Preparation of btv antigen part 2

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MONOCLONAL ANTIBODY SECTION

by Debbra Marcel for July 2013 R&D Meeting

List of activities 1. Preparation of the virus (presented by Naama in Aug 2012)

2. Preparation of Vero cells

3. Inoculation of BTV (BTV-Serotype 1) into Vero cells

4. Identification of virus isolates based on the type of cytopathic effect (CPE).

5. Harvesting & Inactivation of virus

6. Concentration of inactivated BTV

7. Optimization of Bluetongue Antigen by AGID

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Objective The objective of this presentation:

To explain the cell culture process as one of the important

methods in the production of Bluetongue Antigen (BT Antigen)

The objective of performing the production of BT Antigen:

To continuously supply the needs of the antigen in

performing the serological identification of BTV by Agar Gel

Imunodiffusion (AGID) test to the samples received in VRI

(disease surveillance)

To meet our purpose in developing the AGID test kit

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Overview In this process, Bluetongue Virus Serotype-1 (BTV-1) is used as the

antigen for the kit. Preparation of the virus antigen will be done by propagating them in African green monkey kidney (VERO) cells and isolates will be identified based on the type of cytopathic effect (CPE). The cell culture fluid will be harvested after the CPE is reached and then centrifuged to remove complete viral particles and cellular debris. The formalinized BTV antigen will be purified by using dialysis method

This method involves the withdrawal of fluids through dialysis membranes using polyethylene glycol to produce an increasingly concentrated sample within the dialysis membrane.

The preparation of BTV soluble antigen, regardless of serotype, by the method described gave excellent results in both the micro and macro-AGID tests

Original procedure adapted from: J. Clin. Microbiol. 1983 (modified by UMA)

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Activity 2 (Preparation of BTV Antigen AGID)

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Introduction Cell culture

the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment.

may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been established.

Vero cell:

established cell line derived from the kidney of an African green monkey (Cercopithecus aethiops) in the 1960s (Y. Yasumura and Y. Kawakita at the Chiba University in Japan).

one of the most common mammalian continuous cell lines used in research & virology studies, vaccine & antigen production, propagation and study of intracellular bacteria (e.g., Rickettsia spp.; UNIT 3A.4) and parasites (e.g., Neospora), and assessment of the effects of chemicals, toxins and other substances on mammalian cells at the molecular level.

Bluetongue (BT) Antigen :

Soluble antigen used in Agar Gel Immunodiffusion (AGID) test to detect the presence of BTV antibodies in animal sera.

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Requirements of a Tissue Culture Laboratory

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Culture of Animals Cell, 5th Edition, R. Ian Freshney, page 56

Preparing an aseptic environment by GLP

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Culture of Animals Cell, 5th Edition, R. Ian Freshney, page 80

Preparation of Cell Culture Medium

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*antibiotics: Penicillin, Streptomycin, and kanamycin = 100 unit; 100 unit; 50 ug

Maintenance Medium (MEM MM) – 2% FBS 1) Tryptose Phosphate Broth - 10% 2) Fetal Bovine Serum (FBS) - 2 % 3) 7.5% NaHCO3 - 1.5 4) PSK (*antibiotics) - 1% 5) MEM - up to 100%

MEM Minimal Essential Media (basal medium) Preparation of powdered media: 1. Dissolve powder into roughly 95% of total

volume to be made. 2. Add the required amount 3. of sodium bicarbonate. 4. Adjust pH to 0.2 to 0.3 units below the desired

working pH (7.0-7.4) by adding 1N NaOH or 1N HCl with stirring.

5. QS to final volume. Keep container closed until ready to filter.

6. Filter through a 0.2 micron filter (see Picture 1)

Picture 1 Picture 3 Picture 2

Picture 1: The sterilization of MEM (by filtration) Picture 2: MEM before supplemented Picture 3: MEM after supplemented

The MEM basal medium then supplemented with other nutrients according to the proportion desired as follow:

Growth Medium (MEM MM) – 10% FBS 1) Tryptose Phosphate Broth - 10% 2) Fetal Bovine Serum (FBS) - 10 % 3) 7.5% NaHCO3 - 1.5 4) PSK (*antibiotics) - 1% 5) MEM - up to 100%

Thawing Vero cells from liquid nitrogen/-80°c Materials

Vero cell stock, frozen in liquid nitrogen/at -80°C

MEM GM (10% FBS)

15mL conical tubes, sterile

25cm2/50cm2 tissue culture flasks with vented caps, sterile

Serological pipettes, sterile

70% ethanol solution

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Method (Protocol 1, Current protoc. Microb., NIH, 2009 = Maintenance of Vero cell culture)

1. Quickly thaw vial (cryovial) of Vero cells by gently swirling in a 37°C water bath. (Keep the O-ring and cap of the cryovial out of the water)

2. In a laminar flow hood, decontaminate the vial by spraying with 70% ethanol

3. Transfer the Vero cell suspension from the cryovial into a 15mL conical tube containing 10mL of MEM Growth Medium (10%FBS) (after thawing the cells, dilute and remove the DMSO before transferring the cells to tissue culture flasks)

4. Pellet cells by centrifugation at 200 × g for 5 minutes at room temperature.

5. Remove and discard supernatant; resuspend cells in 5-10mL MEM Growth Medium.

6. Transfer Vero cell suspension to tissue culture flask. 7. Incubate flasks in 37°C incubator with 5% CO2. 8. Monitor cells daily or every other day. Change media

every 3-4 days. (When cells reach a >90% confluent monolayer, passage cells into new tissue culture flasks)

IMPORTANT NOTES:

All equipment and solutions coming into contact with cells must be sterile, and proper sterile technique should be used.

All cell culture incubations are performed in a humidified 37°C incubator, with 5% CO2.

All solutions should be warmed to room temperature or 37°C before contacting cells.

Table A.1 Cell medium volume requirements

Checking cells

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1. Cells should be checked microscopically daily to ensure they are healthy and growing as expected. As an attachment cells, Vero cells should be mainly attached to the bottom of the flask, round and plump or elongated in shape and refracting light around their membrane. Media should be pinky orange in colour. If media

2. Discard cells if: • They are detaching in large numbers (attached lines) and/or look shrivelled and grainy/dark in colour. • They are in quiesence (do not appear to be growing at all).

subconfluent confluent

Observing cells

Inverted microscope with phase contrast

detaching quiesence (no growth)

Unhealthy cells Healthy cells

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Sub-culturing/Passaging Cells 1. When the cells are 80-90% confluent , they

should still be in the log phase of growth and need to be sub-cultured/passaged into new sterile culture vessel/flask until we acquire the desired amount of Vero cells for the viral infection process.

2. Warm the fresh culture medium at 37°C water bath or incubator for at least 30 minutes before subculture/passage cells. Then carry out the appropriate procedures (Refer protocol 2, Current protoc. Microb., NIH, 2009 = Maintenance of Vero cell culture)

3. After the sub-culture/passage of cells process done, make sure the flasks are labeled appropriately (cell line, passage level, date, etc)

4. Place flask(s) straight into 37°C CO2 incubator. 5. Write down the details of the sub-culturing in the

culture record log sheet. There should be a separate log sheet for each vial of cells resuscitated and in use.

Figure 1. The growth & maintenance of cell culture.

A. Lag phase = cells grow slowly while recovering from the stress of sub-culturing.

B. Log or exponential phase = cells enter a period of exponential growth, lasts until the entire growth surface is occupied/cell concentration exceeds the capacity of the medium

C. Stationary phase = Cell proliferation slows and stops.

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CELL COUNTING FORMULA:-

C = n x d x 10-4/ml

c = cell concentration (cells/ml)

n = number of cells counted

v = volume counted (ml)

d = dilution factor

Cell Counting . Indication:

a) Clean hemocytometer slide andcoverslip before use. b) Pressing coverslip down onto slide. c) Adding a cell suspension to an assembled slide. d) Longitudinal section of the slide, showing the

position of the cell sample in a 0.1-mm-deep chamber. e) Viewing slide on microscope. f) Magnified view of the total area of the grid. The

central area enclosed by the dotted circle is that area which would be covered by the average 10× objective. This area covers approximately the central 1 × 1 mm square of the grid.

g) Low-power (10× objective) microphotograph showing the 25 smaller 200 × 200-μm squares of a slide, which make up the central 1 × 1-mm square, loaded with cells pretreated with Naphthalene Black (Amido Black).

h) High-power (40× objective) microphotograph of one of the smaller 200-μm squares, bounded by three parallel lines and containing 16 of the smallest (50 × 50 μm) squares. Viable cells are unstained and clear, with a refractile ring around them; nonviable cells are dark and have no refractile ring.

(Complete cell counting procedures by CSIRO, AAHL, 2008)

Fig. 21.1. Using a Hemocytometer Slide

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Viable cells=>90%

Cell contamination

Main cause of contamination:-

Contact with non-sterile supplies, media, or solutions

Particulate or aerosol fallout during culture manipulation, transportation/incubation

Swimming, crawling, growing into cell culture (exposed vessel)

Accidents and mistake

Steps for reducing contamination problems:-

Use good aseptic techniques

Reduce accidents

Keep the laboratory clean/in sterile condition

Routinely monitor for contamination

Use frozen cell repository strategically

Use antibiotic sparingly

Most common contamination

fungus mycoplasma yeast bacteria

If severe cells contamination occur , Vero cells are to be discarded appropriately (separated & autoclaved) and new cell stock from LN2/-80ºc need to be culture again

(start all over again) to reduce spreading of infection & cell mutation (minimise the use of antibiotic/fungizone)

After the Vero cells successfully propagated into the desired amount and reach confluency (monolayer), the cells are ready to proceed to the next step:

“Inoculation of BTV1 (BTV-Serotype 1) into Vero cells” ~ next presentation

Conclusion

Acknowledgement … Monoclonal Antibody Unit

Ali A.R., Nurul Fatiha A. S., Naama T., Fauad T.R.

Preparation of btv antigen part 2

Education

Transcript of Preparation of btv antigen part 2