overview of methodology
aCGH (Array Comparative genomic hybridization)

Direct sequencing method

This is a combination of comparative genomic hybridization(CGH) principle to detect copy number variations (CNV)such as genomic amplification or deletion by comparing
fluorescence-labeled sample-derived DNA with normal cellderived DNA, and microarray technology.

Direct sequence analysis using PCR-amplified DNA as asequencing template.

Atomic absorption spectrophotometry
A technique of element content determination by atomizing
an element sample by putting it in a chemical flame or in aheated graphite tube. To the atoms evaporated in a flame,
an element specific light is emitted, and the amount of lightabsorbed is measured to determine the element content.

Chemiluminescent enzyme immunoassay (CLEIA)
Antibodies linked to the solid phase are reacted with antigens
that are secondarily reacted with enzyme-labeled antibodies.
After adding a chemiluminescent substrate, the emission
intensity is measured.

Chemiluminescent immunoassay (CLIA)

Electro chemiluminescence immunoassay (ECLIA)
Antibody-bound beads are reacted with antigens that aresecondarily reacted with antibodies labeled with rutheniumpyridine complexes, which promotes the electrochemical
reaction. The emission intensity of ruthenium-pyridine
complexes is measured.

Electrophoresis
A technique for analysis of macromolecules based on thefollowing phenomena: When an electric current is applied
to an electrolyte solution in which charged particles
suspended, the particles migrate towards the electrodeof opposite charge. Based on the length of migration, thetarget molecule is determined.
Cellulose acetate membrane, agarose gel, andpolyacrylamide gel are used as support media.

Enzymatic method

Antibodies linked to the solid phase are reacted with antigens
that are secondarily reacted with chemiluminescent-labeled
antibodies. The emission intensity of chemiluminescent
substance is measured.

This technique is based on the same assay principle as
the colorimetry. Analytes are specifically measured using
enzymatic reactions.

Chromogenic substrate method

Enzyme immunoassay (EIA)

By adding heparin, an antithrombin III (AT-III)-heparincomplex is generated. Then, the thrombin inactivation capacity
is measured using a chromogenic substrate for thrombin.

EIA is based on the same assay principle as RIA. Using
either enzyme-labeled antigen or antibody as a labeledsubstance, the antigen-antibody reaction is carried out.
After adding a chromogenic substrate, the enzyme activity
is measured.

Clotting time
Clotting time is the time required for a plasma sample tocoagulate, using plasma without a factor to be tested,thromboplastin, actin, and calcium chloride.

Colorimetry

Complement fixation (CF)A technique based on the principle that complements bindwith antigen-antibody complexes and that they mediate
hemolysis.
When red blood cells coated with hemolysin (sensitized
red blood cells) bind to complements, hemolysis occurs;
however, in the existence of the antigen-antibody
complexes, complements are used, resulting in no hemolysis.
Based on the hemolytic reaction, the presence of a specificantibody is determined.

A technique to measure the enzyme activity of a specificantigen. Using enzyme-labeled antibodies to the target
antigen, the antigen-antibody reaction is carried out. Afteradding a chromogenic substrate, the enzyme activity ismeasured.
The direct method uses enzyme-labeled antibodies thatreact directly with the antigen. The indirect method uses
unlabeled antibodies that react with the antigen first andenzyme-labeled antibodies for the secondary reaction.

Enzyme-linked immunospot (ELISPOT)
In a plate coated with a specific antibody, isolated cells anda specific antigen are added and cultured. Proteins secretedby the cells are reacted with a chromogenic substrate to
generate spots.

Enzyme-linked immunosorbent assay (ELISA)
Antibodies linked to the solid phase are reacted with
antigens that are secondarily reacted with enzyme-labeled
antibodies. After adding a chromogenic substrate, theenzyme activity is measured.

A

overview of methodology

After converting an analyte to a coloring substance,
a visible wavelength is irradiated. The absorbance ismeasured, and color of the analyte is compared with that of
the standard solution.

Enzyme-labeled antibody method

overview of methodology
Flow cytometry

Hemagglutination inhibition (HI)A technique for analysis of individual cells. Cells stainedwith fluorochrome-labeled monoclonal antibodies are flown
in a stream of solution at a high speed and are passed
through the laser beam. The cells are individually analyzed
by forward scatter (size of the cell), 90° side scatter(internal complexity of the cell), and fluorescence intensity(cell surface expression of the antigen).
Two-color flow cytometry uses a combination of fluorochromesfor double staining.

A technique based on the phenomenon that hemagglutinating
activities of viruses are inhibited by antibodies against theviruses. Antigen-antibody complexes are reacted with red
blood cells, and the presence of the antibody against the virus
is determined by whether or not hemagglutination is inhibitedor not.

Fluorescence enzyme immunoassay (FEIA)
One of the techniques of EIA. Using either enzyme-labeled
antigen or antibody as a labeled-substance, the antigenantibody reaction is carried out. After adding a fluorescence
substrate, the fluorescence intensity is measured.

FIA (Fluorescence Immunoassay)
After reacting the target substance with the immobilized
antigen, labeling with a fluorescent substance.
A method in which the antibody or antigen is subjected
to a secondary reaction and the fluorescence intensity ismeasured.

Fluorescence in situ hybridization (FISH)
The target DNA is hybridized with a fluorescent probe.
Using a light with a specific wavelength, the fluorescent site
in the chromosome is detected as a signal by fluorescencemicroscope.
The direct method uses a fluorescent probe to directly bindto the target DNA. The indirect method uses a labeled probe
to bind to the target DNA which is subsequently bound to afluorescence substance for color development.

Fluorescent antibody method (FA)overview of methodology

Using fluorochrome-labeled antibodies to the target
antigen, the antigen-antibody reaction is carried out, andthe fluorescence intensity is measured using fluorescencemicroscope.
The direct method uses fluorochrome-labeled antibodies
that react directly with the antigen. The indirect method
uses antibodies that react with the antigen first and thenfluorochrome-labeled antibodies for the secondary reaction.

Gold colloid method
An optical measurement method using color change
developed by aggregates of colloidal gold particles after
antigen-antibody reaction with gold colloid-labeled
antibodies.

Hemagglutination (HA)
Red blood cell surface antigens are reacted with theantibodies.
After the antigen-antibody reaction is completed, thepresence of the antibody is determined based on appearance
or non-appearance of clots.

B

High performance liquid chromatography (HPLC)A chromatography technique using a liquid mobile phase.
HPLC separates a mixture into components promptly
and accurately, using a column filled with a high-density
absorbent and a high-pressure pump.

Immunochromatography (IC)The antigen or antibody in the specimen and the labeled
antibody or labeled antigen labeled with gold colloidal particles
or the like move on the membrane filter forming an immune
complex, and the immune complex is supplemented with theantibody or antigen immobilized on the membrane filter inadvance, and the detection method is colorized to determine
the result visually or otherwise.

Immunoradiometric assay (IRMA)
IRMA is one of the techniques of RIA. Antibodies linked
to the solid phase are reacted with antigens that aresecondarily reacted with antibodies labeled with radioactive
isotope (RI).
This is also called as the sandwich technique, because thesolid-phase linked antibodies and the RI-labeled antibodies
bind to the antigens, respectively, forming an antigen
sandwich.

Indirect fluorescent antibody method (IFA)
Using fluorochrome-labeled antibodies to the target
antigen, the antigen-antibody reaction is carried out, andthe fluorescence intensity is measured using fluorescencemicroscope.
The direct method uses fluorochrome-labeled antibodies
that react directly with the antigen. The indirect method
uses antibodies that react with the antigen first and thenfluorochrome-labeled antibodies for the secondary reaction.

Invader method
A technique consisting of two steps of homogeneous
isothermal reaction to detect genetic polymorphisms by theuse of cleavase (one of the endonucleases) that specificallyrecognizes and cleaves the triple-stranded DNA structure.

Ion-selective electrodeThe electrode method uses electric charge-transfer reaction
at the liquid interface.
Ion-selective electrode is used to measure ionicconcentrations based on a potential difference which occurs
proportionally to the logarithm of ionic activity in response
to a specific ion.

overview of methodology
Isothermal nucleic acid amplification test

Loop-mediated isothermal amplification (LAMP)

Unlike PCR, this method amplifies nucleic acids at constanttemperature, using strand displacement DNA synthesis
enzymes, etc.

LAMP employs 4 kinds of primers that recognize 6 distinct
sequences of a target gene. By using the chain substitution
reaction, the reaction process proceeds at a constanttemperature.

Latex agglutination (LA)Using antigen- or antibody-coated latex particles(sensitizing latex particles), the antigen-antibody reaction
is carried out. The existence of the antibody or antigen
is determined based on appearance or non-appearance ofclots.

Latex agglutination immunoassay (LA)Using antigen- or antibody-coated latex particles, theantigen-antibody reaction is carried out. The turbidity ofclots formed by the antigen-antibody reaction is measured
by the light transmittancy or light scattering intensity.

MALDI-TOF-MS
An ionization technique that uses laser energy absorbing
matrix to create ions by irradiating a laser to an analyte
mixture without decomposing proteins is known as matrixassisted laser desorption/ionization (MALDI). MALDI
is combined with a technique of time-of-flight mass
spectrometry (TOF-MS) which is based on the principle thatthe ion’s time of flight changes depending on the formed
ion’s mass-to-charge ratio.
MALDI: Matrix-assisted laser desorption/ionization
TOF-MS: Time-of-flight mass spectrometry

Methylation-Specific PCR

Latex photometric immunoassay (LPIA)
Using antigen- or antibody-bound latex particles, theantigen-antibody reaction is carried out. The turbidity ofclots formed by the antigen-antibody reaction is measured
by the light transmittancy by irradiating a near infrared
light.

This is a method to confirm methylation in the target region
by PCR, based on the difference in base sequence between
bisulfite-mediated methylated cytosine and unmethylated
cytosine, utilizing the conversion of unmethylated cytosine
to uracil by bisulfite mediation.

Multiplex ligation-dependent probe amplification (MLPA)

Line immunoassay

Liquid chromatography/mass spectrometry (LC/MS)

Multiplex PCR Fragment Analysis

HPLC is a technique to separate a target substancequickly and accurately, which is combined with a mass
spectrometer as a detector, thereby realizing further
improvement in the detection selectivity and the qualitative
function.

After amplifying multiple target genes, capillaryelectrophoresis is performed on PCR amplicons by sequencer.
Separated according to DNA size, the amplified peaks areanalyzed and identified by the dedicated software with thepredetermined QMVR width as a reference.

Liquid chromatography-tandem mass spectrometry (LC/MS/MS)

Nephelometry

Liquid chromatography separates the target substanceinto compounds using difference in the affinity, which is
combined with the mass spectrometry function that further
divides the compounds by mass difference to isolate,fragment, and detect a specific mass ion.

A technique to determine the light-scattering intensity
by irradiating light to a turbid substance formed by anantigen-antibody reaction.

Liquid (nucleic acid) hybridization

A technique based on neutralization: Through reaction of avirus with specific antibody, loss of infectivity occurs.
The virus and the antibody against the virus are reacted
and inoculated into cell culture that is sensitive to thevirus. The presence of neutralizing antibody is determinedbased on the cytopathogenic effect (CPE).

After isolating rRNA in a liquid phase, hybridization is
performed using a chemiluminescent-labeled DNA probe.
After allowing the hybrid to be attached to the separating
agent, chemiluminescence detection is performed.

Neutralization test (NT)Next generation sequence (NGS) method
A method for simultaneous determination of nucleotide
sequences of a large amount of DNA fragments using a next
generation sequencer.

C

overview of methodology

When antigens are mechanically applied onto a membrane,
specific antibodies are reacted to the antigens, andantibodies labeled with enzymes are subsequently reacted
to detect the antibodies.

A specific probe coupled with a common PCR primer
sequence is hybridized to the target sequence. After PCR
reaction, the amplified products are examined for theirquantitative changes, especially for comparatively major
genomic deletions and duplications.

overview of methodology
Ouchterlony method
Ouchterlony double immunodiffusion

Radioimmunoassay (RIA)

One of the gel-diffusion techniques, also called as
ouchterlony double immunodiffusion, to identify antigen
and antibody reactions. After antibodies and antigens are
diffused in the gel plate, the presence or absence and thenumber of precipitin lines formed by the antigen-antibody
reaction are confirmed.

A target antigen is labeled with radioactive isotope (RI) andbound to its specific antibodies which is then competitively
reacted with an antigen in a specimen. After the antigenantibody reaction, the bound labeled antigens (bound) are
separated from the unbound ones (free), and the antigen
concentration is determined based on the proportion ofradioactivity.
To separate the bound and free antigens (B/F separation),
the following methods are used. Solid-phase method:
Antibodies are linked to the solid phase. Double antibody
method: Antigen-antibody complexes are bound to thesecond antibody and precipitated. Ammonium sulfate
precipitation method: Antigen-antibody complexes are
precipitated by ammonium sulfate. PEG method: Antigenantibody complexes are precipitated by a precipitation
reagent.

Particle agglutination (PA)Using antigen- or antibody-coated gelatin particles(sensitizing particles), etc., the antigen-antibody reaction
is carried out. The existence of the antibody or antigen
is determined based on appearance or non-appearance ofclots.

Passive hemagglutination (PHA)

Radioreceptor assay (RRA)

The surface of red blood cells is coated with antigen toform sensitized red blood cells. Using the sensitized red
blood cells, the antigen-antibody reaction is carried out.
The presence of the antibody is determined based on
appearance or non-appearance of clots.

RRA is based on the same assay principle as RIA. Instead ofan antibody, a receptor is used, and the biological activity
is determined by the reactivity.

Polymerase chain reaction (PCR)

Real-time PCR

A technique to amplify a specific target region of DNA inan exponential manner. By heating DNA molecules, theDNA denatures and becomes single stranded, and then bycooling, the DNA anneals to the double stranded again. By
using this phenomenon, a single-stranded DNA is used asa template to bind the target primer. The DNA synthesis
occurs by repeated cycles of DNA polymerase activity.

Real-time PCR is the one of the nucleic acid amplification
tests using polymerase chain reaction (PCR) as thebasic principle. By the use of oligonucleotide that yields
fluorescence after degradation, the fluorescent signal isidentified for each PCR cycle, which enables the real-timequantification of the target nucleic acid.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

POT method (PCR-based ORF Typing)overview of methodology

A method to determine the genotype (POT type) of a strain
by selecting ORF (Open Reading Frames: gene reading
frames) with different possession states for each strain anddetecting their possession patterns by multiplex PCR.
Comparison of POT types allow strain-level identification,and isolated strain obtained from mass infections are often
of the same POT type.

proton nuclear magnetic resonance (NMR) spectroscopy
The principle of nuclear magnetic resonance (NMR)
spectroscopy is to irradiate a sample placed in a magnetic
field with electromagnetic waves, and record and measure
the frequency at which the nucle in the sample resonate
based on their characteristics as a function of the intensityof the resonance peak.

D

When RNA to be amplified by PCR, complementary DNA(cDNA) is synthesized using reverse transcriptase (RT)using RNA as a template.

Southern blot hybridization
A technique to detect a specific gene by the following
procedures: After restriction enzyme digestion of DNA,DNA is isolated and denatured to a single-stranded form
with electrophoresis. Using capillary action, the singlestranded DNA is transferred to a nylon membrane andhybridized with a target probe to detect the target gene.
This technique is used for analysis of abnormal changes in
DNA in a quantitative and qualitative manner.

Transcription-mediated amplification (TMA)
A technique of RNA amplification using two kinds ofenzymes, two kinds of primers, and substrates.
Extracted RNA allows reverse transcriptase to create
double-stranded DNA. Using the double-stranded DNA asa template, RNA synthesis is repeated by RNA polymerase
transcription to amplify the target region of RNA.

overview of methodology
Transcription Reverse Transcription Concerted reaction (TRC)
A method for real-time detection of RNA amplified in one
step by combining a DNA probe labeled with intercalating
fluorescent dye and constant temperature RNA amplification.

Turbidimetric immunoassay (TIA)
A technique to determine the light transmittancy byirradiating light to a turbid substance formed by anantigen-antibody reaction.

Ultracentrifugation
A technique to separate one type of material from others
and to measure physical properties based on difference inthe specific gravity of proteins using an ultracentrifuge.

Ultraviolet absorption spectrophotometry (UV)This technique is based on the same assay principle as
the colorimetry. Analytes are measured using ultraviolet
wavelengths.
Normally, near-ultraviolet wavelengths between 200 and
400 nm are used.

Various types of bandings
Using cultured lymphocytes or bone marrow cells, cells
in the metaphase are fixed. Then, a stain gives a series oflight and dark stripes (bands) along the chromosome, andanalysis is performed based on the distribution and lightand-dark differences of the stripes.
G-banding uses treatment with trypsin solution and Giemsa
staining. C-banding consists of steps including HCI treatment,
exposure to Ba(OH)2, 2 × SCC, and Giemsa staining. Q-bandinguses a stain called quinacrine mustard and the fluorescencepattern is observed using fluorescence microscopy. In highresolution banding, visualized images of cell division between
the end of the prophase and the beginning of the metaphase areused so that an increased number of bands are observed than innormal conditions.

overview of methodology

Western blot
A technique to detect a specific protein by separating
proteins using electrophoresis, electrical transfer ofproteins to a nitrocellulose membrane, and marking thetarget protein using a proper primary antibody and aproper enzyme-labeled secondary antibody. This technique
is also called as immunoblot.

H-thymidine (TdR) uptake assay

3

A technique based on the phenomenon of lymphocyte
blastogenesis after stimulation by nonself antigens.
Lymphocytes are cultured with a stimulant and3H-thymidine. After the DNA synthesis, the amount of3
H-thymidine uptaken by the cells is determined as theradioactivity. The stimulants include PHA, Con-A, anddrugs.

E