Optical tracking and detection of immunomagnetically selected and aligned cells.

We have developed a platform for cell analysis based on immunomagnetic selection and magnetic alignment of cells in combination with an epi-illumination tracking and detection system. Whole blood was labeled with ferromagnetic nanoparticles and fluorescent probes, and placed in a magnetic field in a chamber. Cells labeled with ferromagnetic nanoparticles moved upward and aligned along ferromagnetic lines deposited by lithographic techniques on an optically transparent surface of the chamber. An epi-illumination system using a 635 nm laser diode as a light source scanned the lines and measured signals obtained from the aligned cells. The cell counts per unit of blood volume obtained with the system correlated well with those obtained from the counts from a standard hematology analyzer and flow cytometer. The cell analysis platform is significantly less complex and more sensitive than current cell analysis equipment and provides additional functionality through its ability to subject the cells to repeated and varied analyses while they remain in a natural environment (i.e., whole blood).

On the mechanism of Clq binding to antibody-I. aggregation and/or distortion of IgG vs combining site-transmitted effects.

It was shown previously that in sheep calcium-dependent anti-GAT there is a subpopulation which reacts with and can be precipitated with poly G [Liberti (1975) Immunochemistry 12, 303-310]. This entire subpopulation was also found to react with the cross-reacting polypeptides GA and GT. Furthermore, from hydrogen exchange experiments, it was found that only the immunizing antigen GAT completely filled the combining sites of these antibodies whereas poly G was shown to occupy an average of 47% of all sites, and GA and GT, 75 and 85% respectively. Since precipitins formed with this subpopulation and each of these antigens should have reasonably similar densities and orientations of 'aggregated' IgG but differing combining sites occupancies, we have used this system to explore the relative role of Fc aggregation and/or IgG distortion vs combining site-transmitted effects on the binding of Clq to antibody. For two preparations of this subpopulation (one of high avidity, the other obtained via poly G-Sepharose and of lower avidity) there is only a 5% difference in the delta G (10.2-10.8 kcal/mole) of Clq-IgG interaction for a change in combining site occupancy of 47-100%. For the high-avidity preparation there is a correlation between delta G and degree of ligand occupancy of combining site. This could reflect combining site-transmitted effects or may be related to small differences in the molecular architecture of these precipitins. Clq saturation curves support the latter notion. In view of the very moderate effect of combining site filling (from 47 to 100%) on Clq-IgG interaction for the high-avidity preparation and the absence of any correlation for the lower-avidity preparation. It appears that an allosteric model for antibody initiation of complement is untenable. Unless combining site-originating contributions are completed when less than 50% of an antibody-binding site is occupied by ligand, it would seem that Clq binding to immune complexes must be governed either by enhanced interactions resulting from Fc clustering which occurs via antibody interactions with antigen or by distortion of the antibody molecular upon ligand binding or some combination thereof.

Isolation and characterization of highly stable rabbit C1q.

Rabbit C1q has been isolated by techniques which retain elements of previous isolation methods along with novel procedures incorporated to yield stable, non-aggregating, highly purified and biologically active C1q. The procedure involves euglobulin preparation by dialysis of serum and two column chromatographies, DEAE-Sephadex and Ultragel ACA 34. The method results in yields of C1q from rabbit sera of 20-30%. Ouchterlony analysis, sedimentation velocity experiments, equilibrium ultracentrifuge molecular weight analysis, SDS, polyacrylamide gel electrophoresis and isoelectric focusing experiments indicate the presence of only a single component, which has all the physical characteristics previously reported for C1q. The preparation has all the biological characteristics of C1q such as the ability to agglutinate IgG-coated latex particles and to restore hemolytic activity to RC1q serum. Preparations obtained by this procedure are stable to freezing (-70 degrees C) for extended periods of time and show no evidence of aggregation upon thawing following periods of storage of up to 6 months. Mild acetylation and handling of preparations for extended periods at 37 degrees C and 45 degrees C indicate no evidence of aggregation or loss of agglutinating and hemolytic activities. Preparations so isolated meet all the criteria required for critical physical-chemical studies of this protein.

A model system illustrating the isolation and enrichment of a rare population of tumour cells in bone marrow.

A model system employing a modified nylon matrix is described for the separation of rare cells titrated into either a leukaemic cell line or normal bone marrow. A 75- to 125-fold enrichment and recovery of the rare cell population was achieved, starting from an initial level of 0.014 to 0.2% of the total population. The rare cell population was identified by pre-labelling with Hoechst 33342, which intercalates into the DNA, and renders cells highly fluorescent. Separation and recovery of cells was totally dependent on the use of a panel of monoclonal antibodies binding to the labelled population. The nylon matrix, precoated with an anti-mouse immunoglobulin, traps the cells coated with monoclonal antibodies, and these can be released simply by gentle manipulation of the matrix. The matrix employed has been shown to not specifically trap committed bone marrow progenitors as determined by CFU-GM, BFU-E and CFU-GEMM assays. The use of this technique should simplify the isolation of rare tumour cells metastasizing to bone marrow.

Quantitative immunochromatographic analysis: theory and application to theophylline immunoassay.

The development of an immunochromatographic technique suitable for rapid analysis of biological fluids is described. Quasi-one-dimensional antibody lattices specific for theophylline were constructed by packing Sepharose beads conjugated with specific antibody into specially designed narrow capillary tubes. The design of these capillary columns was such that they would subtract a preset threshold quantity of antigen (label and analyte) from the total amount presented. Labeled antigen, which appeared in the flowthrough, could then be used to precisely quantitate the analyte present. The ideal format would permit very precise subtraction of 100% of the available antigen up to the threshold amount and none of the remainder. The microcolumn described here comes close to this ideal behavior through the attainment of very high ratios of bound/free antigen. The elevated bound/free ratio could be explained by theoretical analysis of the effect on equilibria of the high antibody concentration in this quasi-one-dimensional system. Lattices containing anti-theophylline antibodies were used to develop a competitive enzyme immunoassay for theophylline which demonstrated a dose-response that was closely similar to that predicted by theoretical treatment. The entire assay procedure was performed in less than 30 min and demonstrated a sensitivity limit of approximately 20 ng/ml. Preliminary studies on clinical serum samples suggest that this assay has potential for the routine analysis of biological fluids.

Solvent effects on the structure of rabbit Clq, a subcomponent of the first component of complement.

The effect of solvent conditions on the conformation of rabbit Clq was studied by both spectroscopic and nonspectroscopic methods. The conformation of Clq in buffered saline solutions at pH 7.4 or 6.0 did not differ significantly from Clq at twice the saline concentration as determined with circular dichroism, difference spectroscopy, and tritium-hydrogen exchange techniques. Addition of calcium to the buffers had no structural effects in any of the conditions examined. Hydrogen exchange experiments performed at pH 7.4 were also unaffected by magnesium, manganese, or ethylenediaminetetraacetic acid. With all the methods used a pH effect was observable between 5.1 and 8.3. From solvent perturbation difference spectroscopy results it was calculated that the equivalent of 10 +/- 2 and 6 +/- 1 mol of tyrosine and tryptophan/mol of Clq, respectively, became exposed at the lower pH. A small positive CD band in the 231 to 235 nm region decreased in wavelength and increased in magnitude as a function of decreasing pH, indicating tyrosine exposure at the lower pH and possibly changes in the collagen-like structure of Clq. Hydrogen exchange experiments indicate a small, but significant, conformation transition occurring in the pH 5 region and a stabilization of conformation between pH 6 to 8. From these results the conformational pH dependence was interpreted as an acid expansion of Clq with a minor conformational transition occurring between pH 5 AND 6. These effects may in part be associated with decreased Clq-Ig interactions which have been observed at the lower pH.

Cell analysis system based on immunomagnetic cell selection and alignment followed by immunofluorescent analysis using compact disk technologies.

BACKGROUND: Although the flow cytometer has become the standard in cell analysis, it has limitations. Recently, we introduced a new cell analysis method based on immunomagnetic selection and aligning of cells. No flow system is needed and cell analysis can be performed in whole blood. METHODS: Whole blood is incubated with fluorescent labels and immunomagnetic nanoparticles. The blood is injected into a capillary that is in a strong magnetic field. The immunomagnetic-labeled cells move upward and align themselves along ferromagnetic lines present on the upper surface of the capillary. An optical focus and tracking system analogous to that used in a conventional compact disk player focuses a 635-nm laser-diode on the magnetically aligned cells. The emitted fluorescence signals are projected on two photomultipliers. Allophycocyanin (APC)-labeled CD4 (CD4-APC) and Cyanin5.5 (Cy5.5)-labeled CD8 (CD8-Cy5.5) antibodies and Oxazine750, all red excited, are used as fluorescent labels. RESULTS: A differential white blood cell count performed in whole blood is obtained using the CD4-APC in combination with Oxazine750. The results are compared with the Technicon-H1 hematology analyzer. Correlation coefficients of 0.91 for neutrophilic granulocytes, 0.93 for lymphocytes, 0.93 for monocytes, and 0.96 for eosinophilic granulocytes were obtained. Immunofluorescence is demonstrated using CD4-APC and CD8-Cy5.5. The absolute counts obtained for CD4+ and CD8+ are compared with the Coulter Epics XL flow cytometer. Correlation coefficients of, respectively, 0.91 and 0.94 were obtained. CONCLUSION: We conclude that our system is as capable as a standard flow cytometer or hematology analyzer for a reliable routine white blood cell analysis, including immunophenotyping, and can be used as an easy-to-handle disposable white blood cell test.