1. Screening and detection of scFV fragments specific to HA and NP protein of H5N1 virus Wu Jie, Ke Changwen, LI Hui, Chen Qiuxia, Zou Lirong, Zhou Jie, Mo Yanling Center for Disease Control and Prevention of Guangdong Province

2. Introduction:   Highly pathogenic avian influenza virus subtype H5N1 infects humans with a high fatality rate and has pandemic potential.   Influenza viruses elude immune responses and antiviral chemotherapeutics through genetic drift and reassortment.

3.   As a result, the development of new strategies that attack a highly conserved viral function to prevent and/or treat influenza infection is being pursued.  The antibody specific to virus have been discovered to have efficacy for the prevention, treatment and diagnosis of virus infection.

4.   The humanised antibody binding to human H5N1 virus specifically was panned from Human scFv Libraries and constructed phage library.  AQSIQ  The project is supported by “Science and Technology Planning Project of AQSIQ”.

5. H5N1 virus belongs to genera A within the family Orthomyxoviridae H5N1 virus belongs to genera A within the family Orthomyxoviridae and has a multipartite, negative-sense, single-stranded RNA genome and a lipid envelope. and has a multipartite, negative-sense, single-stranded RNA genome and a lipid envelope. H5N1 Virus:

6. Monoclonal antibody : An immunoglobulin molecule produced by B-lymphoid cells that combine specifically with an immunogen or antigen. An immunoglobulin molecule produced by B-lymphoid cells that combine specifically with an immunogen or antigen.

8. phage display scFV antibody library Filamentous phages usually contain a genome of single-stranded DNA and infect Gram-negative bacteria. Filamentous phages usually contain a genome of single-stranded DNA and infect Gram-negative bacteria. It can still grow normally with the foreign gene insertion. It can still grow normally with the foreign gene insertion..

9.  pIII protein is minor coating protein, only 3-5 copies of each virion.  PVIII is mainly coating protein, 3000 copies of each virion.

10. The procedure of construction of phage display antibody library:

11. The steps of panning antibody from library ： incubation incubation washing washing elution elution enrichment enrichment

12. Preparation of monoclonal antibody Screening scFV from antibody library The comparison of monoclonal antibody and phage display antibody

13. Items Monoclonal antibody scFV antibody Host cell: hybridomabacterial Screening range : ~10 3 10 7 ~ 10 9 Time: several months several weeks Operation:complicated relative simple Immune injection: necessaryavoidable Humanized antibody: + Cost:highlow Production capacity: limitedunlimited

14. Reagents ：  Human Single Fold scFv Libraries I+J was bought from MRC HGMP Resource Centre ， Cambridge, UK.  HA and NP proteins was expressed by lab.  HRP-anti-M13 antibody and Horse Radish Peroxidase conjugated Protein A was bought from Amersham International plc, Little Chalfont, Buckinghamshire, HP7 9NA, UK.  Gene fragment was sequenced by Invitrogen Corp.  The culture medium and biological reagents is the analytic pure.

15. Methods: 1.The positive scFV screening from phage antibody library ； 2.The amplification of elution ； 3.The detection of positive scFV by ELISA ； 4.The soluable expression of the positive phage and the detection of binding activity. 5.PCR detection of the positive phage. 6.The sequence analysis of the positive scFV.

16. Results ： 1.The result of panning library Round NoThe titration of elution(HA) The titration of elution(NP) Round 1 2.3 x 10 4 CFU/ml1.8 x 10 4 CFU/ml Round 2 4.5 x 10 5 CFU/ml3.9 x 10 5 CFU/ml Round 3 4.3 x 10 6 CFU/ml4.0 x 10 6 CFU/ml

17. 2 ． The detection of positive scFV with ELISA Fig1A. The positive scFV of binding acitivity with HA protein Note ： OD450nm was the average of triplicate experiments

18. Fig1B. The positive scFV of binding acitivity with NP protein Note ： OD450nm was the average of triplicate experiments

19. 3 ． PCR detection of positive scFV ： Light chain gene fragment of 368bp

20. Heavy chain-linker gene fragment of 527bp Heavy-linker-light chain gene fragment of 935bp

21. 4.Sequence and analyses of the positive scFV ： The sequence results showed that there are 11 mutant sites of 47 、 49 、 50 、 51 、 53 、 54 、 56 、 96 、 97 、 98 and 99 in light chain and 6 mutant sites of 44 、 47 、 85 、 86 、 87 、 88 and 89 of scFV binding HA. Only there are 11 mutant sites of 47 、 49 、 50 、 51 、 53 、 54 、 56 、 96 、 97 、 98 and 99 in light chain of scFV binding NP. It presumed that the above mutant AA are the key point of contribution to the binding activity. The sequence results showed that there are 11 mutant sites of 47 、 49 、 50 、 51 、 53 、 54 、 56 、 96 、 97 、 98 and 99 in light chain and 6 mutant sites of 44 、 47 、 85 、 86 、 87 、 88 and 89 of scFV binding HA. Only there are 11 mutant sites of 47 、 49 、 50 、 51 、 53 、 54 、 56 、 96 、 97 、 98 and 99 in light chain of scFV binding NP. It presumed that the above mutant AA are the key point of contribution to the binding activity. Sites of 47-56 of light chain Sites of 96-99 of light chian NP 1 GIYPSGRRTA KLSR NP-2 SIQPEGRYTR LGKR Sites of 47-56 Sites of 96-99 Sites of 44-47 Sites of 85-89 of heavy chain of light chain of heavy chain of heavy chain HA-1 SISSKGQLTS KKRQ SASS RRNRPK HA-2 SITYRGSWTI HSPP MASQ KRHGPW HA-3 PIPLQGSSTL PART MASH GRMQPT

22. The experiment in near future ：  The comparison of the binding activity of scFv to the monoclonal antibody.  The non-specific binding activity of scFv to unrelated proteins.  The measurement of virus neutralization of scFv.  The inhibition activity of H5N1 virus infection to sensitive cell of scFv.

23. scFv would be candidate to the prevention and control of H5N1 Avian influenza dissemination ：  It would be candidate to the diagnostic reagent of H5N1 detection ELISA kit ；  It would be used as colloidal gold rapid detection reagent.  It would be the candidate medicine for H5N1 therapy.