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Immunological Methods
These are one of a large group of tracer
methods based on the use of pairs of
molecules with high binding affinities.
Either the binder or the ligand may be
labeled in these methods. They include
qualitative, quantitative, & localization
techniques.
All are governed by the same simple
binding equilibrium considerations.
Binding Equilibrium
B+L
kf
BL
kr
Ka = [BL]/[B]f[L]f = 1/Kd = kf/kr
where [ ]f is the concentration of free,
unbound reagent in solution
Ka also is the relative time BL remains
as BL vs B + L; typically 1 part in 106 1018 in tracer systems.
Examples of Affinity Couples
-1
Binder
Ligand
~Ka (M )
Antibody
Antigen (Hapten)
103 - 1012
Transport Protein
Hormone, Nutrient
106 - 1010
Protein A or G
Antibody Fc
6
11
8
12
10 - 10
Receptor
Hormone
10 - 10
Lectin
Oligosaccharide
109 - 1011
Chelating Peptide
Metal Ion/Cluster
109 - 1012
Avidin/Streptavidin
Biotin
1015 - 1018
Antibodies Nomenclature
Epitope: the arrangement of sequential or
spacially adjacent chemical groupings that
are the site to which an antibody binds
Paratope: the binding site of an antibody,
accommodates up to ~1000 D
Idiotype: collection of all epitopic sites in or
near the paratope on an immunoglobulin
Allotype: genetically coded differences
between proteins of different individuals of
a species
Haplotype: complete set of alleles at all loci
within a gene complex
Antibody Types
IgM: soluble pentamer of 180kD, (2κ or 2λ light
chains, LC, + 2μ heavy chains, HC)5 + j chain
(15kD); membrane monomer with extended HC
= B cell antigen receptor
IgG: soluble 160kD (2κ or 2λ LC + 2γ HC); major
soluble form
IgA: soluble 150kD (2κ or 2λ LC + 2α HC); major
Ig of intestinal, respiratory, urogenital tracts,
milk & tears
IgD: B-cell membrane-bound 170kD (2κ or 2λ LC
+ 2δ HC); no HC-HC S-S bonds
IgE: soluble 190kD (2κ or 2λ LC + 2ε HC); binds
to mast cells & basophils, high with parasitic
worm infection
Antibody Structure
Immunoglobulin Structure:
http://www.cehs.siu.edu/fix/medmicro/igs.htm
Antibody Structure:
http://cig.salk.edu/bicd_140_W99/lecture11.htm
Biochemistry, Generation of Antibody
Diversity:
http://www.mun.ca/biochem/courses/3107/Topics/
Antibodies.html
Antibody Structure
http://www.liu.edu/
cwis/bklyn/acadres/
facdev/FacultyProj
ects/WebClass/mic
roweb/images/IgM.gif
http://hp.vect
or.co.jp/autho
rs/VA020045/v
rml/igg.jpeg
http://fig.cox.miami.edu/
~cmallery/255/255prot/i
http://www.medscape.com/content/2001/00/40/67/406737/art-jap4103.01.Fig2.jpg
mmunog.jpg
Antibody Production
Immune responses initially form IgM with IgG
later as B-cell maturation proceeds &
somatic mutation causes chain switching.
Late clonal responses & boosters favor high
affinity antibodies.
Immune Response System:
http://www.people.virginia.edu/~rjh9u/imresp.html
Blood Bank Antigens & Antibodies:
http://matcmadison.edu/is/hhps/mlt/mljensen/BloodBank
/lectures/blood_bank_antigens_and_antibodi.htm
Body’s Defenses:
http://home.earthlink.net/~dayvdanls/Immune_lecture
.html#The%20First%20Line
Antibody Production
Polyclonal Antibodies
•the normal result of animal immunization;
•derived from multiple B (plasma) cells;
•usually directed vs multiple epitopes;
•often high affinity binding;
•multiple paratopes allow Ab-Ag aggregates
& precipitates to form.
A unique combination at each bleeding of
each animal ==> limited supplies of any
particular preparation.
Antibody Production
Monoclonal Antibodies
•the result of cloning hybrids of myeloma
cells & B cells from immunized animals;
•derived from single B cells;
•directed vs single epitopes;
•often moderate affinity;
•only forms Ab-Ag aggregates or precipitates
with Ag having repeated epitopes.
Cloning ==> unlimited supplies of a unique
molecular reagent.
Kinetic Considerations & Antibodies
Avidity: IgG Ab have 2 paratopes with identical
affinities. Intact Ab normally binds better than an
Fab fragment as the intact Ab paratopes enjoy
the advantage of proximity & may help to orient
antigenic epitopes relative to the Ab. The 1st
paratope to bind tethers the Ag close to the 2nd
paratope, increasing the likelihood of binding,
effectively raising concentration of the paratope
relative to that in bulk solution.
A similar effect speeds binding to membranebound or immobilized Ab or Ag that have limited
abilities to diffuse or reorient relative to a binding
partner; reducing diffusional dimensions speeds
reactions.
Antibodies as Tracers
Ab can be labeled directly:
•radioactively: 125I substitution on tyr or his,
3H on CHO, 35S or 14C amino acids
•biotin addition, via several chemistries
•enzyme conjugation via several chemistries
•fluorochrome or chromophore additions,
often to amine groups
•metal chelator or metal cluster conjugation
•adsorption to colloidal particles, e.g., noble
metals or latex
•heavy atom or spin label addition
Antibodies as Tracers
Ab can also be labeled indirectly by binding
labeled molecules to sites on Ig molecules:
•Protein A or G binding to Fc
•Anti-allotypic Ab from other species
directed at nonparatopic epitopes (2nd Ab)
•Anti-idiotype Ab directed at unoccupied
paratopes
•Avidin or streptavidin binding to Igconjugated biotin
•Anti-fluorochrome or chromophore Ab
binding to Ig-conjugated fluors or phores
•Lectins binding to CHO sidechains
Conjugation & Linkage Chemistries
Glycoproteins can be labeled using methods & reagents
described in K.L. Campbell, Solid State Assays: Reagents
and Film Technology for Dip-Stick Assays, p. 237-287, in
Albertson & Hazeltine (ed) Non-Radiometric Assays:
Technology and Application in Polypeptide and Steroid
Hormone Detection, Alan R. Liss, Inc.: New York, 1988.
Many newer reagents are described at the following sites.
Molecular Probes, Source of Many Labeling Reagents
Introduction to Cross-Linking Reagents:
http://www.probes.com/handbook/
Pierce Chemical Site, Source of Cross-Linkers & Labels:
http://www.piercenet.com/resources/browse.cfm?fldID=78C
0D44E-A2D3-11D5-9E2A-00508BD9167A&strLit=catalog
Bang’s Laboratories Inc., Source of Particle Labels
http://www.bangslabs.com/index_flash.php
General Protocols: Ligand-Binding Methods
1. Blockade of non-specific binding sites with a
general agent such as a non-immune serum
2. Washing to remove excess reagents
3. Formation of a specific binding complex
4. Washing to remove excess reagents
5. Addition of any visualization reagents
6. Washing &/or visualization by microscopy,
FACS, spectrometry, MRI, radiometry, etc.
Steps other than washes require +/- controls
(ligand or binder absence or prior saturation)
during method development; overall +/- controls
are needed during method application.
Ligand-Binding Methods: Considerations
Quantitative detection of small (< ~1000 D) ligands
requires use of competitive methods involving a limiting
[binder] along with a labeled ligand added in slight excess
of [binder]; a negatively graded signal results as
unlabeled ligand competes for binding with lableled
ligand. The approach can also be applied to large ligands.
Quantitative detection of large ligands can also use noncompetitive methods where a capture agent, in excess of
[ligand], allows a ligand to be immobilized & detected by
addition of an excess of [labeled binder].
Qualitative detection uses an excess of a labeled binder or
ligand to demonstrate presence of the complementary
ligand or binder; one reagent is normally chemically
tethered to a matrix or is a part of a macrostructure such
as a fixed cell.
Competitive Assay: RIA, EIA, FIA, etc.
Non-Competitive Assay: IRMA, EIMA, ...
Assay Error Structure
Assay Precision & Analytical Range
Assay Parallelism
Lectins as Binders & Tracers
Animal Lectins: http://ctld.glycob.ox.ac.uk/ctld/lectins.html
http://www.lectins.de/Lectins_engl.pdf
Plant Lectin Physiology:
http://www.dl.ac.uk/SRS/PX/openday/lectin/flower.html
http://www.biologie.uni-hamburg.de/b-online/e17/17h.htm
Lectin Links: http://plab.ku.dk/tcbh/lectin-links98.htm
http://www.cermav.cnrs.fr/cgi-bin/lectines/menu2.cgi?1C3K
Lectin Crystallography: http://mbu.iisc.ernet.in/~mv/
http://ultr.vub.ac.be/lectins/sugar_site.html
http://www.ucalgary.ca/~ngk/carbo/ctype.html
Polysaccharide Structures:
http://employees.csbsju.edu/hjakubowski/classes/ch331
/cho/complexoligosacch.htm
Lectin Binding Sites
http://www.probes.
com/handbook/fig
ures/0713.html
http://www.vectorlabs.com/infopage.asp?dpID=24&locID=146
Lectins Used in Localization
http://linux.farma.unimi.it/RSPSG/2D/glicopr.html
Con A in complex with mannose core as
seen using Rasmol view of a PDB file.
Multivalency
allows this
lectin to
bridge other
molecules in
tracing
methods just
as IgG & IgM;
this is a
common
feature of
lectins as
well as of
avidin &
carrier
proteins.
Analytical Utility of Lectins: Examples
Lectins as Affinity Separation Reagents:
http://www.galab.com/english/glyco&bio/bioseparation.html
Lectins as Fingerprinting Reagents:
http://www.procognia.com/technology/overview/fingerp
rints.htm