School of Health and Life Sciences

Department of Biochemistry and Microbiology

For
BS in Biochemistry and Biotechnology

BBT316L: Immunology Lab

Practical Guide Book

Module Tutor
Nusrat Zahan Rouf
Lecturer
Mail: nusrat.rouf01@northsouth.edu
Phone: +880-2-55668200, Ext: 6409

Laboratory Officer:
Abdullah All Jaber
Room: SAC414
Mail: abdullah.jaber02@northsouth.edu
Contact: +880-2-55668200, Ext: 6289

PLEASE NOTE THAT IT IS YOUR OWN RESPONSIBILITY TO

ENSURE THAT YOUR ATTENDANCE AT EACH PRACTICAL
Introduction to the
CLASS Immunology:
IS RECORDED EACHPractical
WEEK Classes
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The laboratory course is designed to complement and extend the material delivered to you in
lecture course. To make the most of your practical studies you should:
• Read through the practical beforehand each week

• Work in groups (unless otherwise instructed) but make sure you take an active part in the
practical work – you need to both understand how to carry out procedures and acquire
dexterity to do so effectively!

• Work with safety in mind

• If you have a problem, ask! We are here to help and it is better to check first rather than
plough on regardless

• Carry out data analysis and calculations during the laboratory sessions whenever needed.

• Directed learning: you should address any additional questions for self-study posed in
this workbook in your own time

Code of practice: To create and maintain a safe working environment

STUDENTS WORKING WITH OR IN CLOSE PROXIMITY TO CHEMICALS IN
LABORATORIES ARE REQUIRED TO WEAR LAB COAT AT ALL TIMES

The chemistry laboratory can be a dangerous place. While little that you will be asked to do is
intrinsically dangerous, a majority of the materials that you handle are in some way potentially
dangerous if they are handled carelessly. Perhaps the greatest potential danger in your laboratory
work will be from corrosive substances such as strong acids and bases that readily attack human
tissues.

At the discretion of the laboratory instructor, should you fail to heed warnings about wearing
your lab coat, you may, after two warnings, be dismissed from the laboratory and required to
drop the course. We do not wish to use these penalties. PLEASE do not force us to do so.
However, be assured that ALL STUDENTS will require to wear lab coat when working with
chemicals and we will not hesitate to assess these penalties to make this requirement work.

LABORATORY COATS MUST BE WORN (FASTENED) AT ALL TIMES. ANY

STUDENT WITHOUT SUITABLE PROTECTIVE CLOTHING WILL NOT BE
ALLOWD TO CARRY OUT PRACTICAL WORK WITH CHEMICALS.

STUDENT SHOULD NOT WEAR SLIPPERS OR OPEN SHOES DUIRNG LAB.

STUDENTS SHOULD REMOVE THEIR LAB COATS BEFORE LEAVING THE LAB
ROOM.
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 Safety Guidelines
1. IMMEDIATELY REPORT ANY ACCIDENT OR SAFETY ISSUE TO THE
LABORATORY INSTRUCTOR.

Emergency Procedures

1. If chemicals are spilled:

a. If chemicals are spilled on you or if you notice an incident of chemical spill on another
student: GET help from your instructor. Portions of your body or clothing that have been
in contact with the chemicals should immediately be flushed thoroughly with water. An
eye wash is available in the laboratory. Don’t wait to see if there is burning sensation or
rashes to develop. Rinse the affected body part immediately!
b. Work with the instructor to clean up spills on the bench or floor. Notify students working
nearby of the presence of broken glassware and/or chemicals, and keep people from
walking through the area until cleanup is complete.

2. Wash minor cuts or burns with cold water and get band aids. Report all such incidents, no
matter how minor, to the instructor.

Safety Clothing:

3. Shoes with solid tops (not sandals) must be worn to protect your feet from broken glass or
other sharp materials. Shorts or cut-off pants should not be worn in the lab; it is important to
protect your body with clothing rather than risk skin contact with chemicals or other materials.
Avoid wearing clothing with loose portions, such as open sweaters, baggy cuffs, and hanging
scarves. Loose clothing can catch on glassware and equipment, can drag through spills, and may
in some cases be a fire hazard. Similarly, loose, long hair should be tied back so that it does not
become entangled with equipment, get exposed to chemicals, or provide an impediment to
vision.

Handling Chemicals

4. Do not taste or ingest any laboratory chemicals.

5. Avoid touching chemicals with your hands, and always thoroughly wash your hands after
using chemicals.

6. Work in a fume hood when carrying out reactions that may produce bad odour or gases those
are toxic to inhale. If you need to smell vapour, do not put your nose directly above a flask,
beaker, or other vessel containing chemicals. Hold the vessel at least one foot away, use your
hand to gently and very cautiously divert the vapours toward you.

7. Pipetting by mouth is not permitted. Use mechanical pipettes, pipette aids or the rubber bulbs
provided in the laboratory.
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8. Always add acids to water (its alphabetical!); never add water to acids. Combining acid
and water frequently generates heat; addition of the acid to the water reduces the amount of heat
generated at the point of mixing and provides more water to disperse the heat.

9. LABEL all the flasks, beakers, test tubes and other vessels containing chemicals according to
their contents. This practice helps in proper identification of chemicals during an experiment as
well as it is important for waste disposal procedures.

10. Hold reagent bottles and other vessels containing liquids in a manner so that any drip will be
opposite to the label.

Be careful. If there is any stain from previous drip, try to not touch it.

Always clean off any drip or spill. If necessary, ask for help from your instructor or assistant.

11. Laboratory wastes and residues are to be disposed of in an approved manner. Waste
containers for these will be provided and specific instructions on proper disposal procedures will
be provided by the laboratory instructor for each session.

Laboratory Supervision

12. No unauthorized experiments are permitted. Students may try new experiments only with a
faculty member’s consent.

13. No one should work in the laboratory alone. All laboratory work must be under the
supervision of a faculty member or student laboratory assistant or any authorized person. Any
makeup session will need an approval from laboratory instructor.

Good conduct in the laboratory

14. Food and beverages are not permitted in the laboratory. This includes drinking from water
bottles.

15. Please leave your bags / backpacks in the hallway or outside of the laboratory.

Other Procedures

16. Consult with your instructor before carrying out any procedure that might cause injury from
broken glass, for example, cutting of glass tub or insertion of glass tube into a rubber stopper

17. CLEAN your bench top thoroughly at the end of each laboratory session.
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EXPERIMENTS

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Experiment 1: Identification of Blood Cells from a peripheral blood film
(PBF) under the microscope.

Background:
Blood is sometimes considered to be a fluid connective tissue because of the mesenchymal origin of its
cells and a low ratio of cells to liquid intercellular substance, the blood plasma. Plasma forms about 55%
of blood volume & contains water (95%) & many solutes including proteins, mineral ions, organic
molecules, hormones, enzymes, products of digestion, & waste products for excretion. In human adults
about 5 liter of blood contribute 7-8 % to the body weight of the individual& maintain the pH between
7.35-7.45. It has important transport, distribution, regulatory, & protective functionsin the body.

Blood cells
Erythrocytes (Red Blood Cells):

Erythrocytes do not contain a nucleus. They are typically biconcave disks & the average diameter of the
disk is ~7 µm. Each liter of blood contains about 5x1012 red cells varying with age, gender & state of
health & the lifespan of an erythrocyte in the bloodstream is 100-120 days. The contribution of red blood
cells (erythrocytes) to the total volume of the blood (haematocrit) is about 43%.Erythrocytes function in
the transport of oxygen. They do contain haemoglobin, which fills almost the entire cytoplasm & it is the
oxygen binding protein in erythrocytes, contributes about 30% of the weight of an erythrocyte.

Leukocytes (White Blood Cells):

Leukocytes can be further subdivided into granular leukocytes, i.e. neutrophils, basophils and eosinophils,
and non-granular leukocytes, i.e. monocytes and lymphocytes.

Granular Leukocytes:

The term granulocytes refer to the presence of granules in the cytoplasm of these cells. The granules
correspond to secretory vesicles and lysosomes. Specific granules are the granules which are only found
in one particular type of granulocytes. Granular leukocytes are all approximately the same size: ~ 12-15
µm in diameter. Their nuclei form lobes, and nucleoli cannot be seen. The number of nuclear lobes varies
according to cell type.

Neutrophil:

Neutrophil have a very characteristic nucleus. It is divided into 3-5 lobes which are connected by thin
strands of chromatin. The number of lobes increases with cell age. Up to 7 lobes can be found in very old
neutrophils (hypersegmented cells). In addition to the usual complement of organelles, they also contain
two types of granules. Primary granules (or A granules) contain lysosomal enzymes. Secondary granules
(or B granules), the specific granules of the neutrophils, contain enzymes with strong bactericidal actions.
The specific granules of neutrophils stain only weakly if they are at all visible - they are "neutral", hence
the term neutrophil.
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Basophil:

Basophilic granulocytes have a 2 or 3 lobed nucleus. The lobes are usually not as well defined as in
neutrophilic granulocytes and the nucleus may appear S-shaped. The specific granules of basophils are
stained deeply bluish or reddish-violet. In some smears, their color corresponds closely to the color of the
nucleus which sometimes is difficult to distinguish amongst or behind the granules.They contain heparin,
histamine, lysosomal enzymes and leukotrienes.Heparin and histamine are vasoactive substances. They
dilate the blood vessels, make vessel walls more permeable and prevent blood coagulation. As a
consequence, they facilitate the access of other lymphocytes and of plasma-borne substances of
importance for the immune response (e.g. antibodies) to, e.g., a site of infection.

Eosinophil:

Their nucleus usually has only two lobes. As the term "eosinophil" indicates, these granules are not
neutral but stain red or pink when eosin or a similar dye is used in the staining process.Aside from the
usual complement of organelles eosinophils contain some large rounded vesicles in their cytoplasm.
These granules correspond to the eosinophilic grains that we see in the light microscope. These specific
granules contain, in addition to enzymes that otherwise are found in lysosomes, an electron-dense,
proteinaceous crystal.This crystal is composed of major basic protein (MBP).Eosinophilsphagocytose the
antigen-antibody complexes, and this may prevent the immune system from "overreacting". Their
granules also contain the enzymes histaminase and arylsufatase. These enzymes break down histamine
and leukotrienes, which again may dampen the effects of their release by basophils or mast cells. MBP,
which can also function as a cytotoxin, and its release by eosinophils may be involved in the response of
the body against parasitic infections, which are accompanied by an increase in the number of eosinophils.

Monocytes:

These cells can be slightly larger than granulocytes (~ 12-18 µm in diameter). Their cytoplasm stains
usually somewhat stronger than that of granulocytes, but it does not contain any structures which would
be visible in the light microscope using most traditional stains (a few very fine bluish gains may be
visible in some monocytes). The "textbook" monocyte has a C-shaped nucleus. Monocytes contain
granules (visible in the EM) which in appearance and content correspond to the primary granules of
neutrophils, i.e. the granules correspond to lysosomes.Once monocytes enter the connective tissue they
differentiate into macrophages. At sites of infection macrophages are the dominant cell type after the
death of the invading neutrophils. The phagocytose microorganisms, tissue debris and the dead
neutrophils. Monocytes also give rise to osteoclasts, which are able to dissolve bone.

Lymphocytes:

These cells are very variable in size. In small ones, which are the majority of lymphocytes in the blood,
the nucleus may appear to fill the entire cell. Large lymphocytes have a wider rim of cytoplasm which
surrounds the nucleus. Both the nucleus and the cytoplasm stain blue (and darker than in most other cell
types in the blood). The typical lymphocyte only contains the usual complement of cellular organelles.
Upon exposure to antigens by antigen-presenting cells (e.g. macrophages) and T-helper cells (one special
group of T-lymphocytes) B-lymphocytes differentiate into antibody producing plasma cells.-lymphocytes
7

represent the "cellular arm" of the immune response (cytotoxic T cells) and may attack foreign cells,
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cancer cells and cells infected by, e.g., a virus.

Principal:
The polychromic staining solutions (Leishman, Wright, Giemsa) contain methylene blue &
eosin. These basic & acidic dyes induce multiple colors when applied to cells. Methanol acts as
fixative & also as a solvent. The fixative does not allow any further change in the cell & makes
them adhere to the glass slide. The basic component of white cells (i.e. cytoplasm) is stained
with acidic dye& they are described as acidophilic. The acidic components (e.g. nucleus with
nucleic acid) take blue to purple shades by basic dye & they are called basophilic. The neutral
components of the cell are stained by both the dyes.

Procedure-
Preparation of peripheral blood film (PBF)-

1. Take a grease-free clear glass slide.

2. Transfer a small drop of blood near the edge of the slide.
3. Another slide having smooth, even edge is taken as a spreader.
4. Place the spreader slide at an angle of 45ᵒ. Pull back the spreader until it touches the drop
of blood. Let the blood run along the edge of the spreader.
5. Push the spreader forward to the end of the slide with a smooth movement.
6. Then dried the blood film in the air.

Staining of peripheral blood film (PBF)-

Commonly used stains:

1. Leishman’s stain
2. Geimsa stain
3. Wright’s stain
4. Jenner’s stain

Staining steps:

1. Place air dried blood film in the staining rack.

2. Cover the blood film with Leishman’s stain.
3. Wait for 5 minute. (The cells are fixed by methyl alcohol present in the stain).
4. Add distilled water double amount of the stain.
5. Mix the stain & distilled water by gentle blowing by mouth.
6. Wait for 10 minutes.
7. Wash the stain with water until the film is pink.
8. Place the slide in a slanting position till the film is dry.
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 Observation-

What should you know for the assessment?

1. Detail observation
2. Principle of the Staining process, Interpretations
3. Equipment used to observe this experiment.
Theoretical Knowledge about the Experiment:

1. Why do we need to count Blood Cell Types?

2. Role of Different Blood Cells
3. What are the key features to identify particular blood cell types.
4. What are the Staining Procedures available for identification of Blood Cells
5. Normal Count of Different Blood Cells
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Experiment 2: Determination of blood group (Haemagglutination)

Purpose
1. To determine blood group of a person for blood transfusion and for paternity test.
2. Utilizing in forensic medium for various experimental purpose and for certain blood
diseases.

Background
Blood group is determined by detecting for the presence of antigen on the Red Blood Cells
(RBC). The presence of a specific antigen is examined by the corresponding antibody which
causes agglutination of erythrocytes. Human blood is grouped mainly for two particular groups
of antigens which are more likely to cause blood transfusion reaction. These are ABO group
antigen and Rhesus (Rh) or D antigen.

ABO antigens: The ABO antigen expressed by an individual cell is carbohydrate antigens
(glycoprotein). These are composed of oligosaccharide chains constructed in a stepwise manner
with each sugar being added to the growing chain by a specific enzyme (glycosyltransferase).
There are three types of blood group antigens: O, A and B. They differ slightly in the
composition of carbohydrates (Figure 1).

Figure 1. Structure and production of ABO antigens (Glycoprotein). Prot. represents protein.

Humans with A-type blood do not contain antibody against A-antigen, they possess antibody
against antigen B. In nature, some bacteria and plants express molecules like blood group
antigen. During our early life time we somehow get access to these molecules and our body
generates antibody against those. Same thing is true for humans with B-type blood. The
combination of blood typing antigen and antibody against them in human is presented in the
following table:
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Table 1: ABO blood group

Blood Antigens on erythrocytes Antibodies in serum

type
A A Anti-B

B B Anti-A

AB A and B Neither

O Neither Both Anti-A and Anti-B

Rh antigen: There are six antigens in this group as C, c, D, d, E and e. Of these D and d are the
main antigens. These two provide three groups D, Dd and d. D is Mendelian dominant while d is
recessive. Hence group D and Dd will be Rho positive (Rho +ve) and d will be Rho negative
(Rho –ve). Each of the antigens have antigenic property but only D is strongly antigenic.

Both of these antigens (ABO and Rho) are detected by agglutination test using monoclonal
antibody against them.

Principle

The basic principle of blood grouping is haemagglutination process. Human red blood cells
possessing A and/or B antigen will agglutinate in the presence of antibody directed towards the
antigen. Agglutination of RBC with Anti-A, Anti-B and Anti-AB reagents are a positive test
result and indicates the presence of the corresponding antigen. Absence of agglutination of RBC
with Anti-A, Anti-B, Anti-AB reagents is a negative test result and indicates the absence of the
corresponding antigen.

Human red blood cells possessing Rho (D) antigen will agglutinate in the presence of antibody
directed towards the antigen. Agglutination of RBC with spectrum Anti-D (Rho) plus reagents is
a positive test result and indicates the presence of D (Rho) antigen. No agglutination of RBC
with spectrum Anti-D (Rho) plus reagents is a negative test result and indicates the absence of D
(Rho) antigen.

Materials

Sample: Human blood (Collected from own body)

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Reagents:
1. Reagent A (Anti-A: reagent mouse monoclonal)
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2. Reagent B (Anti-B: reagent mouse monoclonal)

 3. Reagent AB (Anti-AB: reagent mouse monoclonal)
4. Reagent D (Anti-D: reagent mouse monoclonal)

Equipment: Clean test slide, sterile needle, tooth pick

Procedure
1. Mark (circle) the test slide as A, B and D to keep three blood drops distant apart
2. Collect blood from the individual using a sterile needle
3. Take blood drops on the glass slide at A, B, D marked sites
4. Add one drop of each A, B, D reagent to the blood at the corresponding sites
5. Mix the reagent well with the serum by tooth pick
6. Gently stir the slide to visualize the agglutination

Results interpretation
• If agglutination occurred, then it is positive result
• If agglutination did not occur, then it is negative result

Table 2: Interpretation of blood grouping test

1st circle 2nd circle 3rd circle Result

(Anti-A) (Anti-B) (D)
+ - + A+
+ - - A-
- + + B+
- + - B-
+ + + AB+
+ + - AB-
- - + O+
- - - O-

Observation and Result

Based on your observation, record the results in the following table

Table 3: Results of blood grouping test

Blood sample Agglutination Agglutination Agglutination Determination of

after addition of after addition of after addition of blood group
Anti-A to blood Anti-B to blood Anti-D to blood
sample sample sample
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What should you know for the assessment?

1. Detail observation
2. Principle of the process, Interpretations
3. Precaution of this experiment.

Theoretical Knowledge about the Experiment:

1. Why do we need to know our blood type?

2. Who is a universal blood donor and a universal blood recipient?
3. Besides ABO and Rh, are there other types of red blood cell antigens?
4. Explain the genetics behind blood typing?
5. Discuss briefly on ABO blood typing?
6. Which chromosome does the antigens resides?
7. How does blood typing play a role in blood transfusion?
8. Discuss how blood transfusion differs from plasma transfusion?

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Experiment 3: Determination of bacterial antigens by agglutination
(Bacterial agglutination): VDRL (Venereal Disease Research
Laboratory) test
Purpose

To screen for or diagnose an infection with the bacterium Treponema pallidum, which causes the
sexually transmitted disease (STD) syphilis

Background

Syphilis is a venereal (sexually transmitted disease) disease caused by the spirochete

microorganism Treponema pallidum. As the organism cannot be cultured on artificial media, the
diagnosis of syphilis depends on the correlation of clinical data with the detection of specific
antibody by serological tests. Serological screening tests for syphilis using cardiolipin and
lecithin as antigens are simple to perform, but may give rise to a false positive test result because
the test uses non-treponemal agents. False positives occur in 1 to 2 percent of the population and
can be caused by many conditions including, pregnancy, HIV infection, tuberculosis, leprosy and
certain other bacterial infections. Therefore, positive test specimens should be subject to further
serological studies, such as fluorescent treponemal antibody-absorption (FTA-ABS) test,
Treponema pallidum hemagglutination assays (TPHA) etc. Over the long term, latent syphilis
infections can, however, lead to false negative tests.

Principle

VDRL test is done by serological screening test using 0.2% lecithin, 0.03% cardiolipin and 0.9%
cholesterol, against unknown samples. Test antigen is a modified form of VDRL antigen
containing micro-particulate carbon which aids the macroscopic reading. The presence or
absence of a visible flocculation or agglutination indicates the presence or absence of circulating
antibodies in the samples tested.

Materials

Reagent: The VDRL Antigen

Samples: Patient’s serum
Equipment: Glass slides, micropipette, tips

Procedure

1. Bring the test reagents and samples to room temperature

2. Place one drop (50µl) of serum sample into the glass slide
3. Shake slowly the antigen and add 20 µl to the sample under test
4. Mix both drops by rotating for about 8 minutes
5. Inspect presence or absence of agglutination macroscopically under a high intensity lamp
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or strong daylight.
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Results interpretation
• Positive reaction: positive will show large black aggregates
• Slight positive reaction: slight but definite small aggregates. Serum sample weakly
reactive
• Negative reaction: negative will give a smooth grey result

Observation and results

Based on your observation, record the test results in the following table.

Sample Reaction on slide Figure of representative Agglutination

slide reaction

Negative Control

Serum 1

Serum 2

What should you know for the assessment?

1. Detail observation
2. Discussion (advantage and limitation of this process should be included)
3. Precaution of this experiment.

Theoretical Knowledge about the Experiment:

1. When this test is done?

2. What does the test result mean?
3. Is there anything else I should know about false positive result?
4. What are the symptoms of syphilis?
5. What other test should be done for confirmation after the VDRL test?
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Experiment 4: Qualitative and semiquantitative determination of C-reactive
protein in serum by agglutination of latex particles.
Purpose:
To identify the presence of inflammation and to monitor response to treatment for an
inflammatory disorder. Measurement of C-reactive protein aids in evaluation of the amount of
injury to body tissues.

Background
C-reactive protein (CRP) is a serum protein which is synthesized in the liver. Its rate of synthesis
and secretion increases within hours of an acute injury or the onset of inflammation.

CRP was first described in 1930 by Tillet and Francis who showed that the sera of patients
suffering from acute infection precipitated with a non-proteic pneumococcus extract called C
polysaccharide in the presence of calcium ions. The protein which caused this reaction was
therefore called C-reactive protein. Later it was shown that C-reactive protein is frequently found
in the sera of normal persons in very low concentrations, in most cases not exceeding a level of
6mg/l.

However, during the inflammatory process, whether of an infectious or other nature, the titers of
C-reactive protein can reach levels that are far above normal values.

The increase of C-reactive protein occurs in a non-specific way in different kinds of tissular
aggression, as for example in infectous states, rheumatic fever, rheumatoid arthritis, myocardiac
infarct, malignant tumour, abdominal abscess, peritonitis, burns, etc. For this reason a high C-
reactive protein concentration in serum lacks diagnostic value when the patients illness is not
defined. Nevertheless, it is very useful for following-up and monitoring such illness, as well as
for the differential diagnosis in certain cases.

Principle
The latex reagent is a suspension of polystyrene latex particles of uniform size coated with the
IgG fraction of an anti-human CRP specific serum. Latex particles allow visual observation of
the antigen-antibody reaction (CRP-IgG anti-CRP).

If the reaction takes place, due to the presence of C-reactive protein in the serum, the latex
suspension changes its uniform appearance and a clear agglutination becomes evident.

This change occurs because the C-reactive protein present in the serum reacts with the IgG
coated to the latex particles.

When the latex is mixed with the serum, if the serum contains approximately more than 6mg/l or
0.6 mg/dl of C-reactive protein, a clear agglutination will appear.

Results are expressed in mg/l of CRP.

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Components
a) Latex reagent: 2x2.5ml
Suspension of polystyrene latex particles coated with rabbit IgG anti-human CRP,
in a buffer. Contains <0.1% sodium azide.
b) Positive control: 1x1.5ml
Diluted human serum containing more than 6 mg/l of CRP. Ready to use.
Contains <0.1% sodium azide
c) Negative control: 1x1.5ml
Diluted human serum containing more than 6 mg/l of CRP. Ready to use.
Contains <0.1% sodium azide
d) Disposable slides: 18 units (x6)

Sample collection
• Use fresh serum. Samples can be stored at 2-8°C for 8 days. For longer periods, samples
should be frozen (-20°C)
• It is not necessary to inactivate the serum
• As in all serological tests, hemolytic, lipemic or turbid sera may cause incorrect results
and should not be used. Do not use plasma.

PROCEDURE FOR QUALITATIVE TEST

1. Allow the reagents and samples to reach room temperature (20-30°C)

2. Place 50µl of the sample (or one drop of control) onto one section of the slide
3. Shake the reagent vial and add one drop (50µl) of reagent next to the drop of sample
4. Mix both drops with a stirrer covering the whole surface of the slide section
5. Rotate the slide for 2 minutes manually or on a rotary shaker set at 80-100 rpm
6. Observe for agglutination

Interpretation of the results

• The presence of agglutination indicates a content of C-reactive protein in the serum equal
to or greater than 6mg/L
• The absence of agglutination indicates a content of C-reactive protein in the serum of less
than 6mg/L

PROCEDURE FOR SEMIQUANTITATVE TEST

1. Place 50µl of normal saline onto slide 2 through 6

2. Using an automatic pipette, place 50µl of the serum onto slide sections 1 and 2
3. Using the same pipette take in and release the serum and the saline solution on section 2
several times until they are well mixed
4. Take 50µl of the mixture made in section 2 and transfer it to section 3.
5. Repeat the above mentioned operations to obtain a thorough mixing of reagents, through
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section 6 thereafter discarding 50µl

6. Test each dilution as described in the section QUALITATIVE TEST
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 SECTION 1 2 3 4 5 6
Saline (μl) - 50 50 50 50 50
Step 1
Serum (μl) 50 50
Step 2 & 3
Mix and transfer (μl) 50 50 50 50
Step 4 & 5
Dilution 1:1 1:2 1:4 1:8 1:16 1:32
(Serum Dilution)
Latex reagent (μl) 50 50 50 50 50 50
CRP conc. (mg/L) 6 12 24 48 96 192

* Normally CRP conc. in healthy adults is less than 6 mg/L. If the highest dilution tested (1:32)
is positive, prepare additional 2-fold dilutions and repeat Step 2-6.

Interpretation of the results

The approximate titer will correspond to the highest serum dilution that still presents a clearly
visible agglutination.

What should you know for the assessment?

1. Details of the calculation (showing dilution factor) and CRP conc. In the table.
2. If we have obtained positive result in highest dilution tested (1:32) and done
additional 2-fold dilution was done in step two what will be the dilution factor of the
serum from section 1-6?
3. Discussion (advantage and limitation of this process should be included)
4. Precaution of this experiment.

Theoretical Knowledge about the Experiment:

1. When this test is done?

2. What does the test result mean?
3. Is there anything else I should know about false positive result?
4. What are chronic inflammatory diseases?
5. Should everyone have a CRP test?
6. When and why we will do qualitative and semiquantitative CRP test?

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Experiment 5: Qualitative and Semiquantitative determination of specific
antibodies in human sera against Salmonella typhi and S.
paratyphi (Widal test).
Purpose

To diagnose patients of typhoid and paratyphoid fever.

Background

Widal test is a tube agglutination test employed in the serological diagnosis of enteric fever. The
test is named after Georges Fernand Isidore Widal, a French physician and bacteriologist, born
March 9, 1862, Algeria; died January 14, 1929, Paris.

Enteric fever occurs when pathogenic microorganisms like S. typhi, S. paratyphi A, B and C
infect the human body. During the course of disease, the body responds to this antigenic stimulus
by producing antibodies whose titer rises slowly in early stages, to a maximum and then slowly
falls till it is undetectable. Antibodies to Salmonella organisms may be detected in the patient
serum from the second week after onset of infection. Information regarding the titers and
whether or not they are rising or falling can be obtained by performing serological tests using
Widal antigen suspensions. Usually titers of 1:80 and above are taken as diagnostically
significant, however for endemic areas higher cut-offs may need to be established.

Principle

When the colored, smooth, attenuated Widal antigen suspensions are mixed/incubated with
patient serum, anti-salmonella antibodies present in the patient serum react with the antigen
suspensions to give agglutination. Agglutination is a positive test result, indication presence of
anti-salmonella antibodies in the patient serum. No agglutination is a negative test result
indicating absence of anti-salmonella antibodies. The stained antigen suspensions are killed
bacteria, stained to enhance the reading of agglutination tests. The blue stained antigens are
specific to the O (somatic) antigens while the red stained antigens are specific to the H (flagellar)
antigens.

Materials

Equipment: Pipettes, reaction slides with white background, tips, mixing stick

Reagents
• Normal saline solution (0.85% NaCl)
• TO (O antigen of S.typhi)
• TH (H antigen of S.typhi)
19

• AH (H antigen of S.paratyphi A)
• BH (H antigen of S.paratyphi B)
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Procedure
Two types of agglutination techniques are available:
A) The slide test and
B) The tube test

In this experiment, we will perform both qualitative (slide screen) and semiquantitative slide
agglutination test. In both tests, samples were prepared from fresh serum obtained by
centrifugation of clotted blood. The sample may be stored at 2-8 C for 48 hours. For longer
period of time serum must be frozen.

• Slide Screen Method

1. Label circles on the reaction slide

2. Place 50µl of physiological saline onto the first circle
3. Place on drop of patient’s serum t be tested onto next four circles (labeled as TO, TH,
AH and BH)
4. Add one drop of appropriate WIDAL antigen suspension to the reaction circles
containing physiological saline
5. Add one drop of WIDAL antigen suspensions to the reaction circles containing the
patient’s serum
6. Ix contents of each circle uniformly over the entire circle with separate mixing sticks
7. Rock the slide gently back and forth, and observe for agglutination macroscopically
at one minute

• Slide Semiquantitative method (This method is performed on the samples that gave
positive result in the slide screen method).

1. Label different circles on the reaction slides

2. Using a micropipette, dispense 80, 40, 20, 10 and 5µl of undiluted serum onto the
circles on the reaction slides
3. Shake the reagent bottle well and add one drop of undiluted antigen suspensions to
each serum aliquot
4. Mix well using a stirring stick and rotate the slide

Observations and Results

Agglutination seen in any circle is indicative of the following results:

• 80 microlitre serum + 1 drop of reagent mixes then the titre is 1:20
• 40 microlitre serum + 1 drop of reagent mixes then the titre is 1:40
• 20 microlitre serum + 1 drop of reagent mixes then the titre is 1:80
• 10 microlitre serum + 1 drop of reagent mixes then the titre is 1:160
• 5 microlitre serum + 1 drop of reagent mixes then the titre is 1:320
• 2.5 microlitre serum + 1 drop of reagent mixes then the titre is 1:640
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Interpretation of the results

• Slide Screen Method

1. If agglutination occurred, positive
2. If agglutination does not occur, negative

• Slide Semiquantitative Method

The maximum dilution in which the agglutination occurred was the titer of the
specific antibody

What should you know for the assessment?

1. Discussion (advantage and limitation of this process should be included)
2. Limitation and Precaution of this experiment.

Theoretical Knowledge about the Experiment:

1. Write about Salmonella and typhoid?

2. Interpretation of the results
3. What might cause false negative, false positive and delayed or weakly reactions?
4. Write briefly about quantitative and semi quantitative methods?

21
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Experiment 6: Determination of hepatitis B surface antigen (HBsAg) in
human serum by Enzyme-Linked Immunosorbent Assay
(ELISA)

Background:

Hepatitis is an inflammatory disease of the liver that can severely damage the organ. The
diseases can result from non-infectious causes or from infectous viral and bacterial agents. Viral
hepatitis B is endemic throughout the world. The infection is spread primarily through
percutaneous contact with infected blood, e.g., sharing of needles by drug addicts or transfusion
of blood products that have not been screened for HBV. The hepatitis B virus is also found in
virtually every type of human body fluid and has been speared through oral and vaginal contact.
HBV can be transmitted perinatally from mother to child. The incubation period for HBV
average 90 days (range: 40-180 days). Common symptoms include malaise, fever, gastroenteritis
and icterus. HBV infection can to (a) icteric hepatitis, (b) Sub clinical anicteric hepatitis, (c)
Fulminant hepatitis, (d) Chronic active or persistent hepatitis. Over 90% of adults patients with
hepatitis B completely recover from acute illness, approximately 1% die of fulminant hepatitis,
and approximately 6-10% become chronic active or persistent carriers.

The complete HBV is a 42-nm diameter viron composed of an outer surface or envelop that
carries the hepatitis B surface antigen (HBsAg). The envelope surrounds an inner core that
contains the hepatitis B core antigen (HBcAg). Inside the core is the HB- DNA genome. Another
antigen hepatitis B e antigen (HBeAg) is a viral core protein found in the blood stem during
active replication of HBV.

HBsAg is the first serological marker to appear in the circulation, well before clinical symptoms,
and in the viral component usually found in the highest concentration in the serum of HBV
infected patients. It is a heterogeneous antigen. The principle determinant is called ‘a’ and is
common to all types of HBsAg. Other major determinants of the antigen d/y and w/r, this
determinant pairs are mutually exclusive, i.e. only the combinations adw, adr, ayw and ayr are
possible.

Presence of HBsAg in serum may indicate (a) acute HBV infection, (b) chronic HBV infection.
Or (c) sub clinical carrier state. The significance of HBsAg in serum is determined by evaluating
it in relationship to the presence or absence of the other HBV Markers and the clinical
presentation and history of the patient.
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Principle of the assay:

The method for qualitative determination is a direct sandwich assay, illustrated in following
figure based on the ELISA technique (Enzyme-Linked Immunosorbent Assay):

(1) Well coated with anti-HBs antibodies (mouse monoclonal);

(2) HBsAg from sample or control.
(3) Enzyme tracer: Anti-HBs antibody (Sheep) conjugated to horse radish peroxidase (HRP).

The presence of HBsAg allows the enzyme tracer to bind to the solid phase. Enzyme activity is
measured by adding a colorless chromogen/substrate solution. The enzyme action on
chromogen/substrate (H2O2 and TMB) produces a color which is measured as optical density
(OD) with a ELISA reader at 450 nm using 620 as the reference wavelength.

Optical Density (Absorbance) is proportional to the intensity of the color develop. The intensity
of the color is proportional to the HBsAg concentration present in samples or controls.
Therefore, Optical Density (Absorbance) is proportional to the HBsAg concentration present in
samples or controls.
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Materials/Reagents: (Provided in the kit)

1. Coated Wells : 96 wells are pre-coated with monoclonal antibodies specific to HBsAg.

2. Sample Diluent : One bottle containing 5 ml of green/brown buffer containing

detergents and proteins of goat and bovine origin. Mix by inversion
before use.

3. Negative Control : Protein-stabilized buffer tested non-reactive for HBsAg (1 ml).

4. Positive Control : HBsAg diluted in protein-stabilized buffer (1 ml).

5. Conjugate : Red-colored liquid in a white vial containing 6 ml of horseradish-

peroxidase labelled goat antibody to HBsAg in a red buffer containing
proteins of bovine and goat origin. Mix by inversion before use.

6. Substrate : One bottle containing 6 ml of 3,3’,5,5’-tetramethylbenzidine

(TMB) and 6 ml of urea peroxide solution.

7. Stop Solution : One bottle of 6 ml diluted sulfuric acid solution (0.5M H2SO4).

8. Wash Fluid : One bottle containing 30 ml of 20 times working strength. Glycine/

Borate Wash Fluid. Add one volume of Wash Fluid Concentrate
to 19 volumes of distilled water to give the required volume.

Materials/Reagents Required (Not provided in the kit):

1. Stop Solution : 0.5M to 2M Sulphuric Acid.

2. Freshly prepared distilled water
3. Micropipettes and Multichannel micropipettes of appropriate volume.
4. Incubator capable of maintaining the temperature limits defined in the assay protocol with
shaking
6. Microplate Sealer
7. Rim towel or Soft tissue for manual wash
8. Disposable reagent troughs
9. Microplate ELISA Reader
10. Sodium hypochlorite for decontamination.
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Specimen Collection:

Serum, EDTA plasma or citrate plasma samples may be used. Blood collected by venipuncture
should be allowed to clot naturally. Ensure that the serum samples are fully clotted. Remove any
visible particulate matter from the sample by centrifugation.

Specimen and Reagent Storage:

Store samples at 2 to 8°C. Samples which are not analyzed within 72 hours should be processed
to separate serum or plasma and store stored at –20°C or colder. Avoid multiple freeze-thaw
cycles. After thawing, ensure samples are thoroughly mixed before testing.
Upon receipt all reagents or the assay kit should be stored at 2-8 degree C, away from intense
light. Do not freeze.

Please read Analytical Precautions carefully before performing the test:

The addition of the various components of the assay to the wells may be confirmed visually by
examining the plate for the following colors:

Sample Diluent is green/brown in color.

In addition to the Sample or Control the color will change to blue/green.

The color change will vary from sample to sample but some changes should always be
visible.

Conjugate is red in color.

Substrate Solution is initially pink with any reactive wells becoming purple.

On addition of Stop Solution, the purple color of the reactive will changes to orange, whilst
the negatives remain pink.

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Procedure:

Reagents preparation: Allow the reagents to reach room temperature (18-30°C). Check the
Wash buffer concentrate for the presence of salt crystals. If crystals have formed, resolubilize by
warming at 37°C until crystals dissolve. Dilute the Wash buffer (20X) as indicated in the
washing step instructions. Use distilled or deionized water and only clean vessels to dilute the
buffer.

Step 1: Preparation: Mark three wells as Negative control (e.g. B1, C1, D1), two wells as
Positive control (e.g. E1, F1), and one Blank (e.g. A1, neither samples nor HRP-Conjugate
should be added into the Blank well).

Step 2: Adding Diluent: Add 20μl of Specimen Diluent into each well except the Blank.

Step 3: Adding Sample: Add 100μl of Positive control, Negative control, and Specimen into
their respective wells except the Blank.

Step 4: Incubation: Cover the plate with the plate cover and incubate for 60 minutes at 37°C.

Step 5: Adding HRP-Conjugate: At the end of the incubation, remove and discard the plate
cover. Add 50μl HRP-Conjugate into each well except the Blank, and mix by tapping the plate
gently.

Step 6: Incubation: Cover the plate with the plate cover and incubate for 30 minutes at 37°C.

Step 7: Washing: At the end of the incubation, remove and discard the plate cover. Wash each
well 5 times with diluted Wash buffer, allowing the microwells to soak for 30-60 seconds each
time. After the final washing cycle, turn down the plate onto blotting paper or clean towel and
tap it to remove any remaining liquid.

Step 8: TMB Substrate: Add 50μl of Chromogen A and 50μl Chromogen B solutions into each
well including the Blank. Incubate the plate at 37°C for 30 minutes avoiding light. The
enzymatic reaction between the Chromogen solutions and the HRP-Conjugate produces blue
color in Positive control and HBsAg positive sample wells.

Step 9: Stopping Solution: Using a multichannel pipette or manually, add 50μl Stop solution into
each well and mix gently. Intensive yellow color develops in Positive control and anti-HCV
positive sample wells.

Step 10: Measuring the Absorbance: Calibrate the plate reader with the Blank well and read the
absorbance at 450nm. If a dual filter instrument is used, set the reference wavelength at 630nm.
Calculate the cut-off value and evaluate the results.
26

(Note: read the absorbance within 10 minutes after stopping the reaction).
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Quality Control and Calculation of the Results

Each microplate should be considered separately when calculating and interpreting the results of
the assay, regardless of the number of plates concurrently processed. The results are calculated
by relating each specimen absorbance (A) value to the Cut-off value (C.O.) of the plate. If the
Cut-off reading is based on a single filter plate reader, the results should be calculated by
subtracting the Blank well A value from the print report values of specimens and controls. In
case the reading is based on dual filter plate reader, do not subtract the Blank well A value from
the print report values of specimens and controls.

Calculation of the Cut-off value (C.O.) = Nc + 0.06 (Nc= the mean absorbance value for three
negative controls).

Quality control (assay validation): The test results are valid if the Quality Control criteria are
fulfilled. It is recommended that each laboratory must establish appropriate quality control
system with quality control material similar to, or identical with the sample being analyzed.

- The A value of the Blank well, which contains only Chromogen and Stop solution, is < 0.080 at
450 nm.

- The A values of the Positive control must be ≥ 0.800 at 450/630nm or at 450nm after blanking.

- The A values of the Negative control must be < 0.100 at 450/630nm or at 450nm after
blanking.

If one of the Negative control A values does not meet the Quality Control criteria, it should be
discarded and the mean value calculated again using the remaining two values. If more than one
Negative control A values do not meet the Quality Control Range specifications, the test is
invalid and must be repeated.

Interpretations of the Results:

Negative Results (A / C.O. < 1): Specimens giving absorbance less than the Cut-off value are
negative for this assay, which indicates that no hepatitis B virus surface antigen has been
detected with AiDTM HBsAg ELISA, therefore the sample is probably not infected with HBV
and the blood unit does not contain hepatitis B virus surface antigen.
Positive Results (A / C.O. ≥ 1): Specimens giving an absorbance equal to or greater than the
Cut-off value are considered initially reactive, which indicates that hepatitis B virus surface
antigen has probably been detected using AiDTM HBsAg ELISA. All initially reactive
specimens should be retested in duplicates using AiDTM HBsAg ELISA before the final assay
27

results are interpreted. Repeatedly reactive specimens can be considered positive for hepatitis B
Page

virus surface antigen with AiDTM anti-HCV ELISA.

Borderline (A / C.O. = 0.9-1.1): Specimens with absorbance to Cut-off ratio between 0.9 and
1.1 are considered borderline and retesting of these specimens in duplicates is required to
confirm the initial results.
Example:
The presence or absence of HBsAg is determined by comparing the absorbance value of
unknown samples to that of the cut-off value.

 The unknown samples with absorbance values above the grey zone should be considered
reactive for HBsAg
 The unknown samples with absorbance values below the grey zone should be considered
nonreactive
 Samples with absorbance values within ±10% of the cut-off value (grey zone) must be
retested in duplicate in order to confirm the initial result

What should you know for the assessment?

1. Discussion (advantage and limitation of this process should be included)
2. Limitation and Precaution of this experiment.
3. Briefly explain about the observation and interpretation?

Theoretical Knowledge about the Experiment:

1. Explain the components of sandwich ELISA?
2. What might be observed in the negative and positive control and how is the
observation interpreted?
3. Discuss briefly on hepatitis B antigen

28
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Experiment 7: Determination of hepatitis B surface antigen (HBsAg) in

human serum by IMMUNOCHROMATOGRAPHY.

Background:
HBsAg determination device – one step test for HBsAg is a rapid, qualitative, two site sandwich
immunoassay for the detection of Hepatitis B antigen, a marker for Hepatitise B infection, in
serum/plasma specimen.

Blood containing the Hepatitis B virus (HBV) is potentially infectious. Hepatitis B surface
antigen (HBsAg), earlier known as Australia antigen is among the first serological markers that
circulate in the blood of infected persons even two or three weeks prior to appearance of clinical
symptoms. The levels of HBsAg are especially elevated during the symptomatic phase and
decline thereafter. Detection of HBV using HBsAg as a marker to screen blood donors is
essential to reduce the risk of transmission of Hepatitis B by blood transfusion. HBsAg detection
is also useful for screening high risk groups for HBV and for differential diagnosis of Hepatitis
infection. The test device one step test for HBsAg detects the presence of HBsAg in
serum/plasma specimens, qualitative, at concentrations as low as 0.5 ng/ml.

Principle of the assay:

The test device one step test for HBsAg utilize the principle of
IMMUNOCHROMATOGRAPHY, a unique two site immunoassay on a membrane. As a test
sample flows through the membrane assembly of the test device, the colored monoclonal anti-
HBsAg-colloidal gold conjugate complexes with the HBsAg in the sample. The complex tether
on the membrane to the test region where it is immobilized by another monoclonal anti- HBsAg
antiserum coated on the membrane leading to formation of a pink-purple colored band which
conforms a positive test result. Absence of this colored band in the test region indicates a
negative test result. The un-reacted conjugate and unbound complex if any move further on the
membrane and are frequently immobilized by the anti-rabbit anti-serum coated on the membrane
at the control region, forming a pink-purple band. This control band serves to validate the test
result.

Reagents and materials:

Each kit contains-

A. Individual pouches each contain a:
1. Test device: contains membrane assembly predispensed with monoclonal anti-HBsAg
antiserum-colloidal gold conjugate, rabbit IgG colloidal gold conjugate and
monoclonal anti-HBsAg antiserum and anti-rabbit antiserum coated at the respective
regions.
2. Disposable plastic dropper
3. Desiccant pouch
B. Package insert
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Testing procedure and interpretation of results:
1. Bring the sealed puch to room temoerature, open the pouch and remove the devise.
Once opened, the device must be used immediately.
2. Dispense 2 drops of 50µl of serum/plasma specimen into the sample well “S” using
the droper provided
3. At the end of 15 minutes, read the results as follows:
-Negative: Only one pink-purple band appears at the control region “C”
-Positive: In addition to the control band, a pink-purple band also appears at the
test region “T”.
4. The test should be considered invalid if neither the test band nor the control band
appears. Repeat the test with a new device.

Limitations of the test:

1. Though the test device is a reliable screening assay, it should not be used as sole
criterion for diagnosis of HBV infection
2. Interference due to heterophile antibodies, Rheumatoid factors and other
nonanalyte substances in patients serum, capable of binding antibodies
multivalently and providing erroneous analyte detection in immunoassays, has
been reported in various studies
3. Do not compare the intensity of the test lines and the control limes to judge the
concentration of HBsAg in the test specimen
4. Since various tests of HBsAg in their performance characteristics and antibody
composition, their reactivity patterns may differ
5. Testing of pooled samples is not recommended
6. The membrane is laminated with an adhesive tape to prevent surface evaporation.
Air pockets or patches may appear which do not interfere with the test result.
Presence of a band at the test region, even if low in intensity or formation, is a
positive result.
7. Most positive results develop within 15 minutes. However, certain sera sample
may take a longer time to flow. Therefore, Negatives should be confirmed only at
30 minutes. Do not read results after 30 minutes.
8. As with all diagnostic test. A definitive clinical diagnosis should not be based on
30

the result of a single test, but should only be made by the physician after all
clinical and laboratory findings have been evaluated.
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What should you know for the assessment?
1. Discussion (advantage and limitation of this process should be included)
2. Limitation and Precaution of this experiment.
3. Briefly explain about the observation and interpretation?

Theoretical Knowledge about the Experiment:

1. Explain the purpose of each line on the strip ie: S line, T line and C line?
2. Advantages and disadvantages of this test?
3. How do you interpret one line and two lines present on the strip?

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