Recent Developments in Multianalyte Immunoassay
By - 04/25/2009
(1 vote)
Zhifeng Fu 1,2 , Hong Liu 1 , Zhanjun Yang 1 , Huangxian Ju 1 *
1 Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of China ), Department of Chemistry, Nanjing University Nanjing 210093, P.R. China
2 College of Pharmaceutical Sciences, Southwest University , 400716, P.R. China
* For correspondance - hxju@nju.edu.cn
Originally Published in : Current Trends in Biotechnology and Pharmacy (ISSN 0973- 8916 )
Abstract
Multianalyte immunoassay has gained increasing attention due to its high sample throughput, short assay time, low sample consumption and reduced overall cost per assay. Most of the current developed approaches for multianalyte immunoassay are based on spatial-resolved, multilabel or separation mode. This paper reviews the progress of multianalyte immunoassay and its applications in different fields with 90 references. The outlook of this promising technique has been discussed.
Keywords
Immunoassay; Immunosensor; Multianalyte immunoassay; Array; Review
Introduction
As a promising approach for selective and sensitive analysis, immunoassay has recently gained increasing attention in different fields including environmental monitoring, clinical diagnosis, food safety, pharmaceutical analysis and bacteria identification. It is often necessary to monitor or quantitate several components in a complex system. For example, due to the limited specificity and sensitivity of biomarkers for clinical diagnosis, the measurement of a single biomarker is usually insufficient for diagnostic purpose. Some studies have showed that the measurement of biomarkers panel can avoid false positive or false negative results to improve their diagnostic value (1). Traditionally, immunoassay of analytes panel is performed as discrete tests, i.e., one analyte per assay run, and several runs are needed to detect all components in a complex system. Great consumptions of time, reagent and labor limit the application. To dissolve these limitations, multianalyte immunoassay (MAIA) that can measure two or more analytes in a single run has become a long-cherished goal of immunochemist since simultaneous radioimmunoassay of human insulin and growth hormone in serum sample using I-131 and I-125 as labels was reported in 1966 (2). Compared with parallel single-analyte immunoassay methods, MAIA offers some remarkable advantages, such as high sample throughput, improved assay efficiency, low sample consumption and reduced overall cost per assay (3). This review focuses on the progress and applications of MAIA, including spatial- resolved, multilabel and separation modes.
1. Spatial-Resolved Mode
The spatial resolution of different immunoreaction areas using a universal label for fluorescent, chemiluminescent (CL), spectrophotometric, electrochemical, and piezoelectric detections with array detectors including charge-coupled device (CCD) camera and multichannel electrochemical workstation is the most popular MAIA method.
1.1 Optical detection
movement of various solutions and detector for collection of signals produced from positive Antigen and antibody arrays dotted on planar supports, such as multi-well plate, nylon membrane and glass slide, combined with fluorescent probes (4-10) and enzymes (9, 11- 13) as labels are traditionally adopted to perform spatial-resolved MAIA using CCD and laser scanner detector. Weller’s group (14) proposed a parallel affinity sensor array for the rapid analysis of 10 antibiotics in milk. Microscope glass slide modified with (3-glycidyloxypropyl) trimethoxysilane was used for the preparation of hapten microarray and inserted into a flow cell to act as an automated flow-through CL multianalyte immunosensor. After incubation process, the horseradish peroxidase labeled immunocomplexes of the 10 antibiotics generated enhanced CL signals, which were recorded with a CCD camera. The fully automated liquid handling and sample processing enabled one analysis cycle to be completed in less than 5 min. With the similar device and protocol, multiple herbicides (15, 16) and allergen-specific IgE in human serum (17) have been assayed in array mode.
Jiang et al. (18) reported a miniaturized, microfluidic version of serial-dilution fluorescent immunoassay for antibodies in HIV+ human serum. In this assay, serially diluted solutions of serum flowed in channels across orthogonal, parallel strips of HIV ENV proteins (gp41 and pg 120) adsorbed on a polycarbonate membrane. The bound antibodies could be measured using a second, fluorescent labeled antibody. This assay used a microdilutor network to achieve serial dilution and allowed simultaneous, quantitative analysis of multiple analytes with high concentrations on a single chip. Some immunosensors arrays composed of recognition component, fluidics component for samples have been developed at the Navel Research Laboratory USA Sandwich fluoroimmunoassays are performed on the surface of microscope slides previously patterned with stripes of capture antibodies. After both sample and fluorescent tracer antibodies are introduced in a direction perpendicular patterned with stripes of capture antibodies, the immunocomplexes formed can be observed as a checkerboard pattern of fluorescent spots excited by evanescent wave on the surface. These array immunosensors have been successfully applied in MAIA of proteins, bacterias and biohazards (19-27). Barzen et al. (28-30) proposed several optical multianalyte immunosensors for environment pollutants based on flow-injection immunoassay coupled with total internal reflection fluorescent detection. They immobilized haptens on the different areas of transducer surface of the flow cell and determined simultaneously multiple pollutants in a spatial-resolved and competitive mode. Rodriguez-Mozaz et al. (31) simultaneously detected atrazine, isoproturon and estrogen estrone in river water using an immunosensor fabricated with a similar protocol (Figure 1). The performance of the developed immunosensor was evaluated against a well- accepted traditional method based on solid-phase extraction followed by liquid chromatography- mass spectrometry, and the results obtained from the two methods indicated good agreement.
A one-step lateral flow immunoassay on a strip format for the rapid and simultaneous detection of free and total prostate specific antigens (f-PSA and t-PSA) and estimation of f-PSA to t-PSA ratio (f/t-PSA) in serum has been reported (32). Herein, f-PSA or t-PSA is sandwiched between anti-f-PSA or anti-t-PSA monoclonal antibodies parallel immobilized on the strip and a colloidal gold labeled anti-PSA tracer antibody. The presence of f-PSA and t-PSA results in the appearance of two parallel pink colour lines. Two membrane-based competitive immunoassays using gold particles and horseradish peroxidase (HRP) as tracers in lateral flow format have also been developed for MAIAs of carbaryl and endosulfan (33). The visual detection limits for carbaryl and endosulfan are 100 and 10 µg/L with gold and 10 and 1 µg/L with HRP as labels,respectively. Yacoub-George et al. (34) designed a portable multichannel immunosensor for biological warfare agents, which was based on a capillary ELISA technique in combination with a miniaturized fluidics system and used CL as the detection principle (Figure 2). The fluidic system allowed three CL immunoassays to be performed simultaneously within three fused silica capillaries with three photodiodes as detectors. Koch et al. (35) also presented a portable optical multichannel immunosensor for the simultaneous operation of three flow-through capillary enzyme immunoassays. The parallel operation was achieved by stop-flow incubation. When one capillary was in the process of signal collection, the other two were in incubation procedure. This work represented a versatile tool for immunoassay of several biological warfare agents in parallel with only one non-array detector.
The application of a surface plasmon resonance- based biosensor with four flow channels in combination with a mixture of four specific antibodies resulted in a competitive inhibition MAIA for the simultaneous detection of five aminoglycosides in reconstituted skimmed milk (36). Chung et al. (37) developed a sequential method for the analysis of HRP and bovine serum albumin using a surface plasmon resonance biosensor. Non-array fluorescent detector has also been used for spatial-resolved detection of multiple pesticides (38), hormones (39) and proteins (40) by moving the antigens or antibodies immobilized affinity microcolumn and capillary immunosensor with a motorized translational stage. Owing to the relatively complicated detection device, this strategy needs to be further improved.
1.2 Electrochemical detection
Amperometric immunosensor array fabricated with multiple working electrodes sharing one common counter electrode and reference electrode has been successfully used for MAIA of pepsinogens (41), tumor markers (42-45) and hormones (46). CombiMatrix Corporation (47) developed a microarray of individually addressable electrodes using conventional CMOS integrated circuitry. This microarray system provided a host for MAIA due to the large number of electrodes available, which integrated over 1000 electrodes per square centimeter. The results for human á 1 acid glycoprotein, ricin, M13 phage, Bacillus globigii spore, and fluorescein indicated that this method was one of the most sensitive available, with limits of detection in the attomole range. Electrochemical sensor array often suffers from cross-talk due to the diffusion of electroactive product generated at one electrode to a neighboring electrode (43,45). Thus, an enough spatial distance between adjacent electrodes is necessary to cumber the diffusion procedure. Use of double siloxane layer (45) and iridium oxide (42-44) matrix can retard the diffusion of enzyme- generated product to lower cross-talk.
Ju et al. (48,49) proposed two disposable immunosensor arrays for simultaneous electrochemical determination of multiple tumor markers. The low-cost immunosensor arrays were fabricated simply using cellulose acetate membrane to co-immobilize thionine as a mediator and antigens on different working electrodes of a screen-printed chip, on which the immobilized thionine shuttled electrons between HRP labeled to antibodies and the electrodes for enzymatic reduction of H2O2 to produce detectable signals. This chip could avoid the electrochemical and electronic cross-talks between the electrodes, which enabled the arrays to be miniaturized without considering the distance between immunosensors. Kong et al. (50,51) proposed two arrays of eight electrodes for label-free capacitive and conductive immunoassay of liver fibrosis markers using ultrathin á 1 alumina sol-gel films and electrochemically deposited polypyrrole to immobilize antibodies, respectively.
1.3. Mass-sensitive detection
Luo et al. (52) constructed a 2x5 model piezoelectric immunosensor array fabricated with disposable quartz crystals for quantification of microalbumin, 1-microglobulin, á 2 microglobulin, and IgG in urine. With the piezoelectric immunosensor array, 4 urinary proteins could be quantified within 15 min. This method had an analytical interval of 0.01-60 mg/ L. Similarly, a novel simultaneous immunoassay technique has been developed for the determination of complement factors C4 ,C 5,Clq and B factor) by constructing a piezoelectric quartz crystal array system (53). These mass- sensitive piezoelectric immunosensor arrays can provide a convenient label-free approach to MAIA.
1.4. Optical encoding and addressing
Spatial-resolved arrays are typically manufactured by labor-intensive methods requiring high precision such as ink-jet printing, micromachining, photolithography, and photodeposition. Randomly ordered addressable sensor array developed in Walt’s laboratory (54) provided an alternative approach to array fabrication. In this approach, micrometer-sized sensors were produced by immobilizing different recognition molecules on the surface of microparticles encoded using two fluorescent dyes. The addressing procedure was performed by taking the fluorescence intensity at each emission wavelength and then dividing the two values to get the signature of that particular ratio. With this principle, multiple drugs (55), proteins (56-58), biological warfare agents (59) and cytokines (60) were simultaneously detected with CL or fluorescent method and randomly ordered antibodies immobilized copolymer microspheres or metallic particles as microsensors. Theoretically, thousands of antigens or antibodies can be spotted onto one single planar support to screen thousands of analytes. Although this mode can screen large numbers of analytes, accurate quantitative data in these arrays are usually limited or difficult to be obtained (44). Requiring of complicated and expensive spotting technique with high precision also greatly limits its application. Although optical encoding and addressing allows randomly ordered sensor arrays to be identified for MAIA, the encoding process complicates the manipulation. Furthermore, spatial-resolved MAIA is typically performed with expensive array detector such as CCD camera for optical detection or multi- channel workstation for electrochemical measurement (61).
2. Multilabel Mode
The second dominant mode for MAIA isperformed using different labels to tag antibodiesor antigens (one per analyte), includingradioisotopes, enzymes, fluorescent and metalcompounds. Different analytes can be easilydistinguished using these labels by suchparameters as potential, wavelength, decay timeand so on.
2.1 Wavelength resolution
ELISA for MAIA involves labeling the analytes with various enzymes, whose catalyzed reactions can easily be distinguished from each other by absorption spectra (62, 63). Selection of theenzyme labels is a key step in the developmentof an ELISA based MAIA. Blake et al. (62)mentioned that the ideal enzyme labels for MAIAshould meet the following requirements: (i) theenzymes should be readily available,inexpensive, and have high turnover numbers;(ii) each enzyme must be stable under theselected simultaneous assay conditions and noteasily to be interfered by other enzyme or itssubstrate; (iii) all enzymes must have similaroptimal assay conditions; (iv) the assay methodfor each enzyme should be simple, sensitive,rapid, and cheap; (v) all enzymes should notoccur in the practical sample to be assayed, andinterfering factors should be absent; (vi) eachenzyme should contain potentially reactivegroups that allow linking to antigen or antibodywhile retaining the enzyme activity; (vii) thespectra of the products of the enzyme-catalyzedreactions should not overlap with each other. Ihara et al. (64) immobilized a mixture ofantigenic peptides of FAK and c-Myc tonanospheres with red emission, and a mixture ofc-Myc and á catenin to green nanospheres,respectively. As seen in Figure 4 (64), anti-FAKand anti á catenin antibodies could formaggregates with red and green emissions,respectively. The anti-c-Myc antibody couldform aggregate emitting yellow light as a resultof color overlapping. This strategy enabledspecific antibodies to be detected in one-stepprocedure with color-encoded nanospheres.Swartzman et al. (65) proposed a bead-basedtwo-color MAIA for cytokines IL-6 and IL-8using Cy5.5 and Cy 5 as fluorescent labels,respectively. The linear dynamic ranges of themwere 125-4000 pg/mL and 15.6-2000 pg/mL,respectively. This work utilized fluorometricmicrovolume assay technology to image andmeasure bead-bound fluorescence while thebackground fluorescence was ignored.Consequently, no wash steps were required toremove unbound antibody, ligand, andfluorophore. Goldman et al. (66) used antibody-conjugated quantum dots with emissionmaximums at 510, 555, 590, and 610 nm todemonstrate multiplex assays for four proteintoxins present in the same sample. However, adeconvolution of composite spectra was neededto distinguish the overlapping signals.
2.2. Time resolution
The fluorescence of lanthanide chelates has theadvantages of high quantum yield, long decaytime, exceptionally large Stoke’s shift, andnarrow emission peak. Specific chelatefluorescence can be easily distinguished from thesample matrix fluorescence and the scatteredlight, and the fluorescence from differentlanthanides can also be easily discriminated dueto their difference in decay time and emissionwavelength, which makes the lanthanide chelatespreferable to any other probes for developingmultilabel-based time-resolved MAIA. Of the 15lanthanide ions, Eu3+, Sm3+, and Tb3+ are the mostcommonly employed probes, and have beenwidely used for time-resolved fluorescent MAIAof multiple tumor markers (67), hormones (68),recombinant proteins (69) and antibodies (70). Ito et al. (71) developed a simple and rapid time-resolved fluoroimmunoassay for simultaneousdetermination of alpha-fetoprotein (AFP), humanchorionic gonadotropin (hCG) and estriol (E3)using Eu3+ and Sm3+ chelates. In this proposedmethod, a 96-well microtiter plate for AFP andhCG assay and a transferable solid phase platefor E3 assay were combined to perform MAIAof the three analytes with only two probes. Themeasurable ranges for AFP, hCG and E3 were3.91-1000 ng/mL, 877-250 000 IU/L and 0.39-100 ng/mL, respectively.
2.3. Potential resolution
The dual-analyte homogeneous immunoassay ofphenobarbital and phenytoin was carried outsimultaneously at physiological pH by square- wave voltammetry on Nafion-loaded carbonpaste electrode. Phenobarbital and phenytoin (ICPMS) after Eu3+ and Sm3+ were dissociatedfrom the plates with HNO solution. However,were labeled by cobaltocenium salt andferroceneammonium salt with standard redoxpotentials of -1.05V and 0.26 V, respectively.Detection limits of 0.25 and 0.2 mu wereachieved for the two antiepileptic drugs,respectively (72). As seen in Figure 5 (73), anelectrochemical stripping immunoassay protocolusing different inorganic nanocrystal as tracersand magnetic beads as support has beendeveloped for the simultaneous measurementsof proteins. Each biorecognition event yields adistinct voltammetric peak, whose position andsize reflect the identity and concentration of thecorresponding analyte, respectively. Thisprotocol has been used for a simultaneousimmunoassay of â 2 microglobulin, IgG, bovineserum albumin, and C-reactive protein usingZnS, CdS, PbS, and CuS colloidal crystals aslabels, respectively (73). Hayes et al. (74)proposed a MAIA method for human serumalbumin and IgG. Bismuth and indium ions werecoupled to the two proteins through thebifunctional chelating agent diethylenetriamine-pentaacetic acid. Following the competitivereactions between unlabeled and labeled proteinsfor limited amount of specific antibodiesimmobilized on polystyrene, the bound metal ionlabels were released by acidification and detectedby differential pulse anodic strippingvoltammetry with detection limits of 1.8 and 0.6pg/mL for human serum albumin and IgG,respectively.
2.4. M/e resolution
Zhang et al. (75) developed a dual-labelimmunoassay method for the simultaneousdetermination of AFP and free hCG â in humanserum. Monoclonal antibodies immobilized onmicrotiter plates captured AFP and hCG beta,which were detected by Eu3+-labeled AFP andSm3+-labeled hCG â tracer antibodies withinductively coupled plasma mass spectrometry 3this technique could not be used for microarraydetection since it was necessary to dissolve theelemental tags before introducing them into theplasma source. They (76) also reported thedetection of multiple proteins on each spot ofthe immuno-microarray by laser ablationICPMS. AFP, carcinoembryonic antigen (CEA)and human IgG were detected as model proteinsin sandwich format on a microarray with Sm3+-labeled AFP antibody, Eu3+-labeled CEAantibody, and Au nanoparticle-labeled IgGantibody as tracer antibodies. The detection limitswere 0.20, 0.14, and 0.012 ng/mL for AFP, CEA,and human IgG, respectively. This detectionmethod allowed detection of multiple analytesfrom each spot of microarray with a spatialresolution at micrometer range, which couldalleviate the stress to fabricate high-densityarrays.
2.5. Scintillation energy resolution
In 1966, as the founder of MAIA, Morgan (2)proposed an original simultaneousradioimmunoassay of human insulin and growthhormone in serum sample using I-131 and I-125as labels and exploiting the difference inscintillation energy produced from the tworadioisotopes to discriminate the two analyte.Similarly, the simultaneous radioassay ofvitamins (77) and hormones (78) could be carriedout using Co-57 and I-125 as probes. Recently,few attention is paid to the radioimmunoassaybased MAIA due to the damage of radioisotopesto environment and operator.
2.6. Substrate zone resolution
It has been noted that the different labels used inmultilabel mode often need markedly differentoptimal assay conditions, and traditionallysimple combination of multiple labels often leadsto loss of assay performance (2). Furthermore, this mode sometimes suffers from signaloverlapping of different labels (66).Ju et al. (61) designed a substrate zone-resolvedmultianalyte immunosensing system, with whichHRP labeled carcinoma antigen 125 (CA 125)immunocomplex and alkaline phosphataselabeled CEA immunocomplex were sequentiallydetected in their corresponding CL substratezones. This designed technique solved two keyproblems in multilabel mode: one was to obtaindistinguishable CL signals without considerationof wavelength, and the other was to enable eachCL reaction to be catalyzed by the label in itsoptimal assay condition without loss of assayperformance. Unfortunately, as other MAIAmethods based on multilabel mode, thistechnique encountered a difficulty to find moreavailable enzyme labels, which limited thenumber of analytes. In order to overcome thislimitation, this group further proposed a twodimensionalresolution system of channels andsubstrate zones (79). Using CA 125, CA 153, CA199 and CEA as two couples of model analytes,two couples of capture antibodies wereimmobilized in two channels, respectively. Witha sandwich format the CL substrates for alkalinephosphatase and horseradish peroxidase weredelivered into the channels sequentially toperform multiplex immunoassay after the sampleand trace antibodies were introduced into thechannels for on-line incubation. When three orfour channels were used in the flow-throughdevice, the detectable analytes in a single runcould be 6 or 8, respectively, with a 10 s longeranalytical time for each added channel.
3. Separation Mode
Another method coupled with separationtechniques such as capillary electrophoresis (CE)and high-performance liquid chromatography(HPLC) can be used for MAIA. Competitiveimmunoassay combined with fluorescentdetection is generally adopted to perform CEbased MAIA, the analytes includes abused drugs(79, 81) and peptides (82). Obviously thisstrategy often suffers from the adsorption ofimmunoreagents on inner wall of the capillary,which can be prevented by optimization ofseparation buffer type and pH that allowesapplication of high electric field (82). An on-linecoupling of a label-free reflectometricinterference spectroscopic biosensor to a HPLCsystem has been described for MAIA of fourpesticides (83). In this system a highly crossreactiveantibody against the four pesticides isused to bind the pesticides. The eluate of theHPLC is mixed continuously with the antibodies,and the presence of antigens is detected by areduction of the antibody binding to thetransducer.Roda et al. (84) proposed a field-flowfractionation (FFF)-CL based solid-phasecompetitive immunoassay, in which micrometersizedbeads coated with the capture antibodywere used as solid phase, and analyte-HRPconjugate was used as tracer. Once thecompetitive immunoreaction took place withinthe injection loop of the system, the antibodyboundtracer was separated from tracer insolution in a few minutes by means of FFF. FFFbasedMAIA could be developed by use of beadswith different sizes (1-50 mu), each coated withthe specific antibody for one analyte. The beadscould be fractionated by FFF before CL signalscollection to realize detection of multipleanalytes in a single run.The thermosensitive poly (Nisopropylacrylamide)(PNIP) and magnetic beadshave been widely utilized as the separationcarriers for immunoassays. A fast homogeneousimmunoreaction as well as a simpleheterogeneous separation process is carried outfor MAIA in the light of some certaincharacteristics of water-soluble PNIP andmagnetic beads, and thus, lower nonspecificaffinity and higher sensitivity are accomplished(85). The results of CL detection of IgG and IgAindicate the detection limits as low as 2.0 and1.5 ng/mL, respectively.
4. Cross-Reactivity
Cross-reactivity is a crucial analytical parameterregarding specificity and reliability of MAIA,which is frequently encountered in MAIA ofsmall molecule analytes. In many cases, theantibodies recognize a variety of analogs andmetabolites of the target analyte, for example,some s-triazines and their metabolites withsimilar structures shown in Figure 6 (86). Evenmonoclonal antibodies are often unable todiscriminate absolutely molecular analogs withsmall structural differences. Efforts to derivemonoclonal antibodies to small analytesgenerally produce panels of antibodies that differin their cross-reactivity for the primary targetanalyte and its analogs and metabolites.Antibodies arrays combined with somechemometric means inclusive of neural networkare often used to overcome the difficulty in exactquantitation resulted from the cross-reactivity(86-90).
5. Conclusion and Outlook
In recent yeas, MAIA has attracted considerableinterest due to its outstanding advantage in assayspeed, cost and labor consuming. So far thespatial-resolved mode has been the most popularmode due to its high analyte throughput and largeinformation amount. The further work needs todevelop arrays with higher density and simplerpreparation protocol using cheaper array detector.Most of the multilabel mode based methods focuson using of lanthanide chelates as labels andtime-resolved fluorescent detection. More labelswith higher signal resolution degree and lessrequirement to assay condition are urgentlyneeded. Various resolution methods in time,space, substrates, reactants, labels and detectionmethods will be designed and developed forMAIA in the future. Military application andenvironment monitor are anxious tominiaturized, integrated and portable MAIAsystem fit for field application. MAIA systemwith high sample throughput and rapid assayspeed has great application potential in diseasescreen.
Acknowledgements
This research was financially supported by theNational Science Funds for Distinguished YoungScholars (20325518) and Creative ResearchGroups (20521503), the Key Program(20535010) from the National Natural ScienceFoundation of China and the Science Foundationof Jiangsu (BS2006006, BS2006074).
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Fig 1 : Scheme of flow-injection immunosensor used for detection of multiple pollutants in river water.
Fig 2 : Scheme representing the arrangement of the fluidics components of the CL multichannel immunosensor for biological warfare agents.
Fig 3 : Schematic diagrams of (A) screen-printed four-electrode system and (B) preparation of immunosensor array and MAIA procedure: (a) Nylon sheet, (b) silver ink, (c) graphite auxiliary
electrode, (d) Ag/AgCl reference electrode, (e) W1, (f) W2 and (g) insulating dielectric.
Fig 4 : Schematic illustration of the MAIA using selective aggregation of antigenic peptide-modi-fied nanospheres.
Fig 5 : Multiprotein electro-chemical stripping immunoassayprotocol using different inorganicnanocrystal tracers: (A) introduc-tion of antibody-immobilizedmagnetic beads, (B) capture of theantigens to the antibodies-immo-bilized magnetic beads, (C) cap-ture of the nanocrystal-labeledsecondary antibodies and forma-tion of sandwich immuno-complexes, (D) dissolution ofnanocrystals and electrochemicalstripping detection.
Fig 6 : Some s-triazines and their metabolites with similar structures.