PRODUCTION, CHARACTERIZATION
AND USE OF CHICKEN IGY MONOCLONAL ANTIBODIES
Presented by: MUFIDATUR ROSYIDAH
(126090100111012)
BACKGROUND
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
IMMUNOGLOBULIN STRUCTURE
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).
METHOD
CHICKEN IGY ISOLATION
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 twiceWashing by Buffer
Protein testImmunobindi
ng 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 BluePositive 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
Membrane
+ 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 gel
Protein in membran strips
Incubated in appropriate mAb solution
Immunoenzyme reaction
SDS-PAGE and immunoblotting
2 gr mAbs 1F5/3G2 diluted in 0,1 M Na2CO3
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
RESULT AND DISCUSSION
MONOCLONAL ANTIBODY PRODUCTION
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 : sparrow7 : chicken, 8 : duck, 9 : goose, 10 : quail, 11: rabbit, 12 : pig, 13: cattle14 : horse, 15 : mouse, 16 : human.
4E4 clones
3C10 clones
IF5 clones
2F10 clones
Commercial polyclonal HRP-
conjugated rabbit anti-chIgY
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
MAPPING OF IGY EPITOPE
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
DETECTION OF IGY ANTIBODIES
Fig. 4. Detection of IgY antibodies specific for in vivo expressedMycoplasma 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
COMPARISON OF COMMERCIAL CONJUGATE AND HRP-CONJUGATED
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