1. Presented by: MUFIDATUR ROSYIDAH (126090100111012)

2. 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

3. 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).

5. 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

6. 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

7. 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...

8.  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

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

10. 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

11. 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

12. 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

14. 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

15. 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

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

18. 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

19. 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