Chapter Ⅴ
Immunotechnology
Section A
Antibodies as research and
diagnostic tools
抗体作为研究和诊断的工具
A variety of assays have been developed which
provide specific qualitative and quantitative
measurement of Ag or Ab,both of which are often of
considerable research and clinical relevance,Ab to an
organism in the serum of a patient demonstrates infe-
ction by the organism,Ab with defined specificity is
used to determine the presence of disease associated
antigens in a patient,As tools in molecular and cellular
research,Abs permit localization and characterization of
Ags.
Key notes
Methods for measuring antigen-antibody reactions
have been well established and include those that have
direct biologic relevance (Table 1),The combination of
Ab with biologically active Ag (virus,toxin,enzyme
and hormone) can be detected by neutralization of the
virus infection,toxicity,enzymatic and hormone
activity,respectively,Precipitation and agglutination
have also been adapted fo development of several
useful assays.
Antibody and assays
Table 1,Effects of combination of antigen and antibody
Agglutination Antigenic particle + specific Ab results in aggregation
of particles
Precipitation Soluble Ag + specific Ab results in lattice formation
and precipitation
C Activation Ag in solution or on particle + specific Ab results in
activation of C
Cytolysis Cell + anti-cell Ab + C may result in lysis of the cell
Opsonization Antigenic particle + Ab + C enhances phagocytosis by
Mo,M0,PMNs
Neutralization Toxins,viruses,enzymes,etc,+ specific Abs may
result in their inactivation
* C,Complement; Mo,monocytes; M0,macrophages; Ab,antibody;
PMNs,polymorpho-nuclear cells
A variety of other assays have been developed
which provide specific qualitative and
quantitative measurement of Ag or Ab for both
research and diagnostic purposes,
Since the immune system recognizes and remembers
virtually all Ag that are introduced into an individual,
assays which demonstrate the presence of Ab to an
organism in the serum of a patient have become a
standard way of determining that the patient has had
contact with,was infected by,the organism
Diagnostic
purpose
e.g the presence of Ab to HIV in the serum of a
patient usually means that the patient has been
infected with HIV,
Diagnose for AIDs
Alternatively,Abs with defined specificity (e.g,
(to Ags associated with cancer cells) can be used
to determine the presence of disease associated
Ags in a patient,
Abs are also extremely important tools in
molecular and cellular research as they
permit the localization and characterization
of Ags.
Research
Section B
precipitation and agglutination
沉淀和凝集
Combination of Ab with Ag results in lattice
formation and precipitation if there is sufficient Ag
and Ab (equivalence),These reactions are the basis
for qualitative and quantitative assays for Ag or Ab,
including radial immunodiffusion（辐射免疫扩散）
and immunoelectrophoresis（免疫电泳）,
Key notes
Precipitation assays
The interaction of surface Ags on insoluble
particles (e.g,cells) with specific Ab to these Ags
results in agglutination of the particles,
Agglutination can be used to determine blood
types; the presence of Ab to bacteria in serum is an
indication of previous or current infection; and in
the Coomb's test autoantibodies to erythrocytes can
be assayed.
Key notes
Agglutination assays
As previously described,when there is both
sufficient Ag and sufficient Ab,the combination of Ag
and Ab proceeds until large aggregates are formed
which are insoluble in water and precipitate
(equivalence),The extent to which a lattice forms
depends on the relative amounts of Ag and Ab present.
1,Precipitation assays
Lattice formation,and precipitation are the basis for
several qualitative and quantitative assays for Ag or Ab,
These assays are done in semisolid gels into which holes
are cut for Ag and/or for Ab and diffusion occurs until
Ag and Ab are at equivalence and precipitate,
In radial immunodiffusion,Ab (e.g,horse anti-
human IgG) is incorporated into the gel and Ag (e.g,
human serum) is placed in a hole cut in the gel,Ag
diffuses radially out of the well into the gel and
interacts with the Ab forming a ring of precipitation,
the diameter of which is related to the concentration of
the Ag (Fig,1).
Radial
immunodiffusion
Fig,1,Measurement ofAg by precipitation In gels,Ab-containing gel is
placed on a glass or plastic surface,Holes are cut in the gel and filled
with Ag which diffuses radially out of the well and interacts with the Ab
in the gel,Soluble complexes are initially formed but as more Ag
diffuses equivalence is reached resulting in a lattice and precipitation,
The diameter of the precipitation ring Is related to the concentration of
the Ag and,using known standards,can be quantitated and compared
with the levels of Ag in other samples.
Similar assays have been developed in which a
voltage gradient (electrophoresis) is used to speed up
movement of Ag into the Ab containing gel (rocket
immunoelectrophoresis火箭免疫电泳 ).
In immunoelectrophoresis,Ags (e.g,serum) are
placed in a well cut in a gel (without Ab) and
electrophoresed,after which a trough is cut in the gel
into which Abs (e.g,horse anti-human) are placed,
The Abs diffuse laterally（横向扩散） to meet
diffusing Ag,and lattice formation and precipitation
occur permitting determination of the nature of the
Ags (Fig,2).
immunoelectrophoresis
Fig.2 identification of antigens using gel
electrophoresis,Ag(Ee.g,serum)is placed
in a well cut in a gel and subjected to a
voltage gradient which causes the various
antigens to migrate different distances
through the gel dependent on their
charge,After electrophoresis,a trough is
cut in the gel into which antibodies (e.g,
horse anti-human serum) are placed,The
antibodies diffuse laterally from the until
they meet Ag diffusing from its location
after elecphoresis of define3d standards,
the identity of fthe Ag can be determined.
Agglutination involves the interaction of surface
Ags on insoluble particles (e.g,cells) and specific
Ab to these Ags (Fig,3),Ab thus links together
(agglutinates) insoluble particles,Much smaller
amounts of Ab suffice to produce agglutination
than are needed for precipitation,For this reason,
agglutination rather than precipitation may be used
to determine blood types or if Ab to bacteria is
present
2,Agglutination assays
For this reason,agglutination rather than
precipitation may be used to determine blood
types or if Ab to bacteria is present in blood as
an indication of infection with these bacteria,
Since IgM has 10 binding sites,whereas IgG
has two,IgM is much more efficient at
agglutinating particles or cells.
Although Abs are frequently used by themselves to
assay for the presence of an Ag,a second Ab is
sometimes used in what is known as a Coomb's test.
In some instances,such as when an autoantibody has
been produced against a given cell type,the cells will
have human Ab bonded to them,and thus can be
identified by
a second Ab (an Ab to human immunoglobulin)
which will cause agglutination of the cells,In an
indirect Coomb's test,the presence of circulating
Ab to a cell sur face Ag is demonstrated by adding
the patient's serum to test cells (e.g,erythro-cytes)
followed by addition of Ab to human Ab.
Agglutination,Antigen insoluble before adding antibody
Precipitation,Antigen is initially soluble; antibody
binding to it creates a lattice and makes it insoluble
Section C immunoassay
免疫测定
The presence and concentration of a specific Ag or of
an Ab to a specific Ag in solution can be determined by
radioimmunoassays (RIA,放射免疫测定法 ) or enzyme-
linked immunoabsorbent assays (ELISA，酶联免疫吸附测定法 ),Ag attached to a solid surface captures the
Ab with which it reacts and is quantitated using a
labeled second Ab reactive to the first,These assays
permit measurement of a wide variety of Ags as well as
the concentration of Abs specific for a given Ag,such as
those reactive with an infectious organism.
Key notes
ELISA,RIA
Using a fluorescence microscope and Abs labeled with a
fluorescent molecule,tissue sections can be examined for
cells expressing particular Ags (e.g,those which are tumor
associated),Direct or indirect immunofluorescence
techniques permit qualitative and quantitative evaluation
of several different cell associated molecules at the same
time,Flow cytometers rapidly analyze large numbers of
cells in suspension,providing a molecular fingerprint of
the cells,Fluorescence activated cell sorters separate cell
subpopulations for more detailed study.
KEY NOTES
Immunofluorescence and
flow cytometry
Immunoblotting is used to assay for the presence of
molecules in a mixture,Western blot analysis involves
separating molecules by sodium dedecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE),
transferring them to another matrix and detecting the
molecule of interest using ELISA or RIA,This assay is
often used to confirm the presence of Abs to infectious
agents (e.g,HIV) in patient serum,Immunoblotting can
also be used to analyze products of single cells (e.g,
cytokines) and the nature of the producing cell.
Key notes
immunoblotting
The presence of Ab to a particular Ag in the serum of
a patient can be determined using very sensitive
radioimmunoassays (RIA) or enzyme-linked irnmuno-
absorbent assays (ELISA),Such assays (Fig,) are of
particular value in demonstrating Ab to Ags of
infectious agents,e.g,virus,bacteria,etc,
1,ELISA,RIA
The radioallergosorbent test (RAST，放射变应原吸附测定法 ) uses a radiolabeled Ab to human IgE as
detecting ligand and permits the measurement of specific
IgE Ab to an allergen,ELISA and RIA also provide very
specific and sensitive measurement of toxins,drugs,
hormones,pesticides,etc.,not only in serum,but also in
water,foods and other consumer products,Based on
these procedures,assays for nearly any Ag or Ab can be
readily developed.
Radioimmunoassay
（ RIA）
(a) Radioimmunoassay (RIA),
Antigen is incubated on
plastic and small quantities
are adsorbed,Free antigen is
washed away,Test antibody is
added,which may bind to the
Ag,and unbound Ab washed
away,Ab remaining bound to
the Ag is detected by a
radiolabeled ligand (e.g,an
Ab specific for the isotype of
the test Ab,or staphylococ-
cal protein A which binds to
the Fc region of IgG).
Radioallergosorbent test（ RAST）
This measures Ag-specific IgE
in an RIA where the ligand is
a labeled anti-lgE Ab and is
very similar to the standard
RIA.
Enzyme-linked immunoabsorbant assay
This is similar to RIA except
that the ligand (e.g,the Ab that
binds the test Ag) is covalently
coupled to an enzyme such as
peroxidase,This ligand binds
the test Ab and after free ligand
is washed away the bound
ligand is detected by the
addition of substrate which is
acted on by the enzyme to yield
a colored and detectable end
product.
Sandwich ELISA
This is basically the same as
described in RIA and ELISA
except that Ab to the Ag is
first used to coat the plastic
in order to specifically
capture the Ag from a
mixture,A second enzyme or
radioisotopically labeled Ab,
which reacts with an epitope
on the Ag which is different
from that of the first Ab,is
then added for quantitation
of the antigen.
Although it is possible to use ELISA and RIA to
evaluate the presence of an Ag on a cell,this is
usually more conveniently done using Abs to
which a fluorescent marker has been covalently
attached,Moreover,in most cases a mAb is used
and thus is highly specific for a particular molecule
and a particular epitope on that molecple,
Immunofluorescence 免疫荧光
Comparison with ELISA and RIA
This type of assay can be done using an Ab to the
Ag which is directly fluorescent labeled (direct
immunofluorescence) or by first incubating the
unlabeled Ab with the cells (e.g,a mouse mAb to
human T cells) and then,after washing away
unbound Ab,adding a second fluorescent labeled
Ab that reacts with the first Ab (e.g,a goat Ab to
mouse immunoglobulin),
Immunofluorescence 免疫荧光
direct and indirect immunofluorescence
Animal tissues (rat) are frequently used to identify human autoantibodies since
the autoantigens recognized are generally conserved across the species,Patient
serum is added to the tissue sections and the autoantibodies bind to particular
autoantigen(s),After washing,fluorescent antibodies to human IgG are added
and viewed under a fluorescence microscope,A green color shows where the
human antibodies have bound to the tissue autoantigens.
Indirect immunofluorescence assay
This indirect immunofluorescent assay has
two advantages,it has higher sensitivity
andrequires labeling of only one Ab,the
second Ab,because,in the example given,it
can detect (react with) any mouse Ab.
Immunofluorescence 免疫荧光
Advantages of indirect immunofluorescene
Fluorescent Abs to cell surface molecules (e.g,
those which are tumor associated) are very useful
in examining tissue sections for cells expressing
the Ag,
Applications(1)
This assay is done by incubating the tissue section
with the labeled Ab (for direct immunofluorescence
(IF)) or unlabeled Ab,followed by labeled second
Ab and then examining the tissue section using a
fluorescent microscope,
These microscopes irradiate the tissue with a
wavelength of light that excites the fluorescent label on
the Ab to emit light at a different wavelength,This
emitted light can be directly visualized,photographed
and even quantitated.
Moreover,it is possible to analyze a tissue sample
using several different Abs at the same time,as each
Ab could be labeled with a different fluorescent
molecule each of which emit light at a wavelength
distinct from the others,
Application(2)
It is also possible to look for intracellular molecules
(e.g,Abs) by first permeabilizing the cells and then
doing the staining and fluorescence microscopy(先透化处理细胞，然后进行染色和荧光镜检以寻找胞内分子 ).
Thus,one can use this approach to develop a molecular
fingerprint of the cells associated with a tissue.
Application(3)
Although fluorescence microscopy can be,and
is,applied to the analysis of single cell
suspensions,another rather technologically
sophisticated approach,flow cytometry,is most
often used,
flow cytometry（流式细胞术）
This assay uses the same basic staining
procedures as described for fluorescence
microscopy,followed by automated quantitation
of the amount of fluorescence associated with
individual cells,（这种方法采用如用荧光显微镜所用的同一基本染色步骤，随之进行与单个细胞相关的荧光量的自动定量。）
After labeling with fluorescent antibody,cells are passed one at a
time through a laser beam,Photodetectors measure the amount of
fluorescence which is plotted as a histogram showing the
proportion of non-fluorescent (unstained) and fluorescent (stained)
cells,Other detectors simultaneously measure scattered laser light,
which is used to generate a 'dot blot' in which lymphocytes,
monocytes and granulocytes can be discriminated.
Flow cytometry
In particular,the suspension of stained cells is fed to
the flow cytometer which disperses the cells so they
then pass single file through a focused laser beam （细胞排成单一纵列通过聚焦的激光束） which excites
any fluorescent label associated with the cells,Those
stained by the fluorescent Ab emit light that is detected
and quantitated by optical sensors and the intensity of
fluorescence is plotted in histograph form（直方图形式） by a computer,
This machine can analyze 1000 cells per second and
provide quantitative data on the number of molecules
of a particular kind on each cell,It can also analyze
mixtures of cells and provide data on their size and
granularity in addition to their expression of specific
molecules,
Some versions of this machine (fluorescence activated
cell sorter) are also able to separate out cells into
microdroplets and sort those expressing a selected
amount of a particular Ag into a separate tube for
further analysis or culture.
It is possible to combine various separation
and detection procedures for identification and
analysis of Ags and for evaluating the expression
of molecules by single cells,
Immunoblotting（免疫印迹）
Western blot analysis involves separating
Ags by polyacrylamide gel electrophoresis
(PAGE) in the presence of sodium dodecyl
sulfate (SDS) which results in separation of
molecules on the basis of size,
Immunoblotting（免疫印迹）
Procedure（ 1）
These molecules are then transferred to another
matrix (e.g,nitrocellulose) to form a pattern on
the matrix identical to that on the gel,
Immunoblotting（免疫印迹）
Procedure
（ 2）
Enzyme linked Ab to the molecule of interest
is then added,the unbound Ab washed off and
substrate added (see ELISA) for visualization,
（ staining and visualization）
Immunoblotting（免疫印迹）
Procedure
（ 3）
1,This assay permits specific identification of proteins in
a mixture.
2,It is also often used to confirm the presence of Abs to
certain infectious agents (e.g,HIV) in the serum of
patients.
Immunoblotting（免疫印迹）
applications
3,Immunobloting can also be used to assay for the
presence of molecules in a mixture as described for the
sandwich ELISA,
4,This has now.been extended for analysis of products of
single cells,For example,to assay for production of a
cytokine.
Immunoblotting（免疫印迹）
applications
Section D
Affinity chromatography
亲 和 层 析
Specific purification of Ag and Ab
Ab coupled to an insoluble matrix (e.g,agarose)
specifically binds its Ag,which can then be eluted
from the Ab yielding relatively pure Ag in one step,
Similarly,Ag or protein A coupled to an insoluble
matrix permits purification of Ab.
Key notes
Specific purification of Ag and Ab
The specificity of Abs is not only important to the
development of many research and diagnostic assays,
but can,in some instances,be used to purify,or be
purified by,interaction with Ag,
This is because Abs do not form covalent bonds when
they combine with Ag,Ab coupled to an insoluble matrix
(e.g,agarose) specifically binds its Ag,removing it from
a mixture of other molecules,After washing to remove
all unbound molecules,the Ag can be eluted at low pH
and/or at high ionic strength,which break the reversible
bonds holding it to the Ab,
Specific purification of Ag and Ab
Why?
The mechanisms
As this can usually be performed without
damaging the Ag or Ab,it is possible to obtain
relatively pure Ag in one step,Similarly,Ag
coupled to an insoluble matrix permits
purification of Ab from media or serum,
Specific purification of Ag and Ab
Why?
The mechanisms
Ab can also be purified based on its binding by
proteins (e.g,protein A) isolated from some strains of
Staphylococcu saureus,Protein A coupled to agarose
binds IgG Abs which can be eluted by decreasing the pH
and/or by increasing the ionic strength of the eluting
buffer,again without damaging the Ab.
Specific purification of Ag and Ab
The mechanisms
Section E
monoclonal and recombinant antibodies
单 克 隆 抗 体 和 重 组 抗 体
Standardized procedures involving fusion of an
immortal cell (a myeloma tumor cell) with a specific
predetermined Ab-producing B cell have been used to
create hybridoma cells producing monospecific and
monoclonal antibodies (mAbs),These mAbs are
standard research reagents with exten sive diagnostic
and clinical applications.
Key Notes
Monoclonal antibodies
(mAbs)
Most mAbs developed have been mouse,and
although useful as research and diagnostic tools,
they are not ideal therapeutics because of their
immunogenicity in humans,This has been dealt
with by humanizing these murine Abs or by
making fully human mAbs.
Key Notes
mAbs)Humanization and
chimenzation of mAbs
By randomly fusing heavy (H) and light (L)
chain variable (V) region genes from B cells,Fv
libraries containing all binding specificities can be
generated and used as a source for creation of
specific mAbs.
Key Notes
Fv libraries
In 1975,Kohler and Milstein developed a
procedure to create cell lines producing a
predetermined,monospecific and monoclonal Ab,
for which they received the Nobel Prize,This
procedure has been standardized and applied on a
massive scale to the preparation of Abs useful to
many research and clinical efforts,
1,Monoclonal antibodies
Who discovered?
The basic technology involves creation of a hybrid
cell by fusion of an immortal cell (a myeloma tumor
cell) with a specific predetermined Ab-producing B
cell from immunized animals or people,The
resulting hybridoma cell is immortal and synthesizes
homogeneous,specific Ab,The utility of this Ab
depends on its specificity,
1,Monoclonal antibodies
How it happened?
Preparation
of
monoclonal
Abs.
Monoclonal Abs (mAbs) can be made in large
quantities and against virtually every Ag,Thus,
mAbs have become standard research reagents and
have extensive diagnostic and clinical applications.
1,Monoclonal antibodies
Why is so useful?
The vast majority of mAbs have been developed in mice,and
although useful as research and diagnostic tools,they have not
been ideal therapeutic reagents at least partly because of their
immunogenicity in humans,That is,a murine Ab introduced into
a patient will be recognized as foreign by the patient's immune
system and a human anti-mouse Ab (HAMA) response will
develop that compro mises the utility of therapeutic Ab,This has
been dealt with in two basic ways.
2,Humanization and chimerization of mAbs
mAbs 的 人 源 化 和 嵌 合 体
Why needed？
Ways dealt with it：
Humanize murine antibodies
人源化鼠的抗体
Make fully human mAbs
直接制备人的 mAbs
The murine Ab can be genetically modified to be
more human,In particular the constant region of
the murine IgG heavy (H) and of the murine light
(L) chain can be replaced at the DNA level with the
constant regions of human IgG1 H and L chains to
create a chimeric Ab where only the variable (V)
regions are murine,This significantly decreases but
does not eliminate the immunogenicity of the Ab,
2.1 Humanize murine antibodies
Apporoach（ 1）
Fig in last slide,Humanizing and chimerizing mouse
monoclonal antibodies,Chimeric mAbs are created by
replacing the murine genes for the constant region of the
light (L) and heavy (H) chain with the corresponding
human constant region genes,Humanized mAbs are
created by inserting the gene sequences for each of the
hypervariable (Hv) regions of the mouse antibody into
the corresponding place in the genes for the L and H
chains for a human antibody.
Another approach involves sequencing the V
regions of the mouse Ab H and L chains and then
inserting the DNA sequences of the hypervariable
regions of these chains into human IgG H and L
chain genes,The resulting Ab is 95% human with
only the binding regions being murine.
2.1 Humanize murine antibodies
Apporoach（ 2）
Human Abs have been made by fusing human B
cells with myeloma cells,although this has been
very difficult and usually requires immortalizing
the B cells using Epstein-Barr virus before fusing,
2,Make fully liuman mAbs
How？
This approach is not ideal as a virus is used,
the specificity of the mAbs produced is limited
and the yield of the Abs produced is poor,
2,Make fully liuman mAbs
shortcoming
s
More recently,a human antibody mouse has been
created by replacing the genes for mouse
immunoglobulins with genes for human
immunoglobulins,Thus,when the mouse is
immunized it makes fully human Abs against the Ag
and the B cells making these Abs can be fused with
myeloma cells to generate hybridomas making the
human mAb.
2,Make fully liuman mAbs
New way
Another way of preparing monoclonal Abs involves Fv
libraries,In this approach,the H chain V region genes of
a large population of B cells are fused randomly to an
equally large number of L chain V region genes to create
all combinations and thus a vast number of combining
sites (Fv regions),These are cloned into bacteriophage
(viruses that infect bacteria) and selected for their
specificity Thus,Fvs can be expressed in a replicating
bioform and used as a source from which specific mAbs
can be created.
3,Fv libraries
Immunotechnology
Section A
Antibodies as research and
diagnostic tools
抗体作为研究和诊断的工具
A variety of assays have been developed which
provide specific qualitative and quantitative
measurement of Ag or Ab,both of which are often of
considerable research and clinical relevance,Ab to an
organism in the serum of a patient demonstrates infe-
ction by the organism,Ab with defined specificity is
used to determine the presence of disease associated
antigens in a patient,As tools in molecular and cellular
research,Abs permit localization and characterization of
Ags.
Key notes
Methods for measuring antigen-antibody reactions
have been well established and include those that have
direct biologic relevance (Table 1),The combination of
Ab with biologically active Ag (virus,toxin,enzyme
and hormone) can be detected by neutralization of the
virus infection,toxicity,enzymatic and hormone
activity,respectively,Precipitation and agglutination
have also been adapted fo development of several
useful assays.
Antibody and assays
Table 1,Effects of combination of antigen and antibody
Agglutination Antigenic particle + specific Ab results in aggregation
of particles
Precipitation Soluble Ag + specific Ab results in lattice formation
and precipitation
C Activation Ag in solution or on particle + specific Ab results in
activation of C
Cytolysis Cell + anti-cell Ab + C may result in lysis of the cell
Opsonization Antigenic particle + Ab + C enhances phagocytosis by
Mo,M0,PMNs
Neutralization Toxins,viruses,enzymes,etc,+ specific Abs may
result in their inactivation
* C,Complement; Mo,monocytes; M0,macrophages; Ab,antibody;
PMNs,polymorpho-nuclear cells
A variety of other assays have been developed
which provide specific qualitative and
quantitative measurement of Ag or Ab for both
research and diagnostic purposes,
Since the immune system recognizes and remembers
virtually all Ag that are introduced into an individual,
assays which demonstrate the presence of Ab to an
organism in the serum of a patient have become a
standard way of determining that the patient has had
contact with,was infected by,the organism
Diagnostic
purpose
e.g the presence of Ab to HIV in the serum of a
patient usually means that the patient has been
infected with HIV,
Diagnose for AIDs
Alternatively,Abs with defined specificity (e.g,
(to Ags associated with cancer cells) can be used
to determine the presence of disease associated
Ags in a patient,
Abs are also extremely important tools in
molecular and cellular research as they
permit the localization and characterization
of Ags.
Research
Section B
precipitation and agglutination
沉淀和凝集
Combination of Ab with Ag results in lattice
formation and precipitation if there is sufficient Ag
and Ab (equivalence),These reactions are the basis
for qualitative and quantitative assays for Ag or Ab,
including radial immunodiffusion（辐射免疫扩散）
and immunoelectrophoresis（免疫电泳）,
Key notes
Precipitation assays
The interaction of surface Ags on insoluble
particles (e.g,cells) with specific Ab to these Ags
results in agglutination of the particles,
Agglutination can be used to determine blood
types; the presence of Ab to bacteria in serum is an
indication of previous or current infection; and in
the Coomb's test autoantibodies to erythrocytes can
be assayed.
Key notes
Agglutination assays
As previously described,when there is both
sufficient Ag and sufficient Ab,the combination of Ag
and Ab proceeds until large aggregates are formed
which are insoluble in water and precipitate
(equivalence),The extent to which a lattice forms
depends on the relative amounts of Ag and Ab present.
1,Precipitation assays
Lattice formation,and precipitation are the basis for
several qualitative and quantitative assays for Ag or Ab,
These assays are done in semisolid gels into which holes
are cut for Ag and/or for Ab and diffusion occurs until
Ag and Ab are at equivalence and precipitate,
In radial immunodiffusion,Ab (e.g,horse anti-
human IgG) is incorporated into the gel and Ag (e.g,
human serum) is placed in a hole cut in the gel,Ag
diffuses radially out of the well into the gel and
interacts with the Ab forming a ring of precipitation,
the diameter of which is related to the concentration of
the Ag (Fig,1).
Radial
immunodiffusion
Fig,1,Measurement ofAg by precipitation In gels,Ab-containing gel is
placed on a glass or plastic surface,Holes are cut in the gel and filled
with Ag which diffuses radially out of the well and interacts with the Ab
in the gel,Soluble complexes are initially formed but as more Ag
diffuses equivalence is reached resulting in a lattice and precipitation,
The diameter of the precipitation ring Is related to the concentration of
the Ag and,using known standards,can be quantitated and compared
with the levels of Ag in other samples.
Similar assays have been developed in which a
voltage gradient (electrophoresis) is used to speed up
movement of Ag into the Ab containing gel (rocket
immunoelectrophoresis火箭免疫电泳 ).
In immunoelectrophoresis,Ags (e.g,serum) are
placed in a well cut in a gel (without Ab) and
electrophoresed,after which a trough is cut in the gel
into which Abs (e.g,horse anti-human) are placed,
The Abs diffuse laterally（横向扩散） to meet
diffusing Ag,and lattice formation and precipitation
occur permitting determination of the nature of the
Ags (Fig,2).
immunoelectrophoresis
Fig.2 identification of antigens using gel
electrophoresis,Ag(Ee.g,serum)is placed
in a well cut in a gel and subjected to a
voltage gradient which causes the various
antigens to migrate different distances
through the gel dependent on their
charge,After electrophoresis,a trough is
cut in the gel into which antibodies (e.g,
horse anti-human serum) are placed,The
antibodies diffuse laterally from the until
they meet Ag diffusing from its location
after elecphoresis of define3d standards,
the identity of fthe Ag can be determined.
Agglutination involves the interaction of surface
Ags on insoluble particles (e.g,cells) and specific
Ab to these Ags (Fig,3),Ab thus links together
(agglutinates) insoluble particles,Much smaller
amounts of Ab suffice to produce agglutination
than are needed for precipitation,For this reason,
agglutination rather than precipitation may be used
to determine blood types or if Ab to bacteria is
present
2,Agglutination assays
For this reason,agglutination rather than
precipitation may be used to determine blood
types or if Ab to bacteria is present in blood as
an indication of infection with these bacteria,
Since IgM has 10 binding sites,whereas IgG
has two,IgM is much more efficient at
agglutinating particles or cells.
Although Abs are frequently used by themselves to
assay for the presence of an Ag,a second Ab is
sometimes used in what is known as a Coomb's test.
In some instances,such as when an autoantibody has
been produced against a given cell type,the cells will
have human Ab bonded to them,and thus can be
identified by
a second Ab (an Ab to human immunoglobulin)
which will cause agglutination of the cells,In an
indirect Coomb's test,the presence of circulating
Ab to a cell sur face Ag is demonstrated by adding
the patient's serum to test cells (e.g,erythro-cytes)
followed by addition of Ab to human Ab.
Agglutination,Antigen insoluble before adding antibody
Precipitation,Antigen is initially soluble; antibody
binding to it creates a lattice and makes it insoluble
Section C immunoassay
免疫测定
The presence and concentration of a specific Ag or of
an Ab to a specific Ag in solution can be determined by
radioimmunoassays (RIA,放射免疫测定法 ) or enzyme-
linked immunoabsorbent assays (ELISA，酶联免疫吸附测定法 ),Ag attached to a solid surface captures the
Ab with which it reacts and is quantitated using a
labeled second Ab reactive to the first,These assays
permit measurement of a wide variety of Ags as well as
the concentration of Abs specific for a given Ag,such as
those reactive with an infectious organism.
Key notes
ELISA,RIA
Using a fluorescence microscope and Abs labeled with a
fluorescent molecule,tissue sections can be examined for
cells expressing particular Ags (e.g,those which are tumor
associated),Direct or indirect immunofluorescence
techniques permit qualitative and quantitative evaluation
of several different cell associated molecules at the same
time,Flow cytometers rapidly analyze large numbers of
cells in suspension,providing a molecular fingerprint of
the cells,Fluorescence activated cell sorters separate cell
subpopulations for more detailed study.
KEY NOTES
Immunofluorescence and
flow cytometry
Immunoblotting is used to assay for the presence of
molecules in a mixture,Western blot analysis involves
separating molecules by sodium dedecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE),
transferring them to another matrix and detecting the
molecule of interest using ELISA or RIA,This assay is
often used to confirm the presence of Abs to infectious
agents (e.g,HIV) in patient serum,Immunoblotting can
also be used to analyze products of single cells (e.g,
cytokines) and the nature of the producing cell.
Key notes
immunoblotting
The presence of Ab to a particular Ag in the serum of
a patient can be determined using very sensitive
radioimmunoassays (RIA) or enzyme-linked irnmuno-
absorbent assays (ELISA),Such assays (Fig,) are of
particular value in demonstrating Ab to Ags of
infectious agents,e.g,virus,bacteria,etc,
1,ELISA,RIA
The radioallergosorbent test (RAST，放射变应原吸附测定法 ) uses a radiolabeled Ab to human IgE as
detecting ligand and permits the measurement of specific
IgE Ab to an allergen,ELISA and RIA also provide very
specific and sensitive measurement of toxins,drugs,
hormones,pesticides,etc.,not only in serum,but also in
water,foods and other consumer products,Based on
these procedures,assays for nearly any Ag or Ab can be
readily developed.
Radioimmunoassay
（ RIA）
(a) Radioimmunoassay (RIA),
Antigen is incubated on
plastic and small quantities
are adsorbed,Free antigen is
washed away,Test antibody is
added,which may bind to the
Ag,and unbound Ab washed
away,Ab remaining bound to
the Ag is detected by a
radiolabeled ligand (e.g,an
Ab specific for the isotype of
the test Ab,or staphylococ-
cal protein A which binds to
the Fc region of IgG).
Radioallergosorbent test（ RAST）
This measures Ag-specific IgE
in an RIA where the ligand is
a labeled anti-lgE Ab and is
very similar to the standard
RIA.
Enzyme-linked immunoabsorbant assay
This is similar to RIA except
that the ligand (e.g,the Ab that
binds the test Ag) is covalently
coupled to an enzyme such as
peroxidase,This ligand binds
the test Ab and after free ligand
is washed away the bound
ligand is detected by the
addition of substrate which is
acted on by the enzyme to yield
a colored and detectable end
product.
Sandwich ELISA
This is basically the same as
described in RIA and ELISA
except that Ab to the Ag is
first used to coat the plastic
in order to specifically
capture the Ag from a
mixture,A second enzyme or
radioisotopically labeled Ab,
which reacts with an epitope
on the Ag which is different
from that of the first Ab,is
then added for quantitation
of the antigen.
Although it is possible to use ELISA and RIA to
evaluate the presence of an Ag on a cell,this is
usually more conveniently done using Abs to
which a fluorescent marker has been covalently
attached,Moreover,in most cases a mAb is used
and thus is highly specific for a particular molecule
and a particular epitope on that molecple,
Immunofluorescence 免疫荧光
Comparison with ELISA and RIA
This type of assay can be done using an Ab to the
Ag which is directly fluorescent labeled (direct
immunofluorescence) or by first incubating the
unlabeled Ab with the cells (e.g,a mouse mAb to
human T cells) and then,after washing away
unbound Ab,adding a second fluorescent labeled
Ab that reacts with the first Ab (e.g,a goat Ab to
mouse immunoglobulin),
Immunofluorescence 免疫荧光
direct and indirect immunofluorescence
Animal tissues (rat) are frequently used to identify human autoantibodies since
the autoantigens recognized are generally conserved across the species,Patient
serum is added to the tissue sections and the autoantibodies bind to particular
autoantigen(s),After washing,fluorescent antibodies to human IgG are added
and viewed under a fluorescence microscope,A green color shows where the
human antibodies have bound to the tissue autoantigens.
Indirect immunofluorescence assay
This indirect immunofluorescent assay has
two advantages,it has higher sensitivity
andrequires labeling of only one Ab,the
second Ab,because,in the example given,it
can detect (react with) any mouse Ab.
Immunofluorescence 免疫荧光
Advantages of indirect immunofluorescene
Fluorescent Abs to cell surface molecules (e.g,
those which are tumor associated) are very useful
in examining tissue sections for cells expressing
the Ag,
Applications(1)
This assay is done by incubating the tissue section
with the labeled Ab (for direct immunofluorescence
(IF)) or unlabeled Ab,followed by labeled second
Ab and then examining the tissue section using a
fluorescent microscope,
These microscopes irradiate the tissue with a
wavelength of light that excites the fluorescent label on
the Ab to emit light at a different wavelength,This
emitted light can be directly visualized,photographed
and even quantitated.
Moreover,it is possible to analyze a tissue sample
using several different Abs at the same time,as each
Ab could be labeled with a different fluorescent
molecule each of which emit light at a wavelength
distinct from the others,
Application(2)
It is also possible to look for intracellular molecules
(e.g,Abs) by first permeabilizing the cells and then
doing the staining and fluorescence microscopy(先透化处理细胞，然后进行染色和荧光镜检以寻找胞内分子 ).
Thus,one can use this approach to develop a molecular
fingerprint of the cells associated with a tissue.
Application(3)
Although fluorescence microscopy can be,and
is,applied to the analysis of single cell
suspensions,another rather technologically
sophisticated approach,flow cytometry,is most
often used,
flow cytometry（流式细胞术）
This assay uses the same basic staining
procedures as described for fluorescence
microscopy,followed by automated quantitation
of the amount of fluorescence associated with
individual cells,（这种方法采用如用荧光显微镜所用的同一基本染色步骤，随之进行与单个细胞相关的荧光量的自动定量。）
After labeling with fluorescent antibody,cells are passed one at a
time through a laser beam,Photodetectors measure the amount of
fluorescence which is plotted as a histogram showing the
proportion of non-fluorescent (unstained) and fluorescent (stained)
cells,Other detectors simultaneously measure scattered laser light,
which is used to generate a 'dot blot' in which lymphocytes,
monocytes and granulocytes can be discriminated.
Flow cytometry
In particular,the suspension of stained cells is fed to
the flow cytometer which disperses the cells so they
then pass single file through a focused laser beam （细胞排成单一纵列通过聚焦的激光束） which excites
any fluorescent label associated with the cells,Those
stained by the fluorescent Ab emit light that is detected
and quantitated by optical sensors and the intensity of
fluorescence is plotted in histograph form（直方图形式） by a computer,
This machine can analyze 1000 cells per second and
provide quantitative data on the number of molecules
of a particular kind on each cell,It can also analyze
mixtures of cells and provide data on their size and
granularity in addition to their expression of specific
molecules,
Some versions of this machine (fluorescence activated
cell sorter) are also able to separate out cells into
microdroplets and sort those expressing a selected
amount of a particular Ag into a separate tube for
further analysis or culture.
It is possible to combine various separation
and detection procedures for identification and
analysis of Ags and for evaluating the expression
of molecules by single cells,
Immunoblotting（免疫印迹）
Western blot analysis involves separating
Ags by polyacrylamide gel electrophoresis
(PAGE) in the presence of sodium dodecyl
sulfate (SDS) which results in separation of
molecules on the basis of size,
Immunoblotting（免疫印迹）
Procedure（ 1）
These molecules are then transferred to another
matrix (e.g,nitrocellulose) to form a pattern on
the matrix identical to that on the gel,
Immunoblotting（免疫印迹）
Procedure
（ 2）
Enzyme linked Ab to the molecule of interest
is then added,the unbound Ab washed off and
substrate added (see ELISA) for visualization,
（ staining and visualization）
Immunoblotting（免疫印迹）
Procedure
（ 3）
1,This assay permits specific identification of proteins in
a mixture.
2,It is also often used to confirm the presence of Abs to
certain infectious agents (e.g,HIV) in the serum of
patients.
Immunoblotting（免疫印迹）
applications
3,Immunobloting can also be used to assay for the
presence of molecules in a mixture as described for the
sandwich ELISA,
4,This has now.been extended for analysis of products of
single cells,For example,to assay for production of a
cytokine.
Immunoblotting（免疫印迹）
applications
Section D
Affinity chromatography
亲 和 层 析
Specific purification of Ag and Ab
Ab coupled to an insoluble matrix (e.g,agarose)
specifically binds its Ag,which can then be eluted
from the Ab yielding relatively pure Ag in one step,
Similarly,Ag or protein A coupled to an insoluble
matrix permits purification of Ab.
Key notes
Specific purification of Ag and Ab
The specificity of Abs is not only important to the
development of many research and diagnostic assays,
but can,in some instances,be used to purify,or be
purified by,interaction with Ag,
This is because Abs do not form covalent bonds when
they combine with Ag,Ab coupled to an insoluble matrix
(e.g,agarose) specifically binds its Ag,removing it from
a mixture of other molecules,After washing to remove
all unbound molecules,the Ag can be eluted at low pH
and/or at high ionic strength,which break the reversible
bonds holding it to the Ab,
Specific purification of Ag and Ab
Why?
The mechanisms
As this can usually be performed without
damaging the Ag or Ab,it is possible to obtain
relatively pure Ag in one step,Similarly,Ag
coupled to an insoluble matrix permits
purification of Ab from media or serum,
Specific purification of Ag and Ab
Why?
The mechanisms
Ab can also be purified based on its binding by
proteins (e.g,protein A) isolated from some strains of
Staphylococcu saureus,Protein A coupled to agarose
binds IgG Abs which can be eluted by decreasing the pH
and/or by increasing the ionic strength of the eluting
buffer,again without damaging the Ab.
Specific purification of Ag and Ab
The mechanisms
Section E
monoclonal and recombinant antibodies
单 克 隆 抗 体 和 重 组 抗 体
Standardized procedures involving fusion of an
immortal cell (a myeloma tumor cell) with a specific
predetermined Ab-producing B cell have been used to
create hybridoma cells producing monospecific and
monoclonal antibodies (mAbs),These mAbs are
standard research reagents with exten sive diagnostic
and clinical applications.
Key Notes
Monoclonal antibodies
(mAbs)
Most mAbs developed have been mouse,and
although useful as research and diagnostic tools,
they are not ideal therapeutics because of their
immunogenicity in humans,This has been dealt
with by humanizing these murine Abs or by
making fully human mAbs.
Key Notes
mAbs)Humanization and
chimenzation of mAbs
By randomly fusing heavy (H) and light (L)
chain variable (V) region genes from B cells,Fv
libraries containing all binding specificities can be
generated and used as a source for creation of
specific mAbs.
Key Notes
Fv libraries
In 1975,Kohler and Milstein developed a
procedure to create cell lines producing a
predetermined,monospecific and monoclonal Ab,
for which they received the Nobel Prize,This
procedure has been standardized and applied on a
massive scale to the preparation of Abs useful to
many research and clinical efforts,
1,Monoclonal antibodies
Who discovered?
The basic technology involves creation of a hybrid
cell by fusion of an immortal cell (a myeloma tumor
cell) with a specific predetermined Ab-producing B
cell from immunized animals or people,The
resulting hybridoma cell is immortal and synthesizes
homogeneous,specific Ab,The utility of this Ab
depends on its specificity,
1,Monoclonal antibodies
How it happened?
Preparation
of
monoclonal
Abs.
Monoclonal Abs (mAbs) can be made in large
quantities and against virtually every Ag,Thus,
mAbs have become standard research reagents and
have extensive diagnostic and clinical applications.
1,Monoclonal antibodies
Why is so useful?
The vast majority of mAbs have been developed in mice,and
although useful as research and diagnostic tools,they have not
been ideal therapeutic reagents at least partly because of their
immunogenicity in humans,That is,a murine Ab introduced into
a patient will be recognized as foreign by the patient's immune
system and a human anti-mouse Ab (HAMA) response will
develop that compro mises the utility of therapeutic Ab,This has
been dealt with in two basic ways.
2,Humanization and chimerization of mAbs
mAbs 的 人 源 化 和 嵌 合 体
Why needed？
Ways dealt with it：
Humanize murine antibodies
人源化鼠的抗体
Make fully human mAbs
直接制备人的 mAbs
The murine Ab can be genetically modified to be
more human,In particular the constant region of
the murine IgG heavy (H) and of the murine light
(L) chain can be replaced at the DNA level with the
constant regions of human IgG1 H and L chains to
create a chimeric Ab where only the variable (V)
regions are murine,This significantly decreases but
does not eliminate the immunogenicity of the Ab,
2.1 Humanize murine antibodies
Apporoach（ 1）
Fig in last slide,Humanizing and chimerizing mouse
monoclonal antibodies,Chimeric mAbs are created by
replacing the murine genes for the constant region of the
light (L) and heavy (H) chain with the corresponding
human constant region genes,Humanized mAbs are
created by inserting the gene sequences for each of the
hypervariable (Hv) regions of the mouse antibody into
the corresponding place in the genes for the L and H
chains for a human antibody.
Another approach involves sequencing the V
regions of the mouse Ab H and L chains and then
inserting the DNA sequences of the hypervariable
regions of these chains into human IgG H and L
chain genes,The resulting Ab is 95% human with
only the binding regions being murine.
2.1 Humanize murine antibodies
Apporoach（ 2）
Human Abs have been made by fusing human B
cells with myeloma cells,although this has been
very difficult and usually requires immortalizing
the B cells using Epstein-Barr virus before fusing,
2,Make fully liuman mAbs
How？
This approach is not ideal as a virus is used,
the specificity of the mAbs produced is limited
and the yield of the Abs produced is poor,
2,Make fully liuman mAbs
shortcoming
s
More recently,a human antibody mouse has been
created by replacing the genes for mouse
immunoglobulins with genes for human
immunoglobulins,Thus,when the mouse is
immunized it makes fully human Abs against the Ag
and the B cells making these Abs can be fused with
myeloma cells to generate hybridomas making the
human mAb.
2,Make fully liuman mAbs
New way
Another way of preparing monoclonal Abs involves Fv
libraries,In this approach,the H chain V region genes of
a large population of B cells are fused randomly to an
equally large number of L chain V region genes to create
all combinations and thus a vast number of combining
sites (Fv regions),These are cloned into bacteriophage
(viruses that infect bacteria) and selected for their
specificity Thus,Fvs can be expressed in a replicating
bioform and used as a source from which specific mAbs
can be created.
3,Fv libraries
