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Methodologies Print

Methodology Abbreviations
CRREL 5 I Cr Release
AE Agarose Electrophoresis
bDNA Branched Chain DNA
BROTH DIL Broth Dilution
CALC Calculation
CM Calorimetric
CEIA Capillary Electrophoresis Immunoassay
CZE Capillary Zone Electrophoresis
CELL CNT Cell Count
CL Chemiluminescence
CHROM Chromogenic
INVADER Cleave-based Invader Assay
CLOT DET Clot Dectection
C Colorimetric
CF Complement fixation
D Dialysis
DFA Direct fluorescent antibody
DISK DIFF Disk Diffusion
DNA PROBE DNA Probe Analysis
DD Double Diffusion
ECL or ECL Electrochemiluminescence Immunoassay
EP Electrophoresis
E-TEST Elution Test
ENZ Enzymatic
EIA Enzyme Immunoassay
EMIT Enzyme multiplied immunoassay technique
ELVIS Enzyme-Linked Virus-Inducible System
EQ DIAL Equilibrium Dialysis
Farr RBA Farr Radiobinding Assay
FLO Flocculation
FC Flow cytometry
FPIA Fluorescence polarization immunoassay
FA Fluorescent antibody
FISH Fluorescent In Situ Hybridization
F Fluorimetry
FEIA Fluoroenzyme Immunoassay
FP Freezing Point
GC/MS Gas Chromatography/Mass Spectrometry
GRAVMET Gravimetric
HA Hemagglutination
HF Hematofluorimetry
HPLC High Performance Liquid Chromatography
HC Hybrid Capture II
MAC EIA IgM Antibody Capture EIA
IMAGE Image Analysis
IAA ImmunoArray Assay
IA Immunoassay
IB Immunoblot
WB Immunoblot
IC Immunochemiluminometric Assay
ICMA Immunochromatographic Membrane Assay
IHC Immunohistochemistry
IRMA Immunoradiometric assay
IFA Indirect fluorescent antibody
ICP/MS Inductively Coupled Plasma/ Mass Spectrometry
IR Infrared Spectrum Analysis
INSP Inspection
ICP/OES Ion Coupled Plasma, Optical Emission Spectrophotometry
IEC Ion Exchange Chromatography
ISE Ion Selective Electrode
IEF Isoelectric Focusing
KS Kinetic Spectrophotometry
LIA Latex Immunoassay
LPA Latex Particle Agglutination
LM Light Microscopy
INNO-LIA Line Immunoassay
INNO-LIPA Line Probe Assay
LIPA Line Probe Assay
LC/MS-MS Liquid Chromatography/Tandem Mass Spectrometry
MS Mass Spectrometry
MICRO-IF Micro-Immunofluorescence
MEIA Microparticle enzyme immunoassay
NEPH Nephelometry
NEUT Neutralization
NASBA Nucleic Acid Sequence-Based Amplification
PA Particle Agglutination
PCR Polymerase chain reaction
RID Radial immunodiffusion
RIA Radioimmunoassay
RIPA Radioimmunoprecipitation assay
RADIOMET Radiometric
RGT STRIP Reagent Strip
RT-PCR Real-Time PCR
RIBA Recombinant Immunoblot Assay
SHELL VIAL Shell Vial Culture
SB Southern Blot
S Spectrophotometry
TTGE Temporal Temperature Gradient Gel Electrophoresis
TLC Thin Layer Chromatography
TCA Tissue Culture Assay
TMA Transcription-Mediated Amplification
TTI Tritiated Thymidine Incorporation
TURB Turbidimetry
UREA CLOT Urea Clot Solubility
V Viscosity

Descriptions of Select Methodologies
Capillary Zone Electrophoresis (CZE ) is a techniquesimilar to electrophoresis EP. However the separation in the electric field takes place in a narrow capillary tube without the supporting gel media.The analytes are generally quantitatedusing UV-vis detectors.

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.

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.

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.

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.

Flow cytometry (FC) is a laser-based fluorescence detection method used to characterize cell antigens on large numbers of hematopoetic or lymphoid cells in suspension typically derived from peripheral blood, bone marrow aspirate or lymph node tissue. Flow cytometry instruments use a thin laminar flow of fluid to direct thousands of cells from a small diameter nozzle through a thin beam of laser light of a certain wavelength. The cells are categorized by size forward light scatter, cellular complexity or granularity side scatter and by antigen density fluorescence intensity for each 'CD' clusters of differentiation marker. The antigen distribution for thousands of cells is printed in graphical histograms which a pathologist interprets individually and collectively to determine the phenotype of dominant cell populations in leukemias and lymphomas of various types.

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.

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.

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.

Immunoblot (WB) , 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.

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.

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.

Isoelectric Focusing (IEF) is a technique similar togel electrophoresis, however,isoelectric focusinggives sharper bands then gel electrophoresis. In isoelectric focusing a pH gradient is established on the supportingmedia which is then placed in an electricfield. Zwitterionic molecules such as proteins migrate on the gel until they reach the pH region equal to their isoelectric point.They may be visualized by staining and evaluated qualitatively for te presence of certain proteins.

Liquid Chromatography/Tandem Mass Spectrometry (LC/MS-MS) is a technique combining the principles of chromatography and mass spectrometry. Chromatographic separation of the analytes is based on the difference of their distribution between a mobile liquid phase and a stationary phase.Mass spectrometry measures ions based on the mass to charge m/z ratio. In LCMS-MS,following a liquid chromatography separation, the sample is introduced into a mass spectrometer,wherethe molecules are ionized, fragmented, and the fragments aresorted based on their m/z ratio,then subsequently introduced to a second mass spectrometer for further fragmentation, sorting and quantitation. This technique is highly specific and sensitive.

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.

Nephelometry (NEPH) is used to quantitate antigen by analyzing increases in turbidity, as measured by increasing right anglescatter 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.

Neutralization (NEUT) 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.

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.

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.

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.

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.

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.

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