Department of Biotechnology Engineering and

Food Technology

University Institute of Engineering

LAB MANUAL (20BEP-255)

B.E. Biotechnology, 2nd Year

Semester-4th
(Session: 2021-22)

Immunology LAB
SUBJECT CODE: 21BEH-223
BRANCH: B.E. Biotechnology

Dr. Deepak Kala

Assistant Professor
UIE, Chandigarh University, Gharuan
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 Department Vision

To become a foremost center of excellence in the field of biotechnology engineering by nurturing

students with a potential to invoke a scientific temperament leading to scholastic achievements that
can shape biotechnology into a premier precision tool for the creation of health, wealth, and
prosperity at the global scale.

Department Mission

1. Develop a frontline biotechnology engineering program based on quality education, research,

and training.
2. To train high-quality engineering professionals who can cater to the national and global
challenges in biotechnology while contributing to the continuous improvement of biotechnological
services and products as per industry demands.
3. To offer a vibrant and dynamic ecosystem for knowledge creation, instilling scientific and
technical resources by inculcating the zeal of innovation and creativity in young minds with good
research aptitude.
4. To explore the full potential of biotechnology in advancing, improving, or creating the new
generation of products, processes, and technologies while enhancing efficiency, productivity, cost-
effectiveness, and promoting entrepreneurship.
5. To popularize biotechnology and promote large-scale use of biotechnology through reputed
global collaborations, outreach, and alumni engagement.

Program Outcomes (PO's)

PO1: Engineering Knowledge: Apply the knowledge of mathematics, science, engineering

fundamentals, and an engineering specialization to solve complex engineering problems. 
PO2: Problem Analysis: Identify, formulate, review literature, and analyze complex engineering
problems to reach substantiated conclusions using the first principles of mathematics, natural
sciences, and engineering sciences. 

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PO3: Design/Development of Solutions: Design solutions for complex engineering problems and
design system components or processes that meet the specified needs with appropriate consideration
for public health, safety, and cultural, societal, and environmental considerations. ·
PO4: Conduct Investigations of Complex Problems: Use research-based knowledge and research
methods, including design of experiments, analysis, and interpretation of data, and synthesis of the
information to provide valid conclusions. 
PO5: Modern Tool Usage: Create, select, and apply appropriate techniques, resources, and modern
engineering and IT tools, including prediction and modeling to complex engineering activities to
understand the limitations. 
PO 6: The Engineer and Society: Apply reasoning informed by the contextual knowledge to assess
societal, health, safety, legal and cultural issues and the consequent responsibilities relevant to the
professional engineering practice.
PO 7: Environment and Sustainability: Understand the impact of the professional engineering
solutions in societal and environmental contexts, and demonstrate the knowledge of and need for
sustainable development. 
PO 8 : Ethics: Apply ethical principles and commit to professional ethics and responsibilities and
norms of the engineering practice. · 
PO 9: Individual and Team Work: Function effectively as an individual and a member or leader in
diverse teams and multidisciplinary settings. ·
PO 10: Communication: Communicate effectively on complex engineering activities with the
engineering community and with society at large, such as being able to comprehend and write
effective reports and design documentation, make effective presentations, and give and receive clear
instructions. 
PO 11: Project Management and Finance: Demonstrate knowledge and understanding of the
engineering and management principles and apply these to one's work as a member and leader in a
team, to manage projects and in multidisciplinary environments. 
PO 12: Life-Long Learning: Recognize the need for and have the preparation and ability to engage
in independent and life-long learning in the broadest context of technological change.

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 Program Educational Objectives (PEO’s)

1. Demonstrate the ability to apply biotechnology engineering fundamentals in finding solutions

to industrial problems while harmonizing health, safety, environment, and sustainability.
2. Become employable and successful as engineering professionals in emerging and allied areas
of industrial/medical biotechnology, bioinformatics, microbiology, plant biotechnology and genetics.
3. Design multidisciplinary engineering solutions for biotechnology systems.
4. Pursue higher education and research to solve problems of engineering relevance and to
create opportunities in the new areas of biotechnology.
5. Develop entrepreneurship skills and competency while maintaining a high standard of
professional ethics to serve society.

Program Specific Outcomes (PSO’s)

1. Formulate and execute fundamental and advanced biotechnology engineering solutions at an

industrial scale.
2. Supervise and operate on modern analytical tools, techniques, and instruments of
biotechnology and implement biotechnology engineering solutions to local, national, and global
demands.
3. Determine the gaps in knowledge by connecting interdisciplinary aspects of biotechnology;
test and implement the viable protocols while choosing higher education of the domain.

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SN Title L T P S C CH Course
20BEP-255 Type
1 Immunology Lab 0 0 2 0 1 2 PC

Pre-requisites/ Basic Concepts of Biology

Exposure
Co-requisites NA

Anti-Requisite NA

a. COURSE DESCRIPTION
This course is to provide detail demonstrate isolation, separation and quantification of various cells and
biomolecules of immune system present in blood. Students are able to demonstrate isolation, separation
and quantification of various cells and biomolecules of immune system present in blood

b. COURSE OBJECTIVES
Students should hold knowledge in basic biology and immunology. Students should have read the basic
concepts in molecular and cell biology techniques.

COs Immunology Lab

CO1 To develop the skills to perform the basic practical of Serology

CO2 To provide scientific understanding of diagnosis techniques and detail interpretation of results.

CO3 To acquire the skills of basic and advanced serological testing.

CO4 To understand the concept of Ag-Ab interaction and their application in immunology

CO5 To understand the basics of agglutination reaction part of medical diagnostics used in
haematology and diagnostic microbiology

List of Practical’s
Experiment
Content CO
No.
1 To understand the basic concept of Blood Grouping. CO1

2 Total leukocyte count CO1

3 Blood Film Preparation and identification of cells CO1

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 4 Differential white blood cell count (Differential leukocyte count) CO2

5 Separation of serum & plasma from blood CO2

6 To perform the ELISA of given protein sample CO3

7 Radial Immunodiffusion CO4

8 Rocket Immuno electrophoresis CO4

9 Ouchterlony Double diffusion CO4

10 Agglutination inhibition test CO5

Reference Books:
1. Practical immunology, Frank Hay, 4th Edition , Blackwell Science
2. Medical Microbiology, Anantnarayan
3. Introduction to Practical Biochemistry, D.T. Plummer, Tata MacGraw Hill
4. A Handbook of Practical Immunology – G P Talkwar
5. Text Book of Medical Biochemistry, Praful Godkar. Bahalani Publishers.

COs-POs Mapping

Course
PO1 PO2 PO3 PO4 PO5 PO6 PO7 PO8 PO9 PO10 PO11 PO12 PSO1 PSO2 PSO3
Outcome

CO1 3 2 1 2 1 NA NA NA 1 1 1 NA 2 NA 2

CO2 2 1 1 2 2 NA NA NA 1 1 1 NA 2 NA 2

CO3 3 3 1 3 2 NA NA NA 2 1 2 NA 3 NA 3

CO4 3 3 1 3 2 NA NA NA 3 1 3 NA 3 NA 3

CO5 3 3 1 2 3 NA NA NA 3 1 3 NA 3 NA 3

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 Experiment No. 1

Aim: To understand the basic concept of Blood Grouping.

Theory:

ABO blood grouping system:

 According to the ABO blood group system there are four different kinds of blood groups: A, B, AB
and O (null).

Blood group A

Blood group B

Blood group AB

Blood group O

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Rh factor

Rh (Rhesus) factor is found on the RBC's surface in most

people. Like A and B, this is also an antigen and those who
have it are called Rh+. Those who lack the antigen on the
surface of RBCs are called Rh-. A person with Rh- blood
does not have Rh antibodies naturally in the blood plasma.
But a person with Rh- blood can develop Rh antibodies in the blood plasma if he or she receives
blood from a person with Rh+ blood, whose Rh antigens can trigger the production of Rh antibodies
(as the immune system is triggered by the presence of an unknown antigen in the system). A person
with Rh+ blood can receive blood from a person with Rh- blood without any problem.

Principle: Blood clumping or Agglutination observation.

Compatibility between the blood groups of donor and recipient determines the success of a blood
transfusion. The ABO and Rh blood groups are looked at while conducting the test. In a diagnostic
lab, Monoclonal antibodies are available for A, B and Rh antigen. Monoclonal antibody against
Antigen A (also called Anti-A), comes in a small bottle with droppers; the monoclonal suspension
being BLUE in colour. Anti-B comes in YELLOW colour. Anti-D (monoclonal antibody against Rh)
is colorless. All the colour codes are universal standards. When the monoclonal antibodies are added
one by one to wells that contain the test sample (blood from patient), if the RBCs in that particular
sample carry the corresponding Antigen, clumps can be observed in the corresponding wells. A drop
of blood is left without adding any of the antibodies; it is used as a control in the experiment.

Materials Required:

Monoclonal Antibodies (Anti-A, B and D), Blood Lancet, Alcohol swabs, Tooth picks, Sterile cotton
balls, clean glass slide, Biohazard disposal container

Procedure:

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 1. Set the table with all the materials required. Remember to place the Monoclonal Antibody
(Mab) kit in an Ice tray.

2. Open an Alcohol swab, and rub it at the area from where the blood will be sampled
(fingertip). (Discard the swab)

3. Open the Lancet cover, put pressure at the tip of the finger from where blood will be sampled
(maintain it). Prick the fingertip with the opened Lancet. (Discard the Lancet)

4. As blood starts oozing out, make 1 drop fall on the three depressions of the glass slide. (In
clinical setup, there will be a fourth well used as a control).

5. Place a cotton ball at the site where it was pricked. Using the thumb, put pressure on the area
to stop blood flow.

6. Take the Anti-A (blue) bottle, resuspend the content and use the dropper to place a drop of
the Mab in the 1st spot. Place the bottle back in ice.

7. Take the Anti-B (yellow) bottle, resuspend the content and use the dropper to place a drop of
the Mab in the 2nd spot. Place the bottle back in ice.

8. Take the Anti-D (colorless) bottle, resuspend the content and use the dropper to place a drop
of the Mab in the 3rd spot. Place the bottle back in ice.

9. Take a tooth pick and mix the content in each well. Discard the tooth pick after using in one
well (take a new one for the next well).

10. After mixing, wait for a while to observe the result.

Pre-Lab Questions

1. What are the 8 blood types humans can have using the A-B-O system and Rh factor?

2.What is Universal donor blood type?

3. What is Universal acceptor blood type?

Post-Lab Questions

1. If you have Type AB blood, which antigens would be present on your red blood cells?

2. If you have Type B blood, which antibodies would your body produce?

3. If a person with Type B blood receives a transfusion of Type O blood, what will result?

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 Experiment No. 2

Aim: Total leukocyte count

Introduction

Blood represents about 8% of total body weight. It consists of 3 types of specialized cellular
elements: Erythrocytes (RBCs), Leukocytes (WBCs), Platelets (thrombocytes). These cells are
suspended in complex liquid plasma. Blood performs two major functions

1. Transport through the body: O2 & CO2, Food molecules (Glucose, lipid, aa), Wastes (ex. Urea),
Hormones, heat.

2. Defense of the body against infections & other foreign materials, all WBCs participate in these
defenses.

WBCs (leukocytes) have nuclei and consist of lymphocytes and 3 types of granulocytes whose
cytoplasm contain granules. WBCs count is the count of leukocytes in a volume of blood and
expressed as WBCs/mm³. The number of WBCs is very large, so it is practical to dilute a sample
with diluting solution (2% Glacial acetic acid with methylene blue). This solution will lyse
cytoplasmic membrane, and leave the nuclei of WBCs.

The Hemacytometer contains 2 Neubauer counting chamber. Each chamber contains:

 *4 WBC counting squares

 *Each contains 16 squares

Materials required:

1. Blood sample (EDTA anticoagulated blood or capillary blood)

2. WBCs diluting pipette

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 3. Diluting solution (2% AA with methylene blue)

4. Hemocytometer

5. Microscope

Principle

Whole blood is diluted with a 2% acetic acid solution, which hemolyzes mature erythrocytes and
facilitates leukocyte counting. The standard dilution for leukocyte counts is 1:20. The dilution is
mixed well and incubated to permit lysis of the erythrocytes. Following the incubation period, the
dilution is mounted on a Hemocytometer. The cells are allowed to settle and then are counted in
specific areas of the Hemocytometer chamber under the microscope. The number of leukocytes is
calculated per µL (x 109/L) of blood.

Procedure

1. Clean the hemocytometer and cover glass by flooding them with 70% alcohol. Dry
thoroughly with gauze or tissue; do not allow the alcohol to dry on the hemocytometer. Be
sure to remove all lint. Place the cover glass in position over the ruled area.
2. Put the cover slip or glass slip on the top of grid area in the Chamber (use air tight technique).
3. Dilute your sample: 1: 20 for WBC count
4. Load your sample into the loading area in the chamber
5. Count the cells in the 4 large squares for WBC
6. Calculate the number of cells counted / µL.

Dilution:

1:20 dilution or 1:50 (ex: chronic leukemia)

(1+19=20)

(50µL of blood + 950 µL diluent)

Calculations

Cells/ µL = no. of cells in 1 large square x Dilution factor

volume factor (0.4)

Dilution factor= reciprocal of dilution (20)

Volume factor = (area * depth) = (4*0.1) = 0.4 mm3

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Pre-Lab Questions

1. What is a Neubauer’s Chamber?

2. Explain in detail about different types of WBCs.

3. What is the formula for calculating total numbers of WBCs?

Post-Lab Questions

1. What is the composition of WBC diluting Fluid (Turk’s Fluid)?

2. What are the normal values of white blood cells?

3. What are the precautions to be taken while performing total leucocyte count by hemocytometer?

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 Experiment No. 3

Aim: Blood Film Preparation and identification of cells

Theory:
Blood film enables us to evaluate WBC, RBC, and PLT morphology, also, allows us to perform
differential WBC count, furthermore estimation of WBC and platelets counts can be done on blood
films. Blood films are made on glass microscopic slides.
Principle:
A Romanowsky stain is a stain combination consisting of eosin Y or eosin B with methylene blue
and/or any of its oxidation’s products. Such stains produce the typical purple coloration of leukocyte
nuclei and neutrophilic granules as well as the numerous blues and pinks found in other cell types.
.Methyl alcohol is used as both a solvent and fixative in this procedure

Various stains for peripheral blood film:  Romanowsky stains are universally employed for
staining of blood films. All Romanowsky combinations have two essential ingredients i.e.
methylene blue and eosin or azure. • Methylene blue is the basic dye and has affinity for acidic
component of the cell (i.e. nucleus) and eosin/azure is the acidic dye and has affinity for basic
component of cell (i.e. cytoplasm). • Most Romanowsky stains are prepared in methyl alcohol
so that they combine fixation and staining.

Sample:
EDTA anticoagulated blood, slides, distilled water, cotton, antiseptic, stain
Procedure:
1- Use clean standard size glass slides (3-inch x 1 inch = 7.5 cm x 2.5 cm), wiped from dust
just immediately before use.
2- Place a small drop of well mixed anticoagulated whole blood, in the center line of the
slide, about 1.5 to 2 cm from one end.
3- Immediately, without delay, with the aid of a second clean slide with uniform smooth
edges (spreader slide), with a 45 degrees angle, move back so blood drop will spread

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 along the edge of the spreader slide, when this occurs, spread, or smear the film by a
quick and uniform forward motion of the spreader.

Staining Blood Films with Romanowsky Stains

Blood films are stained so that morphology of blood cells become more easily viewed,
identified, and evaluated. In addition, blood films may be examined for the presence of blood
parasites (Malaria, Trypanosoma, Babesia). Furthermore, stained blood films can provide important
information about a patient’s health, they may lead to a diagnosis or verify a diagnosis, or they may
rule out a diagnosis. Evaluation of stained blood films also may lead to the decision of performing
other hematology special blood stain procedures in order to identify specific cell components.
The widely and popular used Romanowsky stains are:
 Leishman Stain
 Wright’s Stain
Leishman Stain Procedure:
1- Let the films be air dried.

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 2- Put the films on a staining trough rack.
3- Flood the slides with the stain.
4- After 2 minutes (or more, if the stain in newly prepared), add double volume of water,
and blow to mix the stain with water, until a shiny layer is seen.
5- After 5-7 minutes, wash with a stream of water.
6- Wipe the back of the slides with gauze.
7- Set the films in upright position on a filter paper to dry.
8- Read the blood films microscopically.

If delay in staining blood films may occur, fix the films in absolute
methanol, for 1-2 minutes, but do not stain the slides until completely
dried.

Observation:

Romanowsky Stain Blood Cell Characteristics

No. Cell Structure Staining characteristics

1 Red cells Red or pinkish red

2 Nuclei of all cell types Purple/violet

3 Lymphocyte cytoplasm Blue

4 Monocyte cytoplasm Grayish blue

5 Platelets cytoplasm Light blue

6 Neutrophilic granules Violet-pink

7 Eosinophilic granules Orange-red

8 Basophilic granules Purplish black/ Deep blue

9 Platelets granules Purple

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Precautions:
1. Before preparing the films, you must check that blood samples are free from clots, and
this is done with two wooden applicator sticks. If clots are present the specimen is
unsatisfactory.
2. With large blood drops, increase the spreading angle.
3. With small blood drops, decrease the spreading angle.

Pre-Lab Questions

1. What is the function of Romanowsky stain?

2. What are the granulocyte cells?

3. What are the precautions we should take during the blood film preparation experiment?

Post-Lab Questions

1. What are the staining characteristic of nuclei of all cell types?

2. What are the staining characteristic of neutrophilic and eosinophilic granules?

3. What are the staining characteristic of monocyte and lymphocyte cytoplasm?

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 Experiment 4

Aim: Differential white blood cell count (Differential leukocyte count)

Theory

The white blood cell differential count determines the number of each type of white blood cell,
present in the blood. It can be expressed as a percentage (relative numbers of each type of WBC in
relationship to the total WBC).
Blood smear

The preparation and examination of the peripheral blood smear is one of the most frequently
requested tests in the hematology laboratory. Blood smear must be prepared correctly and examined
in such a way as to provide the physician with an accurate interpretation.

Principle:
A Romanowsky stain is any stain combination consisting of eosin Y or eosin B with methylene blue
and/or any of its oxidations products. Such stains produce the typical purple coloration of leukocyte
nuclei and neutrophilic granules as well as the numerous blues and pinks found in o
Procedure

1- Place a drop of blood from the finger, about 2 mm in diameter in the central line of a slide
about 1- 2 cm from one end.
2- The spreader is placed at an angle of 45 degrees to the slide and then moved back to make
contact with the drop.
3- The drop should spread out quickly along the line of contact of the spreader with the slide.
4- The drop should be of such size that the film is 3- 4 cm in length.
5- The film should be dried rapidly. A good blood film preparation will be thick at the drop end
and thin film at the opposite end.
6- The thickness of the spread when pulling the smear is determined by the :
a- Angle of the spreader slide.
b- Size of the blood drop.
c- Speed of spreading.

Staining

Leishman’s stain

Method of staining

1- Pour few drops (about 8) on the slide, wait for two minutes.

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 2- Add double the amount (16 drops) of buffered water. Mix by rocking and wait for 7- 10
minutes.
3- The stain is flooded off with distilled water and this should be complete in 2- 3 second, and
allow it to dry.
4- The blood film is fixed with methyl alcohol for 2 minutes.
5- Pour Giemsa stain diluted 1:9 with buffer over the smear for 8 -10 minutes.
6- Wash off and dry.

Examination of a blood film

There are several necessary steps in the examination of a peripheral blood smear:

1- Under the low- power (10 x), determine the overall staining quality of the blood smear.
2- Determine if there is a good distribution of cells on the smear.
3- Find an optimal area foe the detailed examination and enumeration of cells.

The count

The dry and stained film is examined without a coverslip under oil immersion objective. For
differential leucocyte counts choose an area where the morphology of the cells is clearly visible. Do
differential count by moving the slide in area including the central and peripheral of the smear. A
total of 100 cells should be counted in which every white cell seen must be recorded in a table under
the following heading: Neutrophil, Basophil, Eosinophil, Monocyte and Lymphocyte.

Then find the percentage of each type.

Neutrophil Eosinophil Basophil

Lymphocyte Monocyte

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Pre-Lab Questions

1. What is the significance of DLC (Differential leukocyte count)?

2. A normal differential White Blood Cell count would have what percentage of Neutrophils?

3. A normal differential White Blood Cell count would have what percentage of Lymphocytes?

Post-Lab Questions

1. Elevated Numbers of Neutrophils might indicate what condition?

2. Elevated Numbers of Basophils might indicate what condition?

3. Elevated Numbers of Monocytes might indicate what condition?

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 Experiment No. 5

Aim: Separation of serum & plasma from blood

Theory:

Blood plasma is the liquid component of blood, in which the blood cells are suspended. It makes up
about 60% of total blood volume. It is composed of mostly water (90% by volume), and contains
dissolved proteins, glucose, clotting factors, mineral ions, hormones and carbon dioxide (plasma
being the main medium for excretory product transportation). Plasma is the supernatant fluid
obtained when anti-coagulated blood has been centrifuged. The blood is mixed with an appropriate
amount of anticoagulant like heparin, oxalate or ethylenediaminetetraacetic acid (EDTA). This
preparation should be mixed immediately and thoroughly to avoid clotting.

Blood serum is blood plasma without fibrinogen or the other clotting factors. Serum is clearer than
plasma because of fewer proteins. Proteins are sometimes considered as interfering substances in
some tests as they react with the reagent and thereby yield inaccurate results. Serum is the preferred
specimen in clinical testing as the interference that may be caused by a plasma specimen because of
the presence of an anticoagulant, is eliminated.

Principle:

Blood serum can be defined in a number of ways. Clear, watery fluid of the blood that separates
when blood clots or blood plasma from which the protein fibrinogen or clotting factors, has been
removed.  Clotting factors are the proteins which causes the clotting of blood. when the blood is
allowed to clot after its withdrawn from a vein, the clot slowly shrinks and a clear watery  fluid
squeezed out from the clot is known  as serum.
 

Materials and Equipment:

Human blood sample, Eppendorf tubes containing anticoagulant (EDTA, heparin etc.), pipettes of
appropriate volumes (sterile) · Centrifuge tubes, Benchtop centrifuge with swing-out rotor and
appropriate carriers

Procedure:

Separation of plasma

1) Blood will be collected into EDTA tubes and centrifuged (2000 rpm) at 4 degrees centigrade for
20 minutes.

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2) After centrifugation using clean pipette place 1.0ml of plasma into 1.5ml Eppendorf tube.

3) Freeze immediately at –80 degree freezer.

Separation of Serum

1. A 10 ml tube of whole blood will be collected from each patient.

2. Allow samples to clot for one hour at room temperature

3. Centrifuge for 10 minutes at approximately 3000 rpm.

4. Using clean pipette collect the serum isolated.

5. Immediately freeze vials of serum at –80-degree freezer.

Precautions:

1. Gently invert the tube of blood to mix contents.

2. Carefully open blood tube (universal precautions; gloves, eye protection)
3. With clean pipette tip aliquot appropriate amount of whole blood into clean tubes.
4. Use appropriate amount of anticoagulant.

Pre-Lab Questions

1. What is the difference between serum and plasma?

2. Explain the colour coding of different blood collection tubes?

3. What are the functions of plasma and serum?

Post-Lab Questions

1. What is the storage condition for serum samples?

2. What are the different analysis we can perform using serum and plasm samples?

3. What are the universal precautions must be used when working with blood?

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 Experiment No. 6

Aim: To perform the ELISA of given protein sample (To determine the antigen concentration by
Antigen Capture ELISA method)

Theory: ELISA is the basic assay technique, known as enzyme-linked immunosorbent assay (also
referred to as EIA: Enzyme Immunoassay) that is carried out to detect and measure antibodies,
hormones, peptides and proteins in the blood.
Antibodies are blood proteins produced in response to a specific antigen. It helps to examine the
presence of antibodies in the body, in case of certain infectious diseases.
ELISA is a distinguished analysis compared to other antibody-assays as it yields quantitative results
and separation of non-specific and specific interactions that take place through serial binding to solid
surfaces, which is normally a polystyrene multiwell plate.

Types Of ELISA
ELISA tests can be classified into three types depending upon the different methods used for binding
between antigen and antibodies, namely:
 Indirect ELISA – Antigen is coated to the microtiter well
 Sandwich ELISA – Antibody is coated on the microtiter well
 Competitive ELISA – Microtiter well which is antigen-coated is filled with the antigen-
antibody mixture.

Indirect ELISA
 Indirect ELISA detects the presence of an antibody in a sample.
 The antigen is attached to the wells of the microtiter plate.
 A sample containing the antibodies is added to the antigen-coated wells for binding with the
antigen.
 The free primary antibodies are washed away and the antigen-antibody complex is detected
by adding a secondary antibody conjugated with an enzyme that can bind with the primary
antibody.
 All the free secondary antibodies are washed away. A specific substrate is added which gives
a coloured product.
 The absorbance of the coloured product is measured by spectrophotometry.

Sandwich ELISA
 Sandwich ELISA helps to detect the presence of antigen in a sample.
 The microtitre well is coated by the antibody.

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  The sample containing the antigen is added to the well and washed to remove free antigens.
 Then an enzyme-linked secondary antibody, which binds to another epitope on the antigen is
added. The well is washed to remove any free secondary antibodies.
 The enzyme-specific substrate is added to the plate to form a coloured product, which can be
measured.

Competitive ELISA
 Competitive ELISA helps to detect antigen concentration in a sample.
 The microtitre wells are coated with the antigen.
 Antibodies are incubated in a solution having the antigen.
 The solution of the antigen-antibody complex is added to the microtitre wells. The well is
then washed to remove any unbound antibodies.
 More the concentration of antigen in the sample, lesser the free antibodies available to
interact with the antigen, which is coated in the well.
 The enzyme-linked secondary antibody is added to detect the number of primary antibodies
present in the well.
 The concentration is then determined by spectrophotometry.

Principle of ELISA: ELISA works on the principle that specific antibodies bind the target antigen
and detect the presence and quantity of antigens binding. In order to increase the sensitivity and
precision of the assay, the plate must be coated with antibodies with high affinity. ELISA can
provide a useful measurement of antigen-antibody concentration.

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Materials and Method:

This kit can be used for the determination of antigen concentration bound to immobilized antibody
followed by binding of the antigen to the labelled secondary antigen and its detection by using
appropriate substrate.

Materials required but not provided:

Glass wares: Measuring cylinder, Test Tubes
Reagents: Distilled water
Other requirements: Blotting paper, Micropipette, Tips
Prepare the reagents as indicated below before starting each experiment:
Preparation of sample diluent: Take 1 ml of blocking buffer and make up the volume to 30 ml with
1X PBST. Use this to dilute standard antigen and HRP labeled antigen.
Preparation of dilutions of standard antigen: Concentration of standard antigen is 1 mg/ml; dilute this
to get a range of concentrations using sample diluent, as follows:

24
Procedure:

Day 1: Coating of wells with antibody

1. Dilute 50 µl of antibody with 4.95 ml of coating buffer. Concentration of the diluted antibody is
1µg/ml.

2. Pipette 200 µl of diluted antibody into each well of the microtiter plate (24 wells). Tap or shake
the plate so that the antibody solution is evenly distributed over the bottom of each well.

3. Incubate the wells overnight at 4°C. Day 2: Blocking the residual binding sites on the wells.

4. Discard the well contents. Rinse the wells with distilled water for three times by draining out the
water after each rinse.

5. Fill each well with 200 µl of blocking buffer and incubate at room temperature for 1 hour.

6. Rinse the plate three times (as given above) with distilled water. Drain out the water completely by
tapping the plate on a blotting paper.

Addition of antigen to the wells

7. Prepare standard and test antigen dilutions as given above.

8. Add 100 µl of standard antigen, test samples and 1X PBST to the coated wells (in duplicates) as
shown in the following picture.

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Addition of HRP labeled antigen

9. Dilute 3 µl of HRP labeled antigen with 3 ml of sample diluent

10. Add 100 µl of HRP labeled antigen to all the wells.

11. Incubate at room temperature for 30 minutes.

12. Discard the well contents; fill the wells with 1X PBST, allow it to stand for 3 minutes, discard the
contents

Repeat this step twice.

Addition of substrate and measurement of absorbance

15. Dilute required amount of 10 X TMB/H2O2 (substrate) solutions to 1X using distilled water.

16. Add 200 µl of 1X substrate to each well.

17. Incubate at room temperature for 10 minutes.

18. Add 100 µl of 1X stop solution to each well.

19. Transfer the contents of each well to individual tubes containing 2 ml of 1X stop solution.

20. Prepare substrate blank by adding 200 µl of 1X substrate solution to 2.1 ml of 1X stop solution.

21. Read the absorbance at 450 nm after blanking the spectrophotometer with substrate blank.

26
Observation and Result: Look for the development of blue colour in the wells at the end of the
experiment. Read the absorbance at 450 nm after blanking the spectrophotometer with substrate
blank and record the readings as follows:

Calculation of antigen concentration in test sample: Calculate the average A450 for each of the
samples (standard and test) and plot A450 of standards on Y axis (linear scale) versus the
concentration of antigen in µg/ml on X axis (log scale) on a semi-log graph sheet as shown below:

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Pre-Lab Questions

1. What does ELISA stand for?

2. Why are enzymes used in this immunoassay?

3. What do you understand by sensitivity and specificity of an assay?

Post-Lab Questions

1. Why do you need to assay positive and negative control samples as well as your own

experimental samples?

2. What happened to the proteins in the plastic well if the sample contained the antigen?

3. Why did you need to wash the wells after each step?

28
 Experiment 7

Aim: To study the immunodiffusion technique by Single Radial Immunodiffusion.

Introduction: Single Radial Immunodiffusion, also known as Mancini technique, is a quantitative

immunodiffusion technique used to detect the concentration of antigen by measuring the diameter of
the precipitin ring formed by the interaction of the antigen and the antibody at optimal concentration.
In this method the antibody is incorporated into the agarose gel whereas the antigen diffuses into it in
a radial pattern.

Principle: Single Radial Immunodiffusion is used extensively for the quantitative estimation of
antigen. Here the antigen-antibody reaction is made more sensitive by the addition of antiserum into
the agarose gel and loading the antigen sample in the well. As the antigen diffuses into the agarose
radially in all directions, it’s concentration continuously falls until the equivalence point is reached at
which the antigen concentration is in equal proportion to that of the antibody present in the agarose
gel. At this point ring of precipitation (‘precipitin ring’) is formed around the well. The diameter of

29
the precipitin ring is proportional to the concentration of antigen. With increasing concentration of
antigen, precipitin rings with larger diameter are formed.

The size of the precipitin rings depends on

 Antigen concentration in the sample well

 Antibody concentration in the agarose gel
 Size of the sample well
 Volume of the sample

Thus, by having various concentrations of a standard antigen, standard curve can be obtained from
which one can determine the amount of an antigen in an unknown sample. Thus, this is a quantitative
test. If more than one ring appears in the test, more than one antigen/antibody reaction may have
occurred. This could be due to a mixture of antigens or antibodies.

This test is commonly used in the clinical laboratory for the determination of immunoglobulin levels
in patient samples.

Kit Contents:

The Kit can be used to perform single radial Immunodiffusion technique.

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Materials Required But Not Provided:

Glass wares: Conical flask, Measuring cylinder, Beaker

Reagents: Distilled water, alcohol

Other requirements: Incubator (37oC), Microwave or Bunsen burner, Vortex mixer, spatula,
Micropipettes,

Tips, Moist chamber (box with wet cotton)

Procedure:

1. Prepare 10 ml of 1% agarose gel (as give in the important instructions). Take 6 ml of this gel
solution in a clean test tube.

2. Allow the solution to cool down to 55-60oC and add 80 µl of antiserum to 6 ml of agarose
solution. Mix well for uniform distribution of the antibody.

3. Pour agarose solution containing the antiserum on to a grease free glass plate placed on a
horizontal surface. Allow the gel to set for 30 minutes.

4. Place the glass plate on the template provided.

5. Punch wells with the help of gel puncher corresponding to the markings on the template. Use
gentle suction to avoid forming rugged wells.

6. Add 10 µl of the given standard antigen and test antigen samples to the wells. A. Standard
Antigen A (3.75 mg/ml) B. Standard Antigen B (7.5 mg/ml) C. Standard Antigen C (15 mg/ml) D.
Standard Antigen D (30 mg/ml) E. Test Antigen 1 F. Test Antigen 2

31
7. Incubate the glass plate in a moist chamber overnight at 37oC.

Observation and Result:

Observe for precipitin rings surrounding the antigen wells (Fig 3). Mark the edges of the precipitin
rings and measure the diameter of the rings as shown in table 2.

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Interpretation: The diameter of the precipitin ring depends upon the concentration of antigens
loaded in the wells. By plotting the graph of concentration of antigens versus diameter of the
corresponding precipitin ring one can calculate the concentration of any test antigen.

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Pre-Lab Questions

1. What is the significance of RID test?

2. What do you understand by qualitative and quantitative estimation?

3. What is prozone of effect?

Post-Lab Questions

1. Enlist the limitations of RID.

2. How does the area of equivalence and ring of precipitation occur?

3. Precipitin ring is proportional to?

4. How is a standard curve obtained?

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 Experiment 8

Aim: To study the technique of Rocket Immunoelectrophoresis for determination of the

concentration of antigen in unknown sample.

Introduction: Rocket Immunoelectrophoresis, also known as electro-immunodiffusion, is a simple,

quick and reproducible method for determining the concentration of antigen in an unknown sample.
This quantitative one-dimensional immunoelectrophoresis method involves a comparison of antigen
sample of unknown concentration with a series of dilutions of a known concentration of the antigen
and requires a monospecific antibody against the antigen under investigation. In this method, antigen
migrates from the well through agarose gel containing antiserum, forming rocket shaped precipitin
peaks. The height of this peak is proportional to the concentration of the antigen loaded in the
corresponding well.

Principle: In Rocket Immunoelectrophoresis, negatively charged antigen samples are

electrophoresed in an agarose gel containing antibody which is specific to that antigen. As the
antigen moves out of the well and enters the agarose gel, it combines with the antibody to form
immune complex which is visible as white. Because the antigen is migrated through the gel under the
influence of an applied electric current, it moves in one direction. During the initial phase there is
considerable antigen excess over antibody and no visible precipitation occurs. However, as the
antigen sample migrates further through the agarose gel, more antibody molecules are encountered
that interact with the antigen to form immune complex. When these immune complexes become
large enough to be retained within the gel, movement of the antigen stops. The area of precipitin has
the shape of a rocket and its height is proportional to the concentration of antigen in the
corresponding well.

35
Materials Required But Not Provided:

Glass wares: Conical flask, Measuring cylinder, Beaker Reagents: Sterile distilled water, alcohol.
Other requirements: Incubator (37oC), Microwave or Bunsen burner, Vortex mixer, spatula,
Micropipettes, Tips, Moist chamber (box with wet cotton).

Procedure:

1. Prepare 15 ml of 1 % agarose (as given in important instructions).

2. Cool the solution to 55-60o C and add 250 l of antiserum to 13 ml of agarose solution. Mix well
for uniform distribution of antibody.

3. Pour agarose solution containing the antiserum on to a grease free glass plate placed on a
horizontal surface. Allow the gel to set for 30 minutes.

4. Place the glass plate on the template provided.

5. Punch wells with the help of gel puncher. Use gentle suction to avoid forming rugged wells.

6. Add 10 l of the given standard antigen and test antigen samples to the wells. A. Standard
Antigen A (1.87 mg/ml) B. Standard Antigen B (0.94 mg/ml) C. Standard Antigen C (0.47 mg/ml)
D. Standard Antigen D (0.23 mg/ml) E. Test Antigen 1 F. Test Antigen 2.

36
 7. Pour 1X TBE buffer into the electrophoresis tank such that it just covers the gel. Note: The
remaining 1X TBE buffer can be stored at room temperature.

8. Electrophorese at 80-120 volts and 60-70 mA, until the blue dye travels 3-4 cm from the well. Do
not electrophorese beyond 3 hours, as it is likely to generate heat.

9. Incubate the glass plate in a moist chamber overnight at 37oC.

Observation and Result: Observe for precipitin peaks in the shape of ‘Rocket’ formed against a
dark background. Mark the tip of the precipitin peaks and measure the peak height from the upper
edge of the well to the tip of the peak as shown in table 2.

37
Interpretation: The height of the precipitin peak depends on the concentration of antigens loaded in
the corresponding wells. By plotting the graph of concentration of antigens versus length of the
precipitin peaks one can calculate the concentration of test antigen.

Pre-Lab Questions
1. What is the principal of immunoelectrophoresis?
2. What are the advantages of immunoelectrophoresis?
3. What are the disadvantages of immunoelectrophoresis?

Post-Lab Questions
1. How we can calculate the concentration of antigen in rocket immunoelectrophoresis?
2. How standard graph is obtained in immunoelectrophoresis?
3. What is zone of equivalence?

38
 Experiment 9

Aim: To study the reaction pattern of an antigen with a set of antibodies by Ouchterlony Double
Diffusion method.

Introduction: Immunodiffusion in gels encompasses a variety of techniques, which are useful for
the analysis of antigens and antibodies. Gel immunodiffusion can be classified into two groups:

1. Single Immunodiffusion 2. Double Immunodiffusion

In the Ouchterlony double diffusion, both the antigen and the antibody diffuse toward each other in a
semisolid medium to a point till their optimum concentration is reached. A band of precipitation
occurs at this point. The qualitative Ouchterlony Test can simultaneously monitor multiple Antibody-
Antigen system and can be used to identify particular antigens in a preparation. This procedure was
developed by Örjan Ouchterlony.

Principle: When soluble antigen and antibody samples are placed in adjacent wells in agarose gel,
they diffuse radially into the agarose gel and set up two opposing concentration gradients between
the wells. Once the gradients reach to an optimal proportion, interactions of the corresponding
molecules occur and a line of precipitation will form. Using such a technique, the antigenic
relationship between two antigens can be analyzed. Distinct precipitation line patterns are formed
against the same anti-sera depending on whether two antigens share all antigenic epitopes or partially
share their antigenic epitopes or do not share their antigenic epitopes at all.

The Ouchterlony test also can be used to estimate the relative concentration of antigens. When an
antigen has a relatively higher concentration, the equivalent zone will be formed a little bit away
from the antigen well. When an antigen has a relatively lower concentration, the equivalent zone will
be formed a little bit closer the antigen well. The pattern of lines that form can be interpreted to
determine the relationship between the antigens and antibodies.

Pattern of Identity:

39
X Pattern of identity occurs when the antigens in the two wells are identical and specific for the
antibody in the antiserum present in the third well. The concentration of the two antigens been the
same, they will diffuse at the same rate resulting in a smooth line of precipitate. The antibodies
cannot distinguish between the two antigens i.e. the two antigens are immunologically identical as
shown in Fig 1.

Pattern of Partial Identity:

Y Pattern of partial identity occurs when the antigens in the two wells share some epitopes which are
same for both, yet each of the two antigens also have unique epitopes. In this case antiserum contains
polyclonal antibodies specific for each epitope. When one of the antigens has some of the same
epitopes compared to other, the polyclonal antibody population will respond differently to the two
antigens and the precipitin line formed for each antigen will be different. The ‘spur’ is thought to
result from the determinants present in one antigen but lacking in the other antigen (refer to Fig 1). A
similar pattern of partial identity is observed if the antibodies are cross reactive with an epitope on
one of the antigens that is similar, but not identical to that present on the other antigen.

Pattern of Non-Identity:

Z Pattern of non-identity occurs when the antigens in the two wells are totally different. They are
neither cross reactive, nor do they have any epitopes which are same. In this case the antiserum
containing the antibodies is heterogeneous as some of the antibodies react with antigen in one well
while some react with antigen present in the other well. So, the two antigens are immunologically
unrelated as far as that antiserum is concerned (refer to Fig 1).

Kit Contents:

This kit can be used to determine the relationship between the antigens and antibodies using
Ouchterlony Double Diffusion technique (Antigen-Antibody Pattern).

Materials Required But Not Provided:

Glass wares: Measuring cylinder, Beaker Reagents: Alcohol.

40
Other requirements: Incubator (37oC), Microwave or Bunsen burner, Vortex mixer, spatula,
Micropipettes, Tips, Moist chamber (box with wet cotton)

Procedure:

1. Prepare 10 ml of 1% agarose (as given in important instructions).

2. Cool the solution to 55-60oC and pour 5 ml/plate on to grease free glass plates placed on a
horizontal surface. Allow the gel to set for 30 minutes.

3. Place the glass plate on the template provided.

4. Punch wells with the help of the gel puncher corresponding to the markings on the template. Use
gentle suction to avoid forming of rugged wells.

5. Add 10 µl each of the antiserum and the corresponding antigens to the wells as shown in fig 2.

6. Keep the glass plate in a moist chamber overnight at 37oC.

7. After incubation, observe for opaque precipitin lines between the antigen and antiserum wells.

Observation and Result: Observe for presence of precipitin lines between antigen and antisera
wells. Note the pattern of precipitin line observed in each case.

Interpretation: When antigen and antibody meet in optimal proportions a precipitation line is
formed. In Ouchterlony Double Diffusion (Antigen Antibody Pattern), three patterns of precipitin
lines can be observed.

1. If pattern X or pattern of identity is observed between the antigens and the antiserum, it indicates
that the antigens are immunologically identical.

41
2. If pattern Y or pattern of partial identity is observed, it indicates that the antigens are partially
similar or cross-reactive.

3. If pattern Z or pattern of non-identity is observed, it indicates that there is no cross-reaction

between the antigens. i.e. the two antigens are immunologically unrelated.

Pre-Lab Questions

1. What is the significance of immunodiffusion?

2. What is the difference between single and double immunodiffusion?

3. What is the application of ODD?

Post-Lab Questions

1. What are the different antigen-antibody patterns observed in a typical Ouchterlony double
diffusion experiment?

2. How Ouchterlony double diffusion helpful in evolutionary studies?

3. What are the major precautions we should take during ODD experiments?

42
 Experiment 10

Aim: To learn the technique of latex agglutination.

Introduction: Agglutination is a reaction of clumping together of antigen-bearing cells,

microorganisms or particles in the presence of specific antibodies (agglutinins) in a suspension.
Reaction time for agglutination to occur is shorter compared to other antigen-antibody interactions.
Latex agglutination makes use of latex particles which are built from different organic materials to a
desired diameter, and may be functionalized with chemical groups to facilitate attachment of
molecules. Latex agglutination tests have been in use since 1956 to detect a wide range of analytes in
the clinical laboratory. The first description of a test based on latex agglutination was the
‘Rheumatoid Factor Test’ proposed by Singer and Plotz in 1956. It can be used for detection of both
antigen and antibody.

Principle: In latex agglutination, antibodies are adsorbed to the latex particles (under appropriate
ionic and alkaline pH conditions) by binding to the Fc region of antibodies leaving Fab region free to
interact with antigen present in the applied specimen. The use of smaller latex particle has improved
the sensitivity and reagent longevity of latex agglutination.

Applications of Latex Agglutination Tests:

Latex agglutination tests are very popular in clinical laboratories. These tests are applied to the
detection of many infectious diseases.

To detect microbial and viral infections, autoimmune diseases, hormones, drugs and serum proteins.

To check for certain antibodies or antigens in a variety of bodily fluids including saliva, urine,
cerebrospinal fluid, and blood.

Latex Agglutination Teaching Kit applies the above principle for rapid screening of multiple samples
for infection, microbial contamination or microbial identification. For ease of handling, the Latex
Agglutination Teaching Kit is provided with ready to use reagents. The kit gives direct results in
form of visible agglutination. In this kit three solutions are provided, i.e. Antigen solution, Negative
control & Positive control. The Antigen solution (preserved with 0.099% sodium azide) contains

43
inactivated antigens reactive with Test reagent and non-reactive with Control reagent. The Negative
control contains latex particles coated with non-specific antibodies preserved in 0.099% sodium
azide. The Positive control contains latex particles coated with specific antibodies preserved in
0.099% sodium azide.

Kit Contents:

Procedure:

1. Before starting the experiment, gently mix all the bottles provided in this kit.

2. Add 1 drop of Solution A (25µL) into the circles marked as 1 and 2 of a clean dry agglutination
card.

3. Add 1 drop of Solution B (25µL) into circle 1.

4. Add 1 drop of Solution C (25µL) to circle 2.

5. Spread the drops over the area of both the circles using fresh mixing stick for each circle.

6. Rock the card gently (approximately two to three minutes) and observe for agglutination. An
agglutination reaction is indicated by visible aggregation of the latex particles.

7. The circles marked as 3, 4 and 5, 6 can be used similarly 8. After performing the experiment,
discard the slides and mixing sticks.

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Pre-Lab Questions
1. What is agglutination reaction?
2. What is the application of latex agglutination test?
3. What are the advantages and disadvantages of latex agglutination test?

Post-Lab Questions
1. What are the limitations of agglutination test?
2. What is the mode of action of Acriflavine on agglutination tests to differentiate rough vs smooth
bacteria?
3. What is modified agglutination test (MAT)

45