Immunologic MethodsPart One

DefinitionsPart Two

Antigen-Antibody Reactions

CLS 420Clinical Immunology and Molecular DiagnosticsKathy Trudell MLS SBB(ASCP)CM

ktrudell@NebraskaMed.com

Objectives• Discuss the following as related to immunology

testing:– Sensitivity– Specificity– Cross-reactivity– Screening test– Confirmatory test– Equivalence zone– Postzone– Prozone

Objectives• Explain how the following physical factors

can affect antigen-antibody reactions:– Concentration ratio of antigen and antibody – Ionic strength– pH– Reaction time– Temperature

• Describe heat inactivation of patient serum, including method and purpose.

Immunology vs. Serology

– The study of host reactions to foreign substances.

– The study of serum; in particular, the study of antibodies in serum and other body fluids.

Sensitivity and SpecificityReference Test # of Patients

Negative Negative 550Positive Positive 370Negative Positive 5Positive Negative 2

• Sensitivity = number of true positive tests / total number of patients with disease {true pos + false neg}

[370 / (370+2)] x100 = 99.46%• Specificity = number of true negative tests / total number of

healthy individuals {true neg + false pos} [550 / (550+5)] x100 = 99.10%

Cross Reactivity• Positive reaction with

substance that is structurally similar to analyte.

Predictive Value

• Predictive Value – Positive: the probability that a positive test result is associated with disease

• Predictive Value – Negative: the probability that a normal test result is not associated with disease.

# of patients

Test Result Value

Cut-off value

“Normal” Disease

Screen vs. Confirm

• Screening Test: A test used to detect disease.

• Confirmatory Test: Run to validate the results of the initial screening test.

Antigen/ Antibody Reactions

Equilibrium

• Antigen / antibody reactions are readily reversible.

• Free Ag + Free Ab Ag-Ab complex

Affinity• Antigen and antibody bind with “Lock and Key”

fit.• Affinity – the attractive force between an Fab

piece of an antibody and a single epitope on an antigen.

Cross Reactivity• For which antigen will

the antibody have greater affinity?– The antigen that

stimulated antibody production.

Avidity

• How “tightly” the antigen and antibody bind.

• The sum of forces binding multivalent antibody to multivalent antigen.– Number Fab pieces– Number of identical epitopes

YY YY

Y

Y

YY

YYY

YWhich Ag/Ab combo will have the greatest avidity?

Antigen / Antibody Reactions• May be visualized when lattice structures form.

– Sensitization– Agglutination or precipitation

YY Y

Y

Y

Y

Zone of Equivalence

Y

Y

Y

YY

Y Y

Y

Y

Y

Y

Y

YY

YY

Y

YY

Y

Y Y

Y

YY

Y

Y

Prozone – antibody excess

Postzone-antigen excess

Antigen/antibody reactions are influenced by:

Concentration Ratio of Ag/Ab

Y

Y

Y

YY

Y Y

Y

Y

Y

Y

Y

YY

YY

Y

YY

Y

Y Y

Y

YY

Y

Y

Prozone – antibody excess

Postzone-antigen excess

Zone of Equivalence

Ionic Strength

• Shielding

• Zeta Potential

Y

-

+

++

+

++

-

--

-++

--

--

- -

-

- -

-+

+++

+

--

-

--

-

-

--

+ +

++

--

---

Y YYYYYJ

Zeta Potential

Other items that influence Ag/Ab binding:

• pH• Reaction time• Temperature• Number of antigens

Given that and are alleles, if there is an anti- antibody, which

of these cells will yield the stronger reaction with the

antibody?

Homozygous Heterozygous

Location of Antigens• Free antigens and cell

surface antigens are more available to react than those buried within the membrane or within the cell.

• Interference in ag/ab binding due to the position of other antigens

YY

YY

Heat Inactivation• Complement that is naturally present in a

patient’s serum may interfere with tests that use complement as a reagent.

• When a test needs a controlled amount of complement, the patient’s serum is heated at 56oC for 30 minutes to inactivate the patient’s complement.

• A known quantity of complement may then be added back to the test system.

We can use this knowledge of antigen/antibody behavior when

designing test methods…

Basic immunologic methods will be discussed next!