Clinical Chemistry - Analytical Chemistry (ACS Publications)

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Anal. Chem. 1997, 69, 165R-229R

Clinical Chemistry David J. Anderson,* Baochuan Guo, Yan Xu, and Lily M. Ng

Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115 Larry J. Kricka

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Kristen J. Skogerboe

Department of Chemistry, Seattle University, Broadway and Madison, 900 Broadway, Seattle, Washington 98122 David S. Hage

Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588 Larry Schoeff

Department of Pathology, School of Medicine, University of Utah, 50 North Medical Drive, Salt Lake City, Utah 84132 Joseph Wang

Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003 Lori J. Sokoll and Daniel W. Chan

Departments of Pathology and Oncology, Johns Hopkins University, 600 North Wolfe Street, Meyer B-121, Baltimore, Maryland 21287 Kory M. Ward and Katherine A. Davis

School of Allied Medical Professions and the Department of Pathology, The Ohio State University, 1583 Perry Street, Columbus, Ohio 43210 Review Contents Immunoassays (David S. Hage) General Books and Reviews Theory of Immunoassays Antibodies, Immunoassay Supports and Related Reagents Radioimmunoassays Enzyme Immunoassays Fluorescence Immunoassays Chemiluminescence Immunoassays Nonlabeled Immunoassays Miscellaneous Immunoassays Capillary Electrophoresis (Yan Xu) Reviews Diagnosis of Metabolic Disorders DNA and RNA Analysis Drug Monitoring Protein Analysis Single-Cell Analysis CE-Based Immunoassays High-Performance Liquid Chromatography (David J. Anderson) Books and General Reviews Reviews of Bioanalytes Significant Articles in Clinical or Biomedical Analysis Columns, Packing Materials, and Column Design S0003-2700(97)00008-5 CCC: $14.00

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© 1997 American Chemical Society

Electroanalysis and Biosensors (Joseph Wang) Enzyme Electrodes Immunosensors DNA Biosensors Optical Sensors Ion-Selective Electrodes Voltammetry and Amperometry Mass Spectrometry (Baochuan Guo) New Techniques Protein Characterization DNA Characterization Quantitative Analysis Perspective Chemiluminescence and Bioluminescence (Larry J. Kricka) New Reagents and Analytical Reactions Clinical Assays Immunoassays Nucleic Acid Assays Cellular CL and Luminol- and Lucigenin-Enhanced CL CL and BL Detection Coupled to Separation Techniques Optical Biosensors and Imaging Assays Reporter Gene-Based Assays Forensic Applications Luminometers, Analyzers, Reagents, and Kits

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Molecular Biology Techniques in Clinical Chemistry (Kristen J. Skogerboe) Nucleic Acid in Clinical Microbiology and Virology Testing DNA Testing and Cancer Genetics DNA Testing and Genetic Disease Diagnosis Emerging Analytical Technologies in Nucleic Acid Detection Infrared Spectroscopy (Lily M. Ng) Reviews Noninvasive in Vivo Monitoring Diagnostic Applications of IR Spectroscopy Laboratory Analyses Summary Clinical Instrumentation (General Chemistry Analyzers) (Larry Schoeff) Preanalytical Phase (Sample Processing) Analytical Phase Postanalytical (Data Management) Conclusion Clinical Instrumentation (Immunoassay Analyzers) (Lori J. Sokoll and Daniel W. Chan) Automated Immunoassay Systems Future Trends Clinical Instrumentation (Point-of-Care Testing Analyzers) (Kory M. Ward and Katherine A. Davis) Overview of Point-of-Care Testing Instrumentation Blood Glucose Meters Blood Gas, Electrolyte, and General Chemistry Analyzers Hematology and Coagulation Analyzers Other Chemistry POCT Analyzers The Future Literature Cited

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This review is divided into 11 sections as given in the table of contents. The review covers the application of analytical techniques to clinical and biomedical analysis. The time period covered by the review is from the Chemical Abstracts dates of October 1, 1994 to October 1, 1996, unless specified otherwise. IMMUNOASSAYS Immunoassays are a diverse group of methods that represent a significant fraction of both the routine and specialized testing that is performed in modern clinical laboratories. In this article, an immunoassay is defined as an analytical technique that uses antibodies or antibody-related reagents for the determination of sample components. The selective nature of antibody binding allows the use of these reagents in the development of methods that are highly selective and that can often be used directly with even complex matrixes, like blood or urine. The combined use of antibodies along with readily detectable labels (e.g., radioisotopes or enzymes) also provides many immunoassays with extremely low limits of detection. These characteristics, along with the relatively low cost generally associated with these methods, have continued to make immunoassays the method of choice for a wide range of clinical applications. Many of the general trends reported previously in the field of immunoassays (A1-A3) have continued throughout the period of this review. Examples include an emphasis on nonisotopic labels, more specific reagents, and improved formats for automating or performing immunoassays. This article will discuss recent advances in the theory and analytical methodology of immunoassays, as reported in articles appearing between January 1995 and 166R

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December 1996. In this review, immunoassays are categorized primarily according to the type of label that is present (e.g., radioimmunoassays, enzyme immunoassays, etc.). Additional sections are provided on more general topics, such as the theory of immunoassays or advances in immunoassay reagents. Related items, including immunosensors and commercial immunoassay instrumentation, will be discussed in later sections. General Books and Reviews. Several articles and books discussing the general subject of immunoassays appeared during this review period. A text edited by Diamandis and Christopoulos presented an overview of various topics of interest in this field (A4). Included in this book were sections on the history and future prospects for immunoassays (A5), the development of inhouse immunoassays (A6), and immunoassay automation (A7). Approaches to the automation of immunoassays were also covered in a book edited by Chan (A8) and a related review article (A9). Rubach reviewed the theory and principles of immunoassays, means for antibody production, and factors that influence immunoassay performance (A10). Self and Cook provided an overview of advances in immunoassay technology (A11), while Freitag discussed the principles and applications of various formats for immunoanalysis (A12). Theory of Immunoassays. Various papers appeared during this review period on theoretical or practical aspects of immunoassays. Monographs on immunoassay configurations (A13) and the theory of immunoassays (A14) were written by Christopoulos and Diamandis. Little discussed the methods and requirements involved in the validation of immunoassays (A15). Miller and Levinson had a book chapter regarding immunoassay interferences (A16), and an approach for assessing cross-reactivities was presented by Oubina et al. (A17). Brady reviewed the types of standard curves used in immunoassays (A18), while Ndlovu et al. compared various approaches for constructing confidence intervals when using four-parameter logistic models for curve fitting in competitive binding immunoassays (A19). Problems were discussed in the use of such fits for the analysis of multiple unknowns (A20), and discourses appeared on the sources of variability when unknowns were analyzed by enzyme-linked immunosorbent assays (A21) or immunoassays based on nonlinear calibration curves (A22). The dependence of immunoassay detection limits on antibody affinity was studied by O’Connor and co-workers (A23), and Brown et al. presented a new approach for defining and assessing the minimum detectable concentration for an immunoassay (A24, A25). Bergamaschi reviewed the topic of quality assurance for immunoassays (A26), and a personal computer-based system for immunoassay simulation was presented by Mate and co-workers (A27). A method for estimating the normal range of immunoassays for control subjects was reported (A28), as well as a procedure for evaluating carryover on random-access immunoassay instruments (A29). In addition, the role of wash steps and binding format was considered in an immunometric method for small analytes (A30). There were a large number of studies that considered the fundamental processes involved in antibody binding and recognition. For instance, Jemmerson reviewed the effects of conjugation, conformation, and amino acid sequence on the binding of antibodies to peptides and proteins (A31). Leder et al. studied the conformational adaptation of model peptides upon binding to antibodies (A32). An aqueous two-phase liquid-liquid chromatography system was used to compare the surface properties of

antibodies (A33), and a review appeared on the use of site-directed mutagenesis to study antibody binding sites (A34). Jelesarov et al. reviewed the use of titration microcalorimetry in examining the energetics of antibody binding (A35). The relative contributions of enthalpy vs entropy in the interactions of antibodies with lysozyme were investigated by Braden and co-workers (A36), while Gavalda et al. studied the effect of high pressures on the binding of antibodies with β-galactosidase (A37). Magdassi et al. examined the activity and physical properties of surface-active antibodies that were covalently modified with hydrophobic groups (A38). Similarly, Malmsten and co-workers studied the effects of immobilization and increased hydrophobicity on the interactions of antibodies (A39). A model was presented for the assessment of average molecular weights and conditions needed in the formation of branched immune complexes (A40). Furthermore, the kinetics of such complexation was discussed (A41) and work was reported that examined antibody-analyte-induced colloid aggregation (A42), including the coagulation kinetics of antibodycoated latex supports (A43). A method based on thiocyanate elution was reported for estimating the affinity distribution of polyclonal antibodies (A44). Scanning electrochemical microscopy (A45), scanning tunneling microscopy (A46-A48), and scanning force microscopy (A49) were employed as tools for the visualization of immobilized antibodies. In the same fashion, atomic force microscopy was used to monitor the binding of streptavidin with biotin in immunoassay microtiter wells (A50). The characteristics of antibody adsorption to solid surfaces was reviewed (A51) and dealt with in a number of articles examining materials such as silica (A52-A56), latex (A57, A58) or allylamine films (A59). Langmuir films of IgG-class antibodies were studied by fluorometry and ellipsometry (A60); the orientation of IgG in immobilized Langmuir films was also investigated (A61). Many studies qualitatively or quantitatively examined the affinity of antibodies by such techniques as enzyme-linked immunosorbent assays (A62-A67), surface plasmon resonance (A68-A73), quartz crystal microbalances (A72), scanning probe microscopy (A73), affinity sensors (A74), light-addressable potentiometric sensors (A75), optical grating coupler sensors (A76), affinity capillary electrophoresis (A77), and titration calorimetry (A78). In a similar fashion, the kinetics of antibody-solute binding or dissociation were studied using enzyme immunoassays (A79), surface plasmon resonance or resonant mirror biosensors (A80A86), ellipsometry (A87), internal reflection fluorescence (A88, A89), affinity chromatography (A90, A91), solid-phase displacement immunosensors (A92), fluorescence quenching (A93), and planar optical waveguides (A94). Neri et al. reviewed methods for characterizing the thermodynamic and kinetic properties of high-affinity recombinant antibodies (A95). Foote and Eisen discussed the kinetic and affinity limits of antibodies (A96), while Joshi reported a model for the statistical mechanics of antibody binding (A97). Fractal analysis was used to characterize antibody binding kinetics in biosensors (A98-A102). Beumer et al. discussed the importance of convection on the response and precision of microtiter well-based immunoassays (A103). Antibodies, Immunoassay Supports, and Related Reagents. The success of any immunoassay depends heavily on which antibodies, labels, and other reagents are used. Of these, the antibody is the most central and key component, making this an important aspect in the development of immunoassays. A book by Delves (A104) and a book chapter by Banting (A105) covered

various techniques associated with antibody applications. Perry discussed the role that monoclonal antibodies have played in immunoassays (A106), while other papers (A107-A109) presented a summary or discussion of methods for the preparation of monoclonal antibodies. The use of recombinant antibodies in immunoassays was reviewed by Choudary et al. (A110). Bispecific antibodies were the subject of a book edited by Fanger (A111), while Ishikawa discussed topics related to the labeling of antibodies and antigens (A112). Methods were described for the controlled oxidation of antibodies by periodate (A113), the conjugation of antibodies with biotin (A114), and the introduction of lysine chains onto the constant region of Fab fragment light chains (A115). Several articles also examined the use of molecular imprinting to generate artificial surfaces or supports that mimicked antibody binding regions (A116-A118). Supports and coupling methods for immunoassays were two other areas that continued to experience active research. Butler wrote a chapter that reviewed solid-phase immunoassay supports (A119). Two articles examined the use of photoactive surfaces for the patterned immobilization of antibodies (A120, A121), and several papers considered the use of antibodies or protein A in Langmuir-Blodgett films as immunoassay supports (A122A124). Advances in coupling methods included a review on the oriented immobilization of antibodies (A125), along with an article reporting the site-specific attachment of antibody Fab′ fragments to silica (A126). Fluorescence quenching was used to examine the orientation of photoaffinity-labeled antibodies on a silanetreated silica surface (A127). Other developments in immunoassay supports included reports on the use or preparation of carboxylated (A128) or acetal latex (A129), aminopropyl silica (A130), nitrocellulose particles (A131), paracrystalline glycoprotein S-layers (A132), hydrocoated supports (A133), thiophilic gels (A134), polypyrrole-based colloids (A135), and treated nylon (A136) for antibody or antigen immobilization/adsorption. Other reagent-related topics included reviews by Szurdoki et al. (A137) and Goodrow et al. (A138) on factors to consider in the design of haptens for antibody production. Newman and Price discussed various molecular aspects of antibody binding and how these can be used in immunoassay design (A139). A report appeared on the use of starburst dendrimers as matrices for multifunctional immunoassay reagents (A140). An extended heterobifunctional agent was described for use in coupling enzymes with antibodies (A141), and methods were compared for the immobilization of antibodies to carboxylmethyl dextran surfaces (A142). A number of new fusion proteins were developed for use in immunoassays, such as a protein A/maltose-binding protein hybrid (A143) and a single-chain antibody/lanthanidebinding protein for time-resolved fluoroimmunoassays (A144). Radioimmunoassays. Despite the emphasis in recent years on nonisotopic immunoassays, methods based on radiolabels continue to hold an important place in routine and research-related clinical testing. The main techniques included in this group include the competitive binding radioimmunoassay (RIA), the immunoradiometric assay (IRMA), and the scintillation proximity assay (SPA). A review discussing radioimmunoassay methods recently appeared in a book chapter by Chard (A145), while Mansfield et al. discussed the use of microporous membranes in a specific SPA method for cyclic AMP (A146). An optimization strategy for radioimmunoassays was reported by Junghans (A147). Analytical Chemistry, Vol. 69, No. 12, June 15, 1997


There were several reports of new approaches for radiolabeling proteins or antibodies for potential use in immunoassays. For example, Harapanhalli et al. demonstrated the use of 125I-labeled iodo-5-[(4,6-dichlorotriazin-2-yl)amino]fluorescein as a means for radioiodination of a protein via its free amine groups (A148). Neri and co-workers used human casein kinase II and [γ-32P]ATP to phosphorylate and radiolabel antibody fragments expressed in bacteria (A149). In work by Horiuchi et al., 67Ga (half-life, 78 h) was used along with the chelating agent deferoxamine to label antibodies and develop an IRMA method for human growth hormone (A150). The use of short-lived radioisotopes and chelating agents in antibody labeling was similarly examined by Trubetskoy and Torchilin (A151). A discussion also appeared on the preparation of radiolabeled analytes or antibodies by a more traditional approach (i.e., the chloramine-T method) (A152). Enzyme Immunoassays. Methods that use enzymatic labels remained the most popular type of immunoassay during the period of this review. This group of analytical methods includes the enzyme-linked immunosorbent assay (ELISA), the enzymemonitored immunotest (EMIT), the competitive binding enzyme immunoassay (EIA), and the immunoenzymometric assay (IEMA). These and related topics were the subjects of several recent review articles and book chapters. For example, book chapters by Gosling (A153) and Foley (A154) covered the general topic of enzyme immunoassays, and a book edited by Deshpande discussed various topics concerning enzyme immunoassay development (A155). ELISA methods were covered in a book edited by Crowther (A156), as well as in book chapters by Khetarpal and Kumar (A157) and Heer et al. (A158). Various enzymatic systems can be used in enzyme immunoassays, but those based on horseradish peroxidase (HRP), alkaline phosphatase (ALP), or β-galactosidase (GAL) are the most common. Tsang et al. examined how various reaction conditions affect the specific activity of HRP-antibody conjugates prepared by a periodate-mediated coupling method (A159). A related paper compared the activity of HRP-antibody conjugates prepared by the periodate method to those obtained using a glutaraldehyde coupling technique (A160). Jin and co-workers examined the use of R-N,N-bis(carboxymethyl)lysine and nickel ions to modify periodate-treated HRP for the selective labeling of histidine-tagged recombinant proteins (A161). The influence of coupling method on the stability of HRP-antibody conjugates was studied by Presentini and Terrana for the periodate and maleimide-sulfhydryl coupling techniques (A162). Similarly, Nielsen investigated the stability of freeze-dried HRP-antibody conjugates in the presence of various additives or storage vessels (A163). In work with ALP as a label, a comparison was made between a chemiluminescent 1,2-dioxetane phosphate substrate and the chromogenic substrate p-nitrophenyl phosphate (A164). Also, the use of phenacyl phosphate (A165, A166) and lucigenin (A167) were reported as substrates for the chemiluminescent detection of ALP labels (A165, A166). Paek and Kim demonstrated that the different pH optima for HRP and GAL can be used as a basis for dual-label detection in immunoassays (A168). And finally, Haugland discussed a procedure for the coupling of monoclonal antibodies with either HRP, ALP, or GAL (A169). A variety of other enzymatic systems can also be used for detection in immunoassays. Price et al. reviewed several novel enzyme labels, including enzymes from microbial sources and enzymes produced through protein engineering (A170). Recom168R

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binant barnase (A171) and bacterial chitinase (A172) were two specific alternative labels that were studied during this review period. Another unique system that was considered included the combined use of L-lactic dehydrogenase, lactate oxidase, and HRP with NADH and 3,3′,5,5′-tetramethylbenzidine as a signal amplification system for ALP (A173). In addition, acetate kinase was employed as a label along with soluble firefly luciferase to detect the ATP produced by this label in the presence of ADP and acetyl phosphate (A174, A175). An expression immunoassay was also described in which a DNA fragment was employed as a label by using it to produce copies of the enzyme firefly luciferase, which in turn catalyzed the bioluminescent reaction of luciferin in the presence of oxygen and ATP (A176, A177). Other papers in the field of enzyme immunoassays included a book chapter discussing microtiter plate-based methods (A178) and a number of articles related to curve-fitting or data management software for ELISA techniques (A179, A180). The use of a charge-coupled device (CCD) array was described for detection in ELISA methods based on fluorogenic substrates (A181). Immune complex transfer enzyme immunoassays continued to be of interest; papers dealing with this method included a review by Hashidi et al. (A182) and research articles describing its use for such analytes as anti-HTLV-I IgG (A183, A184) and anti-HIV-1 IgG (A185, A186). The relatively new approach of combining the polymerase chain reaction (PCR) with ELISA detection (PCR/ ELISA) also received a great deal of attention, as indicated by reports which used this method for analyzing Mycoplasma (A187, A188), Entamoeba histolytica (A189), parasites of the Leishmania donovani complex (A190), human cytomegalovirus DNA (A191), human papillomavirus (A192), cytokine mRNA (A193), and K-ras mutations (A194). Stevens et al. discussed the use of digoxigeninand biotin-labeled PCR products for coupling ELISA methods with PCR for such assays (A195), while Oroskar described a solid matrix for the ELISA detection of immobilized PCR products (A196). Other developments in the field of enzyme immunoassays included descriptions of an ELISA method for the gas-phase detection of cocaine (A197), a polymer-based enzyme immunoassay for hepatitis B surface antigen (A198), and a means for trapping antibodies on polymerized surfaces (A199). Fluorescence Immunoassays. This group of nonisotopic techniques employs a fluorescent signal for analyte detection. The methods that make up this category are the competitive binding fluoroimmunoassay (FIA), immunofluorometric assay (IFMA), fluorescence polarization immunoassay, and time-resolved fluoroimmunoassay (TR-FIA). These techniques were recently reviewed by Christopoulos and Diamandis (A200). A review also appeared by Langhals on various fluorescent dyes that can be used as labels in such assays (A201). Haugland discussed methods for coupling antibodies with fluorophores and phycobiliproteins (A202), while McCormack et al. examined the effect of fluorophore/antibody ratios on the final binding activity of the resulting immunoconjugate (A203). Numerous reports appeared on the development or use of improved detection and labeling schemes for time-resolved fluoroimmunoassays. For example, Sabbatini et al. (A204, A205) and Hemmila (A206) reviewed several types of lanthanide complexes that can be used in TR-FIA. Two separate groups used labels based on europium chelates of β-diketones (A207, A208). Another group described the use of europium-loaded liposomes and lipid-tagged antibodies for use as reagents in a TR-FIA method

(A209). The use of europium chelates in a homogeneous fluorescence energy-transfer immunoassay was also examined (A210). In addition, Lamture and Wensel reported the preparation of terbium chelates (and related protein conjugates) made with polymers containing large numbers of dipicolinic acid residues (A211). Other work in the area of fluorescence immunoassay labels included the development of fluorescent coumarin-based polymeric dyes that can be conjugated to proteins (A212) and the use of pyrene or dansyl groups in the photoaffinity labeling of antibodies (A213). Fluorescence polarization immunoassays were reported that employed osmium (A214) or ruthenium chelates (A215) as labels. The combined use of a ruthenium chelate and a nonfluorescent absorbing agent as analyte and antibody labels was demonstrated in the development of a fluorescence energytransfer immunoassay (A216). A lanthanide-based fluorescence energy-transfer immunoassay was also examined that employed 4-(arylethynyl)pyridine as the acceptor group and iminobis(acetic acid) as the chelating agent (A217). The potential use of green fluorescent protein in immunotechniques was discussed (A218), as well as the development of near-IR absorbing fluorescent dyes that can be used for antibody labeling (A219). Several advances or modifications in fluorescence immunoassay formats appeared during this review period. One example was work by Merioe et al., who developed a one-step dual-label TR-FIA method for free and total prostate-specific antigen (A220). Bystryak and co-workers developed a homogeneous fluoroimmunoassay based on the photobleaching of liposomes containing the fluorescent dye 1,3-diphenylisobenzofuran; this was done by generating singlet oxygen near the liposome’s surface by antibodies tagged with an irradiated erythrosine label (A221). In other work, Demcheva et al. reported a micelle-stabilized phosphorescent immunoassay based on a palladium-coproporphyrin label (A222). Chemiluminescence Immunoassays. Techniques based on chemiluminescent or bioluminescent labels are another group of nonisotopic immunoassays that continue to experience active use and development. The most common methods in this category are the competitive binding chemiluminescence immunoassay (CIA), the immunochemiluminometric assay (ICMA), and electrochemiluminescence immunoassays. The general subject of chemiluminescence immunoassays was recently reviewed by Kricka (A223), and the related topic of bioluminescence immunoassays was covered in a review by Geiger et al. (A224). Recent developments in the area of electrochemiluminescence were discussed by Hoyle et al. (A225). Some developments in enzyme-based bioluminescence detection that were mentioned earlier under Enzyme Immunoassays included the use of acetate kinase as a label with soluble firefly luciferase for bioluminescence immunoassays (A174, A175), the development of expression immunoassays in which a DNA label was used to encode copies of luciferase for bioluminescent detection (A176, A177), and the use of phenacyl phosphate (A165, A166) or lucigenin (A167) as substrates in immunoassays using ALP labels. Several articles examined aequorin as a label for bioluminescent signal production in immunoassays (A226-A228). Similar work using a proZZ-obelin fusion protein as a bioluminescent tag was reported (A229). One study compared the performance of various bioluminescent and chemiluminescent agents for the immunochemical detection of in situ analytes

(A230). Other papers examined the enhancement of light production by luminol in the presence of synthetic polyelectrolytes (A231) or in the presence of cyclodextrins during the chemiluminescent detection of β-galactosidase using a Cypridina luciferin analog (A232). A number of recent articles explored new formats and assay designs for bioluminescence or chemiluminescence immunoassays. In one such study Matsunaga et al. used bacterial magnetic particles to develop a chemiluminescence immunoassay for immunoglobulin G (A233). Ullman and co-workers described a detection scheme based on luminescent oxygen channeling for a homogeneous immunoassay method (A234). Two other papers reported new or modified imaging devices for dot-blot or microtiter well bio- or chemiluminescence immunoassays (A235, A236). Nonlabeled Immunoassays. Nonlabeled immunoassay methods based on nephelometry, turbidimetry, particle counting, and latex agglutination are still common in clinical laboratories. Other techniques that can be included in this category are immunoprecipitation and immunodiffusion. Nephelometric and turbidometric immunoassays were recently reviewed by Marmer et al. (A237). Ghourchian and Kamo examined the effect of various interfacial properties on a latex agglutination immunoassay monitored with a piezoelectric crystal (A238), and Gualano et al. studied the use of ultrasonic waves to enhance the detection of bacterial antigens by a latex agglutination technique (A239). Miksa and co-workers described the synthesis and properties of beads based on a composite poly[methyl methacrylate-methacrylic acid-2-(hydroxyethyl) methacrylate] latex for use in immunoassays (A240). Methods for improving the stability and reactivity of latex immunoassay beads were also examined (A241). Finally, OrtegaVinuesa et al. used optical absorbance measurements to investigate various factors that influence the agglutination reaction of analytes with F(ab′)2 antibody fragments adsorbed to anionic or cationic latex particles (A242). Miscellaneous Immunoassays. Besides the traditional immunoassay techniques that have already been discussed, a number of alternative types of immunoassays also continue to be of interest. These other types of immunoassays employ a wide variety of different formats, labels, and/or detection schemes. Many of these assays represent areas of active research and are often not yet commercially available. But the potential advantages they offer do give these methods great promise for use in future clinical testing. Techniques based on electrochemical detection represent one important type of alternative immunoassay. Attractive features of such methods include the speed, accuracy, and precision with which many electrochemical measurements can be made. Two ways that electrochemical detection can be used in immunoassays is by directly employing an electrochemically active species as a label or by using an enzyme label that generates a product that can be detected electrochemically. O’Daly and Henkens recently reviewed the use of electrochemical detection in enzyme immunoassays, with an emphasis on environmental applications (A243). A recycling dual-enzyme electrode was reported by Ghindilis et al. for possible use in detecting ferrocene labels in immunoassays (A244). Ferrocene or related derivatives were also employed as tags in homogeneous immunoassays developed for digoxin (A245) and phenytoin (A246). Linear scan polarography was used to detect production of 2,2′-diaminoazobenzene from o-phenyleneAnalytical Chemistry, Vol. 69, No. 12, June 15, 1997


diamine in an HRP-based enzyme immunoassay (A247), while amperometry along with flow injection analysis was used to monitor the generation of hydroquinone from p-hydroxyphenyl phosphate in an ALP-based enzyme immunoassay method (A248). Ho et al. developed an HRP enzyme electrode for detecting the activity of ALP labels in an immunoassay for thyroid-stimulating hormone (A249). In addition, two reports appeared in which ALP labels for enzyme immunoassays were monitored by a Nafion electrode used in the presence of an anionic substrate and cationic electroactive product (A250, A251). An indium chelate was employed as a label in a heterogeneous immunoassay for human serum albumin, with the indium in the final analyte-antibody complex being released under acidic conditions and detected by anodic stripping voltammetry (A252). Other recent developments included work with cyclic voltammetry and lipid bilayers for monitoring immunochemical reactions (A253), the use of a gold surface as both an electrode and support in a nonseparation sandwich enzyme immunoassay (A254), and the development of a fast amperometric enzyme immunoassay based on the formation of iodine from iodide in the presence of an HRP label (A255). Liposome immunoassays are another group of techniques that continue to be an active area of research. In these methods, liposomes are used to encapsulate a large amount of a traditional immunoassay label, such as a fluorescent compound or enzyme, that is later released as a result of antigen-antibody binding at the liposome’s surface. The use of antigen-coupled liposomes for the detection of specific antibodies was reviewed by Katoh et al. (A256) and discussed in a number of research articles (A257A260). Detection of low molecular weight analytes can also be performed, as described in recent methods for estriol (A261) and d dimer (A262, A263). Rongen discussed the use of biotin- and streptavidin-labeled liposomes for cytokine immunoassays (A264), while Hansen et al. evaluated and compared a number of methods for coupling antibodies to liposomes (A265). Maruyama and coworkers prepared liposomes with amphipathic poly(ethylene glycol)s that were conjugated through their carboxyl groups to antibodies (A266, A267), and Shahinian and Silvius reported a new method coupling antibody Fab′ fragments to liposomes (A268). In addition, the use of a digital color sensor was demonstrated in the quantitation of liposome immunomigration assays (A269). Another class of alternative immunoassays are those that are based on flow injection analysis or liquid chromatography. Potential advantages of such flow-based immunoassays include their speed, precision, and ease of automation. The on-line coupling of immunoassays to high-performance liquid chromatography was discussed by Irth and co-workers (A270). The use of chromatographic systems to perform ELISA-type measurements was examined by Johns and co-workers (A271), while Hitzmann et al. (A272, A273) and Beyer et al. (A274) described flow injection analysis systems for performing immunoassays. Yoshikawa et al. reported a sandwich immunoassay for human serum albumin based on high-performance affinity chromatography and fluorescent-labeled antibodies (A275). Assays based on immunoaffinity chromatography were also reported for the analysis of various antibiotics or sulfonamides (A276), as well as remnantlike particle-cholesterol (A277). Related immunoassays based on flow injection analysis systems were reported for theophylline (A278), allergens (A279), immunoglobulin G (A280-A282), and adrenocorticotropic hormone (A283). Some particularly unique 170R

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approaches included a solid-phase entrapment technique to separate free vs antibody-bound analyte (A284) and bilayer lipid membranes as electrochemical detectors in flow-based immunoassays (A285). Systems were reported that combined immunoaffinity chromatography, reversed-phase liquid chromatography (RPLC) and a postcolumn immunodetection scheme for the analysis of human methionyl granulyte colony stimulating factor (A286) or bovine growth hormone releasing factor (A287); in a similar fashion, a size exclusion column and immunodetection method were used by Vanderlaan and co-workers in an assay for acetylcholinesterase (A288). Tandem immunoaffinity and RPLC columns were also employed in an assay for ∆-9-tetrahydrocannabinol (A289). The combination of immunoaffinity columns with RPLC and detection by mass spectrometry was used in the analysis of LSD analogs or metabolites (A290) and dexamethasone (A291). Capillary electrophoresis (CE) is another separation method that has been explored for use in the development of immunoassays. Most such methods make use of the electrophoretic system for the separation of the free and antibody-bound analyte fractions. Some attractive features of these techniques are their speed and small sample size requirements. The topic of CE-based immunoassays was recently reviewed by Pritchett et al. (A292). Specific examples reported in the literature included competitive binding immunoassays for angiotensin II (A293), digoxin (A294), scrapie prion protein (A295), theophylline (A296, A297), ethosuximide, paracetamol, salicylate, and quinidine (A297). A CE competitive binding immunoassay for cortisol was also reported (A298, A299) and later modified for use in a microchip format (A300). Evangelista et al. described a CE-based immunoassay for the simultaneous analysis of morphine, phenylcyclidine, benzoylecgonine, and THC-COOH (A301). Schultz and co-workers examined the use of CE in the immunoanalysis of insulin content and insulin secretion from single islets of Langerhans (A302). Other strategies that were examined included the preconcentration or binding of analytes on antibody-coated protein G columns prior to separation by CE (A303) and the preconcentration of analytes by immobilized antibodies within CE capillaries (A304, A305). A large number of other additional immunoassay formats were also reported. For example, the detection of simultaneous analytes by immunoassays was discussed in a book chapter by Kricka (A306), and the use of miniaturized microspot systems for multianalyte detection was reviewed by Ekins and Chu (A307). Specific illustrations of multianalyte assays included the detection of R-fetoprotein and human chorionic gonadotropin by flow cytometry (A308); the detection of myoglobin and creatine kinaseMB by an immunochromatographic assay (A309); and the analysis of thyroid-stimulating hormone, human chorionic gonadotropin, and β-galactosidase by DNA-labeled antibodies and PCR (A310). The use of PCR to monitor DNA labels in immunoassays was also the subject of a review by Niemeyer and Blohm (A311) and research articles by Sano et al. (A312) and Joerger et al. (A313). The use of nonradioactive iodine as a label for immunoassays was reported based on a reduction/oxidation detection scheme involving cerium(IV) and antimony(III) (A314). In addition, Mn(III)tetrakis(sulfophenyl)porphine was used a mimetic enzyme label in an immunoassay for R-fetoprotein (A315). Photothermal deflection spectroscopy was used as a detection technique in immunoassays for carcinoembryonic antigen (A316) and R-fetoprotein (A317). Several papers considered the use of mass

spectrometry for detection in immunoassays (A318-A323), particularly methods employing matrix-assisted laser desorption/ ionization (A319-A323) or electrospray ionization (A323). Reviews or articles appeared on the subjects of thin-film immunoassays (A324) and noncompetitive immunoassays for small analytes (A325, A326). A new optical immunoassay method was described for the screening of group B streptococcal antigen (A327), and an immunoassay technique based on water-soluble conductive polymers was developed (A328). Lammers et al. examined the use of calorimetry to monitor immunochemical reactions (A329), while Kauvar discussed the theory and use of pattern recognition in immunoassays (A330). Attenuated total reflection (A331) and surface second harmonic generation were both considered as approaches for detection in immunoassays (A332). Continued work in the development of immunoassay methods employing metal carbonyl complexes as labels was also reported (A333). CAPILLARY ELECTROPHORESIS This review covers the application developments of capillary electrophoresis in the field of clinical chemistry during the period of October 1994 to October 1996. The search method for the literature has been CA Selects for Capillary Electrophoresis from the Chemical Abstracts Service. Since the 1994 review, there has been a rapid expansion in publication that dealt with the use of CE in routine hospital laboratories and in specialized clinical settings. This review is not intended to be comprehensive of all published papers in the review period; rather, the author has tried to select those papers that the author feels are significant to clinical diagnosis of diseases. Reviews. In addition to the previous topical review covering the literature cited by Chemical Abstracts from October 1992 to October 1994 (B1), there were numerous clinical chemistry-related reviews published during this review period. The topics of these reviews include the general clinical applications (B2-B6), the analyses of proteins (B7-B9), DNAs (B10-B12), drugs (B13, B14), neuropeptides (B15) and single cells (B16-B18), the coupling of CE with immunoassay (B19), microdialysis (B20) and mass spectrometry (B21), and the optimization of precision in quantitative analysis (B22). Diagnosis of Metabolic Disorders. (1) Arthritis. Grimshaw and co-workers evaluated the use of CE for quantitative analysis of hyaluronan in human synovial fluid. The polymeric hyaluronan was first hydrolyzed to tetrasaccharide by testicular hyaluronidase. Then, the product together with an internal standard was separated and detected by CE at 200 nm. They found that the changes in tetrasaccharide concentration correlated with the arthritic disease state of a joint (B23, B24). (2) Adenylosuccinate Lyase Deficiency. Adenylosuccinate lyase (ASase) defect causes secondary autism and psychomotor retardation in early childhood. In all body fluids of these patients, two succinylpurine metabolites (succinyladenosine and succinylaminoimidazole carboxamide riboside) can be found that are normally not detectable by conventional methods. Gross and coworkers developed a CE method for screening the disease. Untreated urine was injected into a fused-silica capillary filled with borate buffer (pH 8.63) and separated at 20 kV. The two succinylpurine metabolites were detected at 254 nm from urine of patients with ASase deficiency but not from the control samples. Their migration times were 13.36 and 13.60 min (B25).

(3) Cobalamin Deficiency. Methylmalonic acid (MMA) elevation is an established marker of cobalamin (vitamin B12) deficiency. Marsh and Nuttall reported a rapid assay for MMA in urine by capillary zone electrophoresis (CZE) with indirect photometric detection (B26). In this method, phthalic acid was used as the background electrolyte and absorbance was monitored at 210 nm. Sample preparation consisted of an ethyl acetate extraction, evaporation of the ethyl acetate, and resuspension in distilled water. The CE run time was