special topic 2: (modified) Immunodiagnostics

Immunodiagnostics

Field applying immunologic principles using antigen-antibody reactions for diagnosis, monitoring, and prognosis

Molecular diagnostics

Technique analyzing biological markers in the genome and proteome to diagnose disease, monitor progression, detect risk, and determine effective therapy

Direct detection

Identification of the pathogen or structure itself using microscopy and staining

Serological tests

Detection of antibodies or antigens using blood or serum

Nucleic acid methods

Detection of DNA or mRNA using PCR-based techniques

Protein methods

Detection of specific proteins using ELISA and Western blot

Screening tests

Performed on clinically healthy individuals for early detection and determination of true positive or true negative

Diagnostic tests

Performed on clinically diseased individuals to confirm disease presence

Confirmatory tests

Verification tests considered the gold standard

Sensitivity

True positive rate; proportion of diseased patients correctly identified; high sensitivity reduces false negatives

Specificity

True negative rate; proportion of disease-free patients correctly identified; high specificity reduces false positives

False positive

Test indicates disease when the person does not have it (e.g., melanoma screening error)

False negative

Test indicates no disease when disease is present (e.g., pregnancy, TB, Lyme, drug tests)

HIV testing strategy

Uses ELISA as screening and Western blot or immunofluorescence as confirmatory; both must be reactive for positive diagnosis

Window period

Time between initial infection and reliable detection; depends on seroconversion; infected person may test negative but still transmit disease

Antigens

Foreign molecular structures triggering immune response

Microbial antigens

Bacterial, viral, fungal, protozoal, and helminthic parasites

Nonmicrobial antigens

Cell surface antigens and autoantigens

Epitopes

Specific antigenic determinants recognized by antibodies

Haptens

Small molecules not immunogenic unless bound to a carrier protein

Immunohistochemistry

Use of enzyme-conjugated antibodies to visualize and localize antigens in tissue

Immunohistochemistry vs immunocytochemistry

IHC applies to tissues; immunocytochemistry applies to individual cells

Principle of IHC

Determines antigen expression and protein localization in tissues

IHC applications

Cancer diagnostics and infectious disease detection

HER2 example

Overexpression in breast tissue associated with breast cancer and used for diagnosis and patient status

Polyclonal antibodies

Bind multiple epitopes; strong staining; higher false positive risk

Monoclonal antibodies

Bind single epitope; high specificity; lower false positives; weaker staining

Pooled monoclonal antibodies

Combine specificity and strong staining; limited availability; must not bind noncompetitively

Secondary antibodies

Species-specific antibodies (e.g., anti-rabbit IgG from goat) labeled with HRP or biotin

ELISA

Solid-phase enzyme immunoassay using antibodies and color change to detect antigens or antibodies; used in diagnosis, quality control, and research

Direct ELISA

Antigen binds solid phase; enzyme-labeled antibody added; limited by crude samples and protein competition

Indirect ELISA

Primary antibody binds antigen; enzyme-labeled secondary antibody amplifies signal

Sandwich ELISA

Capture antibody binds antigen then detection antibody; high specificity and sensitivity

PCR

Rapid in vitro amplification of specific DNA segments developed by Kary B. Mullis based on denaturation, annealing, and extension cycles

PCR machine

Thermal cycler controlling temperature changes

PCR mix components

Template DNA, primers, DNA polymerase, dNTPs, buffer, and magnesium ions

Applications of PCR

Mutation screening, genotyping, known mutation assays, and allelic discrimination by size or restriction enzymes

ARDRA

Allelic discrimination using artificially introduced restriction sites

Primer dimers

Cause reduced efficiency, inaccurate quantification, misleading expression levels, amplification failure, and wasted reagents

Gel electrophoresis

Separation of nucleic acids or proteins by electric field

Agarose gel electrophoresis

DNA analysis after PCR or restriction digestion

Sample preparation

DNA mixed with 6X loading buffer containing tracking dye to increase density and ensure well loading

Ethidium bromide

DNA intercalating UV-fluorescent mutagen requiring gloves

SYBR Green/Gold

Highly sensitive cyanine dye binding dsDNA with green fluorescence

GelRed

Less toxic ethidium bromide alternative with higher sensitivity

Western blot

Analytical molecular biology technique used in immunogenetics