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