Experiment No.

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INTRODUCTION TO IMMUNOLOGY AND SEROLOGY

Objectives:
1. To be able to define different glossary of terms.
2. To be able to familiarize with the different equipment used in serology laboratory
3. To be able to discuss the different principles of serologic reactions

1. Definition of Terms – give the concise definition of the following commonly used
terms in serology laboratory. a. Agglutination
The visible clumping of particles (e.g., red blood cells or
a. agglutination bacteria) caused by the binding of antibodies to antigens.
b. allergy
c. anmnestic response b. Allergy
An abnormal immune reaction to normally harmless
d. antibody substances (allergens), leading to hypersensitivity
e. antigen symptoms.

f. complement c. Anamnestic Response

g. autoimmune diseases The enhanced and rapid immune response triggered upon
re-exposure to an antigen due to immunological memory.
h. enzyme immunoassay
i. epitope d. Antibody
A protein (immunoglobulin) produced by plasma cells that
j. immunoglobulins specifically binds to antigens to neutralize or mark them for
k. immunohematology destruction.
l. immunosuppression e. Antigen
m. lymphokine Any substance (foreign or endogenous) that stimulates an
n. monoclonal antibody immune response and reacts with specific antibodies.

o. plasma cell f. Complement

p. polyclonal antibody A group of proteins in the blood plasma that enhance
immune responses by promoting lysis, opsonization, and
q. precipitation inflammation.
r. primary lymphoid organs
g. Autoimmune Diseases
s. radio immunoassay Conditions in which the immune system mistakenly attacks
t. secondary lymphoid organs the body’s own tissues, recognizing them as foreign.
u. seroconversion h. Enzyme Immunoassay (EIA)
v. serology A diagnostic test using enzyme-labeled antibodies or
antigens to detect the presence of specific substances
w. thymus (e.g., antibodies, hormones).
x. titer
i. Epitope
The specific part of an antigen that is recognized and
bound by an antibody or T-cell receptor.

j. Immunoglobulins
Proteins (antibodies) produced by B cells that play a
central role in immune responses by targeting specific
antigens.

k. Immunohematology
The study of antigen-antibody reactions related to blood,
focusing on blood typing, transfusion compatibility, and
hemolytic diseases.

l. Immunosuppression
1 often
The reduction or inhibition of the immune response,
induced to prevent organ rejection or treat autoimmune
disorders.
2. Illustrate and give the functions of the different equipment used in serology
laboratory
a. serological pipets
b. automatic pipets
c. Widal plate
d. water bath
e. serofuge
f. Rh typing box
g. incubator
h. magnifying glass
i. concave slides
j. microscope

3. Differentiate specific immunity from non specific immunity.

4. Enumerate, illustrate and discuss the functions of the five types of
immunoglobulin.
5. Differentiate passive and active immunity.
6. Differentiate artificial and natural immunity.
7. Give at least 5 examples of serological tests and give its clinical significance.
m. Lymphokine
A signaling molecule (cytokine) produced by lymphocytes that mediates immune
responses and cell communication.

n. Monoclonal Antibody
An antibody produced by a single clone of cells, highly specific to one epitope, used in
diagnostics and therapeutics.

o. Plasma Cell
A differentiated B lymphocyte that produces and secretes large quantities of antibodies.

p. Polyclonal Antibody
A mixture of antibodies produced by different B cell clones that target multiple epitopes of
an antigen.

q. Precipitation
The formation of a solid or insoluble immune complex when soluble antigens bind to
antibodies.

r. Primary Lymphoid Organs

Organs where lymphocytes are produced and mature, such as the bone marrow and
thymus.

s. Radioimmunoassay (RIA)
A sensitive laboratory technique that uses radioactively labeled antigens or antibodies to
measure specific substances in a sample.

t. Secondary Lymphoid Organs

Organs where immune responses are initiated, such as lymph nodes, spleen, and
mucosal-associated lymphoid tissues.

u. Seroconversion
The development of detectable specific antibodies in the blood following infection or
immunization.

v. Serology
The study of serum and immune responses, focusing on antigen-antibody interactions for
diagnostic purposes.

w. Thymus
A primary lymphoid organ where T lymphocytes mature and develop.2

x. Titer
The concentration or amount of specific antibodies in a blood sample, determined by serial
dilution.
 Experiment No. 2
PREPARATION OF RED CELL SUSPENSION

Red cell suspensions are prepared from the whole blood samples or from packed
cells. Only fresh blood samples are used as hemolysis is likely to occur in older blood
samples. Care should be taken so as not to hemolyze the red cells during preparation. Red
cell suspensions are used in various serologic techniques and diagnostic tests.

Objective
- to learn how to prepare a desired concentration of cell suspension

Apparatus/material
- graduated centrifuge tube
- pasteur pipets
- serologic pipets, 5 ml
- test tubes

Reagents
- blood in EDTA tube
- NSS

Test procedure
1. Pipet 5 ml of blood into graduated centrifuge tube.
2. Fill the tube to the uppermost calibration with normal saline.
3. Centrifuge for 5 minutes at a speed sufficient to pack the cells (1,500 – 1,800
rpm)
4. Remove the supernatant fluid by pouring as quickly as possible or by aspiration.
5. Re-suspended the red cells in 9 or 10 volumes of NSS by gentle shaking and
again centrifuge. Repeat this step for three times. This process is called by
washing.
6. After the last centrifugation read and record the pack cell volume (pcv) before
removing the supernatant fluid.
7. Decant completely the supernate as possible without disturbing the pcv.
8. Calculate the required amount of NSS to be added to PCV for any desired
concentration of cell suspension by using the following formula:

Total volume: pcv

--------- x 100
desired cell conc.

ml of NSS to be added=total volume – pcv

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 Illustrate the results

Questions:
1. Why is NSS the diluent usually used in red cell suspension procedure?
2. Why is it necessary to wash red cells with NSS for three times prior to use?
3. Why is it necessary to prepare red cell suspension for some serological tests?
4. Calculate the amount of NSS to be added if a 2% cell suspension is desired
and the PCV was 0.2.

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 Experiment No. 3
SERIAL DILUTION PREPARATION

In serological analysis, a measure of antibody concentrations in test serum is

usually made by simply diluting the serum in progressive increments with a suitable
diluent; the unit of measurement is then expressed in terms of the titer of the antibody in
the test serum.

Objective: To understand adequately the mechanics and importance of preparing serial

dilutions

Apparatus:
10 Kahn tubes
test tube rack
2 serological pipets

Reagents:
NSS
red cell suspension

Test Procedure
1. Label test tubes 1 through 10 and place them in a test tube rack.

2. Pipet 0.8 ml of NSS into the 1st test tube. To the remaining tubes deliver 0.5 ml of
NSS.

3. Add 0.2 ml of serum to the 1st tube and mix.

4. Transfer 0.5 ml of the diluted serum from tube 1 to tube 2 and mix well.

5. Transfer 0.5 ml of the contents from tube 2 to 3 and mix. Continue this process
from tube 3 to the last tube in the series. After mixing the contents of the last tube,
discard 0.5 ml from this tube.

6. Add 0.5 ml of red cell suspension (2 or 5%) to all the tubes.

7. Mix and centrifuge for 1 minute at 1,500 rpm.

8. Gently dislodge the button of cells. Observe the reaction macroscopically.

Clumping of cells is the visible reaction.

Notes:
1. When transferring from a low to a high dilution, it is important to use separate
pipets throughout the titration. The small quantity of diluted serum adhering to the

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 outside of the pipet can in many cases, alter drastically the accuracy of the desired
higher concentration. For this reaction, the use of separate pipets is preferred.
2. Alternately, instead of using a clean pipet for each dilution, the original pipet may
be used throughout by rinsing it three times between each dilution. It is important
than the outside of the pipet be wiped off between tubes.
3. The mixing of serum and saline can be conveniently performed by alternately
drawing up the diluted serum into the pipet and expelling it.

Observation:
Illustrate the results.

Computations
Show your computations in each tube.

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 Experiment No. 4
AGGLUTINATION REACTION

Name _________________________ Lab. Batch______ Group_____ Sub Group______

Objectives:
• To be able to distinguish agglutination reaction from other serological reactions
• To be able to demonstrate the agglutination reaction
• To be able to correlate agglutination reaction with other known serological tests

Materials Needed:
• Red Cell Suspension
• Plasma
• Kahn Tubes
• Serological Pipettes (0.1 ml)
• Pasteur Pipettes

Procedure:
1. Refer to experiment no. 2 with regards to the procedure of serial dilution.
2. Exchange plasma from the other sub-group. See to it that the red cell suspension
and the plasma are of different blood type.
3. After the final dilution, centrifuge all the tubes for 15 seconds and observe for
agglutination.
4. Compute the final dilution and determine the titer. (If the agglutination is not
visible macroscopically, you may observe it under HPO).
5. Illustrate the result.

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 Serial Dilution Procedure

Tube No. 1 2 3 4 5 6 7 8 9 10

Saline ml .8 .5 .5 .5 .5 .5 .5 .5 .5 .5

five ml of
Serum ml .2 1:5 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280

Dilution after 1:5 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560
Serum transfer

Cell .5 .5 .5 .5 .5 .5 .5 .5 .5 .5
suspension ml

Total volume 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
ml

Final Dilution 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2500 1:5120
of serum

Questions
1. Give the importance of having serial dilutions.
2. Give the general formula used in determining the dilution factor of a liquid.
3. What causes prozone phenomenon?

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Computations:

Questions to answers:
1. Describe the antigen and antibody used in agglutination reaction.
2. What are the other types of serological reactions?
3. What is a titer? Give the importance of determining the titer.
4. Give examples of serological tests that uses agglutination as the basic principle.
Give the clinical significance of such tests.
5. Differentiate agglutination reaction from precipitation reaction.

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 Experiment No. 5
PRECIPITATION TEST

Precipitation involves the interaction of antigen with antibody in correct

proportion resulting in a visible precipitate. The antibodies can either be polyclonal or
monoclonal. The simplest form of precipitin reaction would be layering of antigen in
solution over a small volume of anti-serum. Precipitation occurs at the interface of the
two reagent, forming a ring.

OBJECTIVES
• To be able to perform the precipitation properly
• To understand the clinical significance of precipitation reaction

MATERIALS
2 – 3% red cell suspension of different blood types and plasma
(O and B; O and A; and A; AB and B; or A and B but never AB and O)

Test tube
Pasteur pipette
Serological pipette with aspirator

PROCEDURES
1. Collect blood to anti-coagulated tube.
2. Prepare 2-3% RCS.
3. Prepare 2 ml plasma. See to it the RCS and plasma are of different blood types.
4. Add 2 ml of 2-3% RCS over 2 ml of plasma.
5. Stand for 15-30 minutes.
6. Observed for the reaction.

Illustrate the result:

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QUESTIONS:
1. Describe the antigen and antibody used in precipitation reaction. Differentiate
your answer from that of the agglutination reaction.
2. What is a zone of equivalence? Illustrate how it can give an optimum antigen and
antibody reaction.
3. What is a prozone phenomenon? Describe how it can affect the serological
reaction.
4. What is a postzone phenomenon? Describe how it can affect the serological
reaction.
5. One of the most accurate methods that involve precipitation reaction is an
immunodiffusion assay. Enumerate all types of immunodiffusion assays and
illustrate the principles involved.
6. What is the rationale in layering the antigen slowly over the plasma?
7. Give the different serological tests that involved principle of precipitation
reaction.

YOU MAY ATTACHED ANOTHER SETS OF PAPER BUT NEVER WRITE AT THE
BACK.

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 Experiment No. 12
FEBRILE AGGLUTINATION TEST

These groups of tests are designed for the detection and determination of
antibodies against certain bacteria. They serve as important aid in the diagnosis of certain
bacterial infections associated with persistent pyrexia.

OBJECTIVE:
• To detect the presence of agglutination antibodies from the serum of patient
suspected of typhoid fever (Widal Test) and of Rickettsial infection (Weil-Felix
Test).

PRINCIPLE:
• Human serum containing antibodies specific for an organism, under appropriate
conditions, visibly agglutinate a suspension of that organism.

APPARATUS:
• Widal plate
• Serological pipettes 0.1 mL
• Applicator sticks

REAGENTS:
• Salmonella “O” antigens (Groups A, B, C, D)
• Salmonella “H” antigens
• Proteus antigens (OX2, OXk, OX19)

TEST PROCEDURE:

1. Using a 0.1 mL of serological pipettes, deliver 0.08, 0.04, 0.01 and 0.005 mL
quantities of serum to each of the wells in the Widal plate from left to right.
2. Shake the antigen bottle to insure a uniform suspension.
3. Add one drop of antigen suspension just below each quantity of serum.
4. Mix the serum and antigen well using a wooden applicator stick. Use separate
stick for each serum quantity or use the same stick and proceed from right to left.
5. Gently tilt the slide back and forth for no more than one minute. A rotator
machine may be used for this purpose.
6. Observe for agglutination using any good indirect light. The degree of
agglutination observed is recorded as follows:
4+ = 100% of the organisms are agglutinated
3+ = 75% of the organisms are agglutinated
2+ = 50% of the organisms are agglutinated
1+ = 25% of the organisms are agglutinated

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 + = less than 25% of the organisms are agglutinated
- = no agglutination is observed

Interpretation of the results:

1. A single negative test does not exclude the absence of infection.
2. A single positive test is of no diagnostic significance unless the titer is sufficiently
high.

OBSERVATIONS:

Widel Test
Antigen 1:20 1:40 1:80 1:160 1:320 Significance

Weil Felix
Antigen 1:20 1:40 1:80 1:160 1:320 Significance

Interpretation for Widal Test:

Interpretation for Weil-Felix

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Illustration of the Results:
Widal Test

Weil-Felix Test

Questions:
1. What is the clinical significance of Widal and Weil-Felix test?
2. What are the different methods of preparation of these bacterial antigens?
3. Give the clinical significance of the rise in titers of the following antigen:
a. O
b. H
c. A
d. B
e. Vi
f. K
g. OxK

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