Separation Analyses
Electrophoresis (EP) is a technique for separation of ionic
molecules (principally proteins) by the differential migration through
a gel according to the size and ionic charge of the molecules in an
electrical field. Smaller molecules with a more negative charge will
travel faster and further through the gel toward the anode of an electrophoretic
cell when high voltage is applied. Similar molecules will group on
the gel. They may be visualized by staining and quantitated, in relative
terms, using densitometers which continuously monitor the photometric
density of the resulting stain.
Flow cytometry (FC) is an emerging
technique which holds great promise for the separation, classification
and quantitation of blood cells and antibodies which affect blood
cells. Complex computerized instruments are used to pass a monocellular
stream of cells, platelets or other microscopic particulate elements
through a beam of laser light. The cells are categorized first by
size and then computer analyzed to sort the mixture of cellular elements
into cell type by size. In addition, monoclonal antibodies to specific
cell surface markers are conjugated to fluorescent dyes and each cell
displaying appropriate fluorescent light emission is counted. Tabulation
of counted data in conjunction with size analysis enables determination
of relative percentages of each specific cellular subset for which
monoclonal antibody conjugates are utilized, even when the size of
the cell is identical to other subset species.
Soluble Antigen-Antibody Reaction Assays
Immunodiffusion (ID), also called Double diffusion (DD) or
the Ouchterlony technique, is the classical procedure used to detect
the presence of antibodies and determine their specificity by visualization
of "lines of identity" (precipitin lines). These precipitin
lines (precipitated antigen-antibody complexes) form where the binding
concentrations of antigen and antibody are equivalent. Patient serum
diffuses from one well through the gel and reacts with a known specific
antigen (or antibody) which diffuses through the gel from a second
well. DD is strictly qualitative, although the density of the precipitin
line and the distance of the line from the sample well may give some
indication of the antibody concentration.
Radial immunodiffusion (RID) is a
quantitative variation of the Ouchterlony technique (immunodiffusion)
in which the agar gel contains evenly distributed antigen (or antibody)
and its counterpart from the test sample diffuses into the gel from
a single well resulting in a circular precipitin line around the sample
well. The diameter of the precipitin ring is proportional to the concentration
of the antibody (or antigen) present in the test sample. By comparing
the diameter of the test specimen precipitin ring to known standards,
a relatively insensitive estimation of the concentration of specific
antibody or antigen can be achieved.
Counterimmunoelectrophoresis (CIE)
is a procedure in which oppositely charged antigen and antibody are
propelled toward each other by an electrical field. This reduces the
time necessary for visualization of the antigen-antibody reaction
from 18-24 hours in ID to less than one hour and also substantially
increases the sensitivity of the analysis. CIE has the capability
of detecting concentrations of antigen/antibody 10 times smaller than
the lowest concentrations measurable by DD or ID.
Immunoelectrophoresis (IEP) is a
two-step procedure which first involves the electrophoretic separation
of proteins, followed by the linear diffusion of antibodies into the
electrophoretic gel from a trough which extends through the length
of the gel adjacent to the electrophoretic path. The antigen-antibody
reactions produce precipitin arcs at positions where equivalence occurs.
Although quantitation is subjective, an experienced eye can determine
not only the presence of the antigen but, through visual comparison
to normal control sera, may discriminate relative increases or decreases
of antigen by gauging the length and density of the precipitin arcs
at positions established for specific antigens using known standards.
Immunofixation (IFIX) is a powerful
enhancement of immunoelectrophoresis in which a series of post-electrophoretic
gel slabs are layered with cellulose-acetate gels saturated with specific
antibodies. The resulting antigen-antibody complexes fixed on the
second gel may then be stained, allowing sensitive and specific qualitative
visual identification of paraproteins by electrophoretic position.
Particulate Antigen-Antibody Reaction Assays
Direct agglutination (DA) is a general term for techniques
which use the agglutination (macroscopic clumping) of particulate
reagents as an indicator of the presence of an antigen-antibody reaction.
Examples (HA, LA and CoA) follow.
Hemagglutination (HA) is a technique
for detecting specific antibodies which, when present, will cause
antigen-coated reagent erythrocytes to agglutinate. Crude quantitation
of the antibodies can be achieved by performing a serial dilution
of the patient serum and noting the highest dilution (titer) at which
agglutination is still present.
Latex agglutination (LA), also known
as latex particle agglutination, for detection of antibodies is identical
to HA in principle, but the substitution of smaller, antigen-coated
latex particles for erythrocytes results in improved sensitivity and
reagent longevity. Alternatively, antibodies can be absorbed to the
latex particles (under appropriate ionic and pH conditions) by binding
to the Fc region of antibodies, leaving the Fab region free to interact
with antigens present in the applied specimens. This phenomenon has
made LA a popular technique for detecting antigens as well.
Coagglutination (CoA) is similar
to the LA technique for detecting antigen (described above). Protein
A, a uniformly distributed cell wall component of Staphylococcus
aureus, is able to bind to the Fc region of most IgG isotype antibodies
leaving the Fab region free to interact with antigens present in the
applied specimens. The visible agglutination of the S. aureus
particles indicates the antigen-antibody reaction.
Hemagglutination inhibition (HI),
also abbreviated HAI, is a variation of the HA technique. Some viral
antigens, when coated on erythrocytes, spontaneously cause agglutination
in the absence of antibody. In these situations, the specific antigen-antibody
reaction actually prevents the agglutination of reagent RBCs. HAI
cannot differentiate between isotypes of specific antibodies (IgG,
IgA or IgM) although positive HAI analysis of specimens treated with
Staphylococcus aureus Protein A (discussed above under CoA)
to remove the IgG isotype antibodies has been used to imply the presence
of specific IgM antibodies to the specific viral antigen. The crude
quantitation of the specific antibodies is possible using serial dilution
(titer).
Nephelometry (NEPH) is used to quantitate
antigen by analyzing increases in turbidity, as measured by increasing
scatter of laser light. The interaction of specific antibodies in
the reagent with the antigen from the sample results in the formation
of antigen-antibody complexes which are rendered insoluble by the
presence of precipitating reagents. Most modern nephelometers compare
the rate of formation of antigen-antibody complexes (determined by
computer analysis of laser light scatter data) to that of known antigenic
standards in order to measure precisely the protein antigens (some
of which are actually immunoglobulins) present in moderate concentrations.
RBC Lytic Assays for Detecting Antigen-Antibody Reactions
Complement fixation (CF) is an exacting, complex yet
sensitive procedure that detects the presence of a specific antigen-antibody
reaction by causing the in vitro activation of complement via
the classical pathway. If complement is not fixed, lysis of the pre-antibody-coated
reagent erythrocytes occurs. Again, crude quantitation of antibodies
is possible by determining the highest dilution (titer) at which lysis
does not occur. The differentiation of specific antibody isotype is
not possible.
Neutralization (Nt) is similar to
complement fixation but is applicable only in certain pathogenic situations
where the antibody being measured is directed against a hemolysin
(a bacterial toxin capable of directly lysing erythrocytes). In these
situations, the hemolysin and reagent erythrocytes are added, and
if the antibody to the hemolysin is present, the lysis of RBCs will
not occur. As in CF, crude quantitation is afforded by serial dilution
which may be quantitatively compared to established standard material
dilutions.
Immunohistochemical Assays
Fluorescent antibody (FA) assay is a general term for procedures
which utilize the visual detection of fluorescent dyes coupled (conjugated)
to antibodies which react with the antigen when present using fluorescent
microscopy. FA allows a competent technologist to identify visually
the site of the antigen-antibody reaction thereby rendering significant
specificity. Variations are further explained below (DFA, IFA, ACIF,
ABIF and Micro-IF).
Direct fluorescent antibody (DFA)
is the straightforward detection of antigens using fluorescently labeled
antigen-specific antibody. Because detection of the antigen in a substrate
of patient sample (cellular smear, fluid or patient- inoculated culture
medium) is the goal, DFA is seldom quantitative.
Indirect fluorescent antibody (IFA)
is the detection of antibodies to specific antigenic material in the
substrate using fluorescent microscopy. Using fluorescently conjugated
antibodies which are specific for a particular isotype of antibody,
it is possible to distinguish IgG, IgA and IgM isotypes of specific
antibodies using IFA. This sensitive technique is highly specific
in well-trained hands and recent developments in the establishment
of internationally recognized standard materials have led to accurate
quantitation of antibody concentrations through endpoint titration
(the highest serial dilution of specimen at which specific fluorescence
remains) and through measuring visual intensity of fluorescence compared
to known reference standard material.
Anticomplement immunofluorescence
(ACIF) is a technique used to make certain indirect fluorescent antibody
techniques more specific and sensitive. Here the fluorescent dye is
conjugated to antibody directed at complement and then added to a
complement-fixing complex of antigen and patient antibody.
Avidin-biotin immunofluorescence
(ABIF) holds promise for more sensitive and specific amplification
of indirect fluorescent antibody procedures. Antibody to the patient's
specific antibodies is labeled with biotin, a compound capable of
specifically binding avidin in high concentrations. Fluorescently
labeled avidin is then added and fluorescent microscopy is used to
detect the presence of the complexes.
Micro-immunofluorescence (Micro-IF)
is really multiple IFA. Several different substrates are arranged
in specific locations on a single microscope slide well allowing a
rapid, simultaneous IFA on each substrate.
Immunoperoxidase (IP) assays are analogous
to IFA in that antibody presence is identified on antigenic substrates
visually. However, in the indirect IP instead of fluorescent dye-antibody
conjugates, enzyme-antibody conjugates (principally peroxidase enzymes)
are reacted with their corresponding substrates to produce a product
which can be seen with a light microscope, eliminating the requirement
for costly fluorescent microscopic equipment.
Immunocytochemical assay (ICA) involves
the computerized assessment of microscopic fields following DFA, IFA
or indirect or direct IP analysis of biopsy tissue from the patient.
In addition to improved specificity with the removal of operator subjectivity,
the quantifiability of results through computer data analysis of color,
intensity and concentration has only begun to be realized.
Immunoassay Procedures
Radioimmunoassay (RIA) uses fixed-dose, low-level, radioactive-isotope-
labeled antigen ("tracer") to compete with unlabeled antigen
from the patient specimen for a fixed number of antibody binding sites.
Traditional RIA is done with specific antibodies in liquid solution.
Solid-phase RIA involves the use of antibody bound to solid support
(e.g., tubes, glass beads or plastic fins). The amount of antigen
in the specimen is determined by comparing the bound radioactivity
with a standard curve.
Immunoradiometric assay (IRMA) uses
low-level radioactively labeled specific antibody to quantitate low
concentration compounds. In IRMA, a first antibody is presented on
solid-phase (coated on tubes or beads). After binding the antigen
present in the sample, a second radioactively labeled antibody is
added. Radioactivity remaining after washing the solid phase is proportional
to the concentration of antigen present in the sample and is quantitated
by comparison to a standard curve.
Radioallergosorbent test (RAST)
is the name given to an in vitro technique which detects the
presence of IgE (and IgG) antibodies to allergens, proteins which
may give rise to hypersensitivity reactions seen in allergies. Allergens
are coated on a complex carbohydrate matrix called a sorbent. Antibodies
specific for the allergen being tested bind to the allergen and, if
present, are detected by a low-level radioactively labeled antibody
to either human IgE or IgG, depending upon the isotype being tested.
Fluorescence polarization immunoassay
(FPIA) is a technique which takes advantage of the increased polarization
(non-random propagation of emission) of fluorescent light emissions
when a fluorescently labeled antigen is bound by reagent antibody.
The higher the concentration of unlabeled patient antigen present
in the test mixture, the less bound fluorescent antigen is present
and, consequently, the lower the polarization of the fluorescent light
emission. Standard calibration yields quantitative results.
Chemiluminescence assays (CIA), including
a subcategory using bioluminescence (biologically derived chemiluminescence
agents), use the generation of light from oxidative chemical reactions
as an indicator of the quantity of unbound luminescently labeled antigen.
This allows quantitation of unlabeled antigen from patient specimens
in a variety of homogeneous (single phase) or heterogeneous (multiple
phase) immunoassay techniques.
Enzyme immunoassay (EIA) is the general
term for an expanding technical arsenal of testing which allows a
full range of quantitative analyses for both antigen and antibodies.
These tests use color-changed products of enzyme-substrate interaction
(or inhibition) to measure the antigen-antibody reaction. Examples
of EIA procedures (EMIT, ELISA, MAC, MEIA) follow.
Enzyme multiplied immunoassay technique
(EMIT) is a homogeneous (single phase) EIA procedure in which the
antigen being measured competes for a limited number of antibody binding
sites with enzyme labeled antigen. The reagent antibody has the ability
to block enzymatic activity when bound with the reagent enzyme-antigen
complex preventing it's formation of product in the presence of substrate.
The free antigen- enzyme complexes resulting from competition with
measured antigen in the sample forms color-change products proportional
to the concentration of antigen present in the specimen.
Enzyme-linked immunosorbent assay
(ELISA) is a sensitive, heterogenous (multiple phase) analytical technique
for quantitation of antigen or antibody in which enzyme-labeled antibody
or antigen is bound to a solid support (e.g., tubes, beads, microtiter
plate wells, plastic tines or fins). After addition of patient specimen
and substrate, antigen, antibody or complex are detected by a color
change indicating the presence of the product of an enzyme-substrate
reaction. Direct ELISA is a technique for measuring antigen using
competition for antibody binding sites between enzyme- labeled antigen
and patient antigen. Indirect ELISA, or enzyme immunometric assay,
measures antibody concentrations using bound antigen to interact with
specimen antibodies. Enzyme-labeled reagent antibodies can be isotype-specific
(i.e., capable of determining the presence of IgG, IgA, IgM or IgE
classes which react with the antigen of interest). The specificity
of indirect ELISA assays for IgM isotypes in some infectious diseases
is limited by false-positive results due to IgM rheumatoid factor
in the presence of IgG-specific antibodies.
IgM antibody capture ELISA (MAC ELISA)
has been developed to impart significant improvement in assay specificity
to indirect ELISA procedures for IgM isotype antibodies. Solid-phase
support (usually microtiter plate wells) are coated with anti-human
IgM antibodies capable of binding all IgM isotype antibodies present
in the specimen. Reagent antigen is then added, followed by enzyme-labeled
antigen- specific antibodies. If IgM antibodies specific for the antigen
in question are present, the "sandwich" complex will result
in enzymatic color-change proportional to the concentration of IgM-specific
antibody present. This technique appears to be the method of choice
in many highly specific and more sensitive assays for IgM infectious
disease antibodies.
Microparticle enzyme immunoassay
(MEIA) is a technique in which the solid-phase support consists of
very small microparticles in liquid suspension. Specific reagent antibodies
are covalently bound to the microparticles. Antigen, if present, is
then "sandwiched" between bound antibodies and antigen-specific,
enzyme- labeled antibodies. Antigen-antibody complexes are detected
and quantitated by analysis of fluorescence from the enzyme-substrate
interaction.
Radioimmunoprecipitation assay (RIPA)
is the term used to describe the qualitative assay used as a confirmatory
procedure for some antibodies to viral antigens. Viral-infected cell
cultures are radioactively labeled and lysed to yield radiolabeled
antigen fragments. Specific antibodies, if present, will bind these
antigen fragments and the resulting antigen-antibody complexes are
precipitated using protein A, boiled to free the immune complexes
which are then separated by electrophoresis. The pattern of antigenic
moieties to which antibodies are present may then be detected using
autoradiography (the exposure of sensitive X-ray film by the radioactive
emissions of the bound, labeled antigens). Comparison to labeled molecular
weight standards electrophoresed in the same run allows determination
of the molecular weight "bands" of antigen to which antibodies
are present.
Techniques in Molecular Biology
DNA "dot-blot" hybridization (DOT-BLOT) is
a rapid technique used to detect the presence of a specific DNA in
a specimen. Dots, or spots of the DNA containing sample are placed
onto a nitrocellulose membrane and fixed. This membrane is then hybridized
to a radioactively labeled DNA segment of known sequence, specific
for the pathogenic DNA being tested. If the pathogenic DNA is present
in the specimen, complementary DNA sequences present on the membrane
will hybridize, or anneal, producing a double-stranded DNA segment
with the radioactive label incorporated into the molecule. The presence
of radioactivity is detected by autoradiography.
Polymerase chain reaction (PCR) is
a highly efficient method to amplify low levels of specific DNA sequences
in a sample to reach the threshold of detection. Two short DNA "primers",
oligonucleotides (small portions of a single DNA strand) specific
for the pathogenic DNA sought whose sequence flanks that section of
DNA to be amplified, are used. Repeated cycles of DNA denaturation
(separation of the double DNA strands), primer annealing (recombination
of the double-stranded structure) and extension of the primed DNA
sequence (by the enzyme DNA polymerase in the presence of added purine
and pyrimidine bases) are performed. Each cycle doubles the amount
of specific DNA sequence present and results in an exponential accumulation
of the DNA fragment being amplified. The reaction products are hybridized
to a radioactively labeled DNA segment complementary to a short sequence
of the amplified DNA. Following electrophoresis, the radiolabeled
product of specific size is detected by autoradiography.
Reverse transcriptase PCR (RT-PCR)
is a technique used to amplify RNA targets. The specimen containing
the target RNA (e.g., HIV-1 RNA, Hepatitis C Virus RNA) is subjected
to reverse transcription to make complementary DNA (cDNA), which is
then, in turn, amplified by PCR.
Southern blot (SB) describes the technique
first developed by the Scottish molecular biologist Edward M. Southern
which now bears his name. Specimen DNA is denatured, treated with
restriction enzymes to result in DNA fragments and then the single-stranded
DNA fragments are separated by electrophoresis. The electrophoretically
separated fragments are then blotted to a nitrocellulose membrane,
retaining their electrophoretic position, and hybridized with radiolabeled
single- stranded DNA fragments with sequences complementary to those
being sought. The resulting double-stranded DNA bearing the radiolabel
is then, if present, detected by autoradiography.
Northern blot (NB) uses techniques
similar to the Southern blot described above. Messenger-RNA from the
specimen is separated by electrophoresis and blotted to a specially
modified paper support to result in covalent fixing of the mRNA in
the electrophoretic positions. Radiolabeled single-stranded DNA fragments
complementary to the specific mRNA being sought are then hybridized
to the bound m-RNA. If the specific mRNA is present, the radioactivity
is detected by autoradiography. The derivation of this technique from
the Southern blot used for DNA detection has led to the common usage
of the term "Northern blot" for the detection of specific
mRNA.
Immunoblot, commonly referred to as
"Western blot" (WB) because of the similarity to the procedures
described above, is used to detect antibodies to specific epitopes
of electrophoretically separated subspecies of antigens. Electrophoresis
of antigenic material yields separation of the antigenic components
by molecular weight. Blotting of the separated antigen to nitrocellulose,
retaining the electrophoretic position, and reacting it with patient
specimen will result in the binding of specific antibodies, if present,
to each antigenic "band". Electrophoresis of known molecular
weight standards allows for the determination of the molecular weight
of each antigenic band to which antibodies may be produced. These
antibodies are then detected using EIA reactions which characterize
antibody specificity. This technique is often used to confirm the
specificity of antibodies which are detected by ELISA screening procedures.
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