MOLECULAR DIAGNOSTIC TOOLS:

IMMUNOLOGICAL APPROACHES TO
DETECT PROTEIN BIOMARKERS OF
DISEASE

DR. FAIZA NADEEM

HEALTH BIOTECHNOLOGY
BT-502
LEARNING OBJECTIVES
• Enzyme-Linked Immunosorbent Assays
• Measuring Disease-Associated Proteins by Sandwich
ELISA
• Diagnosing Autoimmune Diseases by an Indirect ELISA
• Immunoassays for Infectious Disease
• Protein Arrays To Detect Polygenic Diseases
• Immunoassays for Protein Conformation-Specific
Disorders
 WHY PROTEIN AS A BIOMARKERS??
critical role in all
• metabolism, communication, defense, reproduction,
cellular processes transport, motility

• control over protein production; cells function normally

Regulatory
proteins

• diseased tissue; alterations in protein composition or levels of specific proteins

Dysregulation

• more accurately quantified by protein rather than mRNA levels

Abnormal levels of gene
expression

• altered protein conformation (e.g., prion diseases and some neurodegenerative diseases)
irregularities in
posttranslational
modification
WHY IMMUNOLOGICAL APPROACHES??
unique
shape and
challenge for
very low
development of a
cellular
diagnostic assay
levels of a
protein

Antibodies
high affinity and bind to
specificity target
proteins

Immunolo
sensitivity, specificity,
gical
and simplicity for
approache
diagnostic assays
s
AGGLUTINATION
Enzyme-linked immunosorbent assays (ELISAs)
cancers, autoimmune diseases, allergies,
infectious diseases

clinical samples; blood, urine, or tissues

high-throughput assays

Detection on the activity of an enzyme;

covalently bound to an antibody
INDIRECT ELISA
 SECONDARY ANTIBODY

anti-human • raised in another animal by injecting the animal with

immunoglobulin human immunoglobulin

• SA covalently bound (conjugated) to enzyme such as

enzyme
alkaline phosphatase or horseradish peroxidase
SANDWICH ELISA
Disease-Associated Proteins by Sandwich ELISA
monitor progression Other cancerous &
not recommended as
and recurrence of non-cancerous
a screening test
ovarian carcinoma disorders
• protein CA125 in • lung, pancreatic, • Low level at early
serum breast, cervical, stages of the
• high-molecular- colorectal cancers disease
weight • pelvic • monitor a patient’s
glycoprotein inflammatory response to
• higher levels in disease, hepatic treatment
50% of women disorders,
with ovarian cancer nonmalignant
ovarian cysts
 cDNA microarrays

elevated in sera from

quantitative
patients with early
polymerase chain
and late-stage
reaction (PCR)
ovarian cancer

Human Epididymis
Protein 4 (HE4)
Mouse monoclonal
antibodies generated expressed in ovarian
against two different carcinomas
HE4 epitopes
not normal tissue or
benign ovarian
tumors
biotinylated
(B)
monoclonal
antibodies,
streptavidin
(S)
Sandwich ELISA to monitor progression or
recurrence of ovarian cancer

HE4 levels more than 25%

HE4 is quantified
significant to recurrence
spectrophotometrically
or disease progression

decrease magnitude
suggests a positive
response to treatment
AUTOIMMUNE DISEASES; INDIRECT ELISA
• celiac disease, type 1 diabetes, lupus
erythematosus, rheumatoid arthritis

• Indirect ELISAs to diagnose autoimmune diseases

• autoantibodies produced against a self-protein

are detected in patient blood samples
RHEUMATOID ARTHRITIS
• chronic, systemic inflammation of the joints, mainly
the synovial (flexible) joints
• Rheumatoid factor: autoantibody targets the Fc
region of immunoglobulin G (IgG) antibodies and
contributes to rheumatoid arthritis
• commonly used as a diagnostic biomarker to
differentiate rheumatoid arthritis from other forms
of arthritis and other inflammatory conditions
Indirect ELISA For Rheumatoid Arthritis
Diagnosis of Rheumatoid
Arthritis

• Other ELISAs; target rheumatoid factor IgM and

IgA; accurate diagnosis of rheumatoid arthritis
• Early diagnosis to prevent irreversible joint
damage
• managed in the early stages by administering anti-
rheumatic drugs
 IMMUNOASSAYS FOR INFECTIOUS
DISEASE
• Clinical laboratories identify pathogenic microbes
• Growth and isolation of the pathogen, slower than
immunological & molecular methods
• slow grower & fastidious; intracellular bacterium
Chlamydia trachomatis
• Viruses propagated only in host cells
• Immunological detection methods; detect specific
viral, bacterial, fungal, or protozoan pathogens in
body fluids and tissues
IMMUNOASSAYS FOR INFECTIOUS DISEASE
• infectious disease; target proteins or antibodies
• no differentiation b/w current and past infections
• donated blood screened for several pathogens, HIV, hepatitis
virus, human T-lymphotropic virus, Treponema pallidum
(syphilis)
• ELISAs; hepatitis B surface antigen, for antibodies against both
hepatitis B and C viruses
• Viral surface antigens; antibodies appear up to several months
later
 DIAGNOSIS OF HEPATITIS B VIRUS BY ELISA

A sandwich ELISA detects presence of hepatitis An indirect ELISA detects the presence of
B surface antigen in blood anti-hepatitis B virus antibodies in blood,
is often used to confirm a positive result
Protein Arrays To Detect Polygenic Diseases
• ELISA; single target protein; for some diagnoses, more
informative to measure multiple target proteins in a single assay
• Analysis of proteomes; to identify and quantify protein changes
in diseased tissue versus normal tissue; useful for diagnosis of
polygenic diseases (breast cancer, Alzheimer disease, type 1
diabetes, cardiovascular disease)
• Protein microarrays are multiplex immunoassays; detect multiple
biomarkers in a clinical sample
• Biomarkers; subsets of proteins in complex clinical samples such
as biopsied tumor tissue
• commercially available protein microarrays; allergies,
autoimmune diseases, infections
DIAGNOSTIC ALLERGEN
MICROARRAYS

The array format enables detection of hundreds of allergens in a single assay

LIQUID BEAD ASSAY

protein biomarkers with increased risk of cardiovascular disease

Reverse-phase Protein Microarrays
• specific proteins in clinical samples; the samples, cell
lysates or tissue slices, immobilized in a single spot
on a support
• Several samples from different patients, spotted on
the microarray, probed with a single antibody to
detect a specific target protein
• The advantage a large number of clinical samples can
be processed at one time
Reverse-phase Protein
Microarrays
Immunoassays for Protein
Conformation-Specific Disorders
• human neurological disorders arise as a
consequence of protein misfolding that
leads to protein aggregation and cell death
• Parkinson disease, Alzheimer disease, prion
diseases
• Clinical diagnosis of Alzheimer disease is
poor, although 1% of the population
between 60 and 65 years old and 30% of the
population over 80 years old may develop it
Immunoassays for Protein
Conformation-Specific Disorders
• Two hallmarks of Alzheimer disease (i) neurofibrillary tangles of the
cytoskeletal protein accumulate within nerve cell bodies (ii) dense extracellular
aggregates of insoluble proteins called amyloid plaques that develop at the
ends of inflamed nerves
• The principal protein of an amyloid plaque is a small protein called Aβ (β-
amyloid protein)
• All Aβ proteins are derived from the β-amyloid precursor protein (APP) by
proteolytic cleavage
• Abnormal cleavage of APP results in production of Aβ40 and Aβ42 and alters
protein folding, causing exposed regions of the protein to self-interact; hence,
the proteins aggregate in amyloid plaques
• Diagnosis of Alzheimer disease using immunological methods may exploit the
development of antibodies that differentiate between the conformations of
the disease-associated Aβ proteins and normal APP
READING MATERIALS
BOOKS:
Medical Biotechnology, 2013, 1ST edition
Bernard R. Glick, Cheryl L. Patten, Terry L. Delovitch, American Society of
Microbiology
Biotechnology in Medical Sciences, 2014,
Firdos Alam Khan, Taylor and Francis, CRC Press
VIDEO:
https://www.youtube.com/watch?v=EvzA9n-Sklo
RESEARCH ARTICLES:
Ahmed, S., Ning, J., Peng, D., Chen, T., Ahmad, I., Ali, A., ... & Yuan, Z. (2020).
Current advances in immunoassays for the detection of antibiotics residues: a
review. Food and Agricultural Immunology, 31(1), 268-290.