antibody applications

What are the forces involved in antibody binding?

Ab-Ag binding is reversible because of involvement of non-covalent interactions; weaker than covalent bonds.

Allows the dissociations and reassociation under specific conditions.

What is antibody affinity? What influences it?

Refers to strength of attraction between Ag-Ab.

Influenced by the balance between attractive-repulsive forces.

• single Fab — 1 antigen binding 1 epitope.

What is antibody avidity? What influences it?

Refers to the overall strength of the interaction between and antibody and antigen when multiple binding sites involved.

Influenced by antibody valency — # of available binding sites.

What would have higher avidity? IgG or IgM?

IgM

Describe the kinetics of Ab-Ag binding

Affinity and avidity are established by kinetic studies that measure the equilibrium constant of the reversible reaction.

What is specificity?

Ability to discriminate between similar epitopes.

What is cross-reactivity?

A lack of specificity; when antibody to one antigen binds to another.

What is an example of specificity and cross-reactivity?

Patients who contract oral herpes will make antibodies which also recognize genital herpes → false positive; potential misdiagnosis.

What is a Fab?

Antigen binding fragment of an antibody.

What does papain protease do?

Cleaves at the hinge region above the disulfide bond connecting the two heavy chains.

Describe Fabs and their significance in the lab

• Papain protease activity results in 2 Fab and 1 Fc.

• Used in experiments to block cell receptors; stick but no downstream effect

• Fabs are used to block antibodies from non-specifically binding other antibodies.

What does serine protease do?

Cuts at the Fc region, below the hinge and the disulfide bonds connecting the heavy chains.

Describe the F(ab’) 2 fragments and their lab significance

• 1 F(ab’)₂, 1 pFc (partial)

• Hinge is intact

• Small IgGs with better tissue penetration (for staining)

• F(ab’)₂ provides ‘cleaner’ cell staining

Describe the role of Igs in glycosylation and conjugation; provide examples of applications

Igs are glycoproteins. Proper addition of sugar residues is important for Ig function, but also useful as sites of chemical conjugation.

Antibodies can be conjugated with radioactive isotopes, therapeutic drugs, dyes, enzymes, etc.

Why can’t antibodies be made in bacteria?

What are Mabs and their clinical applications?

• Lab made antibodies that recognize specific pathogens or cells

• Derived from single B-cell clone

• Highly specific and high-affinity → good for lab diagnostics

• High affinity → enhances effectiveness, shortens duration of infection when used as therapy

Describe the traditional approach of using Mabs

Describe the traditional approach to Mabs

Describe modern approach to Mabs

What do the suffixes of Mabs mean?

Murine (0% human)

Chimeric (65% human)

Humanized (>90% human)

Human (100% human)

-omab

-ximab

-zumab

-umab

What is serotyping?

Method used to differentiate between microorganisms based on their cell surface antigens.

Commonly used to identify strains of bacteria, viruses, and other pathogens.

What is serology?

Diagnostic identification of antibodies in blood serum.

Used to detect and measure the presence of specific antibodies or antigens in patient blood.

What is a clinical application of an agglutination assay?

• blood sample tested for presence of Ab using Ag of known specificity

• (+) = colour change or agglutination

• macroscopic; molecular observation rarely observed

What is serum protein electro phoresis?

SEPE is a technique used to separate and analyse the different proteins present in a blood serum sample

What are the uses of SEPE?

• diagnosing blood disorders

• assessing immune function

• detecting kidney and liver function

Increased gamma globulins suggests chronic infection, autoimmune disorder, some blood cancers.

Describe a western blot

Electrophoresus separates proteins by size,

• primary Ab binds the protein

• secondary Ab amplify the signal, bound by dye/enzyme

What is ELISA? Its principle?

ELISA is a technique used to detect and quantify proteins (hormones/Abs)

Uses antigen-antibody interactions to measure the presence of target molecule.

What are the applications of ELISA?

• Disease diagnosis (HIV, hepatitis)

• Hormone levels measurement

• protein quantification in research

What is direct ELISA?

Detects analyte using conjugated primary antibody.

What is indirect ELISA?

Primary Ab binds to analyte, followed by an enzyme-linked secondary Ab

What is sandwich ELISA?

Analyte captured between two Abs, the capture Ab and the detection Ab.

What are potential adverse effects of a Group A strep infection?

GAS can cause acute infection, but also severe illness b/c of septicemia.

After infection, some people may develop rheumatic fever and post-streptococcal glomerulophritis (kidney failure)

How does serotyping work on Group A strep?

Serotyped based on M-protein.

What is a a titre?

Dilute Ab/Ag until no reaction observed to amount of Ab.

What does the presence of IgM or IgG suggest?

IgM — recent exposure

IgG — previous or sustained exposure

What is a paired sera?

First test performed during acute phase (1 week of signs + symptoms) and again during convalescence (2+ weeks) to confirm inprovement

What is the purpose of performing paired sera tests simulaneously?

To avoid error. Run on the same day using same reagents.

What is case definition?

Case definition for each disease defined by medical authorities. Describes test results that indicate confirmed infection.

What is a window period?

The delay between infection and seroconversion.

During this period, serological tests and nucleic acid tests cannot predict disease outcome.

What are the issues with serotyping syphilis?

Cannot be cultured in lab.

• immunofluorescence staining is possible, but requires patient to have lesion

• PCR promising, but currently no commercial assay available