39


unassociated mouse targeting secondary antibody 2


AbmIgG AbmIgG* or 1 *. The very small peak


eluting at 2.6 min can either be a dimer of 2


Abfitc: 2 2AbmIgG AbmIgG*. If it is 1 recognises FITC antigen. Summary


in contrast i) retards the linear velocity of non-analytes (impurities), ii) is prevented from interacting with analytes, and iii) has no impact on analyte retention.


). Ps


When MASC separations are carried out with a size exclusion column i) the entire analysis of a sample, irrespective of sample complexity can be achieved with a single mobile phase volume, ii) separations are achieved by isocratic elution, iii) column recycling is unnecessary, and iv) analyte carryover is precluded by using new analyte sequestering transport phase in each analysis.


A limitation of MASC is in distinguishing between analytes and transport particles at the point of detection. Analytes and an ASTP can have indistinguishable absorbance properties. That problem was addressed herein via a fluorescent sandwich format much as in sandwich type immunological assays. Detection of the FITC targeting antibody 1


Abfitc


creating the fluorescent ASTP~FITC: 1 2AbmIgG


for example was achieved by Abfitc


:


* sandwich. This approach has the advantage of adding yet another level of selectivity during detection but increases the analytical complexity and cost of the analysis.


Acknowledgments


The authors gratefully acknowledge support of this work through an NIH Phase II Phase II SBIR grant (5R44GM116663) entitled ‘Sample Preparation; The Achilles Heel of Mass Spectrometry Based Diagnostics II’.


Mobile affinity sorbent chromatography (MASC) will be most useful in routine separations involving small numbers of analytes. The most powerful feature of the method is the simultaneous use of two partitioning phases that cause analytes of interest to elute in the column void volume ahead of impurities. One of these phases is a nanoparticulate transport phase of ~2 mDa that i) binds analytes with high affinity and selectivity based on their 3D structure, ii) causes analyte to elute from columns ahead of impurities, and iii) precludes interactions of analytes with the column stationary phase (Ps


Abfitc * the primary antibody no longer : References


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