Method for the detection and multiplex quantification of analytes in a sample, using microspheres
A technology of microspheres and analytes, applied in the direction of measuring devices, analytical materials, instruments, etc.
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Embodiment 1
[0110] Example 1: Identification of microsphere families by FCM as a function of size
[0111] The microspheres listed below were mixed in similar ratios and analyzed on an EPICS XL flow cytometer (Coulter) (Figure 5).
[0112] • Flow cytometric analysis of a mixture of 6 populations of 2, 3.1 , 6, 7.6, 10.2 and 15.1 μιη size beads showed that single peaks for each population of beads could be distinguished by double discrete analysis. Parameters (FS log / SS log) were measured after logarithmic amplification (FS for forward light scatter; SS for side light scatter).
[0113] refer to
[0114] • Flow cytometric analysis of the mixture of 4 populations of beads of approximately 3, 8, 10 and 15 μm in size indicated that the single peaks of each population of beads could be easily distinguished by FS log / SS log and it is possible that multi-peaks do not represent interference.
[0115] refer to
[0116] see Figures 5A to 5C
Embodiment 2
[0117] Example 2: Combining the Effects of Size and Fluorescence to Distinguish Multi(6) Family Microspheres by FCM
[0118] 4.4 μm microspheres carrying red fluorescence (measured on the FL4 detector) were added to the mixture of Example 1. The combination of the 4.4 μm microspheres and the above mixture made it possible to recognize 2 additional families; the above 3 μm microspheres The difference in forward scatter (log FS) between spheres and 4.4 μm microspheres was still too small to be easily distinguished (control Figure 5A and B). The introduction of FL4 as a relevant parameter can complete the distinction of 4.4 μm microspheres from all other microspheres (especially 3 μm), and can be used to distinguish 2 groups of 4.4 μm microspheres from each other (Figure 6).
Embodiment 3
[0119] Example 3: Functionalization of 8, 10 and 15 μm microspheres by passive absorption
[0120] IgG purification:
[0121] Polyclonal sera were generated from rabbits immunized against the model bacteria B. globigii, B. pseudomallei or Y. pestis or against the model soluble antigens ovalbumin (OVA) and ricin A chain (Ricin). Rabbit immunoglobulin G (IgG) was purified by protein G affinity chromatography.
[0122] Briefly, 50 ml of diluted serum was loaded onto a high flow column (Pharmacia) containing 5 ml of protein G Sepharose 4 in Na 2 HPO 4 Pre-equilibrated in buffer, pH=7. Ligated IgG was subsequently eluted with 0.1M glycine / HCl buffer, pH 2.7, and immediately neutralized with Tris / HCl buffer, pH=9.
[0123] The eluate was dialyzed against 150 mM PBS buffer, pH = 7.2, 4°C, and concentrated by reverse osmosis. The IgG concentration was measured by reading the absorbance at 280nm (ε0.1% at 280nm = 1.41)
[0124] Preparation of 8, 10 and 15 μm beads:
[0125] 1 ml...
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