5BM069: Immunology Practical Assessment - Questions and Answers

This document contains the answers to a practical assessment in immunology, specifically focusing on two practicals: haemagglutination and immuno-fluorescence assay (IFA). The haemagglutination section addresses questions about antibody titre in rows A and B, the nature of anti-SRBC antibody, the mechanism of haemagglutination, and how complement causes cell lysis. The IFA section explores the purpose of ethanol fixation, the function of a humidity chamber, the roles of PBS and BSA, the meaning of antibody components (goat, anti-human, FITC conjugate), and whether the performed method is direct or indirect immunoassay. The answers provided detail the underlying principles and procedures involved in each technique, offering a comprehensive understanding of the immunological concepts and experimental protocols.

5BM069: Haemagglutination and Haemolysis practical - (Wet Practical)
Questions
Please answer the following 5 questions in the space provided and submit on CANVAS, each
question is worth equal marks.
1) What is the antibody titre for row A?
Antibody's titre is the process of sample serum dilution aid to conduct the positive
test reaction with clear observation of antigen-antibody interaction. For
identification of virus presence and antibody effect.
Row A is having to 12 column containing Anti-SRBC antibody dilution with PBS
(phosphate buffer saline).
Each and every wells are containing 25 μL inactive complement along with the
25μL sheep red blood.
So for row A antibody titer is serial dilution of Anti-SRBC antibody along with
inactive complement and sheep red blood. To test Agglutination in the blood at
different concentration of Anti-SRBC antibody.
(2 Marks)
2) What is the antibody titre for row B?
For row B Antibody titre is the serial dilution of Anti-SRBC antibody together
with the Active complement and sheep red blood, to test hemolysis.
In row the purpose of the antibody titer is to analyse the function of antibody's and
presence of the pathogen in the blood by the aid of active complement protein

which causes the cell lysis with aid of pathogens with antibody's.
From the result it has been proved that in lower dilution of anti body cell is not
lysed and at higher to minimum concentration of antibody hemolysis accrued.
(2 Marks)
3) In point 2 of the protocol where it says ‘Add 200 μl anti-SRBC antibody to column 1
of rows A and B’. What is an anti-SRBC antibody?
Anti-SRBC antibody is the produce from the animals by immunising them with
SRBC. Immunization is the process where individual or animal get vaccinated
with the SRBC in order to force lymphocytes to produce antibody B cell against
SRBC. Then produced antibody's from the vaccinated animal is extracted from as
a Anti-SRBC antibody's.
(2 Marks)
4) Why does haemagglutination occur?

Haemagglutination is occur in the presence of pathogen or viruses for example
influenza virus. It basically occur when surface protein of the virus such as the
glycoprotein (hemagglutinin) react with red blood cells, causes the coagulation of
red blood cells along with the formation of lattice.
(2 Marks)
5) How does complement cause cell lysis?
The important role of complement proteins is the killing of pathogens by pathogen
cell lysis.
It bids to pathogens cell wall and make it attracting for opsonization, where
phagocytic cells destruct it.
Complement protein get bind to the target cell, Phagocytic cells posse the
complement protein binding respecter on it, so it engulf the target cell and causes
the cell lysis.

(2 Marks)

5BM069: Immuno-fluorescence (IFA) - (Dry Practical)
Questions
Please answer the following 5 questions in the space provided and submit on CANVAS, each
question is worth equal marks.
1) In point 4 of the protocol where it says ‘Slides are then fixed for 10 min in ice cold
absolute ethanol’. What was the purpose of this step?
The purpose of slide fixation with ice cold absolute ethanol is to make the sample
tissue or cell use full for log term practical test work.
Where slide fixation prevents the enzymatic changes, protein activities in order to
make it same as the life sate for log time.
It aid to prevent the hardness of the cells and tissues.
For the correct microscopy analysis and morphological study of the cells and
tissues material it is crucial to conduct slide fixation.
(2 Marks)

2) In point 6 of the protocol where it says ‘to form a humidity chamber’. What was the
purpose of this step?
Humidity chamber is make by aid of flat plastic box along with the water and
rails. Bottom of the box is fill with the water and rails provide the support to the
slides and make them away from the water.
Main purpose of the humidity chamber is to provide the appropriate humid
environment to the sample and to prevent the dryness from the slides.
In lab there is another facility of electronic humidity chamber which also aid to
provide the appropriate environment to sample.
(2 Marks)
3) In point 7 of the protocol where it says ‘Block the tissue section by adding two drops
of PBS buffer containing 3% BSA’. What is PBS and BSA and why are they used?
Is use for preventing enzymatic changes in the tissues section from the open

environment.
PBS is the Phosphate-buffered saline which is use in the biological research,
basically it aid to mange the appropriate pH level. This is the mix solution sodium
chloride, potassium chloride, potassium dihydrogen phosphate, and disodium
hydrogen phosphate, Its a water based salt solution.
BSA (Bovine serum albumin) is the serum globular protein extracted from the
cows, it is used by the researchers and biological student in multiple projects as it
make the sample tissues and cells stable and do not interfere with biological
activities of cells.
(2 Marks)
4) The primary antibody from the patient sample targeted the dsDNA in the Crithidia
luciliae. The secondary antibody was a goat anti-human FITC conjugate. What do the
words ‘goat’ ‘anti-human’ and ‘FITC conjugate’ tell you about the antibody?
'Anti-human' is the human IgG antibody.
FITC conjugate is the antibody against human IgG.
It means the antibody against human IgG which can be use in
Immunofluorescence.

(2 Marks)
5) Is the method you have just performed an example of a direct or an indirect immuno-
assay and why? (See Fig. 1)
Here in this practical indirect immunoassay is performed.
IIFT (Indirect immuno-fluorescence test) most specific immunoassay use for the
detection of anti-dsDNA antibody.
Anti-double stranded DNA test use to study lupus or diagnose the individual after
the positive result of (ANA) anti-nuclear antibody test.
(2 Marks)
END OF QUESTIONS