1. Chapter 5 Immunoglobulin

2. Contents Introduction SectionⅠ Molecular Structure of Ig
SectionⅡ Characteristics and Functions of the 5 Classes of Ig
Section Ⅲ Fc Receptors for Ab Molecules
Section Ⅳ Biological Activity of Ab
SectionⅤ Immunogenicity of Ig
Section Ⅵ Artificial Ab

3. Concepts
Antibody (Ab): Glycoprotein molecules that are produced by plasma cells and can combine with the corresponding Ag specifically are called Ab.
Ab is produced by B cells in the response to a stimulation of Ag.
Ab possesses a high degree of specificity and affinity

4. Immunoglobulin (Ig):
It refers to all globulins that possess the activity of Ab or show a similar structure to Ab
Therefore, All Abs are Igs, but not all Igs possess the functions of Abs

5. Other Concepts
- Globulin
Antiserum
Humoral Immunity
sIg and mIg(BCR)

6. SectionⅠ Molecular Structure of Ig

7. Ⅰ. Basic structure
Ig is composed of four polypeptide chains joined by S-S bonds.
inter-chain disulfide bonds (S-S)
intra-chain disulfide bonds (S-S)

8. It shows “T” or “Y” shape.

9. 1. H and L chain: . Heavy chains (H): . Light chains (L):
450 ~ 550 aa,
50 ~ 75 KD
. Light chains (L):
214 aa, 25 KD

10. Two terminal ends for each peptide chain
“N” terminal end
“C” terminal end
L chains attach to H chains
from “N” end
“N”
“C”

11. 2. classes and types of Ig
(1) According to the differences of H chains
(amino acid composition, sequence)
Igs can be divided into 5 classes
Five classes of H Chain:     
Five classes of Igs: IgG IgA IgM IgD IgE
subclasses
IgG1~ IgG4
IgA1, IgA2

13.  :  20:1 (in mice); 2:1 (in human)
(2) According to the differences of L chains:
Two types of L chain: , 
 :  20:1 (in mice); 2:1 (in human)
 1~  4

14. 3. Two regions of each peptide chain
(1) Constant region (C)
(2) Variable region (V)
(3) Hinge region

15. (1) Constant region ( C ) (2) Variable region ( V )
3. Two regions of each peptide chain
(1) Constant region ( C )
CH: 3/4 or 4/5 (,) of H chain from the c end
CL: 1/2 of L chain from the c end
(2) Variable region ( V )
CH: 1/4 or 1/5 (,) of H chain from the N end
CL: 1/2 of L chain from the N end

16. (2) Variable region ( V ):
Hypervariable region(HVR)
There are three highly diversity stretches within the V egion,
they are called HVR.

17. Framework region(FR): FR1-FR4

18. Ag-binding sites

19. Complementarity determining regions(CDR)

20. Complementarity determining regions(CDR) L: CDR1, CDR2, CDR3
(2) Variable region (V)
Complementarity determining regions(CDR)
L: CDR1, CDR2, CDR3
H: CDR1, CDR2, CDR3

21. Idiotype of Ig
Igs produced by different B cells possess unique structure respectively in hypervariable region (HVR), the unique structure of Ig is called idiotype or idiotypic determinant

22. In fact:
HVR
CDR
Idiotype
are in the same sites of Ig

23. (3) Hinge region:
Flexible and suitable for CDR of Ig binding to antigenic determinants.
Sensitive to proteolytic enzyme
IgM, IgE

24. Other structures of Ig
Joining chain(J)
Secretory piece(SP)

25. Joining chain(J ) :
Produced by plasma cells
Functions:linker, to compose dimer、pentamer or polymer(IgA, IgM)

26. Secretory piece( SP): IgA . Produced by mucosa epithelial cells
. Secretory IgA (sIgA)
. Functions: protect sIgA, resist proteolysis in extra secretory liquid.
IgA

27. Ⅱ. Domains of Ig

28. 1. Domain :
Polypeptide chains of Ig are folded into a globular structure by intra chain s-s bond within each 110aa region which is called a domain

29. 2. Domains of Ig L chain(2) : VL, CL H chain(4~5): VH, CH1, CH2, CH3
CH4(in IgM,IgE)
hinge region

31. 3. Function of each domain
VH, VL: antigen-binding site
CH1, CL: allogeneic marker
CH2/CH3: complement-fixing site,
permeate placenta(IgG)
CH3/CH4: cell-binding site
Hinge region :flexible and suitable for CDR of Ig binding to antigenic determinants

32. Ⅲ. Hydrolytic fragments of Ig
Ig can be digested by papain and pepsin
Position
Fragments
Function

33. 1. Digested by papain Position:
near the S-S bonds of H inter-chains fromthe N end
Fragments:
2Fab:fragment antigen-binding
Fc:fragment crystallizable
Function:
Fab: recognize and bind Ag Fc:
(1) fix complement
(2) crossing the placenta
(3) bind to FcR in different cells

34. 2. Digested by pepsin Position:
near the S-S bond of H inter-chains from the C end
Fragments and function :
F(ab′)2: bind antigen(2 valence)
pFc′: no function

35. Significance Decrease the immunogenicity of Ig for clinical treatment
 Elucidating the relationships between the structure and function of Igs
Decrease the immunogenicity of Ig for clinical treatment

36. SectionⅡ Characteristics and Functions of the 5 Classes of Igs

37. Ⅰ. IgG
1. Highest concentration in serum (75% of total Ig)

38. 2. Four subclasses: IgG1, IgG2, IgG3, IgG4

39. 3. Unique Ig that can pass through placenta
4. Half-life is longer( days )
5. Starts to be produced at 2-3 month after birth and reach the level of adult at 5 years old

40. 6. Functions of IgG: Against bacteria and virus,neutralize toxin
Combine with the Fc receptor(FcγR)
Activate complement
Combine with SPA
Some belong to the auto-antibodies
Take part in type Ⅱ and Ⅲ hypersensitivity

41. 1. Highest MW：pentamer（90 KD）,10 valences
Ⅱ. IgM
1. Highest MW：pentamer（90 KD）,10 valences

42. 2. Half-life is shorter(4~5 days) 3. The first Ig to be synthesized
Appear in the early stage after infection
Be produced during fetus
The first mIg of the B cells, act as the antigen receptors(BCR)

43. 4. Functions:
IgM is more effective in binding Ag and activating C, and play an important role in anti-infection
Natural Ab for blood-type antigen
Auto-antibody: rheumatoid factor(RF)
Take part in type Ⅱ and Ⅲ hypersensitivity

44. Ⅲ. IgA 1. Two types Serum type ：monomer 2. Two subclasses：IgA1，IgA2
Secretary type（sIgA）: dimer，trimer or polymer
2. Two subclasses：IgA1，IgA2

45. 3. To be produced at 4 months after birth
4. Exist in almost all body fluid

46. 6. Local mucosal immunity
Immune barrier
Neutralize virus/toxin
Rich in colostrum
Activate C by alternative pathway
Take part in type Ⅲ hypersensitivity

47. Ⅳ. IgD
1. The concentration in serum is low and sensitive to proteinase
2. Act as the antigen receptor on B cells (mIgD):
Regulate the differentiation of B cells

48. Ⅴ. IgE 2.Related to typeⅠpersensitivity
1.Concerntration of IgE in serum is the lowest in normal individual, but is very high in some patients.
2.Related to typeⅠpersensitivity
FcεRⅠ: mast cell, basophil

49. Section Ⅲ Fc Receptors for Ab Molecules

50. IgG---FcR: FcRⅠ(CD64)---phagocyte
FcRⅡ(CD32)---immune complex
FcRⅢ(CD16)---NK, MΦ,T cell
IgE---FcR: FcRⅠ--- mast cell, basophil
FcRⅡ--- macrophage, B cell
IgA---FcαR(CD89)---phagocyte, neutrophil

51. SectionⅣ Biological Activity of Ab

52. 1. Recognize and bind to antigen specifically
2. Fix complement
3. Bind to Fc receptor on some cells
4. Transfer selectively :
.Planceta transfer (IgG)
.Mucosa transfer (sIgA)

53. Affinity and Avidity

54. Neutralization

55. IgM,IgG1~3: classical pathway
IgA,IgG4,IgE:
alternative pathway

56. MAC

57. (1) Opsonization(IgG, IgM):
Enhance the phagocytosis of MΦ

58. (2) ADCC( antibody dependent cell mediated cytotoxicity)

59. (3) Hypersensitivity typeⅠ - mast cell, basophil(FcRⅠ)
FceRI
degranulation
IgE
allergen
inflammation

60. SectionⅤ Immunogenicity of Ig

61. Isotype: CH, CL　

64. Allotype：CH, CL

65. Idiotype: VH, VL
Anti-idiotype antibody

66. SectionⅥ Artificial Ab
Polyclonal Ab
Monoclonal Ab
Gene engineering Ab

67. 1. Polyclonal Ab
A mixture Ab with different specificities and affinities
Generate in a natural response or artificial immunization
Cross reaction

68. Cross-reactivity:
if two antigens share an epitope
an antibody recognizes an unrelated,
but chemically similar, epitope

69. 2. Monoclonal Ab (mAb)
Ab produced by single clone (or one hybridomas clone ) and having a single specificity

70. mAb / McAb Prepared by hybridomas technique:
Immunized spleen cells(B) hybride with myeloma cells----hybridomas

71. Artificial antibodies
POLYCLONAL.
MONOCLONAL.
Derived from different B Lymphocytes cell lines
Derived from a single B cell clone
mAb offer Reproducible, Predictable & Potentially inexhaustible supply of Ab with exquisite specificity
Batch to Batch variation affecting Ab reactivity & titre
NOT Powerful tools for clinical diagnostic tests
Enable the development of secure immunoassay systems.

72. 3.Gene engineering Ab Abs prepared by the method of gene recombination
Chimeric Ab:human Fc bind with mice Fab
Recombinant single chain Ab:VH-linker-VL

74. Human-mouse chimeric Ab