Presented by: MUFIDATUR ROSYIDAH ( )

Chicken Immunoglobulin IgM IgA Abundance in Egg Yolk Benefit to diagnostics of pathogen infection Production, characterization of mAbs, and demonstrated in potential use Same physiological function in birds = IgG in mammal IgY

Fig.1 The structural organization of immunoglobulin. A.Human IgG B. Chicken IgY VH (variable domain of heavy chain); VL (variable domain of light chain); CL (constant domain of light chain); CH 1, CH2, CH3 (constant domain of heavy chain); Cv1, Cv2, Cv3 and Cv 4 (constant domain of chicken heavy chain).

Chicken Egg Yolk Serum Cloroform extraction / kit Diluted with PBS (1:20) Turkey Peafowl Pheasant Parrot Sparrow Pigeon Diluted with PBS (1:50) Duck Goose Quail Diluted with PBS (1:50) Human Rabbit Pig Horse Cow Non-Avian IgM Cross reaction Test

Production of mouse monoclonal antibodies (mAbs) Balb/ c Mouse (8 weeks old) Imunized by chIgY 4x (intraperitonial & intravenal) Spleen cell vs NS-0 Meyloma cell fusion (+PEG 50 %) Selected hybridoma Were cloned twice Washing by Buffer Protein test Immunobinding Assay

chIgY samples applied to NTC membrane strips Blocking (Tween 0,5%) Incubation peroxidase conjugated rabbit anti- chIgY antibodies, 30’ Washing in PBS + substrate true Blue Positive control : blue spot appeared Rinsing strips in distilled water Cont...

 Isotyping of mAbs immunoenzyme assay :  Using isotyping reagent ISO-2 Dot immunobinding assay (DIBA) Cont... Membrane + chIgY Incubated in 1F5/3g2 mAb diluted in PBS In different pH value (3-12) Optimal condition Membrane + chIgY Incubated in 1F5/3g2 mAb 5-45 minutes (increasing time interval was 5 min) Minimal incubation time Membran e + chIgY Incubated in 10 mM periodic acid in 50mM Na-acetate, pH 4,5, 1 (increasing time interval was 5 min) Incubation in mAbs Test Of carbohydrates

chIgY samples were treated 2 % β- mercaptoethanol Apllied to gelProtein in membran stripsIncubated in appropriate mAb solutionImmunoenzyme reaction SDS-PAGE and immunoblotting

2 gr mAbs 1F5/3G2 diluted in 0,1 M Na2CO 3 Diluted in 160 µL glutaraldehyde, overnight Dialyzed again in NaHCO3 0,1 M, pH 9,2 Incubated with 4 gr enzyme, 24 h Blocking + Lysin 0,2 M Conjugation of horseradish peroxidase to 1F5/3G2 mAb Dialyzed in PBS

Tested samples incubated withaMycoplasma synoviae & M. Gallisepticum in agar block, 45’ Diluted in 160 washed in PBS HRP-conjugatedn1F5/3G2 mAbs + IgY (incubated) Washing in PBS, drained and treated with substrate containing DAB Western blotting & Immunoenzyme on reaction Indirect Immunoenzyme Assay

Undiluted and diluted serum (1:10) mix with CNBr Sepharose 4B, coupled with mAbs 1F5/3G2, room temperature, 1 h centrifugation, supernatan were collected Assayed for total IgY Immunoadsoption of IgY

Fig. 2 Reaction of mAb with chIgY, isolataed from chicken egg yolk (IgY) and with avian sera (s, 1-7) or egg yolk (y, 8-10) and eith sea of some mammals (s, 11-16). 1: chicken, 2: turkey, 3: peafowl, 4 : pheasant, 5 : parrot, 6 : sparrow 7 : chicken, 8 : duck, 9 : goose, 10 : quail, 11: rabbit, 12 : pig, 13: cattle 14 : horse, 15 : mouse, 16 : human. 4E4 clones 3C10 clones IF5 clones 2F10 clones Commercial polyclonal HRP- conjugated rabbit anti-chIgY

Fig. 3 Reaction of mAb M1 to HC of chicken IgM in DIBA with avian sera (s) or egg yolk (y). 1 : chicken, 2 : turkey, 3 : peafowl, 4 : pheasant, 5 : japanese quail, 6 : sparrow, 7 : pigeon, 8 : parrot, 9 : duck, 10 : goose

 mAbs chicken IgY use to detection of patogen infection (Micoplacma gallisepticum)  Remove the IgY from yolk egg, to get IgA and IgM

Fig. 4. Detection of IgY antibodies specific for in vivo expressed Mycoplasma gallisepticum antigens using HRP-conjugated 1F5/3G2 mAbs. In IIPA agar blocks with Mycoplasma gallisepticum colonies were incubated in tracheal washing of an infected chicken. As secondary antibody HRP-conjugated 1F5/3G2 mAbs were used. Arrows indicate various (2 and 3) and sectorial (1 and 2) staining depending on variably expressed antigens recognized by local antibodies

Fig. 5. 1F5/3G2 mAb for detection of specific IgY antibodies against protein antigens of three major poultry pathogens using immunoblotting. Panel A, Mycoplasma gallisepticum; panel B, Mycoplasma synoviae; panel C, Newcastle disease virus. After incubation in sera of infected chicken membrane strips were incubated in secondary antibodies: lanes 1, peroxidase conjugated rabbit anti- -chIgY antibodies; lanes 2, HRP-conjugated 1F5/3G2 mAb. Molecular mass is indicated on the left side (in kDa); arrows indicate major immunogenic proteins i.e. haemagglutinins pMGA (panel A) and haemagglutinis of M. synoviae, named MSPB (panel B). Note: HRP-conjugated 1F5/3G2 mAb gave much less background staining, particularly in panel C