Clinical Applications

It is difficult to cover all areas of clinical applications by using antibody detection. However, clinical application of immunodiagnostics can be best demonstrated in available immunoassays for HIV (Nielsen and Bryson, 2000) and hepatitis. Immunoassays have been developed to detect anti-HIV antibodies or viral antigens present in serum, plasma, dried blood spots, urine, and saliva. Assay formats range from EIAs, ELISA-based Western blot assays, IFA assays, and even rapid handheld immunoassays. In general, however, the EIA remains the most widely used serologic test for detecting antibodies to HIV-1. Thus, HIV-1 immunoassays represent the advances in antibody detection technologies to detect and identify infectious agents. Another study comparing ELISA methods with Western blotting, microagglutination, IFA, and FC for detection of antibodies to Francisella tularensis and diagnosis of tularemia is another source to demonstrate the use of antibody detection techniques (Porsch-Ozcurumez et al., 2004). In this study, the combined use of ELISA and confirmatory blotting seems to be the most suitable approach for serodiagnosis of tularemia (Porsch-Ozcurumez et al., 2004).

Immunoanalyzers for broad application range (automation, random access, multiplexing, and high throughput) will help meet the challenges of immunodiagno-sis of infectious diseases. The main focus of this section of clinical application will be general use of recent application and automation in terms of detection method.

EIA Detection

ELISA assays are still the methods of choice for large-scale investigations during outbreak or epidemiological surveillance studies. Because of its relative simplicity and good sensitivity, ELISA has been used for screening large numbers of small-volume samples and has had great impact in epidemiology and in the diagnosis of infection, particularly in the diagnosis of the difficult bacteria and viruses such as West Nile (WN) Virus, not to mention that these assays have been used extensively in AIDS and hepatitis testing.

In a typical ELISA for HIV antibody test, HIV antigens (often a purified viral lysate) attached to a microtiter plate or bead serves as the test platform. The anti-HIV antibody in the sample can be tested by incubating with antigens followed by incubation with labeled conjugate secondary antibody and substrate and detection by using colorimetric method (Nielsen and Bryson, 2000).

Eight EIAs including two single-use EIAs and six plate-type EIAs were evaluated for the detection of IgM and IgG antibodies to Mycoplasma pneumoniae, an important etiologic agent of primary atypical pneumonia in children and adults (Talkington et al., 2004). Interestingly, the two single-use EIA methods were more reliable than the plate-type EIAs.

Serologic testing is the primary method of diagnosing WN virus infection. The recommended immunoassays are the immunoglobulin M (IgM) antibody ELISA and the indirect IgG ELISA (Davis et al., 2001). Positive ELISA results are confirmed by flavivirus plaque reduction neutralization tests (Lindsey et al., 1976). This combination of assays is highly sensitive and specific, but performing a complete panel of ELISAs requires 2 to 3 working days to complete, as overnight incubations are deemed necessary to enhance sensitivity. IFAs may also be used for diagnosis, but they are not suitable for a high throughput of specimens and they are less sensitive than ELISA.

Immunoblotting Method

The cross-reactivity of an antibody is prevented by using high-affinity antibody, thus to improve the quality of an immunoassay. Cross-reactivity could result from an antibody that binds to structurally distinct but similar epitopes present on different antigens or result from an antibody that binds to structurally identical epi-topes on different antigens. This is why confirmatory tests are needed in certain tests such as HIV using more specific assays such as the Western blot (Jackson et al., 1997). The separated HIV-1 proteins are electrotransferred to a nitrocellulose membrane. If antibodies to any of the major HIV-1 antigens are present in the specimen, bands corresponding to the HIV-1 proteins (p) or glycoproteins (gp) such as gp24, gp41, or gp120 will be seen on the nitrocellulose strip. Antibodies can thus be detected by using enzyme-conjugated secondary antibody (to human IgG) and demonstrated by darkly colored lines on the membrane under the substrate.

Other than HIV, the RIBA Strip Immunoblot Assay (SIA) for detecting NS5 and c33c recombinant proteins and c100p, 5-1-1p, and c22p synthetic peptides of hepatitis C virus (HCV) is intended as a supplemental test for human serum or plasma specimens found to be repeatedly reactive in HCV antibody screening test (Martin et al., 1998). Semiautomated or automated processing instrumentation is available for immunoblotting.

Handheld Assay

Handheld immunoassays are on the horizon. Development of self-contained miniaturized devices will allow an immunoassay to be performed in a field or point-of-care setting. The OraQuick HIV-1 immunochromatographic card assay has nearly equivalent sensitivity and specificity for HIV-1 as EIA. Two more rapid assays, one a lateral flow immunoassay device and the other a membrane immunoreac-tive test device, have been approved for non-blood donor diagnostic screening (Ketema et al., 2001, 2005). Lateral flow assays were developed for rapid serodi-agnosis of human brucellosis by using the lateral flow assay to detect antibodies against lipopolysaccharide (LPS) of Brucella-specific capture antigen (Smits et al., 2003).

RIA Application

Although unpopular in the clinical setting, RIA is still available for research settings. One example is the Human Papilloma Virus (HPV) type-specific competitive RIA (cRIA) used to evaluate HPV type-specific antibody titers. Briefly, HPV L1 virus-like particle (VLP) antigens (HPV-6 and HPV-11) are coated onto solidphase polystyrene beads and incubated with equal volumes of sera and diluted Mab, as well as the 125I-labeled secondary antibody (Opalka et al., 2003).

Chemiluminescence

Chemiluminescence will be discussed in the "Automation" section, below. Fluorescence Immunoassay

In addition to TRF (McKie et al., 2002; Peruski et al., 2002), another type of fluorescent technology is fluorescence polarization (FP). FP is a phenomenon seen when polarized light excites a fluorescent dye causing photons to be emitted in the same plane as the exciting light. FP assays can be used for detecting antibodies (Nielson et al., 1996). Because of the limited need for sample processing, FP antibody detection assays are particularly useful for high-throughput screening such as AxSYM (Abbott Laboratories, Abbot Park, IL, USA).

Flow Cytometry

Two distinct sizes of microspheres were used for simultaneous detection of two different antibodies and subsequently expanded to the use of four different sizes of microspheres to detect four different antibodies to cytomegalovirus and herpes simplex virus (McHugh et al., 1988) or antibodies against HIV proteins (Scillian et al., 1989). Size discrimination of microspheres allows simultaneous detection of small numbers of analytes, but the inability to distinguish aggregates of smaller microspheres from larger microspheres limits the extent of multiplexing that can be achieved.

Multiplexed Bead Assay

Diagnosis of infection often requires testing for multiple antibodies. The xMAP technique applications include detection of antibodies to a panel of seven respiratory viruses, including influenza A and B viruses; adenovirus; parainfluenza viruses 1,2, and 3; and respiratory syncytial virus (Martins, 2002), and for Bacillus anthracis anti-protective antigen (PA)-specific immunoglobulin G (anti-PA IgG) (Biagini et al., 2004). When compared with the ELISA method (Quinn et al., 2002), xMAP method for anti-PA IgG had a good positive correlation, better sensitivity and speed, and enhanced dynamic range. It uses smaller sample volume and can be multiplexed, that is, measure more than one analyte simultaneously (Biagini et al., 2003). In addition, the Luminex technology was used to simultaneously measure antibodies to HIV-1 p24, gp160, and gp120 eluted from dried blood-spot specimens from newborns (Bellisario et al., 2001; Faucher et al., 2004), and even the HCV antibody and HBs antigen with HIV antibodies (Lukacs et al., 2005).

Simultaneous measurement of antibodies to 23 pneumococcal capsular polysaccharides (PnPs) was developed recently (Biagini et al., 2003), which showed results similar to another xMAP assay developed for antibodies to PnPs (Pickering et al., 2002a). The assay simultaneously determines serum IgG concentrations to 14 PnPs serotypes. The multiplexed assay showed good overall agreement with a well-established ELISA that is currently recommended for evaluation of pneumo-coccal vaccine immunogenicity.

A Luminex xMAP based technology was compared with ELISA for quan-titation of antibodies to the toxoids of Clostridium tetani (Tet) for tetanus, Corynebacterium diphtheriae (Dip) for diphtheria, and Haemophilus influenzae type b (Hib) polysaccharide. The correlations (R2) between ELISA and Luminex of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively. Both methods detected strong postvaccination responses (Pickering et al., 2002b).

Using xMAP technology (Mandy et al., 2001), a new test was developed to measure antibodies induced by flavivirus infection. This assay is based on a recombinant WN virus envelope (E) glycoprotein antigen (rWNV-E). This first-generation test for serodiagnosis of flavivirus infection provides the basis for multiplex system for simultaneously measuring antibodies to several recombinant flavivirus antigens.

A multiplex assay was developed for detection of strain-specific antibodies against the two variable regions of the G protein of respiratory syncytial virus (RSV), which is the single most important lower respiratory tract pathogen of infants and young children worldwide (Jones et al., 2002).

A West Nile virus recombinant antigen microsphere (suspended-microsphere) diagnostic immunoassay was developed for detection of human anti-flavivirus antibodies (Wong et al., 2004). The microsphere immunofluorescence assay can be performed in less than 3 h on specimens of < 30 |xL. Retrospective testing of 833 sera from New York patients with suspected viral encephalitis demonstrated concordance with results obtained with the traditional ELISA for immunoglobulin G antibodies to WN virus. The assay also detects antibodies to E proteins from related flaviviruses, including St. Louis encephalitis, Japanese encephalitis, and dengue viruses. The new microsphere immunoassay provides a sensitive and rapid alternative to traditional ELISAs.

Cytokines were measured as mediators for or effectors against rotavirus disease, the most common cause of severe gastroenteritis in young children. In a pilot study by using bead-based Luminex assay, an overall increased cytokine response was demonstrated in children with acute rotavirus diarrhea compared with those in control children (Jiang et al., 2003).

Simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles (VLPs) for human papillomavirus (HPV) types 6,11,16, and 18 in 50 ^L of serum was achieved by a multiplexed Luminex assay (Opalka et al., 2003). The HPV competitive immunoassay measures titers of polyclonal antibodies and was found to be as sensitive and precise as the currently used cRIAs.

An advantage of the 96-well plate Luminex assay format is that it avails itself to automation, such as the Tecan Genesis liquid handler to automate the assay. The automation such as Bio-Plex system (Bio-Rad Laboratories, Hercules, CA, USA) employing the Luminex multianalyte profiling technology (xMAP) allows individual and multiplex analysis of up to 100 different analytes in a single microtiter well (Vignali, 2000) and is used for detecting 15 human cytokines (de Jager et al., 2003). A multiplexed bead assay was eveluated for assessment of Epstein-Barr virus im-munologic status using BioPlex 2200 system (Bio-Rad). Concordance between results generated by the BioPlex system and conventional assays showed 97% agreement with conventional heterophile and anti-nuclear antigen assays (Klutts et al., 2004).

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