MEDICAL TECHNOLOGY ASSESSMENT PROGAM 1

IMMUNOSEROLOGY
DISCUSSED BY: MR. MARK MENDROS
ANTIGENS, ANTIBODY AND IMMUNE RESPONSE

 Antigens and antibodies are specific to one another.

 If we target Ag in the px spx sample, we use Ab reagent.
 If we target Ab in the px spx sample, we use Ag reagent.
 2 Basic rule/principle in serologic testing

ANTIGENS

A. DEFINITION:
 Any molecular structure that when introduced is capable of Antibody production.
 Antigens of interest are the ones that able to elicit/induced/stimulate the immune system to produce antibodies.
B. PARTS OF ANTIGEN
 Carrier portion:
o responsible for the molecular weight of the antigen;
o Molecular weights can be expressed in Dalton
 Epitope or Determinant:
o determines the specificity of the antigen;
o This particular portion of antigen is responsible for specific antibody production.
If Ag is simple type – less determinant is needed
If Ag is a complex type – epitope is numerous in number
o Found in the surfaces

C. TWO PROPERTIES OF ANTIGEN

1. IMMUNOGENECITY
 Inherent ability of a substance to induce the specific immune response resulting in the formation of immune lymphocytes or
antibodies.
 If antigen is immunogenic, it means that it is capable of inducing antibody production or immune response or immune
lymphocytes as well

FACTORS THAT AFFECCT IMMUNOGENECITY

A. FOREIGNESS:
 the immunogen must be recognized as foreign or non-self to induce immune response
 Immune system is capable of discriminating self from non-self.
 This unique characteristic of immune system would enable the immune system to be activated whenever foreign
agents/invaders are present in our body.
 Non-self-agents are considered as self-agents, so normally; it does not elicit immune response.
 In foreignness the immunogen must be recognized as foreign/non-self/invader, so that antibody production is produce;
immune response is induce.

ANTIGEN TYPES
Autoantigen:
 “Self-antigen”
 A healthy immune system should not be stimulated/activated by an autoantigen because it is considered as self, part
of the body system.
 Autoantigen should not elicit immune response.
MEDICAL TECHNOLOGY ASSESSMENT PROGAM 1
IMMUNOSEROLOGY
DISCUSSED BY: MR. MARK MENDROS
 If autoantigen is able to stimulate/induce antibody production, autoantibodies will form and attack the self-antigen
with such effect it would cause injury/damage to the cells/tissues/organs that will cause auto immune disorder. –
ABNORMAL.
Alloantigen:
 A type of antigen that is derived from other individual of the same species.
 In Blood Bank Laboratory, alloantigen is one of the common reagents used to detect alloantibody.
 ALLOANTIBODIES are commonly test/detected
 Red blood cell antigens, common cause allloimmunization. Alloantigens of donor that the patient does not have.
Heteroantigen:
 Antigens derived from other species.
 Can be derived from non-human source.
 MOST FOREIGN ANTIGEN.
Heterophile antigen:
 A type of hetero antigen that is produced/obtained from unrelated plants/animals that can induce/elicit a common
immune response/similar immune response.

GRAFT TYPES
 GRAFT – simply known as tissue transplant; tissues have antigens;
Autograft:
 Graft that is obtained from the patient’s own body.
 Example: px who suffered from severe burns to reconstruct body portion.
Isograft/Syngraft:
 Type of tissue transplant that is obtained from identical individuals; from the twin brother/sister.
Allograft:
 Type of graft that is obtained from non-identical individual of the same species.
 MOST COMMON graft that is transplanted/used.
 Liquid – Transfuse; Solid – Transplant
 Blood is only a commonly transfused tissue. Liquid connective tissue
Heterograft/Xenograft:
 Obtain from other species.
 Should have thorough tissue typing because it can cause rejection.
 MOST FOREIGN

B. SIZE
 The larger the molecule, the more immunogenic.
 The number of epitope increases proportionally with the size of the molecule
 Minimum size of antigen to induce immune response: 10,000 daltons or more can actually induce immune response;
if less than 10,000 daltons that antigens is consider as HAPTEN (incomplete antigen; if alone cannot induce immune
response).

Inc. size →Inc. epitope →Enhance immune response

Example:

 ALBUMIN – has a molecular weight of 30-60k daltons; Good antigen because of the molecular weight
 HEMOCYANIN – Excellent antigen; Highly complex; hundred to million daltons; the bigger the antigen, complex, and the
greater the epitope the better in terms of inducing immune response

C. CHEMICAL COMPLEXITY
 Proteins
 The best and strongest antigen;
 Proteins having the peptide bonds are highly stable in the circulation.
 When protein Ag is in the plasma, the longer they stay in the circulation, the more immunogenic they become
because they are recognized by immune cell.
 When antigens are easy and fast to dissociate/disintegrate antigens are unstable.
 MOST STABLE Ag.
 Vaccines have proteins and/albumins, that makes the vaccines highly immunogenic.
 Polysaccharides
 ex: endotoxin, pneumococcal capsule
 Glycoproteins
 like ABO, Rh antigens
MEDICAL TECHNOLOGY ASSESSMENT PROGAM 1
IMMUNOSEROLOGY
DISCUSSED BY: MR. MARK MENDROS
 Polypeptide
 ex: insulin
 Nucleic Acids, Lipids, and Amino Acids
 Least Immunogenic

2. ANTIGEN/SPECIFICITY
 The ability to react specifically with the antibody or cell that caused it to be produced.
 This property is solely based on the Epitope present on the antigen surface.

DEFINITION OF TERMS:

HAPTEN – an incomplete antigen; not immunogenic by itself; it will not elicit immune response, to become immunogenic HAPTEN must be coupled
by a carrier protein to increase molecular weight and stabilize structure; MOST COMMON carrier protein/albumin.

IMMUNOGEN – defined as any substance that can induce an immune response; all immunogens are antigenic but not all antigens are
immunogenic; specific for an antigen that can induce immune response

ALLERGEN – Special class of immunogen that induces hypersensitivity reaction, specifically people that develops this hypersensitivity has the Type
1 hypersensitivity reactions; can cause reaction to some people but not to all;

ADJUVANTS – substances added to an immunogen to enhance immune response; basically, used to enhance reaction of the immune system to
antigen.

Actions of ADJUVANTS:

 Prolongs the retention time of the immunogen in the body; to activate immune system they must recognized first by the immune
cells or the immunocompetent white blood cells.
 Increases the effective size of immunogen; bigger immunogen/substance the more immunogenic it becomes.
 Stimulates the influx of macrophage and/or lymphocytes.

Example of ADJUVANTS:

 CFA (Complete Freunds Adjuvants) – water in oil emulsion of Mycobacterium Butyricum and the Bordatella Pertussis culture (either
of the two)
 LPS (Lipopolysaccharide)
 Aluminum adjuvants

AFFINITY VS. AVIDITY

AFFINITY – the strength of the attraction between an epitope and the antigen combining site (fab portion) of the antibody;

AVIDITY – refers to the strength of interaction between complex antigens and antibodies

SIMILARITY OF AFFINITY AND AVIDITY – They both pertain to the strength of interaction of antigen and antibody

DIFFERENCES OF AFFINITY AND AVIDITY – AFFINITY is simple interaction, Single Epitope + Fab portion of antibody; AVIDITY is a complex
interaction, Multiple Epitopes are interacting with antibody.

ANTIBODIES

A. DEFINITION – Substances, specifically glycoproteins (found in gamma region of the electrophoresis), produced by B lymphocytes or
plasma cells in response to antigenic stimulation
B. ALSO KNOWN AS – Immunoglobulins; Gamma globulins (previously known)

TERMINOLOGIES – can differentiate antibody by these terminologies

 ISOTYPE – the same as the antibody classes; rely on the heavy chain structure present.
o GAMMA - IgG
o ALPHA - IgA
o MU - IgM
o DELTA - IgD
o EPSILON - IgE
 ALLOTYPE – determined by the difference in the antibody structure in the constant region; Amino acid sequence in the constant region
MEDICAL TECHNOLOGY ASSESSMENT PROGAM 1
IMMUNOSEROLOGY
DISCUSSED BY: MR. MARK MENDROS
 IDIOTYPE – determined according to the difference in the variable region of the antibody.

STRUCTURE OF IMMUNOGLOBULIN

* Antibody structure is a “Y-shaped” structure, typical monomer structure.

1) Four (4) polypeptide chains: 2 identical LIGHT chains and 2 identical HEAVY chains.
2) Both light and heavy chains are held together by COVALENT DISULFIDE BONDS.
3) Heavy chains are interconnected by DISULFIDE LINKAGES.
4) Ig has 2 terminal regions:
a. CARBOXYTERMINAL – with constant amino acid sequence (constant region)
b. AMINOTERMINAL – with varying antibody specificity (variable region)

*constant region also known as the Fc portion/region (crystallisable fragment/fragment crystalline)

*variable region also known as the Fab portion/region (Antigen binding fragment); Epitope of antigen interacts.

*Epitope of antigen interacts specifically in the HYPERVARIABLE PORTION of the Fab region.

*2 TYPES OF LIGHT CHAINS (KAPPA AND LAMBDA)

KAPPA – synthesized with the regulation of the gene located in the chromosome 2

LAMBDA – synthesize with the regulation of the gene located in the chromosome 22

*HEAVY CHAIN SYNTHESIS (G,A,M,D,E) - synthesize with the regulation of the gene located in the chromosome 14

5) Treatment with papain (monomer) will produce 3 fragments:

 One Fc fragment – fragment crystalline; involve in the Ig biologic function
BIOLOGIC FUNCTIONS:
1- Complement fixation
2- Placental transfer of antibody
3- Serves as binding site for the cell.
 Two Fab fragments (monovalent) – capable of antigen binding even without the Fc but cannot agglutinate of precipitate
6) Treatment with pepsin results in digestion of Fc fragment, leaving 2 Fab fragments. The Two (2) Fab fragments has two (2) antigen
combining sites (Bivalent), therefore, it is capable of antigen binding, precipitation and agglutination.
MEDICAL TECHNOLOGY ASSESSMENT PROGAM 1
IMMUNOSEROLOGY
DISCUSSED BY: MR. MARK MENDROS
DOMAINS OF IMMUNOGLOBULIN

 VARIABLE REGION
o Responsible for the specificity of the antibody.
o Variable region is the antigen binding site.
o Epitope interaction in hypervariable region
 CH2
o Binds with the complement protein
o C1q (recognition unit) binds in the CH2 in the classical pathway.
 CH3
o Responsible for cytotropic reactions involving macrophage, monocyte, mast cells, cytotoxic killer cells, and B lymphocytes.
 CL
o Responsible for light chain type either Kappa 65% or Lambda 35%
 HINGE REGION
o Located between CH1 and CH2
o Considered as the flexible region of the antibody.
o Hinge region is flexible because rich in amino acid proline is abundant in the hinge region which is responsible for the flexibility of
this particular of the antibody structure.
 J CHAIN
o J – stands for joining chain
o Glycoprotein that is used to linked/connect 2 or more antibody monomers
o Examples of antibodies with J-chain:
 IgM – pentamer
 Secretory IgA - dimer

CLASSES OF IMMUNOLOBULINS

IgG

 Predominant Ig in healthy human serum – this antibody has longest circulation life
 Predominant Antibody in the secondary/anamnestic secondary response
 Provides immunity to the newborn
 IgG is the only antibody that can cross the PLACENTA
 Only IgG1 and IgG3 can actively cross the placenta
 Fixation of complement
 IgG3, Ig1, and Ig2 – can actively bind and fix complement proteins in the classical pathway. In the order of most potent to least potent
 Opsonization – facilitated phagocytosis
 Toxin and virus neutralization
 Participates in agglutination and precipitation reaction
 BEST PRECIPITATING ANTIBODY.
 Small size antibody – can diffuse semi-solid medium
 Considered as warm reacting antibody
 Can optimally react @ 37°C
 Always considered as clinically significant.
 Specifically capable of complement binding in classical pathway
MEDICAL TECHNOLOGY ASSESSMENT PROGAM 1
IMMUNOSEROLOGY
DISCUSSED BY: MR. MARK MENDROS
IgG 4 SUBCLASSES – differentiated based on their concentration in the serum and number of disulfide bonds

 IgG1 – predominant IgG sub class; comprises about 66% of the IgG in the circulation; it has 2 disulfide bonds
 IgG2 – comprises about 23% of the IgG in the circulation; it has 4 disulfide bonds
 IgG3 – 7% of total IgG and has 15 disulfide bonds.
 IgG4 – least in percentage 4% of total IgG and has 2 disulfide bonds

IgM

 Considered as the biggest antibody – pentameric antibody (5 monomers)

 Pentameric Ig
 Primary response antibody
 Predominant antibody in the primary immune response and in the acute stage of disease
 First to appear phylogeny and last to leave in senescence
 It has J chain
 First to appear after a primary antigenic stimulus
 Cold reacting antibody
 Optimal temperature is 4°C
 Most potent antibody that can fix complement protein
 Considered as the BEST AGGLUTINATING ANTIBODY
 First antibody to be expressed on the surface of B cells, because the Mu heavy chain is the First heavy chain to be synthesized in the B
cells

BIOLOGIC FUNCTIONS OF IgM

1. Complement fixation; specifically in complement classical pathway

2. Agglutination reaction – can initiate lattice formation; BEST AGGLUTINATING ANTIBODY
3. Can also participate in OPSONIZATION - Can also facilitate phagocytosis as an OPSONIN
4. Can neutralize toxin

IgA

 Exist as MONOMER and DIMER; It has 2 forms

 In the serum/blood is a MONOMER
 In the secretion it is a DIMER
 It has J chain
 Responsible for mucosal immunity against microbial agent that enters the body through mucosa/skin
 2 subclasses: IgA1 and IgA2
 The only antibody with secretory component
 Secretory component of IgA – is responsible for maintaining the IgA structure in the secretion by preventing enzymatic degradation of the
antibody structure
 Can also be transferred from mother to the baby through the colostrum (breast milk)

IgD

 Function is still unknown but believes to play a role in immunoglobulin

 Found on the surface of B-lymphocytes
 Second antibody to be displayed on the surface of B-cells; second to IgM
 Postulated to be an anti-idiotypic antibody
 Can attack the variable region

IgE

 Least abundant antibody Ig in the serum

 Heat-labile antibody, originally known as Reaginic antibody
 Only antibody that can binds strongly to mast cell and basophil receptor – the only antibody with specific receptor
 Mediates some types of hypersensitivity reactions
 The release of the histamine is due to the binding of IgE with an attached allergen
 Increased in parasitic infection and helminth infection
 Mast cells – located in tissues; basophils – located in circulation/intravascular
MEDICAL TECHNOLOGY ASSESSMENT PROGAM 1
IMMUNOSEROLOGY
DISCUSSED BY: MR. MARK MENDROS
CLASSES OF IMMUNOGLOBULINS

IgG IgA IgM IgD IgE

Molecular weight 150 T 160-400 T 900 T 180 T 190 T
Daltons
Halflife 21-23 DAYS 5-6 DAYS 5 DAYS 2-3 DAYS 2-3 DAYS
Subclasses 4 2 2 - -
Domains 4 4 5 4 5
Activate Yes, except IgG4 Alternative pathway Yes, most efficient No No
Complement
Other name Serum Ig Secretory Ig Pentameric Ig - Reagenic ig
Other notes - can cross the - predominant Ig in - predominant Ig in 1° - involved in B cell - associated w/
placenta except IgG2 secretion immune response activation Immediate
- Major Ig in 2° - with J chain - Has J chain - Susceptible to Hypersensitivity
Immune response - exist as monomer in enzyme degradation - Binds basophils and
serum and dimer in Heat and Acid Labile mast cells
secretion - elevated during
parasitic infections

IMMUNE RESPONSE

IMMUNE RESPONSE
 The reaction of the immune system/immune cells when the body is exposed to foreign agents
 Immune response of an individual is affected by the following factors:
1. OVERALL HEALTH STATUS
 if the person is healthy it follows that the immune response is immediate. If unhealthy the immune
response is delayed/poor
2. AGE –
 NEWBORN INDIVIDUALS, their immune system is not yet fully developed/processed of development/naïve
response;
 GERIATRICS INDIVIDUALS, their immune response is weak/delayed response
3. THE DOSE AND THE ROUTE OF ANTIGEN ADMINISTRATION
 The higher the dose of the antigen, the greater the antigen exposed to the immune system the faster and
stronger immune response.
 Best route of administration (to vaccines): Intravenous (IV) – sure to enters the body but not a common
route.; “Intramuscular (IM) / Subcutaneous” - common
4. GENETIC CONTROL OF THE IMMUNE RESPONSE
 MHC (Major compatibility complex) – genetically controlled a manner of activating and eliciting an immune
response

PRIMARY RESPONSE
 Occurs in four (4) phases after exposure to an antigen
o LAG
 antibody is not yet detectable;
 phase wherein antigen is being processed by the immune cells
 pathogen point of entry
 Primary encounter/first time encounter – longer processing
 if antigen has a re-exposure of same antigen – the antigenic processing is shorter because of memory cells
o LOG/EXPONENTIAL
 Antibody titer increases logarithmical
 Peak titer is seen in Log phase
o PLATEAU/STATIONARY
 Antibody titer stabilizes, because there is a balance between the antibody synthesis versus the antibody
catabolism
o DECLINE
 Antibody catabolism is increasing;
 The titer is decreasing
 IgM is the first antibody to appear in the primary response
MEDICAL TECHNOLOGY ASSESSMENT PROGAM 1
IMMUNOSEROLOGY
DISCUSSED BY: MR. MARK MENDROS

SECONDARY RESPONSE
 Also known as ANAMNESTIC/MEMORY RESPONSE
 Elicited by a second or any subsequent exposure to the same antigen
 The antibody titer is higher
 Exhibits the same four (4) phases as the primary response
 IgG – predominant
 Lag phase is shorter; more rapid immune response
 Attenuated antigens
COMPARISON BETWEEN PRIMARY AND SECONDARY IMMUNE RESPONSE

IMMUNE RESPONSE LAG PHASE PLATEAU PHASE DECLINE PHASE AB PRESENT AB TITER
1 LONGER SHORTER LESS GRADUAL IgM LOWER
2 SHORTER LONGER MORE GRADUAL IgG, IgM HIGHER