IMMUNOLOGY AND SEROLOGY DAY 2 HANZEL V.

TOLENTINO, RMT

A. Laboratory Safety
 Chain of infection – requires three: source, method of transmission and susceptible host
 Universal precautions (UP) - instituted by the CDC in 1985 to protect health-care workers
from exposure to bloodborne pathogens, primarily hepatitis B virus (HBV) and HIV.
 Body substance isolation (BSI) replacement of UP not limited to bloodborne pathogens
and considers all body fluids and moist body substances to be potentially infectious.
 Sodium Hypochlorite – also known as household bleach. Commonly used disinfectant
with dilution of 1:10
 material safety data sheet (MSDS) - All chemicals and reagents containing hazardous ingredients
in a concentration greater than 1 percent are required to have this
 biohazard symbol – color is fluorescent-orange or orange-red

B. Precipitation Reactions
 Precipitation involves combining soluble antigen with soluble antibody to produce
insoluble complexes that are visible.
 Affinity - the initial force of attraction that exists between a single Fab (fragment
antigen-binding) site on an antibody molecule and a single epitope or determinant site
on the corresponding antigen.
 Avidity - represents the sum of all the attractive forces between an antigen and an
antibody. This involves the strength with which a multivalent antibody binds a
multivalent antigen, and it is a measure of the overall stability of an antigen–antibody
complex.

Prozone  zone of equivalence  Post zone

excess Ab equal Ab to Ag excess Ag

 How do we measure or quantify precipitation?

o Turbidimetry
 measure of the turbidity or cloudiness of a solution
 Detection device is put in direct line with the source of light.
o Nephelometry
 measures the light that is scattered at a particular angle from the
incident beam as it passes through a suspension
 Detection device is put in perpendicular line with the source of light
o Immunodiffusion Techniques
 Immunodiffusion without electrophoresis
a. Also known as Passive immunodiffusion
b. Uses agar from seaweed OR agarose gel. These two stabilizes the
diffusion process and make the precipitin band visible.
c. No electricity is present
d. rate of diffusion is affected by the size of the particles, the
temperature, the gel viscosity, and the amount of hydration.
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e. Types of Passive Immunodiffusion:

Single – only one diffuses. Either antibody or antigen

i. Single diffusion:
 Pioneered by James Oudin
 Antibody is mixed with agarose gel and put into
tube. Antigen is layered on top and moves down
in proportion to the amount of antigen present
or layered.
 The distance of precipitin bands from the top of
the gel is directly proportional to amount of
antigen
ii. Radial Immunodiffusion
 Modification of single immunodiffusion
 Antibody is mixed with agarose gel and put into
plate/slide. Antigen is put into the well cut in the
gel. Antigen moves sideways in proportion to the
amount of antigen present or put.
 The area of the ring formed is directly
proportional to amount of antigen.
a. Mancini Method of determining result
 End-point method
 24-72 hours for result
b. Fahey and McKelvey method of
determining result
 Kinetic method
 18 hours for result

Double – two diffuses. Both Ab and Ag

iii. Ouchterlony double diffusion
 Antigen and antibody is put on wells cut in a gel
and diffuses.
 Multi specific antibody is put on the center well.
 Antigens are put on wells surrounding the
antibody.
 Antigen and Antibody diffuses and formation of
arc is observed.
 Arc line is observed if both antigens are the same
 Cross line is observed if antigens are not the
same or not share a similar epitope
 Spur formation is seen for partial similarity.
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 Immunodiffusion with electrophoresis

a. The same with passive diffusion but has the presence of
electrical charge
b. This technique can be applied to both single and double passive
mmuno-diffusion.
c. Types of Immunodiffusion with electrophoresis
i. rocket immunoelectrophoresis
 Adaptation of radial immunodiffusion
 Developed by Laurell in 1960
 The end result is a precipitin line that is conical in
shape, resembling a rocket, hence the other
name rocket immunoelectrophoresis
ii. Immunoelectrophoresis
 A double diffusion
 Two-step process
a. Antigens are separated through
electrophoresis
b. Trough is made between specimens and
antiserum is put in it and incubated from
18 to 24 hrs
 These lines or arcs can be compared in shape,
intensity, and location to that of a normal serum
control to detect abnormalities.

iii. Immunofixation Electrophoresis

 Procedure is same as Immunoelectrophoresis
but instead of putting antiserum in the trough,
antiserum is put directly on the gel’s surface
 Faster results of around 1 hour only.
C. Agglutination
 Aggregation of particles caused by combination with an antibody
 Involves two-step process:
o Sensitization – initial binding between one Ab and one Ag
o Lattice formation – binding of Ab-Ag complex to another Ab-Ag complex
 Ways to increase agglutination:
o Increasing viscosity – by addition of dextran or polyethylene glycol (PEG)
o Using enzymes – reduces surface charge of cells
o Agitating – provides physical means for cells to come closer
o Centrifuging - provides physical means for cells to come closer
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o Altering temperature – temperature must be suited to the type of antibody

o Altering pH – optimal for most reactions is between 6.5 – 7.5 except Anti-M and
anti-P1 which react at lower pH
 Can be classified into several categories:
o Direct agglutination
 occurs when antigens are found naturally on the surface of a
particle/cell.
 Unknown is the presence or absence of antibody
 If the direct agglutination reaction involves red cells, then it is called
hemagglutination.
 Example: Widal test
o Passive agglutination
 occurs when antigens are not found naturally on the surface of a
particle/cell.
 Unknown is the presence or absence of antibody
 Uses particles to increase the size of antigen
a. Latex
b. Red cells
c. Gelatin
d. silicates
 Example: Rheumatoid factor and ANA test.
o Reverse Passive agglutination
 antibody rather than antigen is attached to a carrier particle.
 Uses particles to increase the size of antibody
 Unknown is the presence or absence of antigen
o Agglutination inhibition
 based on competition between particulate and soluble antigens for
limited antibody-combining sites,
 lack of agglutination is an indicator of a positive reaction
 involves haptens that are complexed to proteins; the hapten–protein
conjugate is then attached to a carrier particle.
 Before this is performed, Control must be done first by using viral
antigens.
 Unknown is the presence or absence of antigen
 Hemagglutination inhibition used same principle except used RBC as
particle.
o Coagglutination
 uses bacteria as the inert particles to which antibody is attached
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 Staphylococcus aureus is frequently used because of its protein A on its

surface.
 Unknown is the presence or absence of antigen.
 Antiglobulin-mediated agglutination
o Coomb’s test - technique that detects nonagglutinating antibody by means of
coupling with a second antibody
 Direct coomb’s test
a. used to demonstrate in vivo attachment of antibody or
complement to an individual’s red blood cells.
b. Detects:
i. Autoimmune hemolytic anemia
ii. Hemolytic disease of newborn
iii. Drug induced hemolytic reaction
 Indirect coomb’s test
a. A two-step process
b. Detects:
i. Presence of alloantibody
ii. Crossmatching
iii. Bloodgrouping/typing
D. Labeled Immunoassays
 designed for antigens and antibodies that may be small in size or present in very low
concentrations.
 The presence of such antigens or antibodies is determined indirectly by using a labeled
reactant to detect whether specific binding has taken place.
 Labeled immunoassays are classified into two:
o Competitive assay – labeled antigen and unlabeled patient antigen competes for
limited site on the antibody. Amount of unlabeled patient antigen is inversely
proportional to the detected labeled antigen.
o Noncompetitive assay – an antibody (capture antibody) is fixed on the gel. Then
patient antigen is added. Labeled antibody is then layered on top. Amount of
unlabeled patient antigen is directly proportional to the detected labeled
antibody.
 Labeled immunoassays are classified into two according to complexity of procedure
o Homogenous – does not require washing or separation
o Heterogenous – requires washing or separation
 Types of Immunoassays:
Radioactive
Unlabeled Labeled o Radioimmunoassay
Ag Ag  This is the first type of immunoassay developed by Yalow and Berson
 125
I , 131I , 3H (from most to least popular)

Antibody
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 Unknown is presence or absence of patient’s antigen

 Result is read using Schilling’s counter
 Advantage:
a. Extremely sensitive
 Disadvantage:
a. Expensive to maintain
b. Requires special license
c. Shelf life of radioactive chemicals
d. Health hazard
o Enzyme Immunoassay
 Result is read using spectrophotometer
 Enzyme labels:
a. Horseradish peroxidase,
b. alkaline phosphatase
c. β-D-galactosidase
 Advantage:
a. Cheap
b. Readily available
c. Long shelf life
 Type of enzyme immunoassay
Patient’s Enzyme
i. Competitive enzyme immunoassay (Heterogenous)
Unlabeled Labeled
Ag Ag  Two antigens compete for the binding site on the
antibody fixed on the media.
 Unknown is presence or absence of patient’s
Antibody
Solid Media antigen
ii. Noncompetitive Enzyme Immunoassay (Heterogenous)
 Has higher sensitivity than competitive
Enzyme substrate
Enzyme labeled AHG  Also known as indirect enzyme linked

Patient’s Unlabeled Ab immunosorbent assay or indirect ELISA

Ag  Unknown is the presence or absence patient’s

Solid Media antibody

 Used to test for Hepa A, Hepa B and HIV
iii. Capture assay – (Heterogenous)

Enzyme substrate  Also known as sandwich immunoassays

Enzyme labeled Antibody  Almost the same as indirect elisa but the

Patient’s Unlabeled Ag antibody is the one attached to solid media

Ab  Unknown is presence or absence of patient’s

antigen
Solid Media
 Can undergo hook effect - an unexpected fall in
the amount of measured analyte when an
IMMUNOLOGY AND SEROLOGY DAY 2 HANZEL V. TOLENTINO, RMT

extremely high concentration is present.24 This

typically occurs in antigen excess
iv. Enzyme multiplied immunoassay technique (EMIT) –
(homogenous)
 developed by the Syva Corporation
 Unknown is presence or absence of patient’s
antigen

 Enzyme immunoassay procedures were developed more leading to

creation of rapid assays:
a. Membrane-cassette assays
i. The membrane or media is nitrocellulose
b. Immunochromatography
i. Example: test kit for hepatitis B, pregnancy, troponin test
kit
o Fluorescent Immunoassay (immunofluorescent assay)
 Uses fluorophores or fluorochromes, they are organic molecules that
emits light after absorbing energy from incident light.
 Example: fluorescein (green), rhodamine (red)
 Result is read using fluorescent microscope, spectrofluorometer, flow
cytometer or fluorometer.
 Can be classified into two:
a. Direct immunofluorescent assay

Ab with fluorophores + Patient i. Best suited in detection of antigen in tissue

Ag then observe/read under
microscope

b. Indirect immunofluorescent assay

Fluorophores labeled AHG
i. More sensitive than direct immunofluorescent assay.
Patient’s Unlabeled Ab
ii. same principle as indirect elisa
Ag
Solid Media on Slide

o Chemiluminiscent immunoassay
 Emission of light caused by a chemical reaction, commonly oxidation.
 Most common examples:
a. luminol,
b. acridinium
c. esters,
d. ruthenium derivatives,
e. nitrophenyl oxalates
IMMUNOLOGY AND SEROLOGY DAY 2 HANZEL V. TOLENTINO, RMT

E. Molecular Diagnostics
 These techniques are based on the detection of specific nucleic acid sequences in
microorganisms or particular cells
 Molecular techniques used in lab
o enzymatic cleavage of nucleic acids,
o gel electrophoresis,
o enzymatic amplification of target sequences,
o hybridization with nucleic acid probes.
 Humans have 23 pairs of chromosomes. (22 pair of autosome and 1 pair of sex
chromosome)
S D
 southern blot – DNA
 Northern blow – RNA
N R
 Western blot – Protein W P
 DNA sequencing is considered the “gold standard” for many molecular applications
 The polymerase chain reaction (PCR) is capable of amplifying tiny quantities of nucleic
acid up to levels that can be later detected with various strategies
 Sequence of DNA and RNA is sometimes too few or too small that it cant be read by
machine hence amplification is needed. Below is some ways to amplify target sequence:
o Polymerase Chain Reaction
o Nucleic acid sequence based amplification
o Strand displacement amplification
o transcription mediated amplification
o ligase chain reaction
F. Flow Cytometer and Lab Automation
 automated system in which single cells in a fluid suspension are analyzed in terms of
their intrinsic light-scattering characteristics and are simultaneously evaluated for
extrinsic properties (i.e., the presence of specific surface proteins) using fluorescent
labeled antibodies or probes
 Major components of a flow cytometer:
o include the fluidics,
o laser light source
 light source to be used should be match with fluorochromes to be used.
 Argon that emits at 488nm is commonly used.
 Light is measured at forward angle (cell size) or orthogonal right angle
(cell granularity or intracellular complexity)
o optics (photodetectors)
o computer for data analysis and management
 specimen considerations:
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o can use whole blood, bone marrow aspirate, and fluid aspirates. Can also use
tissue specimens but cells should be disintegrated first.
o Use EDTA or Heparin as anticoagulant