Anti-galactosyl Natural Anti-galactosyl Natural Antibodies in the Patients with Antibodies in the Patients with

Antibody Deficiency Antibody Deficiency

M. Hamanová, V. Thon, J. Litzman, J. Lokaj

University Centre for Primary Immunodeficiencies

Department of Clinical Immunology and Allergology

St. Anne University Hospital, Masaryk UniversityBrno, Czech Republic

Natural AntibodiesNatural Antibodies

• Natural Antibodies against Natural Antibodies against carbohydrate antigens are thougt carbohydrate antigens are thougt to be produced through life as to be produced through life as a polyclonal response to antigenic a polyclonal response to antigenic stimulation by gastrointestinal stimulation by gastrointestinal bacteria but their origin is bacteria but their origin is uncertain.uncertain.

New possibility:New possibility:

GalGal1-3Gal1-3Gal was identified as the epitope of rabbit erythrocytes.

XenoagglutininsXenoagglutinins were determied by direct

HEMAGGLUTINATION test with rabbit erytrocytesrabbit erytrocytes..

„Classical investigation“ - rabbit

Natural AntibodiesNatural Antibodies::

Characterization of Natural AntibodiesCharacterization of Natural Antibodies

Natural antibodies against carbohydrate antigenes:

• Xenoreactive IgM Ab = 1,8 – 4,1%, resp. 3,9 – 8,0% of total IgM

• Xenoreactive IgG Ab = 1 – 2,4% of total IgG

McMorrow, I. M., Comrack, C. A., Sachs, D. H., DerSimonian, H. (1997). Heterogeneity of human anti-pig natural antibodies cross-reactive with the Gal(1,3)Galactose epitope. Transplantation 64: 501-10.

 

ELISA with antigen Gal1-3Gal in the isotypes IgM, IgG and IgA,

enable to detect „classical natural antibodies against rabbit

erythrocytes“.

Rieben, R., von Allmen, E., Korchagina, E. Y., Nydegger, U. E., Neethling, F. A., Kujundzic, M., Koren, E., Bovin, N. V., Cooper, D. K. C. (1995). Detection, immunoabsorption, and inhibition of cytotoxic activity of anti-Gal antibodies using newly developed substances with synthetic Gal1-3Gal disaccharide epitopes. Xenotransplantation 2: 98-106.

Development of the method - NADevelopment of the method - NA

Developement and determination of

IgMIgM, IgGIgG and IgA anti-IgA anti---galactosyl galactosyl

natural antibodies (NA)natural antibodies (NA)

by ELISA

and comparison with hemagglutination.

ELISA for Natural antibodies Natural antibodies

• antigen: PAA - -disaccharide GalGal1-3Gal1-3Gal

(PAA-Bdi Lectinity Holdings, Inc., Moscow, Russia)4

Gal

Gal

13

O

NH2

OH

CH2OH

NH2

OH

OH

CH2OH

(CH2)3

NH

CH

O

CH2

O

O

O

HN

CH

O

CH2

OH

(CH2)2

ELISA

for

IgM-IgM-, IgG- IgG- and IgA-NAIgA-NA

• healthy donorshealthy donors• CVIDCVID

• neonatesneonates

ELISA

OD

n = 15 dil. 1:40

0

1

2

3

0 IgM IgG IgA

AGGLUTINATION

dilution

1:128

1:641:32

0

64

128

192

256

1

Occurrence of anti- anti-GalGal NANA in healthy donors in healthy donors

Occurrence of anti- anti-GalGal NANA in CVID in CVID

1:81:4<4

dilution

0

64

128

192

256

1

AGGLUTINATION

OD

ELISA

n = 15 dil. 1:40

IgM IgG IgA

0

1

0 IgM IgG IgA

OD

ELISA

Endobulin n = 4 dil. 1:40

0

1

2

3

0 IgM IgG IgA

Occurrence of anti- anti-GalGal NANA in I in IVIGVIG

dilution

1:128

1:64

0

64

128

192

256

1

AGGLUTINATION

ELISA

OD

n = 15 dil. 1:40

0

1

2

3

0

mother

IgM IgG IgA

OD

ELISA

n = 15 dil. 1:40

0

1

2

3

0

cord blood

IgM IgG IgA

Occurrence of anti- anti-GalGal NANAin cord blood in cord blood

0

1

2

0

0

1

2

0

0

1

2

0

0

1

2

0

Occurrence of anti- anti-GalGal NANAin neonates, followed in time in neonates, followed in time

ELISA ELISA ELISA ELISA

OD ODODOD

n = 28 n = 19 n = 14 n = 25

dil. 1:40 dil. 1:40 dil. 1:40 dil. 1:40

IgM IgG IgAIgM IgG IgA IgM IgG IgA IgM IgG IgA

cord blood 6. month 12. month 24. month

With our specific ELISA using GalGal antigenwe determine natural antibodies in different Ig class such as

IgM, IgG and IgA and so specific investigate up to 95% of xenoreactive natural antibodies.

With the new reproducible assay we are able to detect natural antibodies in patients with CVID, particularly in low

concentrations in IgM and IgA isotypes.

Investigation of genetically conserved natural antibodies may permit functional evaluation and characterization

of the differentiation state of B lymfocytes.

As a perspective As a perspective Gal1-3Gal antigen could also be could also be appropriate appropriate

for the detection of natural antibodies of natural antibodies at the cellular level cellular level in ELISPOT assay.