1. IMMUNOASSAYS Basic Concepts & Definitions
Measurement of Antibody Affinity
Quantitative Methods – competitive & noncompetitive assays
Ref: Burtis & Ashwood; Tietz Fundamentals/Textbook
Jan Klein & Vaclav Horejsi; Immunology (1997)
Coleman Lombard Sicard; Fundamental Imm (1992)
Gary D. Christian; Analytical Chem (1994)

2. 1. BASIC CONCEPTS & DEFINITIONS
Immunoassay: use of antibodies to detect analyte
1a. Antibodies
Immunoglobulins that bind to Antigens
5 classes: IgG, IgA, IgM, IgD, IgE
1b. Immunogen
Protein or a substance coupled to a carrier
When introduced into foreign host -> induce Ab to form
1c. Antigen
Any material which can react with Ab
May not induce Ab formation

3. 1d. Antigen-Antibody Binding
Ab molecules have specific binding sites -> bind tightly to Ag -> cause pptn/neutralization/ death
Binding of Ag to Ab due to
van der Waals forces
hydrophobic interactions
charged group attractions
Can measure Antibody affinity: strength of binding between Ab & Ag

4. 2. MEASUREMENT OF ANTIBODY AFFINITY
Binding of Ag to Ab is reversible -> association & dissociation
Ag + Ab <-> AgAb
Law of mass action:
Rate of rxn  to concn of reactants
ka[Ag][Ab] = kd[AgAb]
K = ka/kd = [AgAb]/ [Ag][Ab]
where K is equilibrium constant or affinity constant

5. r/c = nK – rK
r = no. of molecules of bound Ag per Ab molecule
c = concn of free Ag
n = valency of Ab
Plot r/c vs r => Scatchard Plot
Straight line with slope k
x intercept gives n
y intercept gives nK
K (liters/mole) measures affinity of complex

6. Why measure Affinity of an Antibody?
To assess Ab specificity
It influences the functional efficiencies of Abs
eg. high-affinity Abs are very dependable for
various applications:
Diagnostic
Therapeutic
Analytical

7. Labeled Immunochemical Assays
3. QUANTITATIVE METHODS
Read & Understand from Tietz Fundamentals:
Radial Immunidiffusion Immunoassay
Electroimmunoassay
Turbidimetric & Nephelometric Assays
Labeled Immunochemical Assays

8. COMPETITIVE vs NONCOMPETITIVE RXNS
A. Competitive Immunoassays
Used when have limited reagents (Ag)
(i) Simultaneous Competitive Assay
Labels Ag (Ag*) & unlabeled Ag compete for binding to Ab
The probability of Ab binding to Ag* is inversely  to [Ag]
Ab + Ag + Ag* <-> Ab:Ag + A-Ag*

9. (ii) Sequential Competitive Assay
Step 1: unlabeled Ag mixed with excess Ab
-> binding allowed to reach equilibrium
Step 2: Ag* added sequentially -> equilibrate
After separation -> det bound Ag* -> calculate [Ag]
Larger fraction of Ag bound to Ab than in simultaneous assay
If k1 >> k2 ->  in Ab:Ag ->  in Ag* binding
Provide two- to four- fold improvement in detection limit

10. b. Noncompetitive Immunoassays
Used when have excess reagent
Immobilization of Ab to support
Passively adsorption or bind covalently
Direct or indirect attachment (Table 9-3)
ii. Ag allowed to react with Ab -> wash other proteins
iii. Add labeled Ab (conjugate) -> reacts with
bound Ag
Determine bound label ->
[Ag*] or its activity is  [Ag]