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Kindt • Goldsby • Osborne
Kuby IMMUNOLOGY
Sixth Edition
Chapter 5
FIGURE 5-1
B-Cell Development
The events that occur during
maturation in the bone
marrow do not require
antigen, whereas activation
and differentiation of mature
B cells in peripheral lymphoid
organs require antigen.
The labels mIgM and mIgD
refer to membrane-associated
Igs. IgG, IgA, and IgE are
secreted immunoglobulins.
Diversing a Genetic Model Compatible with Ig Structure
•
The vast diversity of
antibody specificities
•
The presence in Ig heavy
and light chains of a
variable region at the
amino terminal end and a
constant region at the
carboxyl-terminal end.
•
The existence of isotypes
with the same antigenic
specificity, which result
from the association of a
given variable region with
different heavy-chain
constant regions.
Genetic Model Compatible with Ig Structure
• Germ line theory : Genome contributed by germ line cells, egg and
sperm, contains a large repertoire of Ig genes =>
• Somatic variation theory: Genome contains a relatively small number of
Ig genes, from which a large number of Abs are generated in the somatic
cells by mutation or recombination. =>
• Identical variable region sequences were associated with both r and m
heavy chain constant regions
이들 두 제안은 항체 구조적인 특성 설명에는 부적절
• Two gene model (Dreyer and Bennett): Two separate genes encode a
single Ig H or L chain, one gene for the V region and the other for the C
region. Two genes must come together at the DNA level to form a
continuous message that can be transcribed and translated into single Ig
H or L chain. Thounds of V region genes are located in the germ line,
whereas only single copies of C-region class subclass genes need exist.
• 이론적인 가설이 당시 기술이 따르지 못해 증명하지를 못하였다.
• 이 가설을 Tonegawa가 증명함으로 항체의 다양성을 이해
1976년, Tonegawa의 Southern blotting 실험 :
The first direct evidence that separate genes encode the V and C regions of Igs
and that the genes are rearranged in the course of B-cell differentiation.
•
배아세포 (embryonic cell) 의 항체 유전자와 adult myeloma B cell의 항체유전자
의 구조가 다름을 증명
•
배아세포와 B cell에서 얻은 DNA를 제한 효소로 절단한 후, agarose gel을 이용한
전기영동 하고, probe를 이용하여 항체 유전자가 어떤 크기의 조각과 결합하는 지
를 조사
•
두 세포에서 같은 probe에 hybridize하는 DNA의 조각의 크기가 다름을 확인
(restriction fragment length polymorphism, RFLP)
•
전구세포에서 B cell로 성숙되는 동안 항체유전자의 구조에 변화
•
B cell은 다양한 종류의 항체 유전자를 보유
: 전구세포단계의 항체유전자 즉 재배열되기 전의 항체 유전자 (germ line DNA)
: B 림프구에 존재하는 완성된 항체유전자 (재배열된 유전자)
: 항체 유전자의 전사된 mRNA 유전자 (cDNA 유전자)
항체 유전자는 V 지역과 C 지역을 구성하는 유전자가 따로 여러 개 존재
FIGURE 5-2 Experimental basis for diagnosis of rearrangement at an immunoglobulin locus.
The number and size of restriction fragments generated by the treatment of DNA with a restriction enzyme are
determined by the sequence of the DNA. The digestion of rearranged DNA with a restriction enzyme(RE) yields a
pattern of restriction fragments that differ from those obtained by digestion of an unrearranged locus with the
same RE. Typically, the fragments are analyzed by the technique of Southern blotting. In this example, a probe
that includes a J gene segment is used to identify RE digestion fragments that include all or portions of this
segment. As shown, rearrangement results in the deletion of a segment of germ-line DNA and the loss of the
restriction sites that it includes. It also results in the joining of gene segments, in this case a V and a J segment,
that are separated in the germ line. Consequently, fragments dependent on the presence of this segment for
their generation are absent from the restriction-enzyme digest of DNA from the rearranged locus. Furthermore,
rearranged DNA gives rise to novel fragments that are absent from digests of DNA in the germ-line configuration.
This can be useful because both B cells and non-B cells have two immunoglobulin loci. One of these is
rearranged, and the other is not. Consequently, unless a genetic accident has resulted in the loss of the germ-line
locus, digestion of DNA from a myeloma or normal B-cell clone will produce a pattern of restriction that includes
all of those in a germ-line digest plus any novel fragments that are generated from the change in DNA sequence
that accompanies rearrangement. Note that only one of the several J gene segments present is shown.
Multigene Organization of Ig Genes
• Cloning and sequencing of the light- and heavy-chain DNA was
accomplished.
• Each multigene family has distinct features
• The k and λ Light chain families contain V, J, and C gene
segments
• Heavy chain family contains V, D, J, and C gene segments.
L---V------D-----J--------C
FIGURE 5-3 Organization of Immunoglobulin Germ-Line Gene
Segments in the Mouse : (a) λ Light Chain, (b) κ Light Chain, and
(c) Heavy Chain
The λ and κ light chains are encoded by V, J and C gene segments. The heavy chain
is encoded by V, D, J and C gene segments. The distances in (kb) separating the
various gene segments in mouse germ-line DNA are shown below each chain
diagram.
FIGURE 5-4
Kappa light-chain gene
rearrangement and RNA
processing events required to
generate a κ light-chain protein
In this example,
rearrangement joins 𝑉κ 23
and 𝐽κ 4.
Heavy-chain gene
rearrangement and RNA
processing events required
to generate finished μ or δ
heavy-chain protein.
Two DNA joinings are
necessary to generate a
functional heavy-chain gene : a
𝐷H to 𝐽H joining and a 𝑉H to
𝐷H 𝐽H joining. In this example,
𝑉H 21, 𝐷H 7, and are 𝐽H joined.
Expression of functional heavychain genes, although generally
similar to expression of lightchain genes, involves differential
RNA processing, which
generates several different
products, including μ or δ heavy
chains. Each C gene is drawn as
a single coding sequence ; in
reality, each is organized as a
series of exons and introns.
(a)
(b)
FIGURE 5-6
Two conserved sequences
in light-chain and heavychain DNA function as
recombination signal
sequences(RSSs).
(a) Both signal sequences
consist of a conserved
palindromic heptamer and
conserved AT-rich
nonamer ; these are
separated by
nonconserved spacers of
12 or 23 base pairs.
(b) The two types of RSSdesignated one-turn RSS
and two-turn RSS-have
characteristic locations
within λ-chain, κ-chain,
and heavy-chain germ-line
DNA. During DNA
rearrangement, gene
segments adjacent to the
one-turn RSS can join only
with segments adjacent to
the two-turn RSS.
Model depicting the general
process of recombination of
immunoglobulin gene segments is
illustrated with 𝑉κ and 𝐽κ .
(a) Deletional joining occurs when the
gene segments to be joined have the
same transcriptional
orientation(indicated by horizontal blue
arrows). This process yields two
products : a rearranged VJ unit that
includes the coding joint and a circular
excision product consisting of the
recombination signal sequences(RSSs),
signal joint, and intervening DNA.
(b) Inversional joining occurs when the
gene segments have opposite
transcriptional orientations. In this case,
the RSSs, signal joint, and intervening
DNA are retained, and the orientation
of one of the joined segments is
inverted.
In both types of recombination, a few
nucleotides may be deleted from or added
to the cut ends of the coding sequences
before they are rejoined.
Circular DNA isolated
from thymocytes in which
the DNA encoding the
chains of the T-cell
receptor(TCR) undergoes
rearrangement in a
process like that involving
the immunoglobulin
genes.
Isolation of this circular
excision product is direct
evidence for the
mechanism of deletional
joining shown in Figure 57
FIGURE 5-9
Junctional flexibility in the joining
of immunoglobulin gene segments
is illustrated with 𝑉κ and 𝐽κ .
In-phase joining(arrows 1, 2 and
3) generates a productive
rearrangement, which can be
translated into protein. Out-ofphase joining(arrows 4 and 5)
leads to a nonproductive
rearrangement that contains stop
codons and is not translated into
protein.
Allelic Exclusion ensures a single
antigenic specificity
• 항체 유전자의 재배열은 항체의 대립유전자 (alleles) 모두에서
동시에 나타나지 않는다 (allelic exclusion)
• 만일 B cell 의 한 allele가 nonproductive 한 rearrangement
를 했다면 다른 allele에서 rearrangement 가 일어난다
• 항체 유전자의 한 allele의 heavy chain gene에서 재배열이 제
대로 일어나 정상적인 heavy chain 단백질이 만들어지면, 다른
allele의 heavy chain gene에서 재배열은 일어나지 않는다
• light chain gene의 재배열에서도 같은 현상이 나타난다
• 그 결과 하나의 B cell에는 한 가지의 heavy chain 단백질과 한
가지의 light chain 단백질만이 만들어진다
• 재배열이 잘못되어 발현이 안되는 B cell은 apoptosis 기작에
의해 사멸
• 하나의 B cell에서는 한 가지의 항체만 생산
FIGURE 5-10
Because of allelic
exclusion, the
immunoglobulin heavyand light-chain genes of
only one parental
chromosome are
expressed per cell.
This process ensures that B
cells possess a single
antigenic specificity. The allele
selected for rearrangement is
chosen randomly. Thus, the
expressed immunoglobulin
may contain one maternal
and one paternal chain or
both chains may derive from
only one parent. Only B cells
and T cells exhibit allelic
exclusion. Asterisks(*) indicate
the expressed alleles.
FIGURE 5-11
Model to account for
allelic exclusion.
Heavy-chain genes rearrange
first, and once a productive
heavy-chain gene
rearrangement occurs, the μ
protein product prevents
rearrangement of the other
heavy-chain allele and initiates
light-chain gene rearrangement.
In the mouse, rearrangement of
κ light-chain genes precedes
rearrangement of the λ genes,
as shown here. In humans,
either κ or λ rearrangement can
proceed once a productive
heavy-chain rearrangement has
occurred. Formation of a
complete immunoglobulin
inhibits further light-chain gene
rearrangement. If a
nonproductive rearrangement
occurs for one allele, then the
cell attempts rearrangement of
the other allele.
Generation of Antibody Diversity
항체의 다양한 특이성은 몇 개 안 되는 항체 유전자의 효율적인 활용
으로 가능
• Germ-line 상태에서 유전자조각을 여러 개 가지고 있다 (multiple
germ-line gene segments)
• VDJ 유전자 조각이 다양하게 재배열되어 조합 (combinatorial
diversity)
• 재조합 시 합쳐지는 부위가 다양하다 ( junctional flexibility)
• 합쳐질 때 새로운 염기들이 첨가된다 (P/N nucleotide addition)
• 체세포 돌연변이에 의하여 V 지역의 염기배열이 바뀐다 (somatic
hypermutation)
• heavy chain과 light chain이 서로 임의로 결합한다 (heavy and
light chain combination)
FIGURE 5-12 Experimental evidence for junctional
flexibility in immunoglobulin-gene rearrangement.
The nucleotide sequences
flanking the coding joints
between 𝑉K 21 and 𝐽K 1 and
the corresponding signal joint
sequences were determined in
four pre-B cell lines. The
sequence constancy in the
signal joints contrasts with the
sequence variability in the
coding joints. Pink and yellow
shading indicate nucleotides
derived from 𝑉K 21 and 𝐽K 1,
respectively, and purple and
orange shading indicate
nucleotides from the two RSSs.
FIGURE 5-13 P-nucleotide and N-nucleotide addition during joining.
(a) If cleavage of the hairpin intermediate yields a double-stranded end on the
coding sequence, then P-nucleotide addition does not occur. In many cases,
however, cleavage yields a single-stranded end. During subsequent repair,
complementary nucleotides are added, called P-nucleotide, to produce
palindromic sequences(indicated by brackets). In this example, four extra base
pairs(blue) are present in the coding joint as the result of P-nucleotide addition.
(b) Besides P-nucleotide addition, addition of random N-nucleotides(light red) by a
terminal deoxynucleotidyl transferase(TdT) can occur during joining of heavychain coding sequences.
Somatic Hypermutation adds Diversity in
Already-rearranged Gene Segments
• 성숙된 B cell은 항원과 반응하게되면 항체를 생산하게 되며, 항
원자극이 되풀이되면 될수록, 만들어지는 항체의 항원 친화력은
이전 보다 점점 좋아지게 된다
친화력의 성숙 (affinity maturation)
• 항체의 친화력의 성숙은 항원에 의하여 활성화된 B cell이 증식
분화하는 동안 항체의 H chain과 L chain 유전자의 V 지역에 돌
연변이가 유도되어 나타난다
: 체세포 돌연변이 (somatic mutation)
: 항체의 V지역 중 특히 hypervariable region (CDR)에 집중
: 친화력이 높은 항체들은 항원에 의하여 자연선택
• 항원 침입이 잦으면 잦을수록 점점 더 좋은 항체들이 만들어져,
자주 침입하는 항원에 대해 보다 효과적인 방어가 가능
• 10-3 per base pair per generation
FIGURE 5-14
Experimental evidence for somatic
mutation in variable regions of
immunoglobulin genes.
The diagram compares the mRNA
sequences of heavy chains and of light
chains from hybridomas specific for the
phOx hapten. The horizontal solid lines
represent the germ-line 𝑉H and 𝑉K Ox-1
sequences ; dashed lines represent
sequences derived from other germ-line
genes. Blue shading shows the areas where
mutations clustered ; the blue circles with
vertical lines indicate locations of mutations
that encode a different amino acid than the
germ-line sequence. These data show that
the frequency of mutation (1) increases in
the course of the primary response(day 7
vs. day 14) and (2) is higher after
secondary and tertiary immunizations than
after primary immunization. Moreover, the
dissociation constant (𝐾d ) of the anti-phOx
antibodies decreases during the transition
from the primary to tertiary response,
indicating an increase in the overall affinity
of the antibody. Note also that most of the
mutations are clustered within CDR1 and
CDR2 of both the heavy and the light
chains.
A final source of diversity is combinatorial assciation of heavy
and light chains
•
•
In human, there is the potential to generate 6624 heavy chain genes
and 375 light genes as a result of variable region gene
rearrangements.
H and L combinations is 2,484,000.
FIGURE 5-15
Immunoglobulin
diversification occurs by gene
conversion in chickens.
In the chicken germ line, the
single functional 𝑉H and
𝑉λ immunoglobulin genes are
preceded by many pseudogenes.
Rearrangement creates a single
functional rearranged V-(D)-J.
Gene conversion introduces
diversity into the V segments of
rearranged V-(D)-J genes using
upstream V pseudogenes as a
template.
Class Switching among Constant-Region Genes
•
•
•
•
•
•
•
항원이 최초로 숙주에게 들어왔을 때에는 IgM 급의 항체가 주로 만
들어지나, 같은 항원이 다시 들어왔을 때에는 다른 급, 주로 IgG급의
항체가 만들어진다 (항체의 급의 전환)
heavy chain의 C 지역이 바뀌어 나타나는 현상
heavy chain의 C지역의 전환은 항체 유전자의 재조합 (DNA
recombination)을 통하여 이루어지나 아직 mechanism은 잘 규명되
어있지 않다.
Switch region: δ chain의 C 지역유전자를 제외한 모든 C 지역 유전
자의 5' (2-3 kb upstram )끝에 있는 보존되어 있는 염기배열
(conserved sequence, GAGCT and TGGG)에 의한 재조합
Recombinase and switch factor (IL-4)
항체 유전자의 급의 전환에는 IL-4와 같은 helper T cell이 만들어
낸 cytokine이 관여하는 것으로 알려져 있다.
Overall, class switching depends on the interplay of
Four elements: switch region,
switch recombinase,
cytokine signals (IL-4)
activation-induced cytidine deaminase (AID)
FIGURE 5-16
Proposed mechanism for
class switching induced by
interleukin-4 in rearranged
immunoglobulin heavy-chain
genes.
A switch site is located
upstream from each
𝐶H segment except 𝐶δ .
Identification of the
indicated circular excision
products containing
portions of the switch sites
suggested that IL-4
induces sequential class
switching from 𝐶μ to 𝐶γ 1
to 𝐶ε .
(a)
(b)
FIGURE 5-17
Experimental demonstration of
the role of the enzyme AID in
class switching and somatic
hypermutation.
(a) AID-expressing (+/-) and AID
knockout (-/-) mice were
immunized twice with a haptencarrier conjugate and the
antihapten antibody responses
measured and plotted in arbitrary
units. IgM responses were detected
in both types of mice. Production
of IgG, which requires class
switching, occurred only in AIDexpressing (+/-) mice.
(b) Messenger RNA encoding the
variable regions of antigen-reactive
antibodies in immunized AIDexpressing and AID knockout mice
was sequenced and the position
and frequency of mutations plotted.
Many mutations are seen in the
AID-expressing mice ; only
background levels of mutation are
seen in the AID knockout mice.
Expression of membrane or secreted Ig
•
분비된 항체(sIg)와 세포막과 결합된 항체(mIg)도 항원결합부위는 서로
완전히 같기 때문에 항원 특이성에 차이가 없다
: 세포막에 결합되어 있던 항체단백질이 단백질분해효소에 의하여 절단
되어 sIg가 만들어지는 것도 아니라 mIg 와 sIg형의 항체는 각각의
mRNA로부터 번역되어 만들어진다.
• primary transcript를 조작하는 방법으로 분비형과 새포막형의 항체를
만든다 (alternative splicing)
: heavy chain 유전자를 δ chain의 C 지역 유전자 뒤까지 전사하여 Cμ
와 Cδ chain 유전자들이 모두 포함된 일차 전사체 RNA를 얻는다
: 이 primary transcript 에 있는 Cμ 전사체의 2개의 poly A site와
alternative splicing을 이용하여 IgM의 memb.과 secreted 형 생산
: 이들 mRNA를 번역하여 각각의 분비형과 세포막형의 heavy chain 단
백질 생산
‧ mRNA 모두 기존의 재배열된 VDJ 유전자의 구조가 변하지 않기 때문
에 생성된 항체의 항원 특이성은 변하지 않는다.
(a)
(b)
FIGURE 5-18
Expression of secreted and
membrane forms of the heavy chain
by alternative RNA processing.
(a) Amino acid sequence of the
carboxyl-terminal end of secreted
and membrane μ heavy chains.
Residues are indicated by the
single-letter amino acidcode.
Hydrophilic and hydrophobic
residues and regions are indicated
by purple and orange, respectively,
and charged amino acids are
indicated with a + or - . The white
regions of the sequences are
identical in both forms.
(b) Structure of the primary transcript
of a rearranged heavy-chain gene
showing the 𝐶μ exons and poly-A
sites. Polyadenylation of the
primary transcript at either site 1 or
site 2 and subsequent
splicing(indicated by V-shaped
lines) generates mRNAs encoding
either secreted or membrane μ
chains.
(a)
(b)
(c)
FIGURE 5-19
Expression of membrane forms
of μ and δ heavy chains by
alternative RNA processing.
(a) Structure of rearranged heavychain gene showing 𝐶μ and
𝐶δ exons and poly-A sites.
(b) Structure of μ 𝑚 transcript and
μ 𝑚 mRNA resulting from
polyadenylation at site 2 and
splicing.
(c) Structure of δ 𝑚 transcript and
δ 𝑚 mRNA resulting from
polyadenylation at site 4 and
splicing. Both processing
pathways can proceed in any
given B cell.
Synthesis, Assembly, and Secretion of Igs
• 전사된 항체의 heavy chain과 light chain의 mRNA는 모두 소포
체 (endoplasmic reticulum, ER)과 결합되어 있는
polyribosome에서 translation (synthesis)
: 5‘쪽에 leader sequence, signal peptide를 coding 하는 L 염
기배열이 있어서, translation 과 동시에 만들어지는 polypeptide
chains들은 ER의 막에 결합, ER 안쪽으로 이동
: ER안쪽으로 이동하면서 leader peptide는 잘려져 나가고, ER
이나 Golgi body에서 heavy chain과 light chain의 assembly가
일어나 완전한 Ig 이 만들어진다
: secretory vesicle을 통하여 세포막 쪽으로 이동하고, 세포막과
vesicle의 융합을 통하여 세포 밖으로 secretion
FIGURE 5-20
Synthesis, assembly, and secretion
of the immunoglobulin molecule.
The heavy and light chains are
synthesized on separate
polyribosomes(polysomes). The
assembly of the chains, the
formation of intrachain and
interchain disulfide linkages, and the
addition of carbohydrate all take
place in the rough endoplasmic
reticulum(RER). Vesicular transport
brings the Ig to the Golgi, which it
transits, departing in vesicles that
fuse with the cell membrane. The
main figure depicts the assembly of
a secreted antibody. The inset
depicts a membrane-bound
antibody, which contains the
carboxyl-terminal transmembrane
segment. This form becomes
anchored in the membrane of
secretory vesicles and is retained in
the cell membrane when the vesicles
fuse with the cell membrane.
FIGURE 5-21
Quality control during
antibody synthesis.
Ig molecules that fail to
fold or assemble properly
remain bound to the
chaperone protein BiP.
Interaction with BiP and
other factors causes the
malformed antibody to be
disassembled, exported
form the ER, marked for
degradation by
conjugation with ubiquitin,
and degraded by the
proteosome.
FIGURE 5-22 Location of promoters(dark red) and enhancers(green) in
mouse heavy-chain, κ light-chain, and λ light-chain germ-line DNA.
Variable-region DNA rearrangement moves an enhancer close enough to a
promoter that the enhancer can activate transcription from the promoter.
The promoters that precede the DH cluster, a number of the C genes, and
the 𝐽λ cluster are omitted from this diagram for clarity.
Antibody Genes and Antibody Engineering
• 항체 단백질을 조작하는 항체 공학 : 단클론 항체의 생산과 변형
• 사람 항체 제조의 필요성 (human antibody)
: 단클론항체는 생쥐의 항체이어서, 인간에게 임상적으로 투여
하게 되면, 이들 항체들이 사람에서 항원으로 인식되어 투여한
항체에 대하여 면역반응이 나타나게되며, 그 결과 혈액내 단클
론 항체를 빠르게 제거시키거나 allegic response를 일으켜 주
사 받은 사람을 괴롭히거나 위험하게 만들 수도 있다 . 이를 해
결하는 방법은 사람 항체 사용
• 항체공학 (antibody engineering)을 이용한 humanized
antibody 의 제조
: 생쥐 단클론항체의 V지역 유전자를 사람의 IgG 항체의 C 지역
에 결합 - 잡종항체 (chimeric antibody)
: chimeric antibody의 V 지역의 아미노산 배열을 CDR 지역만을
제외하고 모두 사람의 V지역 아미노산 배열로 바꾸어 주어 보다
사람의 항체에 가까운 항체 제조
FIGURE 5-23
Antibodies engineered by
recombinant DNA
technology.
(a) Chimeric mouse-human
monoclonal antibody containing
the 𝑉H and 𝑉L domains of a
mouse monoclonal antibody(blue)
and 𝐶L and 𝐶H domains of a
human monoclonal
antibody(gray).
(b) A humanized monoclonal
antibody containing only the
CDRs of a mouse monoclonal
antibody(blue bands) grafted
into the framework regions of a
human monoclonal antibody.
(c) A chimeric monoclonal
antibody in which the terminal Fc
domain is replaced by toxin
chains(white).
(d) A heteroconjugate in which
one half of the mouse antibody
molecule is specific for a tumor
antigen and the other half is
specific for the CD3/T-cell
receptor complex.
FIGURE 5-24
Human antibody from mice bearing a
human artificial chromosome(HAC) that
includes entire human heavy-and lightchain loci.
A human artificial chromosome bearing
the entire unrearranged human heavychain and λ light-chain loci was
introduced into mouse embryonic
stem(ES) cells in which the mouse
heavy-chain and κ and λ light-chain loci
had been knocked out. The modified ES
cells were introduced into blastocysts,
which were transferred to surrogate
mothers and allowed to generate
chimeric mice. Interbreeding of the
chimeric mice produced mice that made
human antibodies but not mouse Ig.
Immunization of these mice allowed the
generation of antigen-specific antiserum
that contained only human antibodies or
the generation of hybridomas that
secreted antigen-specific human
monoclonal antibodies.
(a)
(b)
FIGURE 5-25 Generation of
phage libraries containing
antibody binding sites.
(a) Derivation of single chain
fragment variable(scFv) library.
(b) The scFv library is cloned into
phages that are used to infect
e.coli. Growth of the phageinfected bacteria generates a
phage library that is screened on
antigen-coated plates for the
presence of phage that bind to
the desired antigen. Repeated
cycles of binding-elutionregrowth results in the
enrichment of the phage isolates
for antigen specificity. Clones of
antigen-specific phage can be
isolated and the techniques of
recombinant DNA technology
used to graft the genes for
𝑉H and 𝑉L domains from
selected phage onto the
constant-region scaffolding of an
antibody to engineer a
monoclonal antibody of the
desired antigen specificity.