Laser-Based Particle-Counting Microimmunoassay for the Analysis of Single Human Erythrocytes

Abstract

A particle-counting immunoassay system for ultrasensitive analysis of proteins in a capillary environment has been developed. The assay is based on the agglutination of antibody-coated particles in the presence of an antigen (usually a protein). The particles were electrophoretically migrated in a 20-microns-i.d. capillary past a detection window where a laser beam irradiates continuously. The light scattering events generated by the agglutinated particles were counted while those produced by unreacted particles were electronically rejected. Glucose-6-phosphate dehydrogenase (G6PDH) was chosen as a test compound for the off-column as well as for the on-column versions of this method. A limit of detection of 620 molecules of G6PDH (1 zmol) was found in the on-column assay. The standard deviation between runs was approximately 6%, which is comparable to that of standard immunoassay methods. The application to the determination of G6PDH levels in individual human erythrocytes is presented. A 14-fold cell-to-cell variation was found which can be explained by the age distribution in the red blood cells.

Direct methods for dynamic monitoring of secretions from single cells by capillary electrophoresis and microscopy with laser-induced native fluorescence detection

“…A particle-counting immunoassay using antibody coated latex particles was miniaturized for capillary electrophoresis to analyze glucose-6-phosphate dehydrogenase (GGPDH) using laser scattering detection [47]. In fact, the activity and …”

Determination of different forms of human interferon‐γ in single natural killer cells by capillary electrophoresis with on‐capillary immunoreaction and laser‐induced fluorescence detection

“…For determination of antigens and antibodies the most frequently used method is immunoassay. Only one report considered the use of immunoassay on basis of on-capillary immunoreaction and particle-counting to measure glucose-6-phosphate dehydrogenase (G-6-PDH) in individual human erythrocytes [4]. In this method, G-6-PDH within the cell after lysis reacts with the anti-G-6-PDHcoated latex particles on the capillary for 30 min.…”

“…The light scattering events generated by the agglutinated particles are counted. It can be calculated from the electropherogram in [4] that the number of theoretical plates of the electrophoretic peak is ,4000, which is too low for separation of structure-similar antigens (or antibodies) such as IFN-g. Capillary electrophoretic immunoassay (CEIA) [5][6][7] offers several advantages, such as high selectivity, high separation efficiency, low reagent consumption, and short incubation time. So far the technique is not applied to single-cell analysis.…”

Single cell studies of enzymatic hydrolysis of a tetramethylrhodamine labeled triglucoside in yeast

“…Flow cytometry also provides a rapid method for counting cells and sorting normal and abnormal cell populations (Steinkamp, 1984). Yeung and colleagues (Yeung, 1994;Rosenzweig and Yeung, 1994;Xue and Yeung, 1994) have assayed lactate dehydrogenase and glucose-6-phosphate dehydrogenase in single human erythrocytes using capillary electrophoresis with laser-induced fluorescence detection (CE/ LIF), demonstrating the utility of CE/LIF for single cell analysis. More recently Zare and coworkers (Chiu et al, 1998) have combined CE/LIF with optical trapping to study single secretory vesicles from the atrial gland of the gastopod mollusk Aplysia californica.…”