4. Definition of terms
Amino acid
• Are organic compounds containing amine (-NH2) and
carboxyl(-COOH) functional groups, along with a side chain
(R group) specific to each amino acid
5. Definition of terms…
Protein
• Are large biomolecules, or macromolecules, consisting of one or more
long chains of amino acid residues.
Antigen
• Is a molecule that induces the formation of an antibody and bears one
or more antibody binding sites.
• Which are highly specific topographical regions composed of a small
number of amino acids or monosaccharide units, being known as
antigenic determinant groups or epitopes”
6. Definition of terms…
Epitopes
• Is also known as antigenic determinant, is the part of an
antigen that is recognized by the immune system, specifically
by antibodies, B cells, or T cells.
Antigenicity
• Is the capacity of a chemical structure (either an antigen or
Hapten) to bind specifically with a group of certain products
that have adaptive immunity eg; T cell receptors or antibodies”
7. Definition of terms…
Antibody
• Also known as an immunoglobulin (Ig), is a large Y-shaped
protein produced mainly by plasma cells that is used by the
immune system to neutralize or to identify a foreign
substance such as pathogenic bacteria and viruses”
8. PROTEIN
• “Are large biomolecules, or macromolecules, consisting of one or
more long chains of amino acid residues”.
• When we look at a cell in a microscope or analyse its electrical or
biochemical activity, we are observing the handwork of proteins
• Protein are the main building blocks from which cells are assembled,
and they constitute most of the cell’s dry mass
9. Functions
• Give shape and structure of the cells
• It’s Enzymatic action promote intracellular chemical reactions
• Formation of Protein channels and pumps that control the passage of
nutrients and other small molecules into and out of the cell.
• Cell signalling through signalling protein
10. Functions…
• Some proteins act as motors that propel organelles through the
cytoplasm, and others(proteins) function as components of tiny
molecular machines with precisely calibrated moving parts
• Specialized proteins also act as antibodies, toxins, hormones, elastic
fiber.
• Some act as receptors eg Estrogen receptors
• Storage function
• Gene regulation
11. Genetic control
• Most proteins and enzymes do not work continuously, or at full
speed but Instead their activities are regulated in a coordinated
fashion so the cell can maintain itself in an optimal state.
• The regulation of protein activity occurs at many levels. At one level,
the cell controls the amount of the protein it contains. This can be
done by regulating the expression of the gene that encodes that
protein.
• Also regulating the rate at which the protein is degraded
12. Genetic control…
• DNA is essential in the synthesis of protein
• Genetic code is the nucleotide base sequence on DNA ( and
subsequently on mRNA by transcription) which will be translated into
a sequence of amino acids of the protein to be synthesized
• The code is composed of codons
• Codon is composed of 3 bases ( e.g. ACG or UAG). Each codon is
translated into one amino acid make up 20 amino acid.
14. Protein structures
• Proteins are by far the most structurally complex and functionally
sophisticated molecules.
• Proteins are assembled mainly from a set of 20 different amino acids,
each with different chemical properties.
• A protein molecule is made from a long chain of these amino acids,
held together by covalent peptide bonds.
17. Antigen and antigenicity
• An antigen is a molecule that stimulates an immune response.
• structural molecules/ substances that binds specifically to an antibody
• The modern definition encompasses all substances that can be
recognized by the adaptive immune system
18. Antigen and Antigenicity…
Antigen
“is a molecule that induces the formation of an antibody and bears
one or more antibody binding sites which are highly specific
topographical regions composed of a small number of amino acids or
monosaccharide units, being known as antigenic determinant groups or
epitopes”
19. Antigen and Antigenicity…
Antigenicity is the capacity to act as an antigen,
To react with an antibody, OR to cause the production of antibodies.
“Antigenicity is the capacity of a chemical structure (either an antigen
or Hapten) to bind specifically with a group of certain products that
have adaptive immunity eg; T cell receptors or antibodies”
23. Foreignness
In general, molecules recognized as "self” are not immunogenic; eg we
are tolerant to those self-molecules. To be immunogenic, molecules
must be recognized as "nonself" eg foreign.
24. Molecular size
• The most potent immunogens are proteins with high molecular
weights, eg above 100,000.
• Generally, molecules with molecular weight below 10,000 are weakly
immunogenic, and very small ones, eg, an amino acid, are
nonimmunogenic.
• Certain small molecules, eg, haptens, become immunogenic only
when linked to a carrier protein
25. Chemical properties
• Protein and polysaccharides are most antigenic. Lipids and nucleic
acids are less antigenic.
• Their antigenicity is enhanced by combination with proteins. A certain
amount of chemical complexity is required, for example, amino acid
homopolymers are less immunogenic than heteropolymers containing
two or three different amino acids
26. Antigen determinant(epitopes)
Are the portions of antigen molecules that physically interact with
paratopes (antibody combining sites) of immune response molecules
and therefore actually "determine" antigen specificity.
28. Antigen antibody reactions
• Antigen-antibody reaction, is a specific chemical interaction
between antibodies and antigens during immune reaction.
• It is the binding of an antibody with the antigen that stimulated the
formation of the antibody, resulting in agglutination, precipitation,
complement fixation
29. Antigen-Antibody reactions…
Antibody
• “Also known as an immunoglobulin (Ig), is a large Y-shaped protein
produced mainly by plasma cells that is used by the immune system
to neutralize or to identify a foreign substance such as pathogenic
bacteria and viruses”
• It typically consist of four subunits including two heavy chains and
two light chains
30. Antigen-Antibody reactions…
• The antigens are specifically bound with high affinity by antibodies to
form an antigen-antibody complex.
• The specificity of the binding is due to specific chemical constitution
of each antibody.
33. Applications of antibodies
Applications in medicine : used for
• Diagnosis- detection of specific antibodies for diagnosis.
(E.g.Antibodies which bind to HCG used in pregnancy test kits).
• Therapy- treatment of immune deficiencies such as
hypogammaglobulinemia, whereby, ready-made antibodies are
administered to the patient to induce passive immunity.
• Prenatal therapy- Rh(D) immune globulin antibodies are used in
prenatal treatment to prevent the risk for hemolytic disease of the
newborn.
34. Applications…
Applications in biomedical research
• Western blotting- a blotting paper is exposed to labeled antibodies to
detect proteins.
• Immunosorbent assays (ELISA)- used to detect and quantitate a
particular antigen in blood serum.
35. Applications…
Applications in biomedical research
• Immuno-histo & cyto-chemistry-used for in situ determination of the
presence and the location of proteins.
• Immuno-precipitation assays-antibodies help label and precipitate
target antigens from an aqueous solution.
• In vivo applications-used for neutralization of cell surface receptors
to enable binding to soluble factors.
• Flow cytometry-used for intracellular analysis. This can help detect
proteins in the cytosol, nucleus, and endosomes.
36. Antigen antibody interaction
Are reversible specific non-covalent biochemical reactions:
• Hydrogen bonds
• Electrostatic bonds
• Van der Waal forces
• Hydrophobic bonds
37. The antigen- antibody reaction.
Can be represented by the formula:
K1=constant of association
K2=constant of dissociation
38. The antigen- antibody reaction…
The affinity:
is the strength of the reaction between a single antigenic determinant and a
single combining site on the antibody
or it is the association constant for binding (KA)
KA= k1/k2
Valence: the number of epitopes
Avidity: is the collective affinity of multiple binding sites(affinity+ Valence)
44. Definition…
Immuno histo chemistry (IHC) is a technique :
• used for demonstrating, the location and distribution of antigens of
interest (eg.proteins) in healthy or diseased tissue sections using a
chemical reaction (antibody-antigen interactions.)
45. Definition…
• It combines histological, immunological and biochemical techniques
for the identification of specific tissue components by means of a
specific antigen/antibody reaction tagged with a visible label
46. PRINCIPLE OF IHC
• IHC staining is achieved by using antibodies specifically directed
against the protein of interest (target) .
• Since antibodies are highly specific, thus they recognize and bind only
to the antigen of interest in the tissue section.
47. Visualization
The antibody-antigen interaction is visualized using either
• chromogenic detection, in which an enzyme conjugated to the
antibody catalyzes the conversion of a substrate to produce a colored
precipitate at the location of the antigen.
or
• fluorescent detection, in which a fluorophore is conjugated to the
antibody and can be visualized using fluorescence microscopy.
50. How does it look like?
(a) CD45
(b) CD20
(c) CD138
(d) EBER
51. IHC CAN BE PERFORMED ON
• Formalin fixed paraffin embedded sections
• Frozen section
• Smears
• Imprints
• Cytospins
52. HISTORICAL BACKGROUND
Discover of antibody-The gift of life
The discoveries that led to the development
of immunohistochemistry had their origins in
the 1890s, in bedside research on ‘serum
therapy’ if one injected animals with
attenuated forms of diphtheria bacteria, the
animals would produce ‘anti-toxins
By Von Behring
53. HISTORICAL BACKGROUND…
• 1897 Dr Kraus demonstrated antitoxin reacted with antigens.
• 1920 Heidelberger and Kendall attached a purple azo dye to the
antigen (egg albumin).
• When specific antibody was added, the coloured antigen–antibody
complex precipitated
• 1923 Dr Michael Heidelberger quantified the precipitin text with the
use of dye attached to antigen.
54. HISTORICAL BACKGROUND…
• The Lock and Key structure discovered by Prof Paul Erlich
• Binding properties of antigen and antibody and Specificity
• Dr John Marrack visualized the reaction by attaching dyes to
antibodies
• In 1934, the lattice formation of antigen–antibody complexes was
described by Professor John R. Marrack
55. HISTORICAL BACKGROUND…
• 1941-1945 Dr Alber H Coons developed first fluorescent antibody
labels officially launching light microscope immunohistiochemistry.
56. HISTORICAL BACKGROUND…
• Immuno electromicroscopy was launched in 1959 by Dr S.J Singer
• 1966- 1st developed enzyme labeling instead of fluorescent label.
• I n 1974 IHC was performed for the first time on routine formalin
fixed paraffin embedded sections.
• In 1991 Heat induced antigen retrieval technique in IHC was done
57. Important considerations for IHC
• Fixation
• Antibody selection
• Sectioning
• Antigen Retrieval
• Blocking
• Controls
• Direct method
• Indirect method
• Immunoenzyme
• Fluorescence
• Multiple labeling
You actually need to care about all this
58. When do we need IHC?
Mainly used in research and clinic;
In research:
To localize newly identified proteins in control / experimental tissue
59. When do we need IHC?...
In clinic:
1. To compare level of expression between
• non-treated / treated
• healthy / sick tissue
2. To identify markers relative to disease status
• diagnostic
• prognostic
60. APPLICATION
• The introduction of prognostic and predictive markers has made a
tremendous beneficial impact on patient diagnosis and management
of cancer patient.
• Management of cancer is more it personalized and targeted
• This has been made possible by the gradual development of
immunohistochemical methodologies over the past 70 years
• IHC allowed the identification of specific or highly selective cellular
epitopes in formalin-fixed, paraffin processed tissues with an
antibody and appropriate labelling system.
61. 1.TUMORS OF UNCERTAIN HISTIOGENESIS
• Approach to diagnosis of tumors of uncertain origin,primary as well
as metastatic from unknown primary tumor.
• Selection of antibodies being made is based on clinical history,
morphological features and results of other relevant investigations.
63. EPITHELIAL
• CYTOKERATIN
• EPITHELIAL MEMBRANE ANTIGEN(EMA)-markers of glandular
epithelia
• CARCINOEMBRYOGENIC ANTIGEN (CEA)-markers of glandular
epithelia.
• P63-Marker of squamous and urothelial epithelia.
64. MARKERS OF MUSCLE DIFFERENTIATION
There are 3 types of muscle differentiation
• Skeletal muscle differentiation. Example-Rhabdomyoma,
Rhabdomyosarcoma
• True smooth muscle differentiation: Leiomyoma,Leiomyosarcoma
• Partial smooth muscle differentiation: Myofibroblasts constituting
significant population of cells in healing wounds and stroma reaction
to tumors,nodular fascitis,myofibroblastoma
66. NERVE SHEATH DIFFERENTIATION
S100
• Malignant peripheral nerve sheath tumors show patchy and weak positivity compare to a
strong and uniform +vity in benign.
• Perineural cells are –ve
CLAUDIN 1
• +VE in perineural cells:Perineuromas.
• -VE in schwanommas
GLUT-1- Perineural cells +ve
CD57-Found in NK cells,T cells,oligodendroglial cells and schwann cells
69. MARKERS OF VASCULAR DIFFERENTIATION
1. CD 31-More sensitive and specific
2. CD 34
3. Factor VIII(actual antigen is Vwf)
4. Ulex europaeus 1
5. CD 141(Thrombodulin)
6. Fli-1
7. ERG-New promising vascular marker also positive in prostatic
adenocarcinoma
8. D2-40(podoplanin)-Lymphatic endothelial cells
70. LYMPHOID MARKERS.
1. CD 45- Leukocyte common antigen
2. CD 3- For T cell lineage
3. CD 20- For B cell lineage
4. Markers of Redsternberg cells(RS)- CD30,CD 15
5. Kappa/Lambda for plasmacytoma
71. 2.PREDICTIVE AND PROGNOSTIC MOLECULAR
MARKERS FOR CANCER MEDICINE.
PROGNOSTIC MARKER:
• Aim to objectively evaluate the patient’s overall outcome such as
probability of cancer recurrence after standard treatment.
• The presence or absence of a prognostic marker can be useful for the
selection of patients for treatment but does not directly predict the
response to a treatment.
72. BIOMARKERS TYPE OF CANCER CLINICAL SIGNIFICANCE
BRCA1 BREAST High expression of BRCA1 confers worse prognosis in
untreated patients.
CA19-9 PANCREATIC Higher preoperative CA19-9 levels are associated with
lower resectability,more advanced stage and inferior
survival.
CEA COLORECTAL CA Elevated preoperative CEA levels is associated with poor
prognosis
C-KIT GIST Better prognosis if they harbor a mutation in exon 11 of
the c-KIT gene
ER/ PR BREAST Patients with ER /PR breast tumors have better prognosis
Oncotype
DX
BREAST 21 gene multiplex test used for prognosis to determine
10yr disease recurrence for ER positive,lymphnode
negative breast cancers
VEGF RCC Overexpression is associated with poor prognosis in clear
cell renal carcinoma
73. 3. MARKERS ON ASSESMENT OF INVASION
• Collagen type IV: Component of basement membrane;Anti collagen IV
antibody.
• Basal cells in prostate carcinoma:Anti high molecular weight
cytokeratin (34BE12) and p63 suppresor protein.
• Myoepithelial cells in breast carcinoma: alpha smooth muscle
actin,calponin and p63
• Racemase for prostate carcinoma showing malignant acinar cells
74. 4. PREDICTIVE MARKERS
• Aim to objectively evaluate the likelihood of benefit from a specific
clinical intervention, or the differential outcomes of two or more
interventions, including toxicity.
• Since the year 2000, there have been over 26,000 publications
indexed in PubMed with the joint medical subject headings of
‘neoplasm’ and ‘predictive marker’, and almost 14,000 publications
with ‘neoplasm’ and ‘prognostic marker
75. PREDICTIVE
BIOMARKER
TYPE OF
CANCER
CLINICAL SIGNIFICANCE
BRCA1 BREAST High expression predicts response to chemotherapy
c-KIT GIST Mutation on exon 11 of the c-KIT gene benefit from imatinib and
sunitinib
EGFR NSCLC EGFR1 mutations in patients with NSCLC are predictive for response
to either gefitinib or erlotinib treatment
EGFR CRC predictive factor for response to anti-EGFR1 antibody treatment in
CRC
Her2/neu BREAST overexpressing tumors benefit from treatment with trastuzumab
Her2/neu GASTRIC Expression of Her-2/Neu in gastric cancer is predictive of patient
sensitivity towards treatment with 5-FU, doxorubicin, trastuzumab
and platinum-based chemotherapy
ER
PR
BREAST High cellular expression of ER predicts benefit from tamoxifen based
chemotherapy
76. 5. MARKERS OF PROLIFERATING CELLS
Ki-67 .
• In breast cancer ki 67 identifies high proliferative subset of patients
with ER positive breast cancer who derive greater benefit from
adjuvant chemotherapy
78. 7. NEURODEGENERATIVE DISOEDERS
ANTIBODIES KEY APPLICATIONS LOCATION
Phosphorylated tau Major component of neurofibrillary tangles in
Alzheimer disease and frontotemporal lobar
degeneration
Picks diseas in outer cortical layers
frontal and temporal lobes
Beta amyloid ubiquitin Alzheimer disease cerebral amyloid angiopathy In lewy bodies in substania nigra and
locus cerulus in parkison’s disease
Alpha synuclein Dementia with lewy body disease In substatia nigra
79. 8. BRAIN TRAUMA
• In the last few years, immunohistochemical staining for beta
amyloid precursor protein has been validated as a method to
detect axonal injury within as little as 2–3 h of head injury.
• Immunohistochemical detection of axonal injury can be
useful in establishing timing of a traumatic insult in medico-
legal settings.
80. 9. IHC IN MUSCLE DISEASES
• Such abnormalities involve proteins located in the sarcolemma,
extracellular matrix, cytosol, nucleus, and other sites within muscle
fibers.
• Skeletal muscle biopsy can play a main role in differentiating vascular
dystrophy from non-dystrophic disorders and IHC can assist in
establishing a specific diagnosis of the dystrophies for which specific
protein abnormalities are known.
81. RECENT ADVANCES
• Genogenic IHC for diagnosis example markers to monitor
drug resistance. Example P- glycoprotein a product of
multidrug resistance gene.
• Technician free automation of IHC procedures
• “Pathologist free” microscope image analysis technology for
interpretation of IHC
82. REFERENCES
1. Roitt’s Essential immunology 13th Edition.
2. Protein antigenicity: a static surface property Jiii Novotn , Mark
Handschumacherand RobertE. Bruccoler ImmunologyToday,vol.8,
No. 1, 1987
3. The Structural Basis of Protein Antigenicity: Yvonne Paterson
Department of Microbiology, University of Pennsylvania,
Philadelphia, Pennsylvania 19104 USA.
4. The atomic mobility component of protein antigenicity: A. Tainer,
Elizabeth D. Getzoff, Yvonne Paterson*, Arthur J. Olson and Richard
A. Lerner Departments of Molecular Biology and Immunology*,
Research Institute of Scripps Clinic, La Jolla, California 92037
Editor's Notes
Topography describes the physical features
Hapten: In immunology, a molecule that is incapable, alone, of causing the production of antibodies but which can do so when fastened to a larger antigenic molecule called a carrier. The term "hapten" was taken from the Greek verb "haptein" meaning "to fasten or bind.“
Antibodies are produced by specialized white blood cells called B lymphocytes (or B cells). When an antigen binds to the B-cell surface, it stimulates the B cell to divide and mature into a group of identical cells called a clone.
Plasma cell. ... Plasma cells, also called plasma B cells, are white blood cells that originate in the bone marrow and secrete large quantities of proteins called antibodies in response to being presented specific substances called antigens.
Plasma cells are produced after the division of B cells following the attachment of antigen to the B- cell
Some proteins channels are found in the cell membrane.
A codon is a sequence of three DNA or RNA nucleotides that corresponds with a specific amino acid or stop signal during protein synthesis
start codon is AUG.
Stop codonACG
Secondary:
But this interaction does not involve the side chain but rather the hydrogen bond. DNA is in secondary structure of proteinS
Tertiary:
The tertiary structure is primarily due to interactions between the R groups of the amino acids that make up the protein.
some proteins are made up of multiple polypeptide chains, also known as subunits. When these subunits come together, they give the protein its quaternary structure
COMPLETE ANTIGENS: any antigen capable of stimulating the formation of antibody with which it reacts in vivo or in vitro, as distinguished from incomplete antigen (hapten).
Hapten: In immunology, a molecule that is incapable, alone, of causing the production of antibodies but which can do so when fastened to a larger antigenic molecule called a carrier. The term "hapten" was taken from the Greek verb "haptein" meaning "to fasten or bind.“
Tumor antigen is an antigenic substance produced in tumor cells, i.e., it triggers an immune response in the host
Generally, the greater the phylogenetic distance between two species, the greater the structural (and therefore the antigenic) disparity between them
Some self-components (e.g., corneal tissue and sperm) are effectively sequestered from the immune system, so that if these tissues are injected even into the animal from which they originated, they will function as immunogens.
All 4 levels of protein organization primary, secondary, tertiary and quaternary contribute to the structural complexity of a protein and hence affect its immunogenicity
Complementarity: the state of working usefully together: the state by which antbody and antigen co-agree to bind with each other
Hemolytic disease of the newborn (HDN) is a blood problem in newborn babies. It occurs when your baby's red blood cells break down at a fast rate. It's also called erythroblastosis fetalis. Hemolytic means breaking down of red blood cells.
In situ: at its original/primary position
Antibody opsonization is the process by which the pathogen is marked for ingestion and eliminated by the phagocytes.
Sometimes the staining pattern of a single stain could be different diagnostic context example CD3 (T-cell marker) which is cytoplasmic positivity in precursor T cell neoplasm and membranous positivity in peripheral T cell neoplasm.
Tumor marker, CEA: Carcinoembryonic antigen (CEA) is a protein found in many types of cells but associated with tumors and the developing fetus. ... The most common cancers that elevate CEA are in the colon and rectum. Others: cancer of the pancreas, stomach, breast, lung, and certain types of thyroid and ovarian cancer.
tumor marker listen (TOO-mer MAR-ker) A substance found in tissue or blood or other body fluids that may be a sign of cancer or certain benign (noncancer) conditions. Most tumor markers are made by both normal cells and cancer cells, but they are made in larger amounts by cancer cells. A tumor marker may help to diagnose cancer, plan treatment, or find out how well treatment is working or if cancer has come back. Examples of tumor markers include CA-125 (in ovarian cancer), CA 15-3 (in breast cancer), CEA (in colon cancer), and PSA (in prostate cancer).
Blocking: it help the reagent used for IHC to donot spread away from the tissue area when applied.
Control is done at the beginning of IHC to see if the reagents and the machines to be used are okay. The positive sample is used.
Multiple labeling, using antibodies of different kinds at once to detect the antigens of different kinds found in the body.
Antigen retrival: opening antigens so as antibodies can bind many antigens. It is mainly done by heating.
Ber Ep4 expressed in adenocarcinoma ..to differentiate in effusion cytology lung adenocarcinomafrom mesothelioma.
Mesenchymal neoplasms that are CK +VE
Synovial sarcoma
Epitheloid sarcoma
Chordoma
Myomas are benign tumors that grow from muscle
arcinomas are cancers that develop in epithelial cells, which cover the internal organs and outer surfaces of your body
NON MUSCLE CELLS THAT EXPRESS DESMIN ARE.
Fibroblast reticulum cells of lymphnode
Endometrial stromal cells
Submesothelial fibroblasts
1.DESMIN
Both skeletal and smooth muscle.
Not expressed by myofibroblast.
2.ACTIN:
Divided into muscle and non muscle such as myofibroblast.
Interpretation is quantitative rather than qualitative.
Muscle cells have far more actin than any other cells
3.MYOGLOBIN ;
Found in skeletal and cardiac muscle
:Not in smooth muscle.
Other markers-Myogenin and myoD1
S100-Calcium binding protein.
Named as such due to its 100% solubility in ammonium sulfate.
Useful marker in:Benign and Malignant nerve sheath tumors and melanoma.
Melan A and HMB45 ;CYTOPLASMIC…MITF;NUCLEAR…..S100 BOTH NUCLEAR AND CYTOPLASM.
HMNB45:Monoclonal Ab HMB45 identifies Pmel17 gene product gp 100(present in premelanosomes)
+VE: Immature melanocytes:Melanoma,PEcoma
,-VE in mature melanocytesNevi,resting melanocyte
NB:Desmoplastic and spindle cell melanomas are usually negative.
Less sensitive than melan A and S100 but more specific.
CD 34 NON VASCULAR
Solitary fibrous tumor
GIST
Epitheloid sarcoma
Nerve sheath tumors
Granulocytic sarcoma
GIST: Gastrointestinal track.
CRC-Colorectal tumor,EGFR-Epidermal growth factor receptor,ER Estrogen receptor,NSCLC non small cell lung cancer,PR progesterone
Presence of the makers as predictive makers indicates the probability of being infected with a certain kind of cancer but also indicates the choice of drugs for the treatment of that particular cancer.,
NSCLC: LUNGS
Adjuvant therapy: Treatment that is given in addition to the primary (initial) treatment. Adjuvant treatment is an addition designed to help reach the ultimate goal
In infection you can encounter both antigens and antibodies (produced by the body for defence)
