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 BL

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.

kf

kr

Examples of Affinity Couples

Binder Ligand ~Ka (M-1)

Antibody Antigen (Hapten) 103 - 1012

Transport Protein Hormone, Nutrient 106 - 1010

Protein A or G Antibody Fc 106 - 1011

Receptor Hormone 108 - 1012

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 bindsParatope: the binding site of an antibody, accommodates up to ~1000 DIdiotype: collection of all epitopic sites in or near the paratope on an immunoglobulinAllotype: genetically coded differences between proteins of different individuals of a species

Haplotype: complete set of alleles at all loci within a gene complex

Antibody TypesIgM: 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 formIgA: soluble 150kD (2κ or 2λ LC + 2α HC); major Ig of intestinal, respiratory, urogenital tracts, milk & tearsIgD: 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

http://fig.cox.miami.edu/~cmallery/255/255prot/immunog.jpg

http://hp.vector.co.jp/authors/VA020045/vrml/igg.jpeg

http://www.liu.edu/cwis/bklyn/acadres/facdev/FacultyProjects/WebClass/micro-web/images/IgM.gif

Antibody Structure

http://www.medscape.com/content/2001/00/40/67/406737/art-jap4103.01.Fig2.jpg

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

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.

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 & AntibodiesAvidity: 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.

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

Molecular Probes, Source of Many Labeling ReagentsIntroduction 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=78C0D44E-A2D3-11D5-9E2A-00508BD9167A&strLit=catalog

Bang’s Laboratories Inc., Source of Particle Labelshttp://www.bangslabs.com/index_flash.php

Conjugation & Linkage ChemistriesGlycoproteins 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.

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.

General Protocols: Ligand-Binding Methods

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 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.

Competitive Assay: RIA, EIA, FIA, etc.

Non-Competitive Assay: IRMA, EIMA, ...

Assay Error Structure

Assay Precision & Analytical Range

Assay Parallelism

Animal Lectins: http://ctld.glycob.ox.ac.uk/ctld/lectins.htmlhttp://www.lectins.de/Lectins_engl.pdf

Lectin Links: http://plab.ku.dk/tcbh/lectin-links98.htmhttp://www.cermav.cnrs.fr/cgi-bin/lectines/menu2.cgi?1C3K

Polysaccharide Structures: http://employees.csbsju.edu/hjakubowski/classes/ch331/cho/complexoligosacch.htm

Lectin Crystallography: http://mbu.iisc.ernet.in/~mv/http://ultr.vub.ac.be/lectins/sugar_site.htmlhttp://www.ucalgary.ca/~ngk/carbo/ctype.html

Plant Lectin Physiology: http://www.dl.ac.uk/SRS/PX/openday/lectin/flower.htmlhttp://www.biologie.uni-hamburg.de/b-online/e17/17h.htm

Lectins as Binders & Tracers

http://www.vectorlabs.com/infopage.asp?dpID=24&locID=146

http://www.probes.com/handbook/figures/0713.html

Lectin Binding Sites

http://linux.farma.unimi.it/RSPSG/2D/glicopr.html

Lectins Used in Localization

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.

Lectins as Affinity Separation Reagents:http://www.galab.com/english/glyco&bio/bioseparation.html

Lectins as Fingerprinting Reagents:http://www.procognia.com/technology/overview/fingerprints.htm

Analytical Utility of Lectins: Examples