After graduating with an honours degree in Biochemistry Dr Hills worked for several years as a Clinical Scientist at St Bartholomew's hospital in London. He was awarded his PhD in 2002 from the faculty of Medicine at Queen Mary University of London. He continued his interest in reproductive science at Imperial College London where he worked as a postdoctoral researcher before joining Middlesex in 2004 as a lecturer. Dr Hills has published many high profile original research articles on various aspects of obstetric pathology including pre-eclampsia, recurrent miscarriage, preterm labour and fetal distress as well as several articles in the area of assisted reproduction. Currently, he is research interests include investigating the role of glycosaminoglycans and proteoglycans on the development of placental pathology and breast cancer. Dr Hills teaches a range of topics in biomedical science including clinical biochemistry, cellular and developmental biology as well as statistical analysis. He is author of around 30 peer-reviewed scientific articles and has refereed manuscripts for a variety of journals in the area of reproduction and endocrinology. For more details about 1st international antibody validation forum please check on http://www.stjohnslabs.com/ac_cms/blog

1. Dr Frank Hills

2. Outline
 Applications
 Qualitative: immunodiffusion, IHC, Western blotting
 Quantitative: immunoassay nephelometry, turbimetry, FACS, cell sorting
 Pitfalls
 Specificity/cross-reactivity
 Speed of analysis
 Refinements
 Monoclonal
 Chimeric - FAb fragments
 Non equilibrium assay
 Recent developments
 Assay chip
 Sample prep for mass spectrometry

3. Antibody structure
Antibodies
Aka immunogloboluns (IgG, IgM etc.)

4. Antibody specificity
CH3
Cortisol
MW = 362
CH3
Stress hormone
Used to monitor adrenal function
Diagnosis of adrenal function
e.g. Cushing’s, Addisons
17β-Estradiol
MW = 272
Female sex hormone
Used to monitor ovarian function
Diagnosis of impaired ovarian function
e.g. amenorrhoea, menopause,
IVF
Testosterone
MW = 288
Male sex hormone
Used to monitor testicular function
Diagnosis of impaired testicular function
e.g. Klinefelters syndrome, anorchia, IVF,
hirsutism, polycystic ovary syndrome
CH3
CH3
CH3

5. CH3
Antibody specificity
Cortisol
MW = 362
CH3
Stress hormone
Used to monitor adrenal function
Diagnosis of adrenal function
e.g. Cushing’s, Addisons
17β-Estradiol
MW = 272
Female sex hormone
Used to monitor ovarian function
Diagnosis of impaired ovarian function
e.g. amenorrhoea, menopause,
IVF
Testosterone
MW = 288
Male sex hormone
Used to monitor testicular function
Diagnosis of impaired testicular function
e.g. Klinefelters syndrome, anorchia, IVF,
hirsutism, polycystic ovary syndrome
CH3
CH3
CH3
3 Double bonds 1 double bond

6. Antibody generation
Animal injected with
molecule to be
measured
Animal immune system (B
lymphocytes) generates
specific antibodies
Collect blood
Extract
antibody to
molecule
Immunisation
Collection
Immune response improved by:
Attaching larger, foreign target
Injecting adjuvant

7. Affinity chromatography
Serum added to affinity
column containing molecule to
be detected attached to solid
beads
Some proteins do not
bind to molecule on
beads and pass through
column
Some bind loosely and
others bind tightly
Specific antibodies are
washed off using low pH
buffer

8. Early applications
 Antibodies first used to treat disease in the late 19th
century (diptheria)
 Immunodiffusion (1960s)
 Ouchterlony
 Radial
 Rocket
 Immunohistochemistry
 Western blotting

9. Gel Diffusion
 Antibody or antigen added
to center well.
 Known sample added to
outer wells.
 Wait for bands to form.
 QUALITATIVE only
 Antibody mixed with agar
 Sample added to wells.
 Antigen will diffuse out and
form precipitin ring.
 Diameter proportional to
concentration.

10. Immunohistochemistry
Add substrate
•Colour reaction only where the antibody is present
Enz
Enz Enz
* * *
Brown area:
Coloured substrate
present
enzyme present
2nd antibody present
1st antibody present
Molecule present
Term placenta
Syndecan-1 Isotype control
Blue colour - counterstain
Hills et al (2014)

11. Western blotting
SDS-PAGE separates proteins by size
Proteins blotted on membrane
Antibodies bind to protein of interest
Reveals only the protein of interest

12. Application
•Effects of glycosaminoglycans on villous trophoblast function
Heparin blocks apoptosis
Heparin stimulates EGF receptor activation
Hills et al (2006)

13. Quantitative techniques
 Immunoassay
 Competitive (RIA)
 Direct (ELISA)
 Flow cytometry (FACS)
 Cell sorting (MACS)

14. Enzyme-linked immunosorbant assay (ELISA)
Capture antibody adsorbed onto plate
Serum sample added. Antigen binds to antibody
Enzyme-coupled detector antibody added.
Substrate added
Colour reaction
Absorbance
Antigen

15. Applications of immunoassay
Anim-Nyame et al 2000
Hills et al 2014

16. Flow cytometry

17. Cell sorting (MACS)
Mixture of different cell types
Antibody attached to magnetic bead
Antibody binds to protein only expressed
by one cell type
Cells passed through magnet
Cells which bind antibody are
retained by magnet
Magnet removed
Cells which bind antibody are
removed
MACS to isolate villous trophoblasts from placenta
HLA class I,II,III -ve immunoselection
Confirmed by immunocytochemistry
Hills et al 2012

18. Pitfalls
 Specificity
 Quality of antibody
 Amount of antibody
Amount of antibody
Antibody
bound
Specific binding
Non-specific
binding

19. Refinements
• Specificity (monoclonal and polyclonal antibodies)
• Fusing antibody-producing cells with cancer cells
• Hybridomas produce multiple copies of identical antibodies
• Rapid immunoassays

20. Polyclonal vs. monoclonal
Polyclonal
• Cheap to produce
• Mixed population of antibodies
• May bind to different areas of
target molecule
• Tolerant of small changes in
protein structure (denaturation,
dimerisation, phosphorylation)
Monoclonal
• Expensive to produce
• Single antibody species
• Will only bind single specific
site
• May only recognise a particular
protein form (phosphorylation,
dimersied)
• Infinitely renewable
Polyclonal antibodies
Monoclonal antibodies

21. Rapid immunoassays
Ab + Ag AbAg
60
50
40
30
20
10
0
0 20 40 60 80 100 120
AbAg complex
Time (minutes)
non-equilibrium
equilibrium
• Automated accurate timing allows
rapid non-equilibrium reactions
Detector
*
*
•Nephelometry and turbimetry
(colour reaction not required)

22. New techniques
 Assay chips
 IPP for Proteomic/mass spectrometry analysis

23. Nanodot array luminometric immunoassay (NALIA)
• Antigens or antibodies adsorbed onto underside of membrane on 96 well plate
• 5 x 5 array allows measurement of 10 analytes in duplicate
• In this case,
• autoantibodies analysed
• serum added, drains through membrane
• Autoandibodies bind array
•hrp labelled anti human IgG added
•Chemiluminescent substrate
Patient sample
Autoantibodies
Antigen array

24. Nanodot array luminometric immunoassay (NALIA)
Controls in central
region - + - + -
Array (La, Ro etc
elsewhere)
Economical platform
for a wide range of
multiple analyte
applications

25. Immunoprecipitation for
proteomics
Ab mixed with protein A
coated bead
Bound Ab added to
serum/cell lysate etc
Target protein immobilised
on bead and applied to
SDS-PAGE
Protein of
interest

26. Application – glycan analysis
Zip tips
HILIC (hydrophilic peptides)
C18 (hydrophobic)
MALDI glycan sequencing
-ve mode
• Improving the predictive value of biomarkers
• Isoforms e.g. glycan analysis
• Combines Ab technology with mass spectrometry
Protein of
interest
Trypsin
wash
elute
Cut out bands In-gel digest Further clean-up

27. MALDI Ionisation Technique
Sample mixed with matrix
Laser vaporises and ionises
sample
Ions migrate down a tube
Speed is inversely
proportional to their m/z
ratio
MALDI Glycan
sequencing (-ve mode)

28. Summary
• Antibody technology has been in use for many years
• New technologies are continually developing
• Continue to incorporate antibodies specificity and ease of use
•In our labs we are using antibodies in a variety of ways to:
• Identify novel biomarkers for disease
•Understand mechanisms of actions of specific molecules in order to
• identify novel treatments
Thank you

30. Assay chips
• Antibody immobilised via linker to gold or graphene surface
of biosensor
• Antigen binding causes changes in electrical impedance
Figure 3: The biosensor developed. (a) With the protein G layer. From bottom to top: The golden substrate,
SAM layer, protein G layer and antibodies with attached biomarker, (b) the sensor coated with the antibody Fab
fragment which couples to the electrode through its C-terminal SH group.
• Can be multiplexed
• PoC device