BIOL 135

DIAGNOSIS IN BIOMEDICAL SCIENCE

Lectures 1-2
Investigations into immune system
function

Dr Jessica Hammond
Office B20 Furness. email: jessica.hammond@lancaster.ac.uk
 LEARNING OBJECTIVES

• After attending these lectures and reading the background information, you should
be able to:
• Give an overview of how lymphocytes are able to provide an immune response against a
pathogen
• Give examples of opportunistic infections in AIDS
• Describe the following methods of detection of HIV infection: ELISA,
immunochromatography, latex particle agglutination, RT-PCR, flow cytometry/fluorescence
activated cell sorting.
• Explain how Multiple Myeloma arises and give examples of symptoms.
• Describe the process of cellulose acetate electrophoresis and how it may be used to
diagnose Multiple Myeloma.
• Explain the process of indirect immunofluorescence.
• Describe pernicious anaemia and how it may be diagnosed using indirect
immunofluorescence
 OVERVIEW OF LECTURES 1-2

• A. Introduction to the immune system – revision

• B. Diagnosis of HIV infection

• C. Diagnosis of Multiple Myeloma

• D. Diagnosis of Autoimmune conditions

 THE HUMAN IMMUNE SYSTEM

White blood cells protect your body from infection

These cells have been stained to visualise the nucleus and granules.
 LYMPHOCYTES

• B lymphocytes (B cells)
• T lymphocytes (T cells)
B cell
• Cytotoxic T lymphocytes (TC) receptor
• Helper T lymphocytes (TH) T cell
• Distinguish T and B cells by molecules on their cell receptor
surface CD8
CD4

B-cell TC-cell (CD8+) TH-cell (CD4+)

 T AND B CELL RECEPTOR STRUCTURES

B CELL T CELL

ANTIBODY – ESSENTIALLY A SECRETED B CELL

RECEPTOR
 ANTIBODIES ANTIBODIES
(IMMUNOGLOBULINS) EPITOPE 1
(IMMUNOGLOBULINS) EPITOPE 2

Plasma B-cell Plasma B-cell

B-cell 1 B-cell 2

Memory B-cell epitope 1 Memory B-cell epitope 2

ANTIBODY ELIMINATION OF PATHOGEN
(B-CELL MEDIATED)

Pathogen is OPSONISED
Faster phagocytosis

Antibodies recruit complement proteins

Causes formation of Membrane

Attack Complex

Hole forms in pathogen’s membrane

Pathogen destroyed
T- HELPER CELL IS CRUCIAL IN ACQUIRED IMMUNE
RESPONSE

Tc
Kill
Infected
cells
CYTOKINES

Activated
T-helper cell Antibody
Production
B cell

Cytokines released from activated T-helper cell activate Tc and B cells.

 DIAGNOSIS OF HUMAN IMMUNODEFICIENCY VIRUS (HIV)
INFECTION

HIV virions being

released CD4+ cell
(Helper T cell)

P298 Glencross
Electron micrograph
THE HIV VIRION

HIV infects T Helper cells

How?
-GP120 binds CD4
-GP41 binds CXCR4 on surface
of TH
CD4

HIV destroys T Helper cells

B and Tc cell-mediated
immunity lost
P237 Hall and Yates
AIDS – ACQUIRED IMMUNE DEFICIENCY SYNDROME

Weakened immune system =

Susceptibility to
opportunistic infections

• Toxoplasmosis Toxoplasma gondii

• Tuberculosis
• Pneumonia
• Other Fungal infections
Pneumocystis jiroveci
• Infection-related cancers
e.g. Kaposi’s Sarcoma
Mycobacterium tuberculosis
METHODS FOR DETECTION OF HIV INFECTION

What might be detected?

1. Antibodies to HIV (‘seroconversion’)
develop 2-8 weeks after infection (prior to this = ‘window period’)
Often bind to p24 or gp41
Detection via ELISA, immunochromatography, Latex particle agglutination,

2. VIRAL RNA
Genome of HIV is RNA
RT-PCR (Reverse Transcription Polymerase Chain Reaction) detection

3. FALL IN T HELPER CELL NUMBERS

Flow cytometry and Fluorescence-Activated Cell Sorting
can be used to identify T Helper cells on basis of CD4
1. DETECTION OF ANTIBODIES TO HIV IN THE PATIENT’S
BLOOD
(SEROCONVERSION)

ELISA Immunochromatography Latex particle agglutination

 ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
1 3 1. HIV antigen e.g. p24,
gp41 bound to well
2. Patient serum added.
Anti-HIV antibodies bind
to antigen
3. Anti-human antibody
binds to anti-HIV
antibodies. Enzyme
2 4
linked
4. Substrate added,
converted to coloured
product by enzyme

Wash

P397-8; 447 Glencross

 CHROMOGENIC SUBSTRATES
Antibody conjugated (linked) to:
ALKALINE PHOSPHATASE (AP) ENZYME
Substrate may be BCIP (5-bromo-4-chloro-3-indolyl phosphate)

Can use different enzymes conjugated

to antibody

e.g. Horseradish peroxidase

Colourless Substrates = DAB, TMB, ABTS
Substrate

Detect by light
absorbance
OXIDISING Purple
AGENT product
 IMMUNOCHROMATOGRAPHY

• Detection of antibodies to HIV

• Serum, plasma or whole blood

added to absorbent pad
Control line
• Any anti-HIV antibodies bind HIV HIV test line
Negative result (positive
antigen in test
result)
• HIV test line = antibody which binds Sample port

antigen/antibody complex

• Control line binds HIV antigen Quick, simple and cheap

 LATEX PARTICLE AGGLUTINATION

Latex particle Add blood which may contain

Anti-HIV antibodies

antigen
Latex particle agglutination
Latex particles covered with Visual clumping
HIV antigen e.g. gp41
Quick, simple and cheap
2. DETECTION OF HIV VIRAL
RNA

Useful in ‘window period’ before antibodies to

HIV detectable in serum.
POLYMERASE CHAIN REACTION (PCR)

1. Double stranded DNA is

denatured (separated)
when heated to 95-96oC

2. Primers anneal (bind) to

sites either side of the
target sequence

3. DNA polymerase
catalyses the extension
of target sequence by
joining DNA building
blocks (base + sugar +
phosphate)

4. Cycle repeats (123)

PCR AMPLIFIES A TARGET DNA SEQUENCE
WINDOW PERIOD DETECTION OF HIV USING RT-PCR

DNA copy (cDNA) made of viral RNA DNA copy of cDNA made
using Reverse Transcriptase enzyme by DNA polymerase
Viral RNA cDNA
cDNA

PCR reaction
to amplify DNA

primer

cDNA
Viral RNA

More copies of DNA x 8, x 16 etc.

Can detect 40 copies of HIV RNA per ml of plasma

 3. DETECTION AND MONITORING OF HIV
INFECTION BY ANALYSIS OF T HELPER CELL
NUMBER

Flow cytometry and Fluorescence-

Activated Cell Sorting (FACS)

Disease monitoring
 FLOW CYTOMETRY AND
FLUORESCENCE-ACTIVATED
Blood cells CELL SORTING (FACS)
Blood cells mixed with antibody specific
For CD4, which is engineered to contain
a fluorescent label.
Can be used to distinguish
Cells are streamed through a flow cell
TH cells from other blood cells
by virtue of the CD4 on
the TH cell surface.
Photomultiplier
tubes Therefore, identify
detect light emitted number of T helper cells in
LASER patient’s blood.

Useful in monitoring HIV

infection

Photomultiplier
tubes P402 Glencross
 FLOW CYTOMETRY/FACS
USING MORE THAN ONE
FLUOROCHROME
Mix lymphocytes with range of antibodies • Antibodies engineered to have different
(e.g. to CD4, CD8), each with different fluorescent labels (fluorochromes).
fluorochrome.
• Identification of more than one antigen in
Detection of emitted
the same sample e.g. CD4, CD8.
wavelengths from • Gives you number of Tc and TH etc.
both fluorochromes
Laser 1
Laser 2

Photomultiplier
tubes
FLOW CYTOMETRY/FACS EXAMPLE – PATIENT WITH HIV
P403 Glencross
CD8+ cells labelled with PHYCOERYTHRIN

Normal Tc = 20-45%
CD8+ T lymphocytes
of lymphocytes Normal TH 30-50% of
lymphocytes
(Expect 500-1200 cells/µl
CD4+ T lymphocytes actual here = 170 cells/µl)

<200 CD4+/µl= AIDS

200-500 CD4+/µl=
abnormal

CD4 cells labelled with FLUORESCEIN

 QUIZ

• What sort of cells does HIV infect and why?

• What is seroconversion?

• What does cDNA stand for?

• Is the heavy immunoglobulin chain (T and B cell

receptors) species specific?
 MULTIPLE MYELOMA

•Cancer involving plasma cells

•Symptoms
•Diagnosis – cellulose acetate electrophoresis
 MULTIPLE MYELOMA (MM)

Presenting features
Anaemia:
weakness, tachycardia
Recurrent infection
Bone pain/fractures

Osteoclasts dissolve bone.

Activity up-regulated in MM.
Fractures of long bones, ribs and
vertebrae.

Skull X-ray – several bone lesions

 DIAGNOSIS OF MULTIPLE MYELOMA (MM)

• Cancerous proliferation of a clone of immunoglobulin-producing

plasma cells in the bone marrow.
• Large amount of one immunoglobulin (Ig) produced (PARAPROTEIN).
• Detect via CELLULOSE ACETATE ELECTROPHORESIS.

• Malignant plasma cells stay in bone marrow and activate

OSTEOCLASTS
• Bone broken down

P13 Hall and Yates

P351 Glencross
P273-77 Hoffbrand
DETECTION OF ‘PARAPROTEIN’ IN BLOOD SERUM OF
PERSON WITH MULTIPLE MYELOMA

Cellulose acetate electrophoresis

1 – normal SERUM
2 – multiple myeloma SERUM –
PARAPROTEIN
3 – normal PLASMA
(fibrinogen arrow – confusion with
Paraprotein, so use serum.)

FIBRINOGEN

(Free light chains excreted in urine =

BENCE-JONES PROTEIN)
PARAPROTEIN
 CELLULOSE ACETATE ELECTROPHORESIS

CELLULOSE

CELLULOSE ACETATE

Blocking OH groups in cellulose with acetyl group (to form cellulose acetate) inhibits
protein and water binding. Cellulose acetate made into paper. Proteins migrate
through holes.
P348/9 Glencross
CELLULOSE ACETATE ELECTROPHORESIS PH 8.6

Proteins have overall negative charge at pH 8.6 and migrate

to ANODE. Why?
page 349 Glencross
 AMINO ACID
SIDE
CHAINS
(R GROUP)

-NH2 not
protonated
at pH 8.6 to –NH3+

-COOH
deprotonated at
pH 8.6 to –COO-
VISUALISATION OF PROTEIN IN CELLULOSE ACETATE
ELECTROPHORESIS

• Immerse cellulose acetate in trichloroacetic acid

• Protein reprotonated (-COOH and –NH3+)
• Ponceau S stain added
• Negative charge binds to + protein
• Hydrophobic rings bind any hydrophobic groups in protein
Ponceau S
 AUTOIMMUNITY

Auto = self

Autoimmunity occurs when the bodies’ immune

system fails to recognise ‘self’ and attacks ‘self’
cells.

Focus on pernicious anaemia but principles apply

to other autoimmune diseases such as type I
diabetes.
 PERNICIOUS ANAEMIA

Absorption of vitamin B12 requires combination with INTRINSIC FACTOR (IF)

made by gastric parietal cells

Pernicious anaemia = autoantibodies against gastric parietal cells so less

B12 absorbed

Vitamin B12 needed to make DNA

Red blood cell production requires rapid DNA synthesis.

So, fewer RBC made, less O2 transport = FATIGUE

P136-9 Hall and Yates

 TEST BLOOD SERUM
Autoimmune antibody?
INDIRECT IMMUNO-
FLUORESCENCE

FIXED GASTRIC PARIETAL CELLS

WASH ON GLASS MICROSCOPE SLIDE

Add fluorescently-labelled
Anti-human antibody
Autoimmune antibody binds
antigen in test sample

Anti human antibody

binds autoimmune antibody

Fluorescent label on
anti-human antibody detected
EXAMINE WITH MICROSCOPE
AND UV-LIGHT SOURCE P407 Glencross
 TRANSMITTED LIGHT
FLUORESCENCE
MICROSCOPY

Enables detection of location of

BARRIER Anti-human immunoglobulin which
FILTER has been labelled with fluorescein
(in this case).

Barrier filter – enables visible

fluorescent light to pass to eye
EXCITER
FILTER Exciter filter – maximum amount
of appropriate wavelength light
reaches specimen
P196 Glencross
PERNICIOUS ANAEMIA – INDIRECT
IMMUNOFLUORESCENCE RESULT

Detection of autoimmune
antibodies to stomach parietal
cells on a mouse stomach.

P138 Hall and Yates