IMMUNOLOGY AND SEROLOGY LABORATORY Principles and Experiments:

Activities 1 – 7 1

Activity 1 - Fluorescent microscope – used in immunosero laboratory; for

BASIC LABORATORY APPARATUS AND EQUIPMENT detection of fluorescent antibodies

Test tubes
Serologic pipettes
- Where samples are placed for serial dilutions
- A graduated or measuring glass pipette that has marks all along
its length all the way down to the tip
Thermometer
- Up to 200ul or 2mL
- Indicate or determine temperature
- Viscous fluid (e.g., blood)
o Surface immersion thermometer – incubator,
- Etched ring (2 white ring at the top); blowout pipette
autoclave
o Partial immersion thermometer – water bath, heating
Mohr pipette
block
- Calibration marks do not extend to the tip but at a point above
o Total immersion thermometer – freezer, refrigerator
the tip
- CF
- Fluid is drawn up to a graduation mark and then dispensed to
the zero mark, not completely evacuating the fluid from the F= (C x 1.8) + 32
pipette. - FC
- Not as accurate as volumetric pipette
C= (F - 32) / 1.8
Automatic pipettes/ Micropipettors
- Smaller volumes less than 1mL Volumetric flask/ Graduated Flask
- 1000ul = 1mL - One graduation; 250mL
- Allow fast, repetitive measurement and delivery of solutions of - Used to measure critical/ specific volumes
equal volumes
- Piston-operated devices; air displacement Erlenmeyer flask
- Many graduations
Widal Plate - Measuring and containing non-critical/ non-specific volume of
- For Salmonella Typhoid Fever test; to check if patient’s serum solution
has antibody directed against salmonella antigens
- Has 6 circles: Graduated cylinder
o First two circles: - Have graduation markings
 O antigen – somatic - Used to measure specific/ critical volume
 H antigen – flagellar
o Second two circles: Pasteur pipettes
 Salmonella paratyphi A H antigen (AH) - Glass, and has rubber bulb
 Salmonella paratyphi B H antigen (BH) - Used to aspirate non-specific volumes
o Third two circles:
 Positive control (PC) Beral pipette
 Negative control (NC) - Plastic disposable pipette
- Sample: serum; Reagent: antigen
- Agglutination (clumping of cell) indicates positive reaction.

Water bath
- Temperature control
- Tubes must be regulated at 60 C

Serofuge
- Centrifuge used to separate components based on density
- Less dense – top portion
- More dense – bottom of tube

Rh typing box
- Used to observe agglutination on test tubes, glass slides, or
even on gel part.

Incubator
- Temperature control

Magnifying glass
- Check for agglutination and precipitation; but now, microscope
is used is not visible to naked eye

Concave slides
- Used for agglutination purposes

Microscope
- Bright-field microscope/ Compound microscope – used for
routine testing

Kimberly Anne Hampac | BSMT – 3 | Notre Dame of Marbel University

 IMMUNOLOGY AND SEROLOGY LABORATORY Principles and Experiments:
Activities 1 – 7 2

Activity 2 - Positive APR

AN INTRODUCTION TO THE IMMUNE SYSTEM o An increase in the concentration of serum proteins
that are referred to as acute phase reactants (APR)
accompanies inflammation
IMMUNE SYSTEM  α1 – antitrypsin
- Designed to protect yourself against invading pathogens/  C-reactive protein
foreign materials  Fibrinogen
- Has self-tolerance (able to differentiate self from non-self)  Haptoglobin (HP)
o Recognition of the self-antigens (autoimmunity)  Serum Amyloid A
o Results to Autoimmune diseases if there is no self-  Ceruloplasmin
tolerance  α2 – macroglobin
 Systemic Lupus Erythromatosus (SLE)
 α1 – acid glycoprotein (AGP)
 Rheumatoid Arthritis (RA)
 Complement (C3, C4)
- Immunity - Resistance to infection
 Complement Pathway
Two Types:
1. Innate (inborn, natural, nonspecific) - Classical Complement Pathway
A. 1st line of defense: o Ag-Ab complex
Physical: - Alternative Complement Pathway
 Skin o Lipopolysaccharides (LPS) – characteristic
 mucous membrane components of the cell wall of Gram negative
 cilia bacteria; not found in Gram positive bacteria
 cough reflex o Teichoic acid– a structure found exclusively in gram-
Chemical: positive bacterial cell walls
 pH - Mannose-binding leptin
 stomach acid o Mannose containing microorganism
 secretions (vaginal secretions: lactic acid)
 tears and saliva (lysozymes – directed against  Cytokines (interferons, interleukins)
cell wall of microorganism) o Interferons – secreted by virally-
 sweat infected cell
 mucus o Inflammation
 normal microbiota/ flora o Fever

2. Adaptive (acquired, specific)

C. 3rd line of defense
B. 2nd line of defense
C1. Cellular:
B1. Cellular:
 B lymphocytes
 MNM (Macrophage, Neutrophil/ PMNs, Mast
 T lymphocytes
cell)
o T helper cell – CD4; MHC class II;
 NK cells
APCs
o T cytotoxic cell – CD 8; MHC class
Macrophages found in:
I; nucleated except trophoblast and
 Brain: microglial
sperm
 Lungs: alveolar macrophage, interstitial macrophage
o T regulatory cell
 Liver: Kupffer cells
C2. Humoral:
 Bone: Osteoclasts
 Antibodies (GAMDE) – produced by mature
 Placenta: Hofbauer cells
plasma cells
 Spleen: Littoral cells
 Cytokines
 Kidney: Mesangial cells

B2. Humoral:
IgM
 APRs (C- reactive protein, amyloid A)
- Pentomeric; Largest antibody
o “M” – Malaki
- Millionaire’s antibody
- APR (Acute Phase Reactants)
- Agglutinating antibody
o Non-specific but sensitive; increase concentration
IgG
when there is trauma, injury, or inflammation
- Non-agglutinating antibody
 C-reactive protein
- Smallest antibody
 Transferrin
o “G” – Gamay
 Serum amyloid A
- IgG1 – most effective in crossing the placenta
o IgG1, IgG3, IgG4
- Negative APR
o IgG2 – cannot cross placenta
o Decrease concentration when there is inflammation
- IgG3 – most effective in activating complement fixation
 Albumin
o IgG1, IgG2, IgG3
 Transferrin
o IgG4 – cannot activate complement
 Transthyretin – Retinol-binding protein;
NK cells
transports vitamin A (retinol) and a
- Non-specific
hormone called thyroxine throughout the
- Bridges
body.

Kimberly Anne Hampac | BSMT – 3 | Notre Dame of Marbel University


PAMPs-PRR (soluble/cell-associated)
Indirect: (has opsonization)
PAMPS – soluble PRR molecules – binding to your
PAMPs(microorganism): OPSONIZATION – phagocytosis of PRR-bound
microorganism – proteolytic lysis – lysis of the cell – antigens – ADAPTIVE
IMMUNITY
Direct: (directed to cells)
PAMPS – Cell-associated PRR – phagocytosis of microorganism –
proteolytic lysis – lysis of the cell – antigens – ADAPTIVE IMMUNITY
DAMPs: Danger Associated Molecular Patterns
(alarmins)
A. Necrosis: release of DAMPs (PATHOLOGIC)
Indirect: (has opsonization)
Severe injury, tissue trauma, burns, toxin – NECROSIS (rupture of the
necrotic cell) – release of DAMPs – soluble PRR or cell-associated PRR
PAMPs(microorganism): OPSONIZATION – phagocytosis of PRR-bound
microorganism – proteolytic lysis – lysis of the cell – antigens – ADAPTIVE
IMMUNITY
Direct: (directed to cells)
Severe injury, tissue trauma, burns, toxin – NECROSIS (rupture of the
necrotic cell) – release of DAMPs – soluble PRR or cell-associated PRR –
INFLAMMATION (immune response) – chemokine and cytokine
production
Rubor (redness) – Calor (Heat) – Tumor (swelling) – Dolor (pain) – Functio
Laesa (loss of function)
B. Apoptosis: no release of DAMPs (PHYSIOLOGIC)
Apoptosis (RBC 120 days) – phosphatidylserine – no cell rupture –
Phagocytosis (RES) – no immune response
Kimberly Anne Hampac | BSMT – 3 | Notre Dame of Marbel University
IMMUNOLOGY AND SEROLOGY LABORATORY Principles and Experiments:
Activities 1 – 7 3
Activity 3
SCREENING TEST FOR PHAGOCYTIC ENGULFMENT
PHAGOCYTOSIS
- Elie Metchnikoff
- Greek word: “phagein”, meaning, cell eating
- Endocytosis (2nd line of defense of innate immunity)
- A body defense mechanism wherein specialized cells
(phagocytic cells; MNMs) engulf and destroy foreign particles
- Macrophages and Segmented neutrophils (PMNs)
INFLAMMATION
1. Vascular Response – Characterized by ↑ vascular permeability
(hallmark of inflammation)
a. Rubor (redness) – ↑ blood flow at the site
b. Calor (heat) – transfer of internal heat from the blood to the site
c. Tumor (swelling) – extravasation of protein portion of blood
outside the blood vessel because of ↑ vascular permeability
d. Dolor (pain) – too much pressure on sensory nerves
e. Functio Laesa (loss of function)
2. Cellular Response
Initiation: MRAT
a. Margination – movement of WBC to the endothelial cells;
center to periphery
b. Rolling (selectin) – transient adhesion of WBC to the
endothelial cells with the aid of selectin
c. Adhesion (integrin) – firm attachment of WBC to the
endothelial cells with the aid of integrin
d. Transmigration/ Diapedesis – migration of WBC through the
endothelium to the tissues
e. Chemotaxis (unidirectional/ targeted) – targeted movement
of WBC towards antigens/ bacteria in response to certain
chemicals – chemoattractant (e.g., complement components
– C5a & C3a)
o Chemoattractant guides phagocytes to the site of
injury; without it, movement of WBC will be random
f. Opsonization (IgG & C3b) – coating of pathogens with
opsonins to make it more attractive for ingestion
g. Phagocytosis (CAEPPDD)
o Chemotaxis
o Adherance
o Engulfment
o Phagosome formation
 membrane bound vesicle in a phagocyte
containing the engulfed material
o Phagolysosome formation
 a vacuole formed from the fusion of
phagosome and lysosome in which
microorganisms are killed and digested
o Digestion
o Destruction
3. Cellular Proliferation and Repair
a. Fibroblast (AMP)
o Acidic Mucopolysaccharide (AMP) – counteract
the effect of other substances released by mast cell
and basophil during an immune response to facilitate
resolution or total repair
b. Complete Repair/ Abscess formation/ Granuloma
formation
o Granuloma formation in M. tuberculosis
immunocompromised individulas
o Abscess/ “pus” – dead cells + macrophage/
monocyte + neutrophils
 IMMUNOLOGY AND SEROLOGY LABORATORY Principles and Experiments:
Activities 1 – 7 4

LEUKOCYTES Screening Test for Phagocytic Engulfment

A. Granulocytes
- Neutrophil PRINCIPLE:
o bacterial & fungal infection “Involves the combination and incubation of bacteria and phagocytes,
o complement activation (ACP) which are then examined for the presence of engulfed bacteria.
o principal phagocytic cell (5.4 days) because they are Useful in supporting the diagnosis of impaired neutrophilic function in
the first to respond conjuction with signs and symptoms.”
o circulating (segmenters) & marginating pool
o Marginating pool on blood vessel walls – where half SPECIMEN:
of total neutrophil population is normally found  Coagulase-negative microorganisms
o Circulating (6-10 hours) pool increase immediately o S. saprophyticus
during acute infection o S. epidermidis
o Candida albicans – fungus
- Eosinophil:  Coagulase-positive microorganisms will give false-negative
o less phagocytic activity o S. aureus
o hemostatic regulator of inflammation
o parasitic infection [Major Basic Protein (MBP), 2 METHODS:
Eosinophil Cationic Protein (ECP)] – enzymes Conventional Testing
directed to parasites - Intracellular particles/ ingestible substances that can be
o can only recognize helminths if it is complexed with extracted
IgE - Target cells + live/ heat-killed C. albicans or Staphylococcus
o Trichinela spiralis spp. except SAU (false-negative)
- Giemsa/ Wright-stained smear
- Basophil: - 200 cells are examined and counted for ingested
o less phagocytic activity microorganism in OIO/ 1000x total magnification
o type I hypersensitivity - Results: % target cells showing phagocytosis and number of
o IgE molecules & degranulation (2 IgE molecules to particles per phagocytic cells
activate degranulation)
 Histamine & Heparin: released during
no. of cells with phagocytized material
degranulation x 100
 Mast cell: found in connective tissues; total no. of cells counted
bigger than basophil

The less phagocytic activity is due to:

Flow Cytometry
 Less number in the circulation
- Labeled targets (fluorescent beads coated with opsonins)
o "Never Let Monkey Eating Banana"
o Radioactive materials, fluorescent dyes, enzymes
 Not powerful digestive enzymes
o SAU will have no more interference in this method
o It is not subjective to the medtech that counts the
Dendritic cell – most potent phagocytic cell
smear, so it is more accurate
- Labels:
o Fluorescein-labeled C. albicans
o Fluorescein isothocyanate (FITC)-labeled zymosan
B. Non-granulocytes
o Fluorescein-labeled S. aureus
- Monocyte/ Macrophage:
o Fluorescein-labeled E. coli
o fixed or wandering cells
o Enhanced Green Fluorescence Protein (EGFP)-
o Phagocytosis
expressing E. coli
o Disposal of aged cells
o Bis-carboxyethyl-carboxyfluorescein
 Phosphatidylserine – indicator that
pentaacetoxymethylester-labeled C. albicans
RBCs are for disposal
o Cancer cell surveillance (tumor necrosis factor
alpha – secreted by monocyte/ macrophage
PROCEDURE
directed to cancer cell; can be secreted by NK cells)
1. Heparinized WB
o Tumor rejection
o Heparin is secreted by basophil & mast cell so it is a
natural anticoagualant; it is used to avoid
- Lymphocytes:
interferences to WBC functionality to target cells
o T cell (CD4+ and CD8+)
o B cell (memory or plasma cell)
2. Centrifuge
o NK cell (large granular)/ Null cell
o Separate fluid portion (plasma) to blood cells
 Share common progenitor cell with T cell
but NK cell will not mature in thymus
3. Buffy coat + Broth (coagulase – staph)/ candida albicans
 It is the only lymphocyte that is granular
o “grayish white” in the interphase – buffy coat
 Perforin & granzyme – enzymes
contains: WBC, platelets, and nucleated RBCs
directed to virally-infected/ tumor cells
 CD16 & CD56 – identify NK cell
Why are nucleated RBCs isolated in the buffy coat? – HgB content of
anucleated/ mature RBCs are denser, so they will settle at the bottom of
the tube. Nucleated RBCs do not contain hemoglobin, so they are less
dense. - Hampac

Kimberly Anne Hampac | BSMT – 3 | Notre Dame of Marbel University

 IMMUNOLOGY AND SEROLOGY LABORATORY Principles and Experiments:
Activities 1 – 7 5

4. Incubate at room temperature (Staphylococcus spp.)/ 37 C for GRADING AGGLUTINATION

30 mins (C. albicans) GRADE DESCRIPTION
CELLS SUPERNATE
5. Smear 0 No agglutinates Dark, turbid,
homogenous
6. Giemsa/ Wright Stain W+ Many tiny agglutinates; many free cells Dark, turbid
1+ Many small agglutinates; many free cells Turbid
7. OIO
2+ Many medium-sized agglutinates; Clear
o Has the highest numerical aperture because it needs
moderate free cells
more light, so we use Immersion oil – trap/ contain
3+ Several large agglutinates; few free cells Clear
the light directed to the specimen to the objective, so
it will not diffract to any direction 4+ One large, solid agglutinate; no free cells Clear

OSMOSIS
Normal Saline Solution (0.85% sodium chloride solution)
INTERPRETATION - Some sources claim 0.9%; but 0.85% is the accurate salt conc
 (+) – has phagocyte with phagocytized material; even only one - Isotonic solution
bacteria is phagocytized; bacterial engulfment demonstration - Equal solute and solvent
 (-) – no phagocytized material; no engulfment (neutrophilic - Red cells are unchanged/ normal size (6-8um; 7um to be exact)
dysfuction) - Water consistently enters and exits to and from the cell
o If the person has neutrophil dysfunction:
 Px sample: (-) Hypotonic Solution (>0.85% salt conc)
 Control: (+) - Cells will be larger than usual because water will enter the cell
to compensate for lower salt concentration outside the cell
- Swell cell
- More solvent; dilute
CONSIDERATIONS - Below 0.85% salt conc
- False negative: sample in conventional testing is not fresh and
usage of SAU Hypertonic Solution (<0.85% salt conc)
- It is important to distinguish between granules and cocci - Cells will be smaller than usual because water will exit the cell
- Bacteria must be intracellular to be positive; bacteria outside the to compensate for high salt concentration
WBC is not engulfed, so it is not positive to the test - Shrink cell
- Does not demonstrate that the bacteria is destroyed - More solute; concentrated
- Hyper = higher conc; Above 0.85%

Osmosis
- Movement of a solvent (such as water) through a
semipermeable membrane (as of a living cell) into a solution of
Activity 4 higher solute concentration that tends to equalize the
PREPARATION OF RED CELL SUSPENSION concentrations of solute on the two sides of the membrane.
- In osmosis, water moves from areas of low concentration of
solute to areas of high concentration of solute.
Red Cell Suspension
- Important in maintaining the appropriate serum-to-cell ratio Diffusion
- In diffusion, particles move from an area of higher concentration
to one of lower concentration until equilibrium is reached. In
PRECIPITIN CURVE osmosis, a semipermeable membrane is present, so only the
Prozone phenomenon solvent molecules are free to move to equalize concentration.
- Antibody excess (antiBody – pro)
- No visible agglutination SAMPLE PROBLEMS
- Dilute serum with a buffer PCV
- False-negative Total volume = x 100
desired RCS concentration

Zone of Equivalence PCV =

(Tv) (desired concentration)
- Antigen = Antibody 100
- Visible agglutination PCV
%RCS = x 100
total volume
Postzone phenomenon
- Antigen excess (antiGen– post; after B) mL of NSS to be added = total volume – PCV
- No visible agglutination
- ↑ serum-to-cell ratio; ↑ parts of serum
- Recollection after 2 weeks Volume:
- False-negative 1 L = 1000 mL = 1000 000 uL
1 mL = 1000 uL

SOURCES OF ERROR Mass:

 Hemolysis – no more visible agglutination 1 kg = 1000 g = 1000 000 mg = 1000 000 000 ug
1 g = 1000 mg = 1000 000 ug

Kimberly Anne Hampac | BSMT – 3 | Notre Dame of Marbel University

 IMMUNOLOGY AND SEROLOGY LABORATORY Principles and Experiments:
Activities 1 – 7 6

1. 5mL of 6%; get PCV and NSS volume  False-negative

o Dilute Coombs’ reagent caused by ↑ NSS volume
 Decreased sensitivity and diluted AHG
o Inadequate washing
 Decreased sensitivity and diluted AHG
o NSS not completely aspirated
2. 10mL of 10%; get PCV and NSS volume  Decreased sensitivity and diluted AHG
o NSS pH below 7.2 or decreased
 NSS must be with the pH 7.2 – 7.4
o NSS stored for longer time in plastic container
 ↑ antibody elution

3. 8mL of 15%; get PCV and NSS volume  False-positive

o Overcentrifugation
o Bacterial contamination

Disregarding the volume of the above given examples, what is the

difference among these three test samples in physical appearance?
 Color intensity. 6% is the lightest (low con) while 15% is the
darkest (high conc)

APPLICATIONS
FORWARD TYPING REVERSE TYPING
Detection of ABO antigens Detection of ABO antibodies
Sample: Red cell Serum
Reagent: Antisera RCS (A1, B1)
Interpretation of visible A1 RCS – visible agglutination
agglutination: B1 RBC – no agglutination Activity 5
Blue – A antigen  B blood type (has anti-A, so SERIAL DILUTION PREPARATION
Yellow – B antigen it will agglutinate with A
antigen in A1 RCS) SERIAL DILUTION is performed to know the antibody titer of the
“kung saan merong agglutination, “kung saan wala agglutination, individual. The gretatest dilution of the sample that yields a positive result
yun yung blood type ni Px” yun yung blood type ni Px” is the end point (expressed as fraction). The reciprocal of that fraction is
the titer of the antibody. In serial dilution each dilution is prepared from the
SERUM PLASMA previous dilution.
Fluid portion of coagulated (non Fluid portion of anticoagulated - Microtitration – valuable for any procedure in which dilutions
anticoagulated) blood blood are made and erythrocytes are used as indicator cells
No fibrinogen Has fibrinogen (hemagglutination)
Clear Hazy (presence of proteins) o Colorimetric reactions – performed and quantified
DAT IAT spectrophotometrically with specialized instruments
In vivo In vitro for microtiter plates
No incubation needed Needs incubation (37 C)

Hemagglutination test
- A type of antibody-antigen reaction that will result in the
clamping or agglutination of RBC (e.g., ABO typing and Rh
testing)
ERROR IN WASHING OF RED CELLS
 Group III ABO discrepancy is mostly involved
o Elevated levels of immunoglobulins
o Has fibrinogen
o Cord blood- Wharton’s jelly; wash for 6-8x
 True agglutination vs Pseudoagglutination
o Add one drop of NSS to differentiate
o True agglutination will remain agglutinated
o Pseudoagglutination/ Rouleux formation (stack of
coins) will not clump anymore
ANTIBODY TITER
- Determine the immune status of the individual
- Titer is high when patient is almost or already cured. They
already developed antibody against infection)
- Can be used to monitor mother’s titer for blood group antibodies
during pregnancy
- IgM is seen in acute or present infection. It is the
immunoglobulin in primary response of infection.
- IgG is seen in chronic or past infection. It is a life-long marker.
It is the immunoglobulin in the secondary or anamestic
response.
Why do we centrifuge RBC?
- Centrifugation (1 minute at 1,500-1,800rpm) overcomes the
zeta potential of RBC (net negative charge that makes them
repel each other) to bring them together and accelerate
agglutination

Kimberly Anne Hampac | BSMT – 3 | Notre Dame of Marbel University

 IMMUNOLOGY AND SEROLOGY LABORATORY Principles and Experiments:
Activities 1 – 7 7

Activity 6
SAMPLE PROBLEM PRECIPITATION
Dilution Strength of antigen-antibody agglutination
1 1: 10 4+ PRECIPITATION
2 1:5 3+ - Involves combining soluble antigen with soluble antibody to
3 1:4 1+ produce insoluble antigen-antibody complexes that are visible
4 1:9 0 - First noted by Kraus in 1897, who found that culture filtrates of
5 1:15 0 enteric bacteria would precipitate when they were mixed with
specific antibody.
1. What is the final dilution in each test tube? - Antigen and antibody must have binding sites for one another
1 – 1:10 - Binding characteristics of antibodies are called affinity and
2 – 1:50 avidity, plays an major role along with the law of mass action
3 – 1:200 and the principle of lattice formation.
4 – 1:1800
5 – 1:27000
Affinity – Sensitization Relationship
2. What is the antibody titer of the patient? - Affinity is the initial force of attraction that exist between a single
 The antibody titer of the patient is 200. Fab site (paratope) and the antigenic determinant (epitope).
 Tube 3 is the last tube seen with agglutination. The o Force is non-covalent/ weak; reversible.
reciprocal of its final dilution is the antibody titer.  Hydrogen bond
 Hydrophobic bond
SERIAL DILUTION PROCEDURE  Ionic bond
Tube 1 2 3 4 5 6 7 8 9 10  Van der Waals forces
Number - Affinity will affect sensitization.
mL 0.8 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
saline
mL 0.2 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Avidity – Lattice Formation Relationship
serum - Avidity is the sum of the overall bonds present in the multivalent
Dilution 1:5 1:2 1:2 1:2 1:2 1:2 1:2 1:2 1:2 1:2 antibody and multivalent antigen.
after o Cause the irreversibility of lattice formation
serum - Avidity will affect lattice formation.
transfer
mL of 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
cell
suspens PROCEDURE
ion 1. Add 2mL melted agar to tube. Let it cool. Antibodies (protein) in
Total 1 1 1 1 1 1 1 1 1 1 plasma will denature if it is still hot.
mL 2. Add plasma-containing antibodies (serve as antigen)
Final 1:5 1:1 1:2 1:4 1:8 1: 1: 1: 1: 1: 3. After the agar-containing plasma has solidified, add AHG reagent
dilution 0 0 0 0 16 32 64 12 25 (antibody)
of 0 0 0 80 60 - AHG detects IgG (serve as antigen)
serum 4. Stand for 15-30 minutes at room temperature. AHG reagent will
diffuse to the plasma-containing antibody.
solute serum 0.2 1
Dilution1 = = = = = 1: 5 5. Observe for precipitation.
TV serum+saline 0.2+0.8 5
solute serum 0.5 1  Detection of IgG (non-agglutinating/ incomplete antibodies)
Dilution2 = = = = = 1: 2
TV serum+saline 0.5+0.5 2
Polyspecific AHG reagent Monospecific AHG reagent
TV = ( Vserum + Vsaline + VRCS ) – Vtransferred = 1.5mL-0.5mL = 1mL Detect IgG and C3d Detect either (IgG only / C3d only)
Rabbits “M” for Mouse/ mice
“Dislodge” the button of the cells gently.
- To check if there is agglutination by gently tapping the side of
the tube with your finger. Blood Group Antigen Antibody
- After dislodging, if there are particles/ agglutinates, then the test
A A, H Anti-B
is positive. If unsure, you can check it under the microscope if
B B, H Anti-A
there are clumping of red cells.
AB A, B, H (in small amount) None
O H (plenty) Anti-A, Anti-B, Anti-AB

Nature of Antigen and Antibodies of ABO Blood Group

- H antigen in AB blood group is small in quantity because it is
already converted to A or B antigen.
- Antibodies are mostly IgM. Some are IgG, and IgA.
- Anti-A and Anti-B are mostly IgM in nature. There are some
immune antibodies.
- Anti-AB is mostly IgG in nature.

Kimberly Anne Hampac | BSMT – 3 | Notre Dame of Marbel University

 IMMUNOLOGY AND SEROLOGY LABORATORY Principles and Experiments:
Activities 1 – 7 8

RESULT AND INTERPRETATION

SINGLE LINEAR IMMUNODIFFUSION /
OUDIN IMMUNODIFFUSION

Antibody is incorporated to (bottom)

AHG (antibody) – diffuse/ move
test tube and layered with antigen (top)
(PROZONE)
Antigen – diffuse/ move
Zone of Equivalence – visible
(POST-ZONE)
precipitation
(POST-ZONE)
Zone of Equivalence – visible
Plasma-containing antibody (antigen)
precipitation
- immotile
(PROZONE)
Antibody - Stationary

Blood type O – precipitation is near the center.

- By James Oudin; Precipitation occurred and moved down the
Blood type A and B – precipitation is near the bottom because there are
tube in proportion to the amount of antigen present.
only few Anti-A and Anti-B that are IgG in nature.
Blood type AB – no precipitation because there is no antobody.

SINGLE RADIAL IMMUNODIFFUSION

PRECIPITIN CURVE Prozone

Ab – layered in gel
LEGEND: Post-zone
- Antibody
Ag Zone of Equivalence
- Antigen

Prozone Zone of Equivalence Post-Zone
I. PASSIVE IMMUNODIFFUSION TECHNIQUES
Precipitation of antigen–antibody complexes can also be
determined in a support medium such as a gel. Agar, a high-molecular-
weight complex polysaccharide derived from seaweed, and agarose, a
purified agar, are used for this purpose. Agar and agarose help stabilize
the diffusion process and allow visualization of the precipitin bands.
Reactants are added to the gel, and antigen–antibody combination occurs
by means of diffusion. When no electrical current is used to speed up this
process, it is known as passive immunodiffusion. The rate of diffusion is
affected by the size of the particles, the temperature, the gel viscosity, and
the amount of hydration.
An agar concentration ranging from 0.3 percent to 1.5 percent
allows for diffusion of most reactants. Agarose is preferred to agar,
because agar has a strong negative charge, while agarose has almost
none, so interactions between the gel and the reagents are minimized.
Immunodiffusion reactions can be classified according to the number of
reactants diffusing and the direction of diffusion.
Kimberly Anne Hampac | BSMT – 3 | Notre Dame of Marbel University
- Antibody is layered in gel. Let it dry. Ab is stationary in the gel.
- Cut a well in the center. Place the sample (antigen) in the well.
- As the antigen diffuses out from the well, antigen–antibody
combination occurs in changing proportions until the zone of
equivalence is reached and a stable lattice network is formed in
the gel. The area of the ring obtained is a measure of antigen
concentration, and this can be compared to a standard curve
obtained by using antigens of known concentration.
- Area of the circle is directly proportional to the concentration
- 2 Techniques:
o End-point/ Mancini Method
 Antigen is allowed to diffuse to
completion, and when equivalence is
reached, there is no further change in the
ring diameter.
 Occurs between 24 and 72 hours.
 The square of the diameter is then
directly proportional to the concentration
of the antigen.
o Kinetic/ Fahey and McKelvey method
 Uses measurements taken before the
point of equivalence is reached. In this
case, the diameter is proportional to the
log of the concentration.
 Readings are taken at about 18 hours.
- For either of the two method, it is essential that monospecific
antiserum with a fairly high affinity be used. This increases
the clarity of the precipitation reaction.
- The precision of the assay is directly related to accurate
measurement of samples and standards.

- Used to measure IgG, IgM, IgA, and complement components.
It is simple to perform and requires no instrumentation, but
expensive. Thus, immunodiffusion has largely been replaced by
more sensitive and automated methods such as nephelometry
and enzyme-linked immunosorbent assays except for low
volume analytes such as IgD or IgG subclasses.
OUCHTERLONY METHOD/
DOUBLE RADIAL IMMUNODIFFUSION
- “Double” meaning, two or both antibody and antigen will move.
- Unknown antigens are placed in the outside wells. The
antibodies and antigens all diffuse radially out of the wells.
- Determine whether antigen are the same or identical.
o (A) Serological identity. Antigens are identical,
they will react with the same antibody and the
precipitate line forms a continuous arc/ smooth
arc.
o (B) Nonidentity. Antigens share no identical
determinants, they will react with different
antibodies and two crossed lines are formed.
o (C) Partial identity. Antigen 1a has a determinant in
common with antigen 1, so one of the antibodies
reacts with both antigens, but it is not as complex.
Another antibody that reacts with different
determinants on antigen 1 (absent on antigen 1a)
passes through one precipitation line and forms the
spur on the other line. The spur formed always
points to the simpler antigen with fewer antigenic
determinants
- “identical” does not necessarily mean absolute antigens are
present; it means majority of their antigenic determinants are
the same.
II. ELECTROPHORETIC TECHNIQUES
Diffusion can be combined with electrophoresis to speed up or
sharpen the results. Electrophoresis separates molecules according to
differences in their electric charge when they are placed in an electric field.
A direct current is forced through the gel, causing antigen, antibody, or both
to migrate. As diffusion takes place, distinct precipitin bands are formed.
This technique can be applied to both single and double diffusion.
Anode
Kimberly Anne Hampac | BSMT – 3 | Notre Dame of Marbel University
IMMUNOLOGY AND SEROLOGY LABORATORY Principles and Experiments:
Activities 1 – 7 9
ROCKET IMMUNOELECTROPHORESIS
+ (anode)
Layered antibody
Antigens are placed in wells
- (cathode)
- Antibody is distributed in the gel, and antigen is placed in wells
cut in the gel, just as in RID. However, instead of allowing
diffusion to take place at its own rate, electrophoresis is used to
facilitate migration of the antigen into the agar. When the
antigen diffuses out of the well, precipitation begins.
- As the concentration of antigen changes, there is dissolution
and reformation of the precipitate at ever-increasing distances
from the well. The end result is a precipitin line that is conical
in shape, resembling a rocket.
- The height of the rocket, measured from the well to the apex,
is directly in proportion to the amount of antigen in the sample.
If standards are run, a curve can be constructed to determine
concentrations of unknown specimens.
- It is essential to determine the net charge of the molecules at
the pH used for the test, because this determines the direction
of migration within the gel. Typically, a pH is selected so that
antibodies in the gel do not move, but the antigens become
negatively charged. Antigens then migrate toward the positive
anode. This technique has been used to quantitate
immunoglobulins, using a buffer of pH 8.6
SIMPLE IMMUNOFIXATION
Anode
Layered antibody
Antigens are placed in wells
Cathode
Cathode
 IMMUNOLOGY AND SEROLOGY LABORATORY Principles and Experiments:
Activities 1 – 7 10

COUNTER IMMUNOELECTROPHORESIS
Anode

“Trough” – well; moving antibody

-
- Antibody is layered in the well and antigen are placed in the well.
Electric current will be applied to move antigens (either cathode
or anode). After separation of sample, electric current is applied
again, so the movement of antigens is towards the matrix
containing antibody. Result: precipitin bands
Antigens are placed in wells
*concept relating to Molecular Biology: Anode Cathode
- Horizontal electrophoresis, separation based on isoelectric Cathode
point, when optimum pH is achieved, molecules will assume net
0 charge. Sodium Dodecyl Sulfate is added to coat substance
to make molecules negatively charged (anions) going to one
direction only – anode.
- Vertical electrophoresis is performed. Anions will go to anode.
Stain using fluorescent dye to visualize precipitin bands.

*detailed explanation of the procedure:

- After electrophoresis takes place, antiserum is applied directly
to the gel’s surface. Agarose or cellulose acetate can be used
for this purpose. Immunodiffusion takes place in a shorter time
and results in a higher resolution than when antibody diffuses
from a trough. Because diffusion is only across the thickness of
- Horizontal electrophoresis to separate antigens base on
the gel, approximately 1 mm, the reaction usually takes place
isoelectric point. Instead of layering directly the antibody, trough
in less than 1 hour.
is made where antibody is placed. Result: precipitin arc
- An antibody of known specificity is used to determine whether
- The same principle with simple immunofixation, the only
patient antigen is present. The unknown antigen is placed on
difference is that antibody in counter immunoelectrophoresis is
the gel, electrophoretic separation takes place, and then the
placed in a trough.
reagent antibody is applied. Immunoprecipitates form only
where specific antigen–antibody combination has taken place,
- Immunoelectrophoresis is a double-diffusion technique that
and the complexes have become trapped in the gel. The gel is
incorporates electrophoresis current to enhance results.
washed to remove any nonprecipitating proteins and can then
- Introduced by Grabar and Williams in 1953, this is performed as
be stained for easier visibility. Typically, patient serum is applied
a two-step process and can be used for semiquantitation of a
to six lanes of the gel, and after electrophoresis, five lanes are
wide range of antigens.
overlaid with one each of the following antibodies: antibody to
- Typically, the source of the antigens is serum, which is
gamma, alpha, or mu heavy chains and to kappa or lambda light
electrophoresed to separate out the main protein fractions; then
chains. The sixth lane is overlaid with antibody to all serum
a trough is cut in the gel parallel to the line of separation.
proteins and serves as the reference lane. Reactions in each of
Antiserum is placed in the trough, and the gel is incubated for
the five lanes are compared to the reference lane.
18 to 24 hours. Double diffusion occurs at right angles to the
- Cerebrospinal fluid is the specimen of choice for diagnosing
electrophoretic separation, and precipitin lines develop where
multiple sclerosis, and urine is used to detect the presence of
specific antigen–antibody combination takes place. These lines
Bence-Jones proteins that are found in multiple myeloma.
or arcs can be compared in shape, intensity, and location to that
- Although immunofixation is more sensitive than
of a normal serum control to detect abnormalities. Changes
immunoelectrophoresis, dilutions may be necessary to avoid
include bowing or thickening of bands, or changed mobility.
the zones of antigen excess, which may occur if concentrations
- This procedure has been used as a screening tool for the
of monoclonal antibody are very high.
differentiation of many serum proteins, including the major
- Perhaps one of the best-known adaptations of this technique is
classes of immunoglobulins. It is both a qualitative and a
the Western blot, used as a confirmatory test to detect
semiquantitative technique and has been used in clinical
antibodies to human immunodeficiency virus 1 (HIV-1). A
laboratories for the detection of myelomas, Waldenström’s
mixture of HIV antigens is placed on a gel and electrophoresed
macroglobulinemia, malignant lymphomas, and other
to separate the individual components. The components are
lymphoproliferative disorders.
then transferred to nitrocellulose paper by means of blotting or
- In addition, immunodeficiencies can be detected in this manner,
laying the nitrocellulose over the gel so that the electrophoresis
if no precipitin band is formed for a particular immunoglobulin.
pattern is preserved. Patient serum is applied to the
Deficiencies of complement components can also be identified.
nitrocellulose and allowed to react. The strip is then washed and
- However, this technique is relatively insensitive, with a detection
stained to detect precipitin bands.
limit of approximately 3 to 20 uL concentration. Interpretation
- It is simpler to visualize the reaction on the nitrocellulose, and
can also be difficult and may take considerable experience.
in this manner, antibodies to several antigens can be detected.
Therefore, it has largely been replaced by immunofixation
- This technique is characteristically used to determine the
electrophoresis, which gives quicker results and is easier to
presence of antibodies to organisms of complex antigenic
interpret.
composition. If antibodies to more than one disease-associated
antigen are identified in patient serum, this usually confirms
presence of the suspected disease.

Kimberly Anne Hampac | BSMT – 3 | Notre Dame of Marbel University

 IMMUNOLOGY AND SEROLOGY LABORATORY Principles and Experiments:
Activities 1 – 7 11

Activity 7
AGGLUTINATION LATTICE FORMATION
- Avidity is the sum of all forces in multivalent antigen and
multivalent antibody.
AGGLUTINATION
- Combining soluble antigen/ antibody and insoluble antibody/
antigen to produce visible antigen-antibody complexes.
- Has carrier
- E.g., insoluble antibody with particles such as red cell or latex;
HYPERSENSITIZATION
- Sensitization is the first exposure.
antigen is soluble and vice versa
- Second exposure is the lattice formation. The body recognizes
antigens so antibodies will evoke immune response.
2 phases:
o Signs and symptoms appear.
o Sensitization
o Lattice Formation
PROCEDURE is the same with serial dilution.
Paratope/ Fab Site
- Antigen-binding site of antibody
NOTES TO REMEMBER:
- Anti-sera is the reagent being diluted.
Epitope/ Antigenic Determinant
- Dislodge by tapping against finger.
- Antibody-binding site of antigen
- Use HPO if agglutination is not visible by naked eye.

INTRODUCTION
In 1896, Gruber and Durham published the first report about
the ability of antibody to clump cells, based on observations of agglutination
of bacterial cells by serum. This finding antogave rise to the use of serology
as a tool in the diagnosis of disease and also led to the discovery of the
ABO blood groups. Widal and Sicard developed one of the earliest
diagnostic tests in 1896 for the detection of antibodies occurring in typhoid
fever, brucellosis, and tularemia. Agglutination reactions now have a wide
variety of applications in the detection of both antigens and antibodies.
Such testing is simple to perform and the end points can easily be read
visually.
Agglutination, like precipitation, is a two-step process that
results in the formation of a stable lattice network. The first reaction, called
sensitization, involves antigen–antibody combination through single
antigenic determinants on the particle and is rapid and reversible. The
second step, or lattice formation, is the formation of cross-links that form
the visible aggregates. This represents the stabilization of antigen–
antibody complexes with the binding together of multiple antigenic
determinants.
Sensitization is affected by the nature of the antigens on the
agglutinating particles. If epitopes are sparse or if they are obscured by
other surface molecules, they are less likely to interact with antibody.
Additionally, red blood cells (RBCs) and bacterial cells have a slight
negative surface charge; because like charges tend to repel one another,
it is sometimes difficult to bring such cells together into a lattice formation.

Sensitization must happen before lattice formation.

Paratope
Noncovalent bonds
Epitope

SENSITIZATION
- Affinity is the initial force between the paratope and epitope.
Initial forces in between them are noncovalent bonds
(reversible).
- 1 Antigen = 1 Antibody

Kimberly Anne Hampac | BSMT – 3 | Notre Dame of Marbel University