Immunology

Assignment-II
Submitted to: Nidhi Srivastava

By Piyush Sharma (MAU18PZL008)

Short Answer questions:

1. Zymogens: it refers to a variety of enzyme precursors which are inactive and are activated by protease cleavage
by other enzymes. Zymogen are also called proenzymes.
2. Membrane attack complex: it refers to complex of complement system formed as the terminal step of all the
complement pathways. MAC constitutes proteins from C5 to C9. It helps in the cell lysis by making pores into its
membrane and thus disrupting osmotic barrier causing endosmosis and cell lysis.
3. Cross reactivity: Cross reactivity refers to the ability of antibody and T-cell receptors to bind with more than one
antigens which share a common epitope.
4. Prozone effect: Prozone effect depicts the impairment of Ab and Ag to form Ab-Ag complexes when high
concentration of Ab or Ag is present. If we plot a graph of Ag-Ab complexes against conc. of either Ab or Ag used
it shows an increase in the complex formation first which then becomes flat and then decreases with further
increase of Ab or Ag conc. to give a hook like appearance hence called Hook effect.
90
Conc. of immune complexes

80
70
60
50
40
30
20
10
0
10 20 30 40 50 60 70 80 90 100
Ab conc.

5. Anaphylaxia: it is a type of severe allergic response which is systemic and often fatal. It occurs within seconds of
the exposure to the allergen. It occurs if allergen directly enters the bloodstream or is absorbed through blood
or skin. Symptoms include drop in blood pressure, contraction of smooth muscles leading to defacation,
urination, labored respiration. This happens due to rapid effects of IgE causing degranulation of mast cells
basophils and their systemic effects.

Long answer type question:

1. Activities performed by serum complement proteins: complement system consists of many serum proteins
which take part in either of the complement pathway or terminal pathways these proteins are discussed below
a. Proteins involved in classical pathway:
i. C1: it has 3 parts
 C1q: it initiates the classical pathway by binding to Ab.
 C1r: it is a serine protease and cleaves C1s
 C1s: Also a serine protease and cleaves C2 &C4
ii. C2: it has 2 parts
 C2a: binds with C4b to form C3 convertase (C2a4b); it is a serine protease
 C2b: no role in complement pathway but helps in vasodilation after further processing
 iii. C3: it has 2 parts
 C3a: acts as an anaphylatoxin and mediates inflammatory responses.
 C3b: presents immune complexes, induce opsonization, binds to C2a & C4b and become
C5 convertase
iv. C4: it has 2 parts
 C4a: Unknown
 C4b: binds to cell membrane of microbes, binds to C2a forming C3 convertase.
b. Proteins involved in lectin pathway:
i. MASP1: MBL-associated serine protease 1.
ii. MASP2: act as serine protease which in association with MBL/ficolin, cleaves C4 and C2
iii. MBL: mannose binding lectins or ficolins, binds to glycoproteins on microbial surfaces and
initiates lectin pathway
c. Proteins involved in alternate pathway:
i. C3
 C3a
 C3b: With Bb, forms the C3 convertase with Bb and one more molecule of C3b
(C3bBb3b), acts as C5 convertase
 C3(H2O): refers to a C3 molecule with an hydrolyzed thioester bond which in association
with Bb forms fluid phase C3 convertase
ii. Factor B: binds to C3(H2O) and is cleaved by factor D
 Ba: Involved in inhibition of B- cell proliferation
 Bb: With C3(H2O), acts as fluid-phase C3 convertase; with C3b, acts as cell-bound C3
convertase; with two molecules of C3b, acts as C5 convertase
iii. Factor D: Functional when bound to C3(H2O) or C3b, involved in proteolytic cleavage of Factor B
iv. Properdin: Stabilizes C3 convertase on microbial surface
d. Proteins involved in terminal pathway:
i. C5:
 C5a: Acts as anaphylatoxin and induce inflammation
 C5b: itself a component of MAC binds to cell surface and facilitates binding of successive
MAC components.
ii. C6: MAC component which stabilizes C5b, which in the absence of C6 degrades rapidly
iii. C7: it binds to C5b6 and induce conformational changes which allows it to insert inside cell
membrane
iv. C8: Binds C5bC6C7 to create a small pore in the membrane
v. C9: 10-20 molecules of C9 polymerize and bind to C5bC6C7C8 to make a larger pore in cell
membrane

2. Agglutination reactions and blood typing system: agglutination refers to the immune reactions in which
antibodies having two binding sites can bind to 2 same or different antigens resulting in formation of their
clumps. Ab which exist in dimers and pentamers form larger clumps. Agglutination reactions are helpful in
bioassays such as for detection of specific Ag or Ab. Heame-agglutination refers to agglutination reaction of
blood when treated with antisera.

Blood typing is based on haeme-agglutination reactions: erythrocytes consists of several proteins and
glycoproteins residues on their surface. Mainly 3 Ag are considered in ABO blood typing.

Blood group A has surface antigen A; B has antigen B and antigen H is common for all blood types. All
these are glycoproteins, which is confirmed because addition of sugar residues decreases the affinity of specific
Ab for these antigens. Ab directed to Ag A bind to N-acetylglucosamine residues, those directed to Ag B binds to
galactose residues and Ab directed to H Ag binds to fucose residues.
 These Ag can be used to detect the blood group by adding antisera. Humans possess IgM Ab against
those members of ABH Ag they do not possess. If an individual with A type Ag recognizes B epitopes and
produce Ab against them i.e. anti-b is transfused with an individual having Ag B and anti-A in serum will lead to
reaction of Ant-A with Ag A and anti- b with Ag B causing agglutination reaction.

Blood group Antigen Reaction with Antisera-A Reaction with Antisera-B

A A agglutination No agglutination
B B No agglutination agglutination
AB A,B agglutination agglutination
O none No agglutination No agglutination

A second human blood group typing system is Rh blood group system which stands for the rhesus factor. It includes 5 Ag
on RBC i.e. D, C, c, E & e but this system focuses on presence or absence of antigen D specifically so if a person with
blood group A has antigen D he will be said Rh +ve and blood group becomes A+ and if he lacks D blood group becomes
A –ve. For its detection antisera D is mixed with blood and then is observed for agglutination.

3. Procedure and principle of RIA: Radio-immuno assay is a technique used for quantitative and qualitative
analysis of antigens, antibodies or proteins present in very low conc. in human body fluids.
Principle: RIA is based on the principle of Ab-Ag specificity and radio-emission. Generally the Ag to be
assayed is bound to a plastic surface and a radio labeled Ab specific to that Ag is added and excess ab
washed off. The Radio labelled Ab is allowed to bind with the Ag and then the mixture is observed using
a gamma-counter.

Procedure: RIA can be performed in various ways with following requirements

Microtiter plate, micro-pipette, blocking protein (Casein or BSA), radiolabelled Ab/Ag,

spectrophotometer.

a. Ab specific to the Ag to be tested is applied to microtiter plates to allow them to bind with plastic surface
non-covalently.
b. Excess of these Ab is washed off.
c. Blocking agent is applied to block any exposed plastic site in microtiter wells. Excess is washed of.
d. A row of microtiter plate is made standard by adding unlabeled Ag in known conc. which increase in
consecutive wells.
 e. In rest of the wells sample to be tested is added.
f. Known amount of Radiolabeled Ag is added to each well.
g. More the conc. of unlabeled Ag lesser labelled Ag will bind to the Ab.
h. Then each well is observed with spectrophotometer and compared with the standard.

4. ELISA: it stands for enzyme linked immuno-sorbent assay. It is a widespread technique used for quantitative and
qualitative analysis of Ag or Ab. In this technique detection Ab are conjugated with enzymes which then reacts
with a substrate to give chromatic result analyzed by spectrophotometer.
Types of ELISA:
 Direct ELISA: it is simplest type of ELISA. It is not generally used as lacks accuracy and sensitivity.
Sample containing Ag is added to the microtiter plate wells and is left So that any available Ag
can bind to plastic surface of the wells. A blocking agent is then added to wells for covering any
exposed plastic sites. Excess sample and blocking proteins are washed off. Then an enzyme
linked Ab specific to the Ag to be tested is added in the wells. Excess is washed off. Finally a
substrate is added to the wells to get a chromatic reaction i.e. colorless substrate is converted
into colored product by the action of conjugated enzyme. The colored product is then observed
under spectrophotometer and result is assessed.
 Indirect ELISA: this method is more accurate and specific but is also complex. In this sample is
added in the microtiter plate and allowed to bind, excess is washed off. Blocking agent is added,
 excess is washed off. A primary Ab specific to the antigen is added in the wells and allowed to
bind to Ag, washing off the excess. A secondary Ab enzyme conjugate is added to mixture, this
secondary Ab is specific against the primary Ab. Excess of secondary Ab washed off. Substrate is
added to visualize the result which is observed under spectrophotometer.
 Sandwich ELISA: this is the most widely used ELISA technique. it is highly specific and sensitive.
The wells of microtiter plate are first applied with capture Ab specific to that Ag and is allowed
to bind to surface. Blocking agent is added to block exposed plastic surface. Excess is washed
off. Sample containing Ag is added which binds to the capture Ab to surface, excess is washed
off. Now either a primary Ab conjugated to enzyme can be added or first primary then
secondary (conjugated with enzyme) can be added. Substrate is added to assess the result using
photometer.
 Competitive ELISA: this method is also widely used and is similar to RIA. Known amount of
capture Ab is applied to the wells. A mixture of known conc. of enzyme labelled Ag and sample
with unknown conc. of Ag is added to the wells. Both these Ag i.e. sample Ag (Ags) and enzyme
labelled Ag (Ag*) compete with each other for binding with capture Ab. More the conc. of Ags
less Ag* will bind to the capture Ab, giving lesser color change reaction with the substrate i.e. the
result will be in –ve relation to the observations.

5. Hybridoma technology: it is a technique used in laboratories to produce large number of monoclonal Ab. in this
technique sensitized plasma cells are extracted from an animal model fused with the myeloma cells for
culturing. Monoclonal Ab refers to Ab produced from a single lineage of stimulated B cells and have same
antigen specificity.
Procedure: A mouse model is first incubated with the Ag we want to produce mAb
 The mouse’s immune system will produce Ab against the Ag.
 Spleen cells o the mouse are extracted and plasma B cells are isolated from the spleen.
 These Ab secreting plasma B cells are then fused with the Myeloma cells.
o Fusion of the two cells is done with electric field hence called electrofusion which includes 3 steps
i.e. prealignment, membrane fusion, postalignment. Fusion happens dues to electric field makes the
cells membranes more permeable.
 The fused cells are called hybridomas which possess the properties of both myeloma cells (immortality,
continuous proliferation) and B cells (secretion of Ab).
 Not All the B cells fuse with myeloma cells some remain unfused and some may fuse with another B cell.
Also some myeloma cells remain unfused or may fuse with another myeloma cells.
 For screening of successful hybridomas selection of the fused cells is done. Selection is done with the help of
HAT medium (Hypoxanthin Aminopterin thymidine).
o Principle of selection: unfused B cells and fused B-B cells have short lifespan and do not survive for
long time so leaving us with unfused myeloma cells, fused myeloma-myeloma cells and hybridomas.
Since myeloma cells are mutated they lack a specific enzyme called HGPRT (hypoxanthine guanine
phosphoribosyltransferase). This enzyme catalyzes important reactions of DNA synthesis by Salvage
pathway. Aminopterins are chemicals which inhibit de-novo pathway of DNA synthesis. So since
myeloma cells lack HGPRT they are unable to synthesize DNA by salvage pathway and by growing
them in HAT medium containing Aminopterin one can ensure the inhibition of de-novo pathway.
Thus myeloma and 2 fused myeloma cells are unable to survive when cultured in HAT medium. On
the other hand Hybridoma cells have HGPRT (inherited from the fused B cell partner) survives in
HAT media.
 The hybridoma cells are then isolated and cultured indefinitely separately.