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The Molecular Genetics of Immunoglobulins. Recall Structure . Numerous V region genes are preceded by Leader or signal sequences (60-90 bp ) exons interspersed with introns .

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recall structure
Recall Structure
  • Numerous V region genes are preceded by Leader or signal sequences (60-90 bp) exons interspersed with introns.
  • Heavy chain contains V (Variable), D (Diversity), J (Joining) and C (Constant) region gene segments. V - D - J – C
  • Light chain contains V, J, and C region gene segments. V - J - C
  • Constant region genes are sub-divided into exons encoding domains (CH1,CH2, CH3, CH4)
characteristics of immunoglobulin gene re arrangement
CHARACTERISTICS OF IMMUNOGLOBULIN GENERE-ARRANGEMENT

1. Involves Allelic Exclusion.

– Only one of two parental alleles of Ig is expressed in a B cell.

– Either kappa or lambda light chain is expressed by a B cell (light chain isotype exclusion).

2. Ig rearrangement occurs prior to antigen exposure.

A. Heavy chain re-arrangement

– Re-arrangement occurs in a precise order:

– Heavy chain re-arranges before Light chain.

– D-J joining occurs first to form DJ and is followed by V-DJ joining to form VDJ.

– Just as in light chain the production of μ heavy chain by re-arrangement of one allele inhibits re-arrangement on other allele. If re-arrangement on first allele is non-productive (due to mutations, deletions or frame shifts that generate stop codons), then re-arrangement on the second allele is stimulated.

light chain re arrangement
Light chain re-arrangement
  • Kappa chain (κ) rearranges before lambda (λ) chain V-joining occurs.
  • Productive arrangement on one allele blocks re-arrangement on other allele.
  • If kappa protein is produced, re-arrangement of lambda chain is blocked.
  • Otherwise lambda chain undergoes re-arrangement.
slide9

Questions?

  • How is an infinite diversity of specificity generated from finite amounts of DNA?
  • How can the same specificity of antibody be on the cell surface and secreted?
  • How do V region find J regions and why don’t they join to C regions?
  • How does the DNA break and rejoin?
slide10

Single germline C gene separate from multiple V genes

V

V

V

V

V

V

V

V

V

C

V

V

V

C

Rearranging V and C genes

V

V

Proof of the Dreyer - Bennett hypothesis

Aim: to show multiple V genes and rearrangement to the C gene

slide11

V

V

Germline DNA

V

V

V

V

V

V

V

C

V

V

V

C

Rearranged DNA

V

V

  • DNA restriction enzymes to fragment DNA

Proof of the Dreyer - Bennett hypothesis

  • Tools:
  • cDNA probes to distinguish V from C regions
  • Germline (e.g. placenta) and rearranged B cell DNA (e.g. from a myeloma B cell)
slide12

V

V

V

V

C

V

V

V

V

V

V

V

V

V

V

V

V

V

V

Size fractionate by gel electrophoresis

Blot with a V region probe

Blot with a C region probe

V

V

V

V

C

V

V

V

V

C

V

V

V

V

V

V

C

A range of fragment sizes is generated

V

V

V

V

N.B. This example describes events on only ONE of the chromosomes

Cut germline DNA with restriction enzymes

slide13

V

C

V

C

V

V

V

C

V

V

Size fractionate by gel electrophoresis

Blot with a V region probe

Blot with a C region probe

Size fractionate by gel electrophoresis

Blot with a V region probe

Blot with a C region probe

V

V

V

V

V

V

V

V

V

C

V

V

C

V

V

V

V

V

V

V

V

V

- compare the pattern of bands

with germline DNA

V

V

Evidence for gene recombination

Cut myeloma B cell DNA with restriction enzymes

V and C probes detect the same fragment

Some V regions missing

C fragment is larger cf germline

slide14

L

VL

JL

CL

L

VL

CL

~ 95aa

~ 100aa

~ 95aa

~ 100aa

VL

CL

L

Extra amino acids provided by one of a small set of J or JOINING regions

~ 208aa

Ig gene sequencing complicated the model

Structures of germline VL genes were similar for Vk, and Vl,

However there was an anomaly between germline and rearranged DNA:

Where do the extra 13 amino acids come from?

slide15

L

VL

JL

CL

L

VH

JH

DH

CH

Further diversity in the Ig heavy chain

Heavy chain: between 0 and 8 additional amino acids between JH and CH

The D or DIVERSITY region

Each heavy chain requires three recombination events:

VH to JH, VHJH to DH and VHJHDH to CH

Each light chain requires two recombination events:

VL to JL and VLJL to CL

slide16

VH Locus:

  • 123 VH genes on chromosome 14
  • 40 functional VH genes with products identified
  • 79 pseudo VH genes
  • 4 functional VH genes - with no products identified
  • 24 non-functional, orphan VH sequences on chromosomes 15 & 16

JH Locus:

  • 9 JH genes
  • 6 functional JH genes with products identified
  • 3 pseudo JH genes

DH Locus:

  • 27 DH genes
  • 23 functional DH genes with products identified
  • 4 pseudo DH genes
  • Additional non-functional DH sequences on the chromosome 15 orphan locus
  • reading DH regions in 3 frames functionally increases number of DH regions

Diversity: Multiple Germline Genes

slide17

Vk & Jk Loci:

  • 132 Vk genes on the short arm of chromosome 2
  • 29 functional Vk genes with products identified
  • 87 pseudo Vk genes
  • 15 functional Vk genes - with no products identified
  • 25 orphans Vk genes on the long arm of chromosome 2
  • 5 Jk regions

Vl & Jl Loci:

  • 105 Vl genes on the short arm of chromosome 2
  • 30 functional genes with products identified
  • 56 pseudogenes
  • 6 functional genes - with no products identified
  • 13 relics (<200bp Vl of sequence)
  • 25 orphans on the long arm of chromosome 2
  • 4 Jl regions

Diversity: Multiple germline genes

slide18

LH1-123

VH 1-123

DH1-27

JH 1-9

Cm

Lk1-132

Vk1-132

Jk 1-5

Ck

Ll1-105

Vl1-105

Cl1 Jl1

Cl2 Jl2

Cl3 Jl3

Cl4 Jl4

Genomic organisation of Ig genes

(No.s include pseudogenes etc.)

slide19

Vk

Jk

Ck

Germline

Rearranged

1° transcript

SplicedmRNA

Ig light chain gene rearrangement by somatic recombination

slide20

Questions?

  • How is an infinite diversity of specificity generated from finite amounts of DNA?
  • How can the same specificity of antibody be on the cell surface and secreted?
  • How do V region find J regions and why don’t they join to C regions?
  • How does the DNA break and rejoin?
slide21

Remember

  • Cell surface antigen receptor on B cells
      • Allows B cells to sense their antigenic environment
      • Connects extracellular space with intracellular signalling machinery
  • Secreted antibody
  • Neutralisation
  • Arming/recruiting effector cells
  • Complement fixation

How does the model of recombination allow for

two different forms of the protein?

slide22

Cm

Primary transcript RNA

AAAAA

Each H chain domain (& the hinge) encoded by separate exons

Secretioncodingsequence

Polyadenylation site (secreted)

pAs

Polyadenylation site (membrane)

pAm

Cm1

Cm2

Cm3

Cm4

Membranecodingsequence

The constant region has additional, optional exons

h

slide23

Cm1

h

Cm2

Cm3

Cm4

DNA

Transcription

pAm

Cm1

h

Cm2

Cm3

Cm4

1° transcript

AAAAA

Cleavage & polyadenylation at pAm and RNA splicing

Cm1

h

Cm2

Cm3

Cm4

AAAAA

Protein

Membrane coding sequence encodes transmembrane region

that retains IgM in the cell membrane

Fc

Membrane IgM constant region

mRNA

slide24

h

Cm1

Cm2

Cm3

Cm4

DNA

Transcription

pAs

Cm1

h

Cm2

Cm3

Cm4

1° transcript

AAAAA

Cleavage polyadenylation at pAs and RNA splicing

Cm1

h

Cm2

Cm3

Cm4

AAAAA

mRNA

Protein

Secretion coding sequence encodes the C terminus of soluble, secreted IgM

Fc

Secreted IgM constant region

slide26

VH

DH

JH

C

2x

1x

DIVERSITY

DIVERSITY

Why do V regions not join to J or C regions?

IF the elements of Ig did not assemble in the correct order, diversity of specificity would be severely compromised

Full potential of the H chain for diversity needs V-D-J-C joining - in the correct order

Were V-J joins allowed in the heavy chain, diversity would be reduced due to loss of the imprecise join between the V and D regions

slide28

Vl

Jl

7

23

12

7

9

9

Vk

JH

Jk

9

9

7

12

23

23

7

7

9

D

12

7

7

12

9

9

VH

9

7

23

V, D, J flanking sequences

Sequencing up and down stream of V, D and J elementsConserved sequences of 7, 23, 9 and 12 nucleotides in an arrangement that depended upon the locus

slide29

HEPTAMER - Always contiguous with coding sequence

NONAMER - Separated fromthe heptamer by a 12 or 23 nucleotide spacer

JH

JH

9

9

23

23

7

7

D

D

12

12

7

7

7

7

12

12

9

9

9

9

VH

VH

9

9

7

7

23

23

Recombination signal sequences (RSS)

12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment flanked by a 12mer RSS

slide30

12-mer = one turn

23-mer = two turns

Intervening DNA

of any length

23

12

V

7

9

7

D

J

9

Molecular explanation of the 12-23 rule

slide31

V4

V5

V3

V1

V3

V4

V2

V6

V2

V5

V6

V7

V8

V7

9

V9

D

J

V8

V9

9

23-mer

  • Heptamers and nonamers align back-to-back
  • The shape generated by the RSS’s acts as a target for recombinases

12-mer

7

7

D

J

V1

Molecular explanation of the 12-23 rule

Loop of intervening

DNA is excised

  • An appropriate shape can not be formed if two 23-mer flanked elements attempted to join (i.e. the 12-23 rule)
slide32

V

D

J

9

7

23

12

7

9

V

9

7

23

J

D

7

12

9

V

9

9

7

7

23

23

J

D

7

7

12

12

9

9

Steps of Ig gene recombination

Recombination activating gene products, (RAG1 & RAG 2) and ‘high mobility group proteins’ bind to the RSS

The two RAG1/RAG 2 complexes bind to each other and bring the V region adjacent to the DJ region

  • The recombinase complex makes single stranded nicks in the DNA. The free OH on the 3’ end hydrolyses the phosphodiester bond on the other strand.
  • This seals the nicks to form a hairpin structure at the end of the V and D regions and a flush double strand break at the ends of the heptamers.
  • The recombinase complex remains associated with the break
slide33

V

9

7

23

J

D

7

12

9

The hairpins at the end of the V and D regions are opened, and exonucleases and transferases remove or add random nucleotides to the gap between the V and D region

DNA ligase IV joins the ends of the V and D region to form the coding joint and the two heptamers to form the signal joint.

V

V

9

7

23

J

J

D

D

7

12

9

Steps of Ig gene recombination

A number of other proteins, (Ku70:Ku80, XRCC4 and DNA dependent protein kinases) bind to the hairpins and the heptamer ends.

slide34

7

9

TCCACAGTG

AG GTGTCAC

V

23

V

9

7

23

J

D

12

9

7

AT GTGACAC

TA CACTGTG

J

D

7

12

9

TC

AG

V

U

J

D

AT

TA

U

7

9

TC

AG

CACAGTG

GTGTCAC

V

23

J

D

12

9

7

AT

TA

GTGACAC

CACTGTG

Junctional diversity: P nucleotide additions

The recombinase complex makes single stranded nicks at random sites close to the ends of the V and D region DNA.

The 2nd strand is cleaved and hairpins form between the complimentary bases at ends of the V and D region.

slide35

V3

7

9

CACAGTG

GTGTCAC

23

V2

V4

12

9

7

GTGACAC

CACTGTG

V5

V9

V8

V6

V7

U

U

D

J

AT

TA

U

TC

AG

TC

AG

V

V

U

J

D

AT

TA

Heptamers are ligated by DNA ligase IV

V and D regions juxtaposed

slide36

Regions to be joined are juxtaposed

The nucleotides that flip out, become part of the complementary DNA strand

U

U

D

D

J

J

AT

TA

AT

TA

U

U

TC

AG

TC

AG

V

V

D

J

AT

TA~TA

TC~GA

AG

V

Generation of the palindromic sequence

Endonuclease cleaves single strand at random sites in V and D segment

The nicked strand ‘flips’ out

In terms of G to C and T to A pairing, the ‘new’ nucleotides are palindromic.

The nucleotidesGA and TA were not in the genomic sequence and introduce diversity of sequence at the V to D join.

slide37

CACACCTTA

Complementary bases anneal

TTCTTGCAA

CACACCTTA

TC~GA

V

D

J

TA~TA

Exonucleases nibble back free ends

TTCTTGCAA

D

D

J

J

AT

TA~TA

AT

TA~TA

TC~GA

AG

TC~GA

AG

V

V

DNA polymerases fill in the gaps with complementary nucleotides and DNA ligase IV joins the strands

TC

CACACCTTA

TC~GA

AG

V

V

D

D

J

AT

TA~TA

AG C

TTCTTGCAA

TA

GTTAT AT

Junctional Diversity – N nucleotide additions

Terminal deoxynucleotidyl transferase (TdT) adds nucleotides randomly to the P nucleotide ends of the single-stranded V and D segment DNA

CACTCCTTA

TTCTTGCAA

slide38

V

D

J

TCGACGTTATAT

AGCTGCAATATA

Junctional Diversity

Germline-encoded nucleotides

Palindromic (P) nucleotides - in the germline

Non-template (N) encoded nucleotides - not in the germline

Creates an essentially random sequence between the V region, D region and J region in heavy chains and the V region and J region in light chains.

slide39

Problems?

  • How is an infinite diversity of specificity generated from finite amounts of DNA?Combinatorial Diversity, genomic organisation and Junctional Diversity
  • How can the same specificity of antibody be on the cell surface and secreted?Use of alternative polyadenylation sites
  • How do V region find J regions and why don’t they join to C regions?The 12-23 rule
  • How does the DNA break and rejoin?Imprecisely to allow Junctional Diversity
slide40

Variable addition and subtraction of nucleotides at the junctions between gene segments contributes to diversity in the third hypervariable region

  • Of the three hypervariable loops in the protein chains of immunoglobulins, two are encoded within the V gene segment DNA. The third (HV3 or CDR3) falls at the joint between the V gene segment and the J gene segment, and in the heavy chain is partially encoded by the D gene segment.
  • In both heavy and light chains, the diversity of CDR3 is significantly increased by the addition and deletion of nucleotides at two steps in the formation of the junctions between gene segments. The added nucleotides are known as P-nucleotidesandN-nucleotides
slide41

As the total number of nucleotides added by these processes is random, the added nucleotides often disrupt the reading frame of the coding sequence beyond the joint.

  • Such frameshifts will lead to a nonfunctional protein, and DNA rearrangements leading to such disruptions are known as nonproductive rearrangements.
  • As roughly two in every three rearrangements will be nonproductive, many B cells never succeed in producing functional immunoglobulin molecules, and junctional diversity is therefore achieved only at the expense of considerable wastage.
slide43
Some rearrangements are productive, others are non-productive: frame shift alterations are non-productive

1 in 3 in phase VL to VJ

1 in 3 in phase VH to DHJH

Only 11% of cells mature and leave

slide44

9

23

7

7

12

9

9

9

23

Coding joint

Signal joint

12

V

D

J

7

7

V

D

J

Junctional diversity

Mini-circle of DNA is permanently lost from the genome

Imprecise and random events that occur when the DNA breaks and rejoins allows new nucleotides to be inserted or lost from the sequence at and around the coding joint.

slide45

Looping out works if all V genes are in the same transcriptional orientation

V1

V3

V4

V9

V2

D

D

J

J

V1

D

J

9

7

23

9

23

7

How does recombination occur when a V gene is in opposite orientation to the DJ region?

12

7

9

V1

V3

V4

V9

V2

V4

D

J

12

7

9

Non-deletional recombination

slide46

V4 and DJ in opposite transcriptional orientations

1.

2.

D

D

D

D

J

J

J

J

12

12

12

12

7

7

7

7

9

9

9

9

9

9

9

9

9

23

23

23

23

23

7

7

7

7

7

V4

V4

V4

V4

V4

3.

4.

D

J

12

7

9

Non-deletional recombination

slide47

1.

2.

V4

Heptamer ligation - signal joint formation

D

J

D

J

12

12

12

7

7

7

9

9

9

12

7

3.

9

9

9

9

9

23

23

23

23

7

7

7

7

V4

V to DJ ligation - coding joint formation

D

J

V4

4.

V4

D

J

Fully recombined VDJ regions in same transcriptional orientation

No DNA is deleted

rearrangement of v d and j gene segments is guided by flanking dna sequences1
Rearrangement of V, D, and J gene segments is guided by flanking DNA sequences
  • A system is required to ensure that DNA rearrangements take place at the correct locations relative to the V, D, or J gene segment coding regions.
  • V gene segment joins to a D or J and not to another V.
  • DNArearrangements are in fact guided by conserved noncoding DNA sequences that are found adjacent to the points at which recombination takes place.
  • These sequences consist of a conserved block of seven nucleotides—the heptamer 5′CACAGTG3′—which is always contiguous with the coding sequence, followed by a nonconserved region known as the spacer, which is either 12 or 23 nucleotides long.
  • This is followed by a second conserved block of nine nucleotides—the nonamer 5′ACAAAAACC3′ .
  • The spacer varies in sequence but its conserved length corresponds to one or two turns of the DNA double helix.
  • This brings the heptamer and nonamer sequences to the same side of the DNA helix, where they can be bound by the complex of proteins that catalyzes recombination. The heptamer-spacer-nonamer is called a recombination signal sequence (RSS).
12 23 rule
12/23 Rule
  • Recombination only occurs between gene segments located on the same chromosome.
  • It generally follows the rule that only a gene segment flanked by a RSS with a 12-base pair (bp) spacer can be joined to one flanked by a 23 bp spacer RSS. This is known as the 12/23 rule.
  • For the heavy chain, a DH gene segment can be joined to a JH gene segment and a VH gene segment to a DH gene segment, but VHgene segments cannot be joined to JHgene segments directly, as both VH and JHgene segments are flanked by 23 bp spacers and the DHgene segments have 12 bp spacers on both sides
t he diversity of the immunoglobulin repertoire is generated by four main processes
The diversity of the immunoglobulin repertoire is generated by four main processes
  • Antibody diversity is generated in four main ways.
  • The gene rearrangement that combines two or three gene segments to form a complete V-region exon generates diversity in two ways.
    • First, there are multiple different copies of each type of gene segment, and different combinations of gene segments can be used in different rearrangement events. This combinatorial diversity is responsible for a substantial part of the diversity of the heavy- and light-chain V regions.
    • Second, junctional diversity is introduced at the joints between the different gene segments as a result of addition and subtraction of nucleotides by the recombination process.
  • A third source of diversity is also combinatorial, arising from the many possible different combinations of heavy- and light-chain V regions that pair to form the antigen-binding site in the immunoglobulin molecule.
  • Somatic mutation
rearranged v genes are further diversified by somatic hypermutation
Rearranged V genes are further diversified by somatic hypermutation
  • The mechanisms for generating diversity described so far all take place during the rearrangement of gene segments in the initial development of B cells in the central lymphoid organs.
  • There is an additional mechanism that generates diversity throughout the V region and that operates on B cells in peripheral lymphoid organs after functional immunoglobulin genes have been assembled.
  • This process, known as somatic hypermutation.
  • Introduces point mutations into the V regions of the rearranged heavy- and light-chain genes at a very high rate, giving rise to mutant B-cell receptors on the surface of the B cells.
  • Some of the mutant immunoglobulin molecules bind antigen better than the original B-cell receptors, and B cells expressing them are preferentially selected to mature into antibody-secreting cells. This gives rise to a phenomenon called affinity maturation of the antibody population,
somatic hypermutation
Somatic hypermutation
  • Occurs when B cells respond to antigen along with signals from activated T cells.
  • The immunoglobulin C-region gene, and other genes expressed in the B cell, are not affected, whereas the rearranged VH and VL genes are mutated even if they are nonproductiverearrangements and are not expressed.
  • The pattern of nucleotide base changes in nonproductive V-region genes illustrates the result of somatic hypermutation without selection for enhanced binding to antigen.
slide53

FR1

CDR1

FR2

CDR2

FR3

CDR3

FR4

100

Variability

Wu - Kabat analysis compares point mutations in Ig of different specificity.

80

60

40

20

20

40

60

80

100

120

Amino acid No.

Somatic hypermutation

What about mutation throughout an immune response to a single epitope?

How does this affect the specificity and affinity of the antibody?

slide54

Day 6

Day 12

Day 8

Day 18

Clone 1

Clone 2

Clone 3

Clone 4

Clone 5

Clone 6

Clone 7

Clone 8

Clone 9

Clone 10

Deleterious mutation

CDR1

CDR2

CDR3

CDR1

CDR2

CDR3

CDR1

CDR2

CDR3

CDR1

CDR2

CDR3

Beneficial mutation

Neutral mutation

Somatic hypermutation leads to affinity maturation

Cells with accumulated mutations in the CDR are selected for high antigen binding capacity – thus the affinity matures throughout the course of the response

Lower affinity - Not clonally selected

Higher affinity - Clonally selected

Identical affinity - No influence on clonal selection

Hypermutation is T cell dependent

Mutations focussed on ‘hot spots’ (i.e. the CDRs) due to double stranded breaks repaired by an error prone DNA repair enzyme.

slide55

Organisation of the functional human heavy chain C region genes

Cm

Cd

Cg3

Cg1

Ca1

Cg2

Cg4

Ce

Ca2

J regions

Antibody isotype switching

Throughout an immune response the specificity of an antibody will remain the same (notwithstanding affinity maturation)

The effector function of antibodies throughout a response needs to change drastically as the response progresses.

Antibodies are able to retain variable regions whilst exchanging constant regions that contain the structures that interact with cells.

slide56

Cm

Cd

Cg3

Cg1

Ca1

Cg2

Cg4

Ce

Ca2

Sm

Sg3

Sg1

Sa1

Sg2

Sg4

Se

Sa2

Switch regions

  • Upstream of C regions are repetitive regions of DNA called switch regions. (The exception is the Cd region that has no switch region).
  • The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7.
  • Switching is mechanistically similar in may ways to V(D)J recombination.
  • Isotype switching does not take place in the bone marrow, however, and it will only occur after B cell activation by antigen and interactions with T cells.
slide57

Cm

Cd

Cg3

Cg1

Ca1

Cg2

Cg4

Ce

Ca2

Sg3

Cd

Cd

Cg3

Cm

Cm

Sg1

Cg3

Cg1

VDJ

VDJ

Cg3

VDJ

Ca1

Ca1

IgG3 produced.

Switch from IgM

IgA1 produced.

Switch from IgM

IgA1 produced.

Switch from IgG3

VDJ

VDJ

VDJ

Cg3

Ca1

Ca1

Switch recombination

At each recombination constant regions are deleted from the genome

An IgE - secreting B cell will never be able to switch to IgM, IgD, IgG1-4 or IgA1

summary
Summary
  • Diversity within the immunoglobulin repertoire is achieved by several means.
  • Perhaps the most important factor that enables this extraordinary diversity is that V regions are encoded by separate gene segments, which are brought together by somatic recombination to make a complete V-region gene.
  • Many different V-region gene segments are present in the genome of an individual, and thus provide a heritable source of diversity. Additional diversity, termed combinatorial diversity, results from the random recombination of separate V, D, and J gene segments to form a complete V-region exon. 
summary1
Summary
  • Variability at the joints between segments is increased by the insertion of random numbers of P- and N-nucleotides and by variable deletion of nucleotides at the ends of some coding sequences.
  • The association of different light- and heavy-chain V regions to form the antigen-binding site of an immunoglobulin molecule contributes further diversity.
  • Finally, after an immunoglobulin has been expressed, the coding sequences for its V regions are modified by somatic hypermutation upon stimulation of the B cell by antigen.
  • The combination of all these sources of diversity generates a vast repertoire of antibody specificities from a relatively limited number of genes.
mechanisms for generating antibody diversity
MECHANISMS FOR GENERATING ANTIBODY DIVERSITY
  • Presence of multiple V genes in the germ line.
  • Combinatorial Diversity - due to potentially different associations of different V, D and J gene segments.
  • Junctional Diversity
  • Somatic Hypermutation
  • Random Assortment of H and L chains.
understanding of immunoglobulin structure and formation has opened up a new world of possibilities
Understanding of immunoglobulin structure and formation has opened up a new world of possibilities
  • Monoclonal antibodies
  • Engineering mice with human immune systems
  • Generating chimeric and hybrid antibodies for clinical use
  • Abzymes: antibodies with enzyme capability