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Neutralisation Plate Layout
Controls
Neutralisation Plate Layout Test
Sera
Cytopathic Effect in
Tissue Culture
Conversion Chart
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Plate preparation
1. Prepare the number of microtitre plates required for Reference Serum Controls (RS), Virus Controls (VC) and Test Plates (TP).
On duplicate RS plates label sections A-H 1-4, 5-8 and 9-12. On duplicate VC plates, draw a vertical line after column nine. On one or both, make additional marks to delineate wells A 10-12 (test cell medium only), B 10-12 (diluent only) and C 10-12. (cell controls). Label these plates appropriately (Neutralisation Plate Layout Controls). Choose the most appropriate TP format, and label the remaining plates accordingly (Neutralisation Plate Layout Test Sera).
2. Cover plates with loose-fitting lids to keep them sterile.
3. Add some diluent to a sterile reservoir and keep loosely covered when not required.
4. Add 50 µl of diluent to all wells on the RS plates except where using a weak serum, when row H should be omitted. Add 50 µl to all wells in columns 1-9 on the VC plates. Dispense 150 µl into the diluent controls (wells B,10-12) and 100 µl into the cell controls.
5. Inactivated reference sera should be diluted so that the end-point in neutralisation assays occurs approximately halfway up the microtitre plate when tested with 100 Tissue Culture Infective Doses (TCID50). Store this pre-dilution at -20°C between tests.
6. Thaw reference sera immediately before use and invert several times to mix throughly before dispensing. Add 50 µl to every well in row H of the RS plates, and additionally to row G if using a weak serum. Make serial twofold dilutions by mixing, and transferring 50 µl from row H to G and so on until row A, when 50 µl must be discarded (dilute from row G if using a weak serum).
7. Cover the plates until the next step.
8. Add diluent to the TPs according to the format required:
EXAMPLE:
a) Test large numbers of sera in blocks of six wells at one virus dilution, 16/plate. Add 50 µl of diluent to the first vertical pair of wells (toxicity controls), and 25 µl to the third pair. Manageable numbers of sera may be tested against additional virus dilutions simply by replicating the second and third vertical pairs in the most economical arrangement. When using one virus dilution, add 50 µl of 1/4 test serum to the first and second (1/8 final dilution) vertical pairs and 25 µl to the third (1/16 final). If also testing against other virus dilutions, repeat the additions to the second and third pairs.
or
b) When testing vaccinates or convalescents, etc., use a 6/plate format. For each serum add 50 µl of diluent to rows B-H in duplicate, and 50 µl of 1/4 sera likewise to A and B, then dilute from row B and discard after row H. Alternatively, using a 12/plate format, only dispense 50 µl of diluent into rows B-D and F-H. Add 1/4 test sera in duplicate to rows A and B, dilute as far as row D, and discard. Similarly, add 1/4 sera to rows E and F, dilute as far as row H, and discard.
9. Cover all plates until required.
Reference virus isolates
1. If the last known log10 titre of the reference virus/es, is 10-4.8 ('4.8'), equivalent to 1 TCID50 per unit volume, then subtracting 2.0 gives the dilution 10-2.8 ('2.8) containing approximately 100 TCID50. Together with a twofold (log10 0.3) or fourfold (log10 0.6) step either side, this gives the three virus Challenge Dilutions (CDs) which must all be reacted with the RS.
Unknown sera are assayed against one or more of these CDs.
2. Always start antigen dilution by adding 0.5 ml working stock to 4.5 ml diluent to make a 10-1 step ('1'). To ascertain further dilutions, use the Logarithm to Arithmetic conversion chart and dispense the correct volumes of diluent into bottles.
For example, if the three CDs selected are '2.5' , '2.8' and '3.1', make a log10 1.5 (2.5-1.0) or 1/32 step by adding 0.5 ml of the 10-1 dilution to 15.5 ml of diluent to make the 2.5 dilution. The second and third CDs are simply made by making two further twofold steps. After the third CD, make six fourfold steps to dilute the virus past its 50% end-point.
3. Add 50 µl of the highest virus dilution to every well in column 9 of both VC plates. Similarly, add 50 µl of the next highest step to every well in column 8, and so on until the third CD, which should be dispensed into column 3 of the VC plates and section A-H 9-12 of both RS plates, then to any test serum dilutions if appropriate (but not to toxicity controls). Similarly, add the second or central CD to column 2 of the VC plates and section A-H 5-8 of both RS plates, as well as to the test sera if necessary. Finish by adding the first CD to column 1 of the VC plates, section A-H 1-4 of both RS plates and to the test sera, if applicable.
5. Put the plates into piles of up to four, loosely cover them and incubate at 37°C for approximately 60 minutes.
6. Prepare an appropriate cell culture suspension of the required concentration which should be sufficient to achieve 100% confluency after 24 hours. (It is important that the cells are agitated frequently). A suspension of 10% cells/ml is usually appropriate for IBRS-2 cells but each laboratory should determine its own optimal dilution of indicator cells.
7. Add 150 µl of test cell medium to wells A 10-12 on one VC plate. Dispense 50 µl of cell suspension to the cell controls (C 10-12 on this VC plate), then to every well of the virus and reference sera titrations and of the TPs.
8. Re-stack and loosely cover the plates and return them to the incubator for about 10 minutes.
9. Remove and tightly cover the plates with thin plastic backing tabs, and return them to the incubator.
10. Plates should be incubated at 37ºC for three days and inspected daily for evidence of CPE (Cytopathic Effect in Tissue Culture). Microscope readings may be feasible after 48 hours; the plates may be finally fixed and stained routinely on the third day. Fixation is effected with 10% formalin-saline for 30 minutes. For staining, the plates are immersed in 0.05% methylene blue in 10% formalin for 30 minutes. An alternative fixative/stain solution is a 0.4% (w/v) naphthalene blue black solution in 8% (w/v) citric acid in saline. The plates are rinsed finally in tap water.
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