Production and characterization of anti-flagelin monoclonal antibodies.

HIRIART YANINA,; GONZÁLEZ MACIEL, DOLORES; SERRADELL, MARIA (1,3);; MARTIN RUMBO

Viña del Mar, Chile.

Congreso; IX Congreso of the Latin American Association of Immunology; 2009

&amp;amp;lt;!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:ES-TRAD; mso-fareast-language:FR;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --&amp;amp;gt; <!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:ES-TRAD; mso-fareast-language:ES-TRAD;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Flagellin is recognized by the innate receptors TLR5 and IPAF, being a candidate mucosal. Usually upon purification flagellin can be contaminated with microbial products able to activate other routes of the innate response. We aimed to produce and characterize anti flagellin monoclonal antibodies (MoAb), to improve the purification systems available. BALB/c animals were immunized via s.c. with 1μg FliC (from S. thyphimurium) with Aluminum hidroxide in 300μl PBS (days 0 and 15) and boosted (day 30) with 1μg FliC in 300μl PBS. 4 hybridomes were obtained in different fusions that were subcloned and purified by protein A columns. One MoAb isotype IgG2a (2D11A1) and 3 MoAbs (2B3H10, 4C1H7 and 5B4H2) isotype IgG1 were obtained. Flagellins from other microbial species different from S. typhimurium were not recognized by the panel. FliCΔ3, a variant devoid of the antigenic domain was recognized by 2B3H10 and 4C1H7. Both mabs also blocked the effect of flagellin on Caco 2 CCL20-luciferase reporter line, although 4C1H7 has a weaker effect. Using biotin-MoAb in competitive ELISA we observed no displacement between different MoAb indicating that there was no overlap among recognized epitopes. Concluding, 4 MoAb anti-FliC of suitable specificity to be used in affinity chromatography were obtained.