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,

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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.
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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
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IgM soluble pentamer of 180kD, (2? or 2? light
chains, LC, 2µ heavy chains, HC)5 j chain
(15kD) membrane monomer with extended HC B
cell antigen receptor IgG soluble 160kD (2? or
2? LC 2? HC) major soluble form IgA soluble
150kD (2? or 2? LC 2a HC) major Ig of
intestinal, respiratory, urogenital tracts, milk
tears IgD B-cell membrane-bound 170kD (2? or
2? LC 2d HC) no HC-HC S-S bonds IgE soluble
190kD (2? or 2? LC 2e HC) binds to mast cells
basophils, high with parasitic worm infection
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Immunoglobulin Structure http//www.cehs.siu.edu/
fix/medmicro/igs.htm
Antibody Structure http//cig.salk.edu/bicd_140_W
99/lecture11.htm
Biochemistry, Generation of Antibody
Diversity http//www.mun.ca/biochem/courses/3107/
Topics/Antibodies.html
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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/BloodB
ank/lectures/blood_bank_antigens_and_antibodi.htm
Bodys Defenses http//home.earthlink.net/dayvda
nls/Immune_lecture.htmlThe20First20Line
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• 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 gt limited supplies of any particular
  preparation.

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• 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 gt unlimited supplies of a unique
  molecular reagent.

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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 membrane-bound or
immobilized Ab or Ag that have limited abilities
to diffuse or reorient relative to a binding
partner reducing diffusional dimensions speeds
reactions.
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• 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

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• 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 Ig-conjugated
  biotin
• Anti-fluorochrome or chromophore Ab binding to
  Ig-conjugated fluors or phores
• Lectins binding to CHO sidechains

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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?fldID78C0D44E-A2D3-11D5-9E2A-00508BD9167Ast
rLitcatalog
Bangs Laboratories Inc., Source of Particle
Labels http//www.bangslabs.com/index_flash.php
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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.
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Quantitative detection of small (lt 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 non-competitive
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.
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Animal Lectins http//ctld.glycob.ox.ac.uk/ctld/
lectins.html http//www.lectins.de/Lectins_engl.pd
f
Plant Lectin Physiology http//www.dl.ac.uk/SRS/P
X/openday/lectin/flower.html http//www.biologie.u
ni-hamburg.de/b-online/e17/17h.htm
Lectin Links http//plab.ku.dk/tcbh/lectin-links9
8.htm http//www.cermav.cnrs.fr/cgi-bin/lectines/m
enu2.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/complexoligosac
ch.htm
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http//linux.farma.unimi.it/RSPSG/2D/glicopr.html
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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.
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Lectins as Affinity Separation Reagents http//ww
w.galab.com/english/glycobio/bioseparation.html
Lectins as Fingerprinting Reagents http//www.pro
cognia.com/technology/overview/fingerprints.htm