Sequential sampling method for improving immunoassay sensitivity and kinetics of small volume samples
An immunoassay, volumetric technology, used in measurement devices, scientific instruments, biological testing, etc., to solve problems such as variability, sample loading, and sampling errors
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0083] This example describes a method for single molecule counting using total internal reflection fluorescence (TIRF).
[0084] Glass slides (50 x 75 mm, S&S Optical, New Haven, IN) and coverslips (25 x 50 mm, Corning, NY) to prepare single-molecule sample slides. Cut rectangular channels with tapered ends into double-sided tape (9500PC, 3M, Maplewood, MN) on a cutter plotter. Sandwich tape between the coated slide and coverslip (taking care to prevent possible leaking air bubbles) to create sample wells. There are 6 channels per coverslip with sample wells 14 mm in length and each holds approximately 5.5, 7 or 8 µL of solution, depending on the width of the channel. The sample solution is pipetted into the channel through the wells on the slide at the ends. The washing step is performed by aspirating the buffer at one end and absorbing the spillage into the tissue at the other end.
[0085]All samples were diluted into and washed with HBS-EP buffer (GE Healthcare, Uppsa...
Embodiment 2
[0090] This example describes a model system for single molecule detection in immunoassays.
[0091] Development of a model system mimicking a sandwich immunoassay for microparticle-based experiments with detectable markers that can be eluted. Specifically, 12 1-mL aliquots were prepared by diluting 2-fold from 1024 fM to 1 fM with DNA oligo2 (5'-TTCTCG TGT TCC GTT TGT ACT CTA AGG TGG ATT TTT TTT TT-amino modifier (SEQ ID NO: 2) ; IDT, Coralville, IA) labeled mouse IgG (IgG-oligo2) samples, where the final sample was a buffer-only control. To each sample was added 10 µL of 1%-solid magnetic microparticles (MP) with a diameter of 5 µm that had been directly coated with goat anti-mouse antibody (Abbott Laboratories, Lake Bluff, IL). quilt. After incubating the samples with rotation for 30 min at room temperature, the volume was reduced to 200 µL using magnetic separation and the concentrated MP / IgG-oligo2 complexes were transferred to a 96-well plate. Here, complexes were inc...
Embodiment 3
[0094] This example demonstrates a method for concentrating an analyte present in a biological sample by sample reloading.
[0095] Single-molecule detection methods typically require only small sample volumes. Taking advantage of the small sample volume requirement, a strategy for recycling and reloading fresh aliquots of the same sample stock was developed to concentrate samples prior to detection and enhance assay sensitivity. Samples can be concentrated onto the surface of the slide by incubating each aliquot for only 1-2 minutes and then replacing it with a fresh stock. For example, aliquots of 400 fM A546-oligo1-bt were loaded into SM wells, measured, and then the aliquots were replaced 10 times with fresh aliquots from the stock, after every 2 min incubation Measurement. Clear the previous aliquot from the well by pumping air through the well between reloading, as image 3 A is shown in the schematic diagram. The surface of the well is not allowed to dry out, but an...
PUM
| Property | Measurement | Unit |
|---|---|---|
| volume | aaaaa | aaaaa |
| diameter | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More