Immunochemical Methods in the Clinical Laboratory

Immunochemical Methods in the Clinical Laboratory

Immunochemical Methods in the Clinical Laboratory Roger L. Bertholf, Ph.D., DABCC Chief of Clinical Chemistry & Toxicology, UFHSC/Jacksonville Associate Professor of Pathology, University of Florida College of Medicine Name The Antigen ASCP/Bertholf Early theories of antibody formation

Paul Ehrlich (1854-1915) proposed that antigen combined with pre-existing side-chains on cell surfaces. Ehrlichs theory was the basis for the genetic theory of antibody specificity. The Template theory of antibody formation Karl Landsteiner (1868-1943) was most famous for his discovery of the

A/B/O blood groups and the Rh factor. Established that antigenic specificity was based on recognition of specific molecular structures; he called these haptens; formed the basis for the template theory of antibody formation. Aminobenzene Sulphonate, a Hapten NH2

NH2 NH2 SO3 SO3 SO3 Ortho Meta Para

Classification of immunochemical methods Particle methods Precipitation Immunodiffusion Immunoelectrophoresis Light scattering Nephelometry Turbidimetry Label methods Non-competitive

One-site Two-site Competitive Heterogeneous Homogeneous Properties of the antibody-antigen bond Non-covalent

Reversible Intermolecular forces Coulombic interactions (hydrogen bonds) Hydrophobic interactions van der Waals (London) forces Clonal variation Antibody affinity Ab Ag Ab Ag [ Ab Ag ] Ka

[ Ab][ Ag ] Precipitation of antibody/antigen complexes Detection of the antibody/antigen complex depends on precipitation No label is involved Many precipitation methods are qualitative, but there are quantitative applications, too Factors affecting solubility

Size Charge Temperature Solvent ionic strength Precipitate The precipitin reaction

etc. Zone of equivalence Antibody/Antigen Single radial immunodiffusion Ag Single radial immunodiffusion r

r [ Ag ] Double immunodiffusion rjan Ouchterlony Developed double immunodiffusion technique in 1948 Double immunodiffusion (Ouchterlony) Quantitative double immunodiffusion S3

S4 P S2 S5 S1 Electroimmunodiffusion Why would we want to combine immunodiffusion with electrophoresis? SPEED

Specificity Carl-Bertil Laurell (Lund University, Sweden) Laurell Technique (coagulation factors) Rocket electrophoresis Electroimmunodiffusion + - Immunoelectrophoresis Combines serum protein electrophoresis with

immunometric detection Electrophoresis provides separation Immunoprecipitation provides detection Two related applications: Immunoelectrophoresis Immunofixation electrophoresis Immunoelectrophoresis -human serum Specimen +

Immunoelectrophoresis - + P C P


Immunofixation electrophoresis SPE IgG IgA IgM

Particle methods involving soluble complexes The key physical property is still size Measurement is based on how the large antibody/antigen complexes interact with light The fundamental principle upon which the measurement is made is light scattering Two analytical methods are based on light scattering: Nephelometry and Turbidimetry Light reflection

Molecular size and scattering - + - Distribution of scattered radiation Nephelometry vs. Turbidimetry

0-90 Rate nephelometry Intensity of scattering Rate C1 C2 Time

Additional considerations for quantitative competitive binding immunoassays Response curve Hook effect %Bound label Competitive immunoassay response curve %Bound vs. log concentration Antigen concentration

Logistic equation a %Bound label y c d a d x a c

Slope = b Log antigen concentration b d Logit transformation a %Bound label

y Y logit y ln 1 y y d where y a d d Log antigen concentration Logit y

Logit plot Log antigen concentration %Bound antigen High dose hook effect Antigen concentration Analytical methods using labeled antigens/ antibodies

What is the function of the label? To provide a means by which the free antigens, or antigen/antibody complexes can be detected The label does not necessarily distinguish between free and bound antigens Analytical methods using labeled antigens/ antibodies What are desirable properties of labels? Easily attached to antigen/antibody Easily measured, with high S/N Does not interfere with antibody/antigen reaction Inexpensive/economical/non-toxic

The birth of immunoassay Rosalyn Yalow (1921-) and Solomon Berson described the first radioimmunoassay in 1957. Radioisotope labels Advantages Flexibility

Sensitivity Size Disadvantages Toxicity Shelf life Disposal costs Enzyme labels Advantages Diversity Amplification

Versatility Disadvantages Lability Size Heterogeneity Fluorescent labels Advantages Size Specificity Sensitivity

Disadvantages Hardware Limited selection Background Chemiluminescent labels Advantages Size Sensitivity S/N

Disadvantages Hardware ? Chemiluminescent labels NH 2 O NH 2 N N

O* H + H 2 H 2 O 2 + OH - OO- Pe r ox i da se

O + N2 + 3 H2O O L um i n o l NH 2 COO + COO -

h ( ma x = 4 3 0 nm ) Chemiluminescent labels CH 3 Br - N+ O-

CH 3 N O O + H 2 O 2 + OH - O CO 2 H A c r i d i n i um e s t e r

+ CO 2 + h + CO 2 H Introduction to Heterogeneous Immunoassay What is the distinguishing feature of heterogeneous immunoassays? They require separation of bound and free ligands Do heterogeneous methods have any advantage(s) over

homogeneous methods? Yes What are they? Sensitivity Specificity Heterogeneous immunoassays Competitive Antigen excess Usually involves labeled competing antigen RIA is the prototype

Non-competitive Antibody excess Usually involves secondary labeled antibody ELISA is the prototype Enzyme-linked immunosorbent assay Specimen Substrate

2nd antibody E S E P E E Microtiter well

E E ELISA (variation 1) Specimen Labeled antigen E S E

P E Microtiter well E ELISA (variation 2) Labeled antibody E

Specimen E E E E E E Microtiter well

E Automated heterogeneous immunoassays The ELISA can be automated The separation step is key in the design of automated heterogeneous immunoassays Approaches to automated separation immobilized antibodies capture/filtration magnetic separation Immobilized antibody methods

Coated tube Coated bead Solid phase antibody methods Coated tube methods Specimen Labeled antigen

Wash Coated bead methods Microparticle enzyme immunoassay (MEIA) S P Labeled antibody E

E Glass fiber matrix E Magnetic separation methods Fe Fe

Fe Fe Fe Fe Fe Fe Fe Magnetic separation methods Aspirate/Wash

Fe Fe Fe Fe Fe Electrochemiluminescence immunoassay (Elecsys system)

Flow cell Oxidized Reduced Fe ASCEND (Biosite Triage) ASCEND Wash

ASCEND Developer Solid phase light scattering immunoassay Introduction to Homogeneous Immunoassay What is the distinguishing feature of homogeneous immunoassays? They do not require separation of bound and free ligands Do homogeneous methods have any advantage(s) over heterogeneous methods? Yes

What are they? Speed Adaptability Homogeneous immunoassays Virtually all homogeneous immunoassays are onesite Virtually all homogeneous immunoassays are competitive Virtually all homogeneous immunoassays are designed for small antigens Therapeutic/abused drugs Steroid/peptide hormones

Typical design of a homogeneous immunoassay No signal Signal Enzyme-multiplied immunoassay technique (EMIT) Developed by Syva Corporation (Palo Alto, CA) in 1970s--now owned by Behring Diagnostics Offered an alternative to RIA or HPLC for measuring

therapeutic drugs Sparked the widespread use of TDM Adaptable to virtually any chemistry analyzer Has both quantitative (TDM) and qualitative (DAU) applications; forensic drug testing is the most common use of the EMIT methods EMIT method S Enzyme S

No signal P S Enzyme Signal Signal (enzyme activity) EMIT signal/concentration curve

Functional concentration range Antigen concentration Fluorescence polarization immunoassay (FPIA) Developed by Abbott Diagnostics, about the same time as the EMIT was developed by Syva Roche marketed FPIA methods for the Cobas FARA analyzer, but not have a significant impact on the market Like the EMIT, the first applications were for

therapeutic drugs Currently the most widely used method for TDM Requires an Abbott instrument Molecular electronic energy transitions Singlet E4 E3 E2 Triplet VR

E1 IC A F 10-6-10-9 sec P E0

10-4-10 sec Polarized radiation z x Polarizing filter y

Fluorescence polarization HO O OH O C in

O Fluorescein out (10-6-10-9 sec) Orientation of polarized radiation is maintained! Fluorescence polarization in HO

O O C O OH But. . .

out (10-6-10-9 sec) Rotational frequency 1010 sec-1 Orientation of polarized radiation is NOT maintained! Fluorescence polarization immunoassay Slow rotation HO O


O Polarization maintained O Rapid rotation Polarization lost FPIA signal/concentration curve

Signal (I/I) Functional concentration range Antigen concentration Cloned enzyme donor immunoassay (CEDIA) Developed by Microgenics in 1980s (purchased by BMC, then divested by Roche) Both TDM and DAU applications are available Adaptable to any chemistry analyzer

Currently trails EMIT and FPIA applications in market penetration Cloned enzyme donor Donor Spontaneous Acceptor Monomer (inactive)

Active tetramer Cloned enzyme donor immunoassay Donor Acceptor No activity Donor Acceptor Active enzyme

Signal (enzyme activity) CEDIA signal/concentration curve Functional concentration range Antigen concentration Other approaches to homogeneous immunoassay

Fluorescence methods Electrochemical methods Enzyme methods Enzyme channeling immunoassay Substrate-labeled fluorescence immunoassay S

Enzyme S No signal Fluorescence S Enzyme Signal Fluorescence excitation transfer

immunoassay No signal Signal Electrochemical differential polarographic immunoassay Oxidized Reduced

Prosthetic group immunoassay P S Enzyme No signal P P

Enzyme Signal Enzyme channeling immunoassay Substrate E1 Product 1 E2 Ag

Product 2 Artificial antibodies Immunoglobulins have a limited shelf life Always require refrigeration Denaturation affects affinity, avidity Can we create more stable artificial antibodies? Molecular recognition molecules Molecular imprinting History of molecular imprinting

Linus Pauling (1901-1994) first suggested the possibility of artificial antibodies in 1940 Imparted antigen specificity on native globulin by denaturation and incubation with antigen. O

- Fundamentals of antigen/antibody interaction Cl O NH 3 + N

O- OH NH2 O CH2-CH2-CH2-CH3 Molecular imprinting (Step 1)

Methacrylic acid + Porogen O H3C O NH N N CH3


N Molecular imprinting (Step 2) O H3C O NH N N


CH3 N Molecular imprinting (Step 3) Cross-linking monomer Initiating reagent O H3C O


NH N N CH3 N Molecular imprinting (Step 4) Comparison of MIPs and antibodies Antibodies

MIPs In vivo preparation In vitro preparation Limited stability Unlimited stability Variable specificity

Predictable specificity General applicability Limited applicability Immunoassays using MIPs Therapeutic Drugs: Theophylline, Diazepam, Morphine, Propranolol, Yohimbine (2adrenoceptor antagonist) Hormones: Cortisol, Corticosterone Neuropeptides: Leu5-enkephalin Other: Atrazine, Methyl--glucoside

Aptamers 1014-1015 random sequences Target Oligonucleotide-Target complex Unbound oligonucleotides + Target Aptamer candidates PCR

New oligonucleotide library

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