Chemistry: analytical and immunological testing – Involving diffusion or migration of antigen or antibody
Reexamination Certificate
1999-11-08
2001-11-27
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving diffusion or migration of antigen or antibody
C422S051000, C422S051000, C422S082080, C422S082110, C435S007100, C435S007930, C435S007940, C435S007950, C435S287700, C435S287800, C435S287900, C435S288700, C435S808000, C435S006120, C436S164000, C436S172000, C436S518000, C436S527000, C436S535000, C436S541000, C436S805000
Reexamination Certificate
active
06323042
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is related generally to immunosensors, and more specifically to immunosensors for flow-through displacement immunoassays.
2. Description of the Background Art
Immunoassays exist in a variety of formats that utilize the interaction of antibodies with antigens usually including direct binding, competitive and sandwich assay schemes. The continuous flow immunoassay is a unique displacement assay that measures the dissociation of a fluorescently labeled antigen from an antibody bound on a solid support when the antigen flows past the antibody (U.S. Pat. No. 5,183,740, issued Feb. 2, 1993 to Ligler et al., the entirety of which is incorporated herein by reference for all purposes.) The displacement of the labeled antigen is proportional to the quantity of antigen present in the sample. Sensitivity of the assay is dependent on the dissociation constant of the antibody and the probability of antigen-to-antibody interaction in the flow system.
In previous studies on flow immunoassays using columns of packed beads or porous membranes as substrates for the antibody immobilization, the following parameters have been determined to affect signal magnitude and assay sensitivity:
(1) The affinity of the antibody for the antigen must be as high or higher than the affinity of the antibody for the labeled analog under the conditions of operation of the displacement immunoassay.
(2) There is a minimum number of antibody-labeled antigen complexes that must be present in the assay in order to generate a signal. Past this minimum level, increasing the number of antibodies by increasing the amount of substrate or antibody density increases the signal magnitude and the number of assays that can be performed, but may decrease the antigen sensitivity, probably due to rebinding of labeled antigen to immobilized antibody. The minimum detectable amount of displaced labeled antigen is also a function of detector sensitivity.
(3) For each antigen-antibody pair, there seems to be an optimum flow rate which is probably related to the dissociation constant of the antibody. Increasing the flow rate above this level increases spontaneous dissociation of labeled antigen, decreases antigen-antibody interaction time, and, consequently decreases the displacement efficiency (ratio of the number of moles of antigen added vs. number of moles of labeled antigen displaced). Decreasing the flow rate too much results in poor discrimination of the signal from background due to peak broadening. In general, flow rates of 0.1 to 2.0 ml/min are taught with 0.2 to 1 ml/min being preferable (U.S. Pat. No. 5,183,740, infra). If not for an unacceptably low signal to background ratio, low flow rates would be desirable for the detection of low analyte concentrations in small (e.g., one picoliter to ten microliters) samples.
In Wemhoff et al. (Wemhoff, G. A., S. Y. Rabbany, A. W. Kusterbeck, R. A. Ogert, R. Bredehorst, and F.S. Ligler
J. Immunol. Methods
, 156, 223-230, 1992), the concept of displacement efficiency was introduced as means for comparing assay performance as various parameters were modified. The displacement efficiency is at its maximum when the concentration of antigen added is low relative to the dynamic range for the column being used. The amount of displaced labeled antigen molecules does not exceed the displacement efficiency times the concentration of labeled antigen bound to the immobilized antibody, even when high concentrations of antigen are added. For the packed bed columns, a maximum displacement efficiency of about 0.001 was typical under optimal flow conditions.
U.S. Pat. No. 5,183,740 to Ligler et al. teaches a variety of support media, including capillary tubes. That patent, however, attaches no criticality to the form of the support media or column. Moreover, that patent fails to teach a range of capillary inner diameters and lengths and fails to suggest any relationship between capillary diameter/length and sensitivity.
Finally, obtaining consistent results from sample to sample requires columns that can be manufactured consistently, reliably, and reproducibly. Packing columns with beads is an imprecise process that results in variability among columns.
SUMMARY OF THE INVENTION
Accordingly, it is an object of this invention to improve the sensitivity of a displacement-type flow immunoassay.
It is a another object of the present invention to reduce the amount of sample required for a displacement-type flow immunoassay.
It is a further requirement of the present invention to provide a flow immunoassay support that can be prepared easily and reproducibly.
It is yet another object of the present invention to provide a flow immunoassay support that can be used with integrated optics.
These and other objects are accomplished by a microcapillary-based flow immunoassay. An antibody is immobilized on the interior of a microcapillary tube. The available antigen-binding sites of the antibody are then immunologically bound to a labeled analog of the antigen. When a sample containing the antigen flows through the microcapillary tube, sample antigen displaces the labeled analog.
If the microcapillary functioned as a support for the displacement assay in the same manner as packed beads, the operational parameters for the packed bead columns would indicate that the microcapillary would not produce sufficient signal for measurement. Based on this displacement efficiency and the estimated number of labeled antigen molecules in the capillary, a maximum of 1.4×10
−18
moles of labeled antigen could be displaced at any one sample addition. (The amount of labeled antigen that can be displaced from the capillary is calculated based on the following assumptions: (1) As described for the antibody immobilization chemistry by Bhatia et al.,
Anal. Biochem
, 178, 408-413, 1989), up to 0.66 nm/mm
2
antibody can be immobilized on borosilicate glass. The antibody can bind a small antigen in a 1:1 ratio. (2) The surface area of the capillary is 346 mm
2
. (3) The antibody has a molecular weight of approximately 160,000, and (4) the displacement efficiency is 0.001. Thus, 346 mm
2
*(0.66) ng antibody/mm
2
) * 1 nmole antibody/160,000 ng)*(nmole labeled antigen/1 nmole antibody/mm
2
)*0.001=1.4 ×10
−18
moles of labeled antigen.) Assuming no peak broadening, this amount of antigen in a 100 &mgr;l volume would produce a molarity of 1.4×10
−14
, which would not be detected using a standard HPLC fluorimeter with a sensitivity to the label of approximately 10
−11
molar. Nevertheless, as shown in the Detailed Description of the Preferred Embodiments below, the present invention actually provides enhanced sensitivity over prior art displacement immunoassays.
REFERENCES:
patent: 5183740 (1993-02-01), Ligler et al.
patent: 5741639 (1998-04-01), Ensing et al.
Gauger Paul R.
Ligler Frances S.
Narang Upvan
Chin Christopher L.
Karasek John J.
Reasing Amy L.
The United States of America as represented by the Secretary of
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