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Production, Characterization and Use of Chicken IgY Monoclonal Antibodies

Production, Characterization and Use of Chicken IgY Monoclonal Antibodies. Presented by: MUFIDATUR ROSYIDAH (126090100111012). Background. IgA. Chicken Immunoglobulin. IgM. Abundance in Egg Yolk. IgY. Same physiological function in birds = IgG in mammal.

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Production, Characterization and Use of Chicken IgY Monoclonal Antibodies

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  1. Production, Characterization and Use of Chicken IgY MonoclonalAntibodies Presented by: MUFIDATUR ROSYIDAH (126090100111012)

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

  3. Immunoglobulin Structure Fig.1 The structural organization of immunoglobulin. 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).

  4. Method

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

  6. Production of mouse monoclonal antibodies (mAbs)

  7. Cont... • Rinsing strips in distilled water • Positive control : blue spot appeared

  8. Dot immunobinding assay (DIBA) Cont... • Isotyping of mAbsimmunoenzyme assay : • Usingisotyping reagent ISO-2

  9. SDS-PAGE and immunoblotting

  10. Conjugation of horseradish peroxidase to 1F5/3G2mAb Dialyzed in PBS

  11. Indirect Immunoenzyme Assay

  12. Immunoadsoption of IgY

  13. Result and Discussion

  14. Monoclonal antibody production 4E4 clones 3C10 clones IF5 clones 2F10 clones Commercial polyclonal HRP-conjugated rabbit anti-chIgY 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.

  15. Monoclonal Antibody IgM 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

  16. Mapping of IgY Epitope

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

  18. Detection of IgY antibodies 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 Mycoplasmagallisepticumcolonieswere incubated in tracheal washing of an infected chicken. Assecondary antibody HRP-conjugated 1F5/3G2 mAbs were used. Arrows indicate various (2 and 3) and sectorial (1 and 2) stainingdepending on variably expressed antigens recognized by local antibodies

  19. Comparison of commercial conjugate and HRP-conjugated Fig. 5. 1F5/3G2 mAb for detection of specific IgY antibodies againstprotein antigens of three major poultry pathogens using immunoblotting. Panel A, Mycoplasma gallisepticum; panel B, Mycoplasma synoviae; panel C, Newcastle disease virus. After incubation insera 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

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