 OPERATING PROCEDURE 5 PRECAUTIONS ADVICE 6 HEALTH AND SAFETY PROCEDURES 1 GENERAL INFORMATION The Bio Rad TSE screening assays are performed a sample of 350 40 mg of central nervous tissues CNS The specific anatomical region for detecting PrP in infected ani
6. TeSeE SAP Combi Short Assay Protocol 192 Tests Ref 355 1186 384 Tests Ref 355 1192 768 Tests Ref 355 1191 REAGENT KITS FOR IN VITRO PURIFICATION AND DETECTION OF 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 Ill chapter A to Regulation EC No 999 2001 User s manual TABLE CONTENTS 1 GENERAL INFORMATION 2 TeSeE SAP Combi Kit 2 1 2 2 2 3 Principle Samples Composition of the TeSeE SAP Combi Kits Preparation of Reagents Storage Shelf life Assay Procedure Calculation and Interpretation of the Results Limits of the Test 3 MATERIAL REQUIRED BUT NOT SUPPLIED 4 PRECAUTIONS 5 HYGIENE AND SAFETY INSTRUCTIONS 6 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 TSEs are generally classified according to their etiology as iatrogenic familial and or sporadic Sheep scrapie has been reported in the 18th 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 sus
7. 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 test tubes 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 ul 10 of reagent C into all micro test tubes 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 Deepwell rack adaptor for heating block Ref 359 0134 Remove the micro test tubes 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 C to 30 C and homogenized with a vortex 5 2 seconds Purified samples must be diluted with 125 ul 10 of reagent R6 Diluted
8. ceptible to certain forms of TSE There is compelling evidence supporting that Bovine Spongiform Encephalopathy BSE has passed from cattle to human probably through consumption of contaminated meat In addition to this variant form of Creutzfeldt Jakob disease vCJD other forms in humans include the Kuru and the iatrogenic Creutzfeldt Jakob 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 pathogeneses 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 ly
9. criteria must be confirmed in accordance with the countries national reference laboratory for TSEs or community reference laboratory in exceptional circumstances 3 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 test tubes with caps and appropriate tube rack e Automatic or semiautomatic adjustable pipettes able to distribute volumes between 20 ul and 500 ul Tissue homogenizer Ribolyser TeSeE PRECESS 24 or TeSeE PRECESS 48 Centrifuge adapted to micro test tubes One micro test tube heating block thermostated at 37 C 2 C and one micro test tube heating block thermostated at 100 C 5 C For the semi automatic purification of the sample TeSeE NSP System Detection step e Automatic or semiautomatic adjustable or fixed pipettes able to distribute 50 ul 100 pl 200 ul and 1000 ul 10 ml 20 ml 100 ml graduated test tubes Contaminated waste containers Microplate incubator thermostated at 37 2 Refrigerated chamber at 2 to 8 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
10. cycles Reconstituted conjugate solution R7 with diluted wash solution 8 hours at room temperature 18 to 30 R8 R9 Development solution 6 hours at room temperature 18 C to 30 C always protected from light 2 6 Assay Procedure 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 or TeSeE PRECESS 48 systems 2 Sample grinding Place the tubes in the crown of the homogenizer Ribolyser TeSeE PRECESS 24 or TeSeE PRECESS 48 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 sec 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 ensuring that the temperature of
11. ds 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 contaminated 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 to 30 C 14 or by soaking 20 000 ppm sodium hypochlorite solution for 1 hour at room temperature 18 to 30 or by autoclaving at 134 minimum for at least 18 minutes under 3 bars of pressure Note never autoclave solutions containing sodium hypochlorite solution or reagent B e 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 activit
12. e sample from the obex region using the Bio Rad sample syringe without damaging the tissue 2 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 D 1 Marks black Cutting wy E di sBi Rd O P O0 1 P EH 0 Barrel of the syringe transparent Piston green 3 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 4 OPERATING PROCEDURE Take a sample syringe and pull out the green piston 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 20 Insert the syringe barrel g
13. es 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 one of the following methods by soaking in 1 sodium hydroxide final concentration for 1 hour at room temperature 18 to 30 C by soaking in 20 000 ppm sodium hypochlorite solution for 1 hour at room temperature 18 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 laboratory coat or boiler suit overshoes gloves two pairs mask wi
14. f the TeSeE SAP Combi Kits PRESENTATION LABELLING TYPE OF REAGENTS 355 1186 355 1192 355 1191 STORAGE 192 tests 384 tests 768 tests 1 1 1 Reagent Denaturing solution 55 ml 120 240 ml 2 to 8 C Clarifying solution 1 1 2 vials 5 Reagent Colouring bromophenol blue 65m 120m 120m t0 8 C Resolving buffer 1 vial 1 vial 1 vial E Reagent C Colouring malachite green 7 ml 14 ml 28 ml 2 C to 8 C Proteinase K 1 vial 2 vials 4 vials 2 PK Colouring phenol red 0 5 0 5 0 5m 12 0 t0 8 C R1 Microplate 12 strips of 8 wells coated with an 2plates 4plates 8 plates 2 C to 8 anti PrP monoclonal antibody Wash solution 10 fold concentrated Tris NaCI R2 buffer pH 7 4 Preservative ProClin 300 0 01 1 vial 2 vials 4 vials 2 C 250 ml 250 ml 250 ml to 25 Negative Control PBS buffer pH 7 2 4 vial 2 vials 4 vials R3 supplemented with BSA 2 to 8 C Preservative ProClin 300 0 1 4m 4m 4m Positive Control PBS buffer pH 7 4 supplemented with non infectious synthetic 1 vial 2 vials 4 vials R4 peptide Lyophilized as 4 as 4 m q s 4 mp to 8 Preservative ProClin 300 0 1 Sample diluent PBS buffer pH 7 2 1 vial 1 vial 4 vial R6 supplemented with BSA and phenol red 35 ml 70 ml 140 ml 2 to
15. icate starting from the original homogenate Samples with an optical density greater than or equal to the cut off value are considered to be initially reactive according to the TeSeE SAP Kit instructions 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 Kit instructions when at least one of the 2 measurements is positive greater than or equal to the cut off value The sample is considered to be negative according to the TeSeE SAP Kit instructions when these two values are less than the cut off value Samples retested in duplicate and found to be negative according to the TeSeE SAP Kit instructions but for which one of the 2 values is close to the cut off value cut off value 1096 must be interpreted cautiously 2 8 Limits of the Test Difficulties can be encountered during the grinding step when using dehydrated samples or peripheral tissues necessary the grinding step step No 2 of the procedure may need to be repeated several times for this type of sample A negative result means that the test sample does not contain any PrP detectable by the TeSeE SAP Combi Kits However as very low levels of 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
16. lls coated with the first monoclonal antibody The second monoclonal antibody is bound to peroxidase 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 e Small ruminants and cervids purification of 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 Note other tissues tonsils ileum eyelid can be used for research purposes only Samples are stored at 2 to 8 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 o
17. mals 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 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 2 and scrapie dorsal nucleus of the vagus 3 Spinal cord 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 collected 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 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 th
18. mphoid 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 Test performed with the following reagents and accessories TeSeE SAP Combi Kit 192 tests Ref 355 1186 TeSeE SAP Combi Kit 384 tests Ref 355 1192 TeSeE SAP Combi Kit 768 tests Ref 355 1191 Grinding Tubes 384 tubes Ref 355 1139 Grinding Tubes 768 tubes Ref 355 1137 Calibration syringe and needle x 200 Ref 355 1174 or Filter plates x 50 Ref 355 1179 Deepwell microplates x 50 Ref 359 0132 Medium beads x 2000 Ref 355 1171 Only for peripheral tissues 2 TeSeE SAP Combi Kit 2 1 Principle The reagents of the TeSeE SAP Combi Kit allow purification concentration solubilisation and detection of PrP from samples of tissues obtained from infected animals The TeSeE SAP Assay is an immuno enzymatic technique sandwich format using 2 monoclonal antibodies for the detection of the abnormal prion protein resistant to proteinase K in tissues collected from infected animals The solid phase is composed of 12 strips of 8 polystyrene we
19. o 30 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 2 Reagents to reconstitute Proteinase K Reagent A is the dilution buffer for proteinase K The solution must be prepared in the following way 4 ul of proteinase K in 1 ml of reagent A NUMBER OF SAMPLES REAGENT A PROTEINASE K 2 1 ml 4 ul 10 3ml 12 ul 18 5ml 20 ul 26 7 ml 28 yl 34 9ml 36 ul 42 11ml 44 yl 50 13ml 52 ul 58 15ml 60 ul 66 17 ml 68 ul 74 19 ml 76 yl 82 21 ml 84 ul 90 23 ml 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 After reconstitution homogenize the solution by successive inversions until you obtain a red homogeneous solution 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 R6 Reseal the vial and let stand for approximately 1 minute homogenizing gently and occasionally to facilitate dissolution Conjugate R7 Dilute
20. o Rad 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 R8 R9 into each well and incubate the plate in darkness and at room temperature 18 to 30 for 30 mn 2 mn Do not use adhesive film during this incubation Add 100 ul 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 6 6 9 6 9 8 9 s 6 S el 50 LA reid 03345 09345 09348 09345 03348 QuvMdn MNMOd Man MNMOQ 03348 03348 SOd LHA SOd 1U3A SOd 13A SOd HOH ONDIVHS ONDIVHS 108 108 asia dsv VOLLH3A IVLNOZIHOH 108 ds ONIHLN39 dsv 108 9291 0 LYT EtMd 0 Md LO LV14 SINVN ILVId 0 i E 0 SA eid fdSvWOLLOS Z pou 8191 0 S s 0 HA 08 E IM Sz 008 O SA Aid HSVM L POUR HA 8 2 251 0 114 d z S vezi 8 8
21. osition 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 21 5 PRECAUTIONS ADVICE As for any pipetting device Bio Rad 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 only 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 lf 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 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 procedur
22. otecting clothings gloves and eye face protection If swallowed seek medical advice immediately and show this container or label 6 REFERENCES 1 J GRASSI E COMOY S SIMON C CREMINON Y FROBERT S TRAPMANN H SCHIMMEL S A C HAWKINS J MOYNAGH JP DESLYS G A H WELLS 2001 Rapid Test for the 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 J MOYNAGH 2001 Screening slaughtered cattle for BSE Nature 409 476 477 3 E COMCY 2000 Contribution au d veloppement d un test de diagnostic post mortem des bovins atteints 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 JP DESLYS 1999 Prevention du risque d Encephalopathie Spongiforme Subaigu Trans missible La Revue du Praticien 49 966 970 R KNIGHT 1999 The relationship between new variant Creutzfeldt Jakob Disease and Bovine Spongiform Encephalopathy Vox sanguinis 76 203 208 D DORMONT 1997 Les Agents Transmissibles Non Conventionnels ou prions Virologie 1 11 22 F HILLA M DESBRULAIS S JOINER KCL SIDLE I GOWLAND J COLLINGE LJ DOEY P LANTOS
23. radually 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 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 core 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 as described in the previous section of this document sample mass required for the test e 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 p
24. reagent R7 to 1 10 in the freshly reconstituted wash solution example 0 1 ml of reagent R7 in 0 9 ml of reconstituted wash solution bearing in mind that 1 ml of ready 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 2 5 Storage Shelf life Store the TeSeE SAP Combi Kits at 2 to 8 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 solution when stored at room temperature 18 to 30 must be used within 6 hours The shelf lives 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 A 24 hours at room temperature 18 C to 30 C R2 Diluted wash solution 2 weeks at 2 C to 8 2 hours at room temperature 18 C to 30 C 4 hours at 2 C to 8 C 6 months at 20 C R4 R constitut d positive control It 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
25. samples must be homogenized with vortex 5 sec 2 sec just before distribution into the plate R1 1 Remove the microplate rack and the required number of rows R1 from the protective packaging Replace the unused rows with the desiccated bag in the microplate sachet and hermetically close it 10 11 12 13 14 10 Prepare the positive control R4 as described in chapter 3 4 2 For each series of tests and every single plate distribute 100 ul 10 of control sample into wells in the following order Wells A1 B1 C1 D1 negative control R3 Wells E1 F1 positive control R4 Wells G1 H1 etc sample diluted with reagent R6 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 PW40 PW41 or 1575 Bio Rad 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 ul 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 PW40 PW41 or 1575 Bi
26. t 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 solution 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 5 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 han
27. th 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 22 Web site www bio rad com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 31 3689 6600 Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Malaysia 60 3 2117 5260 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3170 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 Thailand 66 2 6518311 United Kingdom 020 8328 2000 Bio Rad 3 bd Raymond Poincar 92430 Marnes la Coquette France T l 33 1 47 95 60 00 Fax 33 1 47 41 91 33 Rev A 11 2011 Code 862230
28. the manifold Centrifugation technique Take at least 400 ul lt 1000 ul with a 1000 pl tip and transfer 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 ul of filtered samples into another Deepwell the purification plate for the manual protocol or directly place the master plate on board the NSP refer to the TeSeE NSP operator s manual Note At this stage grinding tubes after homogenisation micro test tubes and Deepwell plate after sample transfer can be stored closed At room temperature At 2 C to 8 C At 20 C 18 C to 30 C in ice or refrigerator tor vear for 8 hours for 15 hours y Grinding tubes and Yes Yes Yes micro test tubes Deepwell plate Yes Yes No Frozen samples must be thawed at room temperature 18 to 30 Samples can be submitted to a maximum of 3 freezing thawing cycles Samples must always be homogenized by inversion before use 4
29. the tube returns to room temperature 18 to 30 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 ul 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 ul sample into a 2 ml Eppendorf micro test tube or Deepwell Ref 359 0132 Filter plate method The transfer and the filtration are done separately using a filter plate Ref 355 1179 and a 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 1179 Take at least 400 ul lt 1000 ul with 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 cm 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
30. trol R3 a 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 controls with the individual remaining values Values higher than the mean of the negative controls 4096 OD R3 4096 must be eliminated If one individual value is eliminated in a one additional value can be eliminated in b If no negative control value is eliminated in a two values maximum can be eliminated in b 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 Interpretation of the results Samples with an optical density lower than the cut off value are considered to be negative according to the TeSeE SAP Kit instructions However results situated just below the cut off value cut off value 1096 must be interpreted cautiously and the corresponding samples should be retested in dupl
31. y A laboratory coat overshoes gloves mask with visor or simple mask with safety glasses are required to ensure the operator s safety e 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 wash wastes or any liquid containing biological samples prior to their elimination Reagent B is a dangerous substance classified as nocive gt 25 alcohol according to European legislation Reagents containing 0 196 ProClin 300 are classified as irritating preparations according to European legislation Xn Alcohol 2596 0 196 ProClin 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 pr
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