Separation Analyses
       EP Electrophoresis
       FC Flow Cytometry

Soluble Antigen-Antibody Reaction Assays
        ID Immunodiffusion
        RID Radial immunodiffusion
        CIE Counterimmunoelectrophoresis
        IEP Immunoelectrophoresis
        IFIX Immunofixation

Particulate Antigen-Antibody Reaction Assays
        DA Direct Agglutination
        HA Hemagllutination
        LA Latex Agglutination (Latex Particle Agglutination)
        CoA Coagglutination
        HI Hemagglutination inhibition
        NEPH Nephelometry

RBC Lytic Assays for Detecting Antigen-Antibody Reactions
        CF Complement Fixation
        Nt Neutralization

Immunohistochemical Assays
        FA Fluorescent Antibody
        DFA Direct Fluorescent Antibody
        IFA Indirect Fluorescent Antibody
        ACIF Anticomplement Immunofluorescence
        ABIF Avidin-biotin Immunofluorescence
        Micro-IF Micro-immunofluorescence
        IP Immunoperoxidase
        ICA Immunocytochemical Assay

Immunoassay Procedures
        RIA Radioimmunoassay
        IRMA Immunoradiometric Assay
        RAST Radioallergosorbent Test
        FPIA Fluorescence Polarization Immunoassay
        CIA Chemiluminescence Assays
        EIA Enzyme Immunoassay
        EMIT Enzyme-multiplied Immunoassay
        ELISA Enzyme-linked Immunosorbent Assay
        MAC IgM Antibody Capture
        MEIA Microparticle Enzyme Immunoassay
        RIPA Radioimmunoprecipitation Assay

Techniques in Molecular Biology
        DOT-BLOT DNA Dot-blot Hybridization
        PCR Polymerase Chain Reaction
        RT-PCR Reverse Transcriptase PCR
        SB Southern Blot
        NB Northern Blot
        WB Immunoblot/Western Blot
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.