Multiplexed immunoassays

Cell-Based Bioassay For a cell-based Nab bioassay, treated cells respond directly or indirectly to the drug in a concentration-dependent manner. Possible biological responses of the cells to chug treatment include cell proliferation, apoptosis, phosphorylation, chemokine release, and expression of proteins or genes [15,22,28 30]. These responses may by quantitated by techniques such as immunoassay, multiplex assays [31], flow cytometry [23], and gene expression profiling [32]. 

As an alternative, extremely sensitive detection can be achieved with reporter antibody probes tagged with intensely SERS-active compounds or with enzymes that react with substrates to yield SERS-active products. These methods often involve sandwich immunoassay techniques, which increase the number of required steps but offer the advantages of excellent sensitivity and the potential for label multiplexing. For example, Nie and coworkers recently reported the simultaneous detection of two types of antigens in a... 

King K.D., Vanniere J.M., LeBlanc J.L., Bullock K.E., Anderson G.P., Automated fiber optic biosensor for multiplexed immunoassays, Environ. Sci. Technol. 2000 34 2845-2850. 

Joos Th.O., Stoll D., Templin M.F., Miniaturized multiplexed immunoassays, Curr Opin Chem. Biol. 2001 6 76-80. 

The most common use of protein microarrays is in immunoassays. In particular, antibody-based immunoassays are the main stream of diagnostic assays due to their specificity. The assay usually runs in a multiplexed mode where the antibodies or other capture agents are immobilized and then exposed to a biological sample. There are four immunoassay formats direct binding, sandwich (ELISA), competitive, and displacement. Direct-binding and sandwich assays are the most common. There are some reports on the use of competitive assays and displacement assays, which are usually associated with high surface area/volume systems [72-76],... 

The concept is demonstrated for a simultaneous immunoassay of (32-microglobulin, IgG, bovine serum albumin, and C-reactive protein in connection with ZnS, CdS, PbS, and CuS colloidal crystals, respectively (Fig. 14.6). These nanocrystal labels exhibit similar sensitivity. Such electrochemical coding could be readily multiplexed and scaled up in multiwell microtiter plates to allow simultaneous parallel detection of numerous proteins or samples and is expected to open new opportunities for protein diagnostics and biosecurity. 

Deiss F, LaFratta CN, Symer M, Blicharz TM, Sojic N, Walt DR (2009) Multiplexed sandwich immunoassays using electrochemiluminescence imaging resolved at the single bead level. J Am Chem Soc 131 6088-6089... 

Figure 6.22 PWG assay dose precision profiles of multiplexed three-analyte immunoassay for two sets of experiments. Dose precisions correspond to standard deviations of analyte concentrations that were back-calculated using corresponding dose response curves. (From Pawlak, M. et al., Proteomics, 2, 383-393, 2002. With permission.)...

We have shown in the past few years that due to their finite size (comparable to an average protein), CdSe-ZnS core-shell nanocrystals capped with a thin layer of dihydrolipoic acid ligands provide excellent nanoscale scaffolds ( nanoscaffolds ) for attaching several proteins on their surfaces. QD-protein conjugates were used to design multiplexed immunoassays to detect soluble toxins. 

Templin, M.F., et al. (2004) Protein microarrays and multiplexed sandwich immunoassays what beats the beads Comb Chem High Throughput Screen. 7, 223-9. 

Mullenix MC, Dondero RS, Datta HD, Egholm M, Kingsmore SF, Perlee LT. Rolling circle amplification in multiplex immunoassays. In Demidov W, Broude NE, editors. DNA Amplification—Current Technologies and Applications. Norfolk, UK Horizon Bioscience, 2004 313-331. 

Ray CA, Bowsher RR, Smith WC, et al. Development, validation and implementation of a multiplex immunoassay for the simultaneous determination of five cytokines inhuman semm./. Pharm. Biomed. Anal. (2005) 36 1037-1044. 

Although well designed LC-MS/MS assays generally outperform immunoassays due to their accuracy, sensitivity, precision, and inherent multiplexing capability, they are not free from analytical problems. Besides limitations in selectivity— isobaric analytes cannot be distinguished—sudden and unpredictable ion yield attenuations, often known as ion suppression effect, have to be considered the Achilles heel of quantitative bio-analytical mass spectrometry. Ion yield attenuation is compromising both the accuracy of an assay and its precision. It can easily lead to gross errors in analyte quantification. 

Detection of staphylococcal enterotoxin B (SEB), a causative agent of food poisoning, was achieved by QDs conjugated with polyclonal sheep anti-SEB antibody.57 Moreover, this approach also harbors the possibility of a multiplexed immunoassay (see Fig. 12.3), which was first reported by Goldman et al.58 in 2004 four toxins of interest in food- or water-borne illnesses (cholera toxin, ricin,... 

Blais, B.W., Gaudereault, M., and Phillippe, L.M. 2003. Multiplex enzyme immunoassay system for the simultaneous detection of multiple allergens in foods. Food Control 14 43 17. 

Nichkova, M., Dosev, D., Gee, S.J., Hammock, B.D., and Kennedy, I.M. 2007. Multiplexed immunoassays for proteins using magnetic luminescent nanoparticles for internal calibration. Anal Biochem 369(1) 34 10. 

D protein microarrays performing multiplex immunoassays on a single chip. Anal. Chem. 75, 4368 372. 

In sandwich immunoassays, a pair of antibodies binds to different epitopes of an analyte and one of the antibodies (the so-called capture antibody ) is immobihzed on the substrate. The second antibody is labeled with a probe (e.g. a fluorescent dye), it allows detection of the analyte molecules that bind to the capture antibody. We use this approach when the antigen is a well-characterized protein. Multiplexing is possible by immobilizing the capture antibodies in an array or on individually coded beads for medium- to low-volume amounts of biological liquids. The submicroliter version of this approach, in which capture antibodies are immobilized in stripes and the sample is guided to the capture sites by means of microfluidic networks, is described in Section 3.1 of this chapter. 

Biagini, R.E., Sammons, D.L., Smith, J.P., MacKenzie, B.A., Striley, C.A.F., Semenova, V., Steward-Clark, E., Stamey, K., Freedman, A.E., Quinn, C.P., Snawder, J.E. (2004). Comparison of a multiplexed fluorescent covalent microsphere immunoassay and an enzyme-linked immunosorbent assay for measurement of human immunoglobulin G antibodies to anthrax toxins. Clin. Diag. Lab. Immunol. 11 50-5. 

While a wide range of opportunities exist, such as environmental, clinical, and trace analysis, the principal application for labs-on-a-chip is in the analysis of biological samples. The miniaturized dimensions allow extremely small sample volumes to be analyzed, and a microchip format can allow chemical reaction, mixing, sample manipulation, and multiplexing to be performed. Single-cell analysis, immunoassays, protein and peptide separations, DNA analysis and sequencing, and polymerase chain reactions have all been performed on microchip devices [48]. 

Pataki J, Szabo M, Lantos E, Szekvolgyi L, Molnar M, Hegedus E, Bacso Z, Kappelmayer J, Lustyik G, Szabo G. Biological microbeads for flow-cytometric immunoassays, enzyme titrations, and quantitative PCR. Cytometry Part A 2005 68A 45-52. Martins TB, Augustine NH, Hill HR. Development of a multiplexed fluorescent immunoassay for the quantitation of antibody responses to group A streptococci. J. Immunol. Methods 2006 316 97-106. 

Wang G, Park H-Y, Lipert RJ, Porter MD (2009) Mixed monolayers on gold nanoparticle labels for multiplexed surface-enhanced Raman scattering based immunoassays. Anal Chem 81 9643... 

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