Date : 02/05/2011. Radia ZERGAOUI Engineer in charge of Quality Insurance

Transcription

1 Technical procedure : SOP-T-VV.11.1 Service Production et Purification de Vecteurs Viraux Standard Operating Procedure (SOP) Quantification of physical particles in rlv supernatants by HIV-p24 ELISA assay Date : 02/05/2011 Written by, Function, visa : Marion BRICARD Viral vector Technician Radia ZERGAOUI Engineer in charge of Quality Insurance Index : 0 Written by, Function, visa : Hélène VERGNAULT Viral Vector Manager Page 1 of 10 Authorized by, Function, visa : Pascale BOUILLÉ President and Scientific Director Sommaire Quantification of physical particles in rlv supernatants by HIV-p24 ELISA assay SOP-T-VV Purpose 2 Application domain 3 References 4 Definitions 5 Responsabilities 6 Processus 7 Appendix Operating modes OM1: Samples preparation OM2: Quantification of HIV-p24 proteins by ELISA OM3: Results analysis Annexes AI: Worksheet for titration by HIV-p24 quantification by ELISA AII: HIV-1 P24 ELISA catalog NEK050 One 96-well plate DATE REVIEWING MODIFICATIONS PURPOSE MODIFIED CHAPTERS COPIES FOR USERS TRAINEES For Information Bricard Marion visa Matéo Bruno visa visa Leroy Julien visa Vergnault Hélène visa visa

2 1 Purpose To describe the methods to titrate rlv supernatants by quantifying physical particles HIV-1 p24 ELISA assay. 2 Application domain This technical procedure applies under the general procedure for production and purification of rlv lentiviral vectors. 3 References Perkin-Elmer Kit Procedure: Perkin-Elmer Life Sciences, Inc «HIV-1 p24 ELISA» for detection of HIV-1 p24 Antigen in Cell Culture Supernatant 4 Definitions rlv: lentiviral vector; ELISA: Enzyme Linked Immuno Sorbent Assay HRP: horseradish peroxydase OPD: OrthoPhenyleneDiamine-HCl, substrate which produces a yellow color that is directly proportional to the amount of HIV-1 p24 captured. IP/ml: infectious particles /ml PP/ml: physics particles/ ml p24: a 24kDa protein (p24), immunologically distinct from proteins in most other retroviruses, has been demonstrated to be a major structural core component of HIV-1. The preparation of a mouse monoclonal antibody with high specificity and affinity for this viral protein has allowed the development of an enzyme-linked immunoabsorbent assay (ELISA) for HIV-1 p24. 5 Responsibilities Manipulations performed by the operators in charge of the titration of viral vector supernatants. 6 Process a) Flowchart At each step of the process, checkpoints have been set to control and manage the activity from the beginning to the end. In the flowchart below are shown all steps and checkpoints in this process. CONFIDENTIAL Page 2 of 10

3 Purpose: Titrate rlv supernatants by quantification of protein HIV-1 p24 by ELISA Pilot: Hélène VERGNAULT Titration of lentiviral vectors by p24 ELISA Steering Tasks / Actors Location Documentation Logigram Input data p24 ELISA Kit Viral vectors to titrate 1 A 2 3 B C 4 D 5 Preparation of solutions Extemporaneous preparation Preparation of standard curve Thawing VV to titrate Homogeneization Dilution of VV / Titer estimated/ process of prod-purif Dilution of samples Map plate Capture p24antigen L2 VV Laboratory Vectalys p24 Kit 1 A yes 2 D 5 6 Viral vectors 3 yes B C yes 4 yes 6 Incubation 37 C / 2h 7 7 Wash 8 8 Complexation of p24 antigen by polyclonal Antibody anti p24 biotinylated SOP-T-VV.11.1 E yes E Homogeneization 9 9 Incubation 37 C / 1h Complexation with the conjugate Streptavidin- HRP 11 Activity indicators F 13 Incubation 30min à température ambiante Preparation of substrate OPD in the dark Yellow-orange color Add OPD substrate L1 Laboratory Vectalys F 14 no 13 Output data Viral vectors titrated by p24 ELISA TU/ml 14 Throw Incubation 15min at room temperature Reading of plate Stop réaction 18 Analysis of résults Operateur Vectalys Viral vectors titrated CONFIDENTIAL Page 3 of 10

4 b) Process Operating mode n 1: Sample preparation 1- Purpose and principles To describe the steps of the preparation of samples, solutions and reagents for the p24 ELISA. 2- Reagents, media and solutions Identification Fournisseur Références HIV- p24 ELISA kit Positive control, 200ng/ml Plate Wash Concentrate, 20X Blank DMEM cell culture media Perkin-Elmer PAA NEK050 E Water milliq deionized 3- Equipments Identification Fournisseur Références Antibody-coated microplate 96 well Microplate coated with monoclonal antibody to HIV-1 p24. Corning cm² T-flask (non cell bind) Corning small sterile rack Multichannel pipettor with volume capacity to 300µl Single channel pipettor to deliver µl Epifluorescence Microscope Dutscher Eppendorf Eppendorf Nikon Counting cell slide Fast Read Dutscher Standard BSL 2 lab equipment and materials. CONFIDENTIAL Page 4 of 10

5 4- Experimental protocol Handling under BSC II: biological safety cabinet type II BSL-2+ precautions a) Solutions and reagent preparation 1 Equilibrate all reagents at room temperature (15-30 C) prior the assay. 2 Using an adapted graduated cylinder, dilute Plate Wash Concentrate, 20X to 1X, with deionized water (milliq). Crystals may form in Plate Wash Concentrate if refrigerated. These should be redissolved by gentle warming prior to use. Approximately 1000 ml of diluted (1X) wash solution is needed per plate. Diluted (1X) wash solution should be prepared fresh prior to assay Prepare all other working reagents within 15 minutes of use. Prepare the necessary reagent quantity only, for running the assay. Discard any excess!!! b) Preparation of standard curve Handling under BSC II: biological safety cabinet type II BSL-2+ precautions 1 In an Eppendorf tube identified as A, dilute the 200ng/ml Positive Control concentrate solution to 100pg/ml. Use blank DMEM cell culture media as diluent. Take 10µl of the 200ng/ml Positive Control concentrate and complete with 990µl of blank DMEM. 2 Vortex to homogenize. 3 From tube A (2000pg/ml), perform serial dilutions in blank DMEM cell culture media as shown in the following table1. Tube Concentration (ng/ml) Volume (µl) Volume of DMEM (µl) B de A 500 C 0,5 500 de B 500 D 0,1 200 de C 800 E 0, de D 500 F 0, de E 500 G Table1: Dilutions for the standard curve CONFIDENTIAL Page 5 of 10

6 c) Samples preparation If samples are not assayed on the day of collection, they should be stored frozen at -20 C or below until being tested Multiple freeze-thaw cycles should be avoided Samples should be diluted so that the measured OD falls into the linearity range. It is possible to estimate the required dilution, depending on the obtained or estimated titer (TU/ml) according to the production and purification process of lentiviral vectors 2 Include an internal standard, i.e. a reference sample with a known titer in TU/ml and PP/ml. Handling under BSC II: biological safety cabinet type II BSL-2+ precautions 3 Thaw viral vectors supernatants in a water bath at 37 C. The samples should be thawed just before the experiment. 4 Once completely thawed, mix gently the vectors by vortexing 5 In well identified Eppendorf tubes, dilute the samples in blank DMEM as shown in table 2. Perform 1/10 serial dilutions. The minimal pipetted volume should be 5µL. The final volume should be 200µl. Two dilutions are tested per sample Estimated or measured Dilution factor FD titer (TU/ml) Table 2 : Dilutions of samples to be tested 5- Documentation Describe all the manipulation in the laboratory notebook. CONFIDENTIAL Page 6 of 10

7 Operating mode n 2: Quantification of physical particles in rlv supernatants by HIV-p24 ELISA assay 1- Purpose and principles To describe the methods to quantify physical particles in rlv supernatants by HIV-1 p24 ELISA assay. 2- Reagents, media and solutions Identification Supplier References Lentiviral vectors rlv : samples to test Vectalys SOP-T-VV.3.2 HIV-p24 ELISA - Standard curve solution - Detection antibody, Rabbit polyclonal anti-p24 antibody in PBS - Streptavidin-HRP 100X - Steptavidin- HRP diluent - Substrate diluent - Triton X100 5% - OPD - Wash solution 1X Reference sample Perkin-Elmer Vectalys NEK050 SOP-T-VV.3.2 DMEM uninoculated cell culture media PAA E Disinfectant Désogerme virex Laboratoires ACI Equipments Identification Supplier References Antibody-coated microplate 96 well Microplate coated with monoclonal antibody to HIV-1 p24. Corning 3596 Bottle nick tilted 75cm 2 non cell bind Corning Little sterile rack Multichannel pipettor with volume capacity to 300µl Single channel pipettor to deliver µl Epifluorescence Microscope Dutscher Eppendorf Eppendorf Nikon Counting cell slide Fast Read Dutscher Stopwatch Spectrophotometer, plate reader 490nm Software Microplate Manager 5.2 CONFIDENTIAL Page 7 of 10

8 Standard BSL 2 lab equipment and materials. 4- Experimental protocol Handling under BSC II: biological safety cabinet type II BSL-2+ precautions a) p24 antigen capture Perform the experiment according to the procedure described in the manufacturer manual. Determine the number of Antibody-coated Microplate strips needed for the assay. 1 Arrange the strips on the rack. Nota bene: Each experiment should include: - a negative control : blank DMEM cell culture media, well G cf. OM1b - a positive control : well B cf. OM1b - substrate blank: «Blanc substrat» ou B substrat - reference sample with known titer (PP/ml and TU/ml) 2 Make a pattern of the plate to clearly identify plate wells. 3 Add 20µl Triton X-100 to all wells except B substrat. 4 Add 200µl of all samples of standard curve (including positive and negative controls) in the identified wells. 5 Add 200µl of the 2 dilutions of each sample to be tested in their identified well, without forgetting the reference sample. 6 Mix well by pipeting up and down 7 Seal the plate 8 Incubate for two hours at 37 C (+/-1 C). b) Wash BSL-1 precautions 1 After two hours at 37 C, remove the seal 2 Wash plate with diluted wash solution 1X: - empty the wells in the stainless steel container having disinfectant solution (Désogerme virex ) - add 300 µl/well wash solution 1X with multichannel pipette, to all wells except B substrat. 3 Repeat the washing step four times. Pay attention to empty wells before adding new reagent!!! c) Complexation of p24 antigen by biotinylated polyclonal antibody 1 Add 100µl/well Detector antibody to all wells except B substrat. 2 Seal plate and incubate 60 minutes (+/- 5 min) at 37 C. CONFIDENTIAL Page 8 of 10

9 d) Complexation with Streptavidine-HRP conjugate 1 15 minutes before use, dilute the right quantity of Streptavidine-HRP 100X concentrate to the 1/100 working concentration with Streptavidin HRP diluents. The minimum pipette volume should be 10µl. Mix thoroughly 2 Wash six times with 300µl/well, all wells except B substrat, as describe in b). 3 Add 100µl/well of Streptavidin-HRP 1X to all wells except B substrat. 4 Seal plate and incubate 30 minutes (+/- 5 min) at room temperature (15-30 C). e) Incubation with substrate 1 15 minutes before use, add one OPD tablet, with non-metallic forceps, to 11ml of substrate diluent for each plate assayed. Vortex vigorously to make sure of complete dissolution. Protect from light!!! The OPD substrate solution should be colorless to pale yellow. A yellow-orange color indicates that the reagent is contaminated and must be discarded!!! 2 Wash six times with 300µl/well, all wells except B substrat, as describe in b). 3 Add 100µl/well OPD substrate to all wells including B substrat. 4 Seal plate and incubate 30 minutes (+/- 5 min) at room temperature (15-30 C), in the dark. Do not stop the reaction before reading the O.D. 5 After incubation time, read immediately the absorbance at 490nm, Perform a blank to the plate reader on air. 6 Stop the reaction by adding 100µl of Stop solution to all wells. 5- Documentation Describe all the manipulation in the laboratory notebook. CONFIDENTIAL Page 9 of 10

10 Operating mode n 3: Results analysis 1- Purpose and principles Describe the method to analyse the results of the titration assay of physical particles of rlv supernatants by quantifying HIV-1 p24 proteins by ELISA. 2- Results analysis a) Standard curve 1 For each point of the standard curve corresponding to a known p24 concentration (ng / ml), record the absorbance values obtained at the wavelength of 490nm. 2 Plot the graph: p24 concentration (ng /ml) according to the DO490nm as shown in the example below. 3 Determine the linearity range of the standard curve that corresponds to the party whose regression coefficient is above For this linearity zone, note the equation: Standard curve ng p24/ml OD 0 0,000 0,025 0,035 0,05 0,072 0,1 0,132 0,5 0, ,758 b) Samples analysis 1 For each diluted sample, record the corresponding O.D. Verify that the O.D. value is well within the linearity range!!! 2 For values in the range, calculate the p24 concentration of the diluted sample as follows: p24 concentration (ng/ml) = [p24]diluted = A x (OD 490nm sample - OD 490nm negative control) + B 3 Calculate the initial p24 concentration in the undiluted sample in the equation below: Initial p24 concentration (ng/ml) = [p24] = diluted [p24] x dilution factor FD 4 Knowing that 1pg of p24 corresponds to 1E4 physical particles, calculate the titer according to the following formula: Titer (PP/ml) = [p24] (ng/ml) x 1000 x = [p24] (ng/ml) x Check that the obtained titer for the reference sample is consistent. CONFIDENTIAL Page 10 of 10