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VIRUS NEUTRALISATION

VIRUS NEUTRALISATION. Lab 9. Principle.

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VIRUS NEUTRALISATION

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  1. VIRUS NEUTRALISATION Lab 9

  2. Principle • Neutralisation is the inhibition of infectious virus by antibody. The first requirement for a neutralisation test, therefore, is that an assay of infectious virus is available, either an end-point or a plaque assay. Plaque reduction is preferable since it is more sensitive and precise.

  3. Plaque neutralisation assay • Dilutions of serum (antibody) are made. • A fixed amount of virus is added to each dilution of serum (e.g. 100 PFU). • The reaction is incubated for one hour to allow antibody to bind to virus. • Each reaction mixture is then inoculated onto an indicator cell culture to determine if any infectious virus remains. This is incubated for one hour to allow any virus to infect the cells. • The inoculum is removed and an agar overlay is added. • The cell cultures are incubated for a few days to allow any virus to grow, then the overlay is removed and the cells are fixed and stained. • The number of plaques in each culture is recorded. • The highest dilution of serum that neutralises 50% of the virus is taken as the end-point. The antibody titre is taken as the reciprocal of the dilution. • For example, if the number of plaques in a culture inoculated with 100 PFU of virus plus a serum dilution of 1:256 was 50, the antibody titre of that serum is said to be 256.

  4. 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F 85 88 82 82 83 84 77 78 75 79 77 76 68 66 70 68 67 67 60 59 58 58 59 57 50 49 52 51 47 52 44 42 40 42 43 43 93 93 93 92 93 91 37 36 38 37 37 36 99 99 96 98 97 97 G H Antibody titre is 32 if PFU = 100 8 16 32 64 128 256 512 1024 1048

  5. End-point neutralisation assay • Make dilutions of serum (antibody). • A fixed amount of virus is added to each dilution. • The reaction is incubated for about one hour to allow antibody to bind to, and inactivate, virus. • Each reaction mixture is inoculated onto an indicator cell culture to determine if any infectious virus remains. • The cell cultures are incubated for a few days to allow any virus to grow, then fixed and stained (to view CPE). • The highest dilution of serum that neutralises all of the virus is taken as the end-point. The antibody titre is taken as the reciprocal of the dilution. • For example, if a serum dilution of 1:128 neutralised the virus, the antibody titre of that serum is said to be 128.

  6. Luciferase neutralisation assay • A form of End-point neutralisation assays • Virus is modified to contain a luminescence indicator that is detected micro-beta reader • Luminescence read reflects the titre of the virus. • For example; if the read for 1:625 dilution in a serum sample is 1x107, and the read is 5x106 for another serum sample, this means the second sample inhibited more virus and so contained more neutralising antibodies

  7. Procedure

  8. Development of VNA response VNA detected 32w after experimental infection

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