AGGLUTINATION REACTION Passive Agglutination

- Visible aggregation of particles caused by - Aka indirect agglutination

combination with specific antibody. - Particles are coated with antigens not normally
- Easy to carry out, require no complicated found on their surfaces
equipment, can be performed as needed in the  Erythrocytes
laboratory without having to batch specimens.  Latex
- Agglutinins: antibodies that produce agglutination  Gelatin

Gruber and Durham (1896) Reverse Passive

- Published the first report about the ability of Agglutination
antibody to clump cells, based on observations of - Antibody rather than
agglutination of bacterial cells by serum. antigen is attached to a
Widal and Sicard (1896) carrier particle
- Earliest diagnostic tests in1896 for the detection of
antibodies occurring in typhoid fever, brucellosis,
and tularemia

Agglutination
2 step process:

1. Sensitization – antigen- antibody combination Agglutination Inhibition

through single antigenic determinants.
2. Lattice Formation – formation of cross-links
that form visible aggregates.
- Based on competition between particulate and
soluble antigens for limited antibody combining
sites
- Involves haptens that are complexed to proteins
Lack of agglutination is an indicator of a positive
reaction

Hemagglutination inhibition- RBCs are the

indicator particles.

Types of Agglutination Reaction

Direct Agglutination
- Antigens are found
naturally on a
particle
 LABELED IMMUNOASSAY Types of Labeled Immunoassay
- Designed for antigens and antibody that may be
small in size or in very low concentrations. Radioimmunoassay
- Ag or Ab is determined by indirectly by using a - Pioneered by Yalow and Berson in the late 1950s
labeled reactant to detect whether or not specific - Radioactive substance as a label
binding has taken place. - 1251- most popular
- Detected by gamma counter
Analyte - Disadvantage:
- Substance to be measured  Health hazard
- Typically, a protein  Disposal problems
- Example: bacterial Ag, hormones, drugs, tumor  Short shelf life
markers, specific immunoglobulins  Need for expensive equipment

Formats of Labeled Immunoassay Enzyme Immunoassay

- Enzymes used as labels for immunoassays
Competitive Immunoassay  Horseradish peroxidase
- All reactants mixed  Alkaline peroxidase
together simultaneously  ß-D-galactosidase
- Labeled Ag competes
with unlabeled patient Ag Heterogenous Enzyme Immunoassay
for a limited number of - competes with unlabeled patient Ag for a
Ab-binding limited number of binding sites on

immunoassays
Competitive
- Amount of bound label is antibody molecules that are attached to a

enzyme
inversely proportional to solid phase
the concentration of the - inversely proportional to the concentration
labeled antigen of the test substance.

Non-competitive - so called indirect enzyme-linked

Non-competitive enzyme immunoassay

Immunoassay immunoabsorbent assays (ELISA)

- Ab is first passively - enzyme labeled reagent doe s not
absorbed to a solid phase participate in the initial Ag-Ab binding
- Unknown patient Ag is reaction
then allowed to react with - used to measure Ab production to
and be captured by the infectious agents that are difficult to isolate
solid phase antibody in the lab and for auto-Ab testing
- After washing to remove - quantitation is not necessary easily
unbound Ag, second Ab applied to POC and home testing
with a label is added to
the reaction.
- Label measured is
directly proportional to
the amount of patient Ag.

Heterogamous vs. Homogenous Assay - Aka Sandwich immunoassay

- Ab, rather than Ag, is bound to the solid
Heterogenous phase
Capture Assay

- Require a step to physically separate free from - Ag captured in these assays must have
bound analyte multiple epitopes
- Anzymatic activity is directly proportional
Homogenous to the amount of Ag in test sample
- Do not need a separation step - Hook Effect - unexpected fall in the
amount of measures analyte when an
extremely high concentration is present
 Homogenous Enzyme Immunoassay 1. Direct Immunofluorescent Assay
- Less sensitive than heterogeneous assays 2. Indirect Immunofluorescent Assay
- Competitive assay
- Principle of change in enzyme activity as specific
Ag-Ab combination occurs
- When Ab binds to specific determinant sites on the
Ag, the active site on the enzyme is blocked,
resulting in a measurable loss of activity
- Directly in proportion to the concentration of px Ag
or hapten present in the test solution
Malate dehydrogenase
Glucose-6-phosphate dehydrogenase

Fluorescence Polarization Immunoassays

Rapid Immunoassay
- Membrane based, easy to perform, and give
reproducible results
- Designed primarily for POC or home testing
- Single-use, disposable assays in a plastic
cartridge
- Either Ag or Ab can be coupled to the membrane
- Immunochromatography – most often qualitative
- Based on the change in polarization of fluorescent
light emitted from a labeled molecule when it is
bound by Ab.
- Degree of polarized light reflects the amount of
labeled analyte that is bound
- Degree of fluorescence polarization is inversely
proportional to concentration of the analyte
- Main Problem: separation of the signal on the label
from autofluorescence produced by different
organic substances normally present in serum.
Chemiluminescent Immunoassays
- Emission of light caused by chemical reaction
typically an oxidation reaction, producing, an
excited molecule that decays back to its original
ground state.
 Luminol
 Acridinium esters
 Ruthenium derivatives
 Nitrophenyl oxalates
- Detection systems basically consist of
photomultiplier tubes.

Fluorescent Immunoassay
- Albert Coons (1941) – demonstrated that Ab could
be labeled with molecules that fluoresce
- Fluorophores or Fluorochromes:
o Typically, organic molecules with a ring
structure
o Can absorb energy from an incident light
source and convert that energy into light of
a longer wavelength and lower energy as
the excited electrons return to the ground
state
- Fluorescein- absorbs maximally at 490-495 nm
and emits green color at 520 nm
- Rhodamine (Tetramethy) – absorbs at 550 nm and
emits red light at 585 nm

Immunofluorescent Assay (IFA)

- First used for localization of Ag in cells or tissues
- Fluorescent probe attached to the Ab is detected
under UV light using a fluorescent microscope
- Presence of specific Ag is determined by the
appearance of localized color against a dark
background
 MOLECULAR DIAGNOSTIC TECHNIQUE
CHARACTERISTICS OF DEOXYRIBONUCLEIC
ACID AND RIBONUCLEIC ACID
Deoxyribonucleic acid (DNA)
- carries the primary genetic information within
chromosomes found in each cell
- A macromolecule of nucleotides
- Has 2 Purine bases: Adenine and Guanine
- Has 2 Pyrimidine bases: Cytosine and
Thymine
DNADOUBLE HELIX
Ribonucleic acid (RNA)
- is an intermediate nucleic acid structure that
helps convert the genetic information encoded
within DNA into proteins that are the primary
cellular component
- Also a macromolecule of nucleotides
- Has 2 Purine bases: Adenine and Guanine
- Has 2 Pyrimidine bases: Cytosine and Uracil
NUCLEOSIDEVS NUCLEOTIDE
A nucleotide is a unit composed of a
phosphorylatedribose sugar and a nitrogen
base
A nucleoside is an unphosphorylatedribose
sugar carryinga nitrogen base
DNA NUCLEOTIDES
DNA REPLICATION
- In DNA, the nitrogen base of a nucleotide—
guanine, adenine, cytosine, or thymine—is
attached to the 1′ carbon of the deoxyribose
sugar.
- The phosphate group is attached to the 5’
carbon of the deoxyribose sugar.
RNA SYNTHESIS
RNA NUCLEOTIDES
- Ribose is the primary sugar in RNA.
- Unlike deoxyribonucleotides, which are
hydroxylated on the 3′ carbon, the
phosphorylated ribose sugar in RNA carries
hydroxyl groups on carbons 2′ and 3′.
- Furthermore, thymine is not present in RNA
and is replaced by uracil.
NUCLEIC ACID POLYMERS
- Nucleotides are polymerized by attachment of
the sugar 3′ hydroxyl groups to the 5′
phosphate groups, forming a phosphodiester
bond.
- A chain of nucleotides makes up one strand of
RNA or DNA.
- Most RNA exists without a partner or
complementary strand; they are single
stranded.
- In contrast, DNA is mostly double stranded
and arranged in a double helix
- G pairs with C by three hydrogen bonds.
- A pairs with T by two hydrogen bonds.
- Two bases paired together in this way are
called a base pair (bp).
- The two chains (strands) of the DNA double
helix are held together by hydrogen bonds
between their nucleotide bases.
- Guanine (G) and cytosine (C) are
complementary, that is, they will only hydrogen
bond with each other.
- Similarly, adenine (A) and thymine (T) are
complementary to each other.
- Replication of DNA is semiconservative, that
is, the two strands of the DNA duplex are
separated and each single strand serves as a
template for a newly synthesized
complementary strand.
- Because DNA polymerization proceeds in the
5′ to 3′ direction and the template is read in the
3′ to 5′ direction, one strand (the lagging
strand) has to be read discontinuously.
- RNA synthesis proceeds in a manner that is
chemically similar to that of DNA synthesis
with some exceptions. Unlike DNA synthesis,
RNA synthesis can start de novo without a
primer.
- RNA synthesis is catalyzed by RNA
polymerase, a more error prone, slower
polymerase (50 to 100 bases/second) than
DNA polymerase (1,000 bases/second).
- The bulk of DNA synthesis takes place in the S
phase of the cell cycle, whereas RNA
synthesis occurs throughout the cell cycle and
varies depending on the cellular requirements.
- RNA is copied from almost all of the genome;
however, only about 2% of the RNA-coding
regions are translated into protein. Some
genes code for transfer RNA (tRNA) and
 ribosomal RNA (rRNA), which is required for
protein synthesis
PROTEIN SYNTHESIS
- Messenger RNA (mRNA) is transcribed from
DNA using RNA polymerase.
- The mRNA delivers the information to
ribosomes where protein synthesis takes
place.
- As each amino acid is added, the peptide
chain continues to grow.
- This process, known as translation, is
accomplished with the help of tRNA, which
brings in individual amino acids.
MUTATIONS
- A change in the nucleotide sequence is a
mutation.
- Depending on how frequently they occur,
changes in the nucleotide sequence may also
be referred to as variants or polymorphisms.
- Diseases such as immunodeficiency result
from mutations in genes encoding proteins that
bring about the immune response.
POLYMORPHISMS
- Structurally, mutations and polymorphisms are
the same thing: a change from a reference
amino acid or nucleotide sequence.
- Polymorphisms are defined by their frequency
in a population.
- Alterations in DNA or protein sequences
shared by at least 2% of a population are
considered polymorphisms.
- The different versions of the affected
sequences are referred to as alleles.
- Polymorphic changes may or may not have
phenotypic effects.
- The most highly polymorphic region in the
human genome is the major histocompatibility
complex (MHC) locus on chromosome 6. The
different nucleotide sequences result in
multiple versions or alleles of the human
leukocyte antigen (HLA) genes in the human
population.
- Other highly polymorphic areas of the genome
include the genes coding for the antibody
proteins and antigen receptorproteins in B
cells and T cells, respectively. These
sequences differ from cell to cell, allowing the
generation of a large repertoire of antibodies
and antigen receptors to better match any
foreign antigen.
- Polymorphisms that create, destroy, or
otherwise affect restriction enzyme sites are
detected as restriction fragment length
polymorphisms (RFLPs) or variations that
differ among individuals.
- Mitochondria located in the cytoplasm of
eukaryotic cells carry their own genome.
Polymorphisms are also found in two regions
of mitochondrial DNA sequences
(hypervariable regions). They are used for
maternal inheritance testing because all
maternal relatives share the same
mitochondria and therefore have the same
mitochondrial polymorphisms
MOLECULAR ANALYSIS
STRAND CLEAVAGE METHODS
- It is done by cleaving it at specific locations
through the use of enzymes and then
separating the resulting fragments on the basis
of size and charge through gel electrophoresis.
- Restriction endonucleases are enzymes that
recognize and bind to specific nucleotide
sequences in the DNA so that they will only
separate the DNA at those locations.
- Restriction endonuclease (restriction enzyme)
cleavage methods are highly informative for
investigating small genomes, such as those of
microorganisms, or plasmids.
- There are hundreds of restriction enzymes
with unique binding and cleavage sites. The
restriction enzymes used in the clinical
laboratory (type II restriction enzymes)
recognize palindromic sites. These sites are
 nucleotide sequences that read the same 5′ to - Radioactive nucleotides provide the
3′ on both strands of the DNA. radioactive signals, whereas the
nonradioactive signals are generated by an
antigen–antibody or biotin–avidin binding with
the antibody or avidin conjugated to alkaline
phosphatase or horseradish peroxidase
enzymes that produce color or light when
exposed to the color or light-emitting
substrates.
- Uses of Southern Blot:
 Positive human identification by DNA
fingerprinting was first performed by
Southern blot.
- Restriction enzyme mapping characterizes
 Southern blots can also be used for
DNA by the pattern of fragments generated
detecting deletions in mitochondrial DNA
when the DNA is cut with restriction enzymes.
and gene rearrangements in nuclear
- Fragment A has two sites for the
DNA
endonuclease to cut, resulting in three bands
on the gel. Fragment B has only one site,
resulting in two gel bands. The loss of the
second restriction site in fragment B is caused
by a mutation in the recognition site for the
endonuclease.
- The analysis is applied to epidemiological
studies of microorganisms and identification of
resistance factors carried on
extrachromosomal DNA (plasmids) in the cell.

HYBRIDIZATION METHODS

SOUTHERN BLOT ANALYSIS

- It starts with restriction enzyme digestion of

DNA isolated from the test organism or cells.
ARRAY METHODS
The restriction fragments are then separated
by agarose gel electrophoresis.
- Here, multiple targets or multiple patient
- The separated double-stranded fragments are
samples could be investigated simultaneously.
then denatured, that is, separated into single
- Such techniques are used for the prognosis in
strands in the gel.
leukemia or diagnosis of complex diseases
- The treated DNA is transferred or blotted from
such as autoimmune states, where multiple
the gel onto a nitrocellulose-based membrane.
nucleic acid or protein targets have to be
- At this stage, all of the millions of fragments
detected.
from complex genomes will be present on the
- Dot Blot Hybridization
membrane, whereas only one particular gene
o The first array methodology.
or locus is of interest.
o In this method, instead of cutting and
resolving DNA or RNA on a gel and
blotting it to a membrane for probe
hybridization, a highly specific unlabeled
probe is spotted directly on a solid support.
Then the test sample (nucleic acids or
proteins isolated from cultures, cells, or
body fluids) is labeled and hybridized to
the many immobilized probes.
- Arrays are used to detect amplifications or
deletions in DNA, gene expression (RNA
- The key to the specificity of the Southern blot
synthesis), and SNP, where there is a change
technique is the use of a probe.
in a single base.
- A probeis generally a nucleic acid, several
- Macroarrays-initially performed by manually
hundred to a few thousand bases in length,
spotting probes on nitrocellulose membranes
with a known sequence that is used to identify
and hybridizing labeled test DNA.
the presence of a complementary DNA or RNA
- Microarrays -also known as gene chips, are
sequence in an unknown sample.
used for a variety of applications including
- Probes typically have an attached and
analysis of genetic mutations in malignancies,
detectable signal. The signal can either be
detection of microdeletions, detection of viral
emission of radioactivity, development of color,
resistance mutations, and gene expression
or light.
profiling.
 - Another configuration of array technology is AMPLIFICATION METHODS
bead arrays.
- These assays are based on preparations of - The most frequently used methods in
100 to 400 fluorescent bead populations that molecular diagnostics involve some aspect of
differ by the relative amounts of two distinct amplification, that is, copying of nucleic acids.
dyes that are incorporated into the beads. - The development of the in vitro PCR reaction
- Each bead population is attached to an by Kerry Mullis, greatly facilitated and
antibody or other molecule that will bind broadened the potential applications of gene
specifically to the target molecules or amplification.
sequences. A secondary antibody conjugated
to a fluorescent signal will detect the presence POLYMERASE CHAIN REACTION (PCR)
of the target. - PCR is an in vitro replication procedure that
- Bead assays are used for multiplex detection results in amplification of the target DNA.
of proteins and nucleic acids - PCR reaction includes
- Applications include tissue typing for stem cell o Template(sample) DNA to be copied;
and organ transplantation and respiratory virus o deoxyribonucleotides, which will be joined
panels. together
o oligonucleotide primers to prime the
SOLUTIONS HYBRIDIZATION synthesis of the copies
o DNA polymerase to covalently join the
- In solution hybridization, the probe and the deoxyribonucleotides
target are both in solution. The target must be o a buffer with a pH optimal for the
single stranded in order for hybridization to polymerase activity
work. After probes and denatured targets are
mixed in solution, the bound products are
resolved by gel or capillary electrophoresis.
- In variations of solution hybridization, DNA
probe and RNA targets form hybrids, indicated
by DNA probe:RNA. The target hybrids formed
in solution are detected by capture on a solid
support.
- Two probes are used, both hybridizing to the
target RNA. One probe, the capture probe, has
an attached molecule of biotin that will bind
specifically to streptavidin immobilized on a
solid support. - For many procedures, premixed PCR reagents
- The other probe, which is the detection probe, (deoxyribonucleotides, primers, and buffer) are
is detected by an antibody directed against supplied from manufacturers to which only the
RNA:DNA hybrids or a covalently attached template DNA and, in some cases, enzyme
digoxigenin molecule used to generate are added.
chromogenicor chemiluminescent signal. - The PCR reaction mix is subjected to a
computerized amplification program consisting
FLUORESCENCE IN SITU HYBRIDIZATION (FISH) of a designated number of cycles.
- 3 Step PCR cycle:
- In situ hybridization is an additional technique 1. denaturation step (94°C to 96°C)
that utilizes nucleic acid probes to identify 2. annealing step (50°C to 70°C)
DNA. However, in this case, the target DNA is 3. extension step (68°C to 72°C)
found in intact cells.
- When fluorescence is used to visualize the
hybridization reaction, this is called
fluorescence in situ hybridization, or FISH.
- In addition to identifying many different
chromosomal abnormalities, FISH has been
used to identify T-cell lymphomas, B-cell
malignancies, and graft versus host disease
after transplants. However, FISH is sensitive to
the buffer and temperature conditions of
hybridization (stringency).
- Stringency refers to the conditions that affect
the ability of a probe to correctly bind to a
specific DNA target sequence.
Quantitative PCR (qPCR) between two preexisting DNA
chains—in this case, two primers.
- Formerly known as the Real Time PCR or RT- o LCR requires temperature changes to
PCR. The term is nowQuantitative PCR or drive the denaturation of the template
qPCR in order to avoid confusion with the and ligated primers.
Reverse Transcriptase PCR which is also RT- o LCR was used to detect point
PCR. mutations in a target sequence.
- In qPCR, results can be seen at the end of - Standard Displacement Amplification (SDA)
each cycle, as opposed to PCR where o is another isothermal amplification
measurement only occurs after all cycles are process
completed. o the amplification products are the
- Both DNA and RNA targets are measured by probes
qPCR. For RNA, cDNA made from the RNA o used to detect the amplified probes
using reverse transcriptase is the input have been designed to test for M
material. tuberculosis,C trachomatis,and N
- Widely used applications of qPCR and RT- gonorrhoeae
qPCR include detection of microorganisms,
especially viruses and other pathogens that SIGNAL AMPLIFICATION
are difficult or dangerous to culture in the
laboratory. - In signal amplification, large amounts of signal
- Assessment of tumor-associated gene are bound to the target sequences that are
expression using qPCR and RT-qPCR may present in the sample. Branched DNA (bDNA)
predict cancer recurrence. is an example of a commercially available
signal amplification method.
TRANSCRIPTION-BASED AMPLIFICATION - Here, probes are amplified and not the target
which makes this method be used to quantify
- Kwoh and colleagues developed the first the amount of target actually present.
transcription amplification system in 1989. - The bDNAsignal amplification assay has been
- RNA is the usual target instead of DNA. A applied to the qualitative and quantitative
cDNA copy is synthesized from the target detection of HBV, HCV, and HIV-1.
RNA, after which transcription of the cDNA
produces millions of copies of RNA products.
- In this technology, RNA is the primary product
as well as the target.
- The TMA process has been simplified with the
addition of RNase Hderived from E coli to
degrade the RNA from the DNA and RNA
hybrid, eliminating a heating step required for
denaturation of the DNA and RNA hybrid to
complete the cDNA synthesis.
- Meaning, this is an isothermalprocess,
meaning that the reaction proceeds at a single
temperature.
- Targeting RNA allows for the direct detection
of RNA viruses, such as hepatitis C virus, and
HIV.
- Targeting the RNA of organisms with DNA
genomes, such as Mycobacterium
tuberculosis, is more sensitivethan targeting
the DNA because each microorganism makes
multiple copies of RNA, whereas it has only
one copy of DNA.

PROBE AMPLIFICATION

- The number of target nucleic acid sequences

in a sample is not changed in probe
amplification. Rather, primers are extended or
ligated into many copies of detectable probes.
Examples of probe amplification are ligase
chain reaction (LCR) and SDA.
- Ligase Chain Reaction (LCR)
o LCR amplifies synthetic probes
complementary to target nucleic acid
o LCR uses DNA ligase, an enzyme that
forms one phosphodiester bond