Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1997-10-24
2001-01-16
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C435S007100, C436S066000, C436S067000, C436S522000, C436S524000, C436S531000, C436S534000, C436S805000
Reexamination Certificate
active
06174734
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to hemoglobin, immunology, and blood analysis.
BACKGROUND OF THE INVENTION
In the presence of glucose, hemoglobin (Hb) is glycated by a nonenzymatic reaction stabilized by an Amadori rearrangement. Potential glycation sites are the N-terminal amino acids of the four polypeptide chains, and free &egr;-amino groups of lysine within the chains. The most reactive site, HbA1c, is the N-terminal valine of the &bgr;chain, which accounts for about 60% of all bound glucose. Bound sugars can also appear at any of 44 glycation sites on the &egr;-amino groups within the chains (about 34% of clycations), and the N-terminus of the a-chains (about 6% of glycations). The rate of glycation at any one site is directly proportional to glycation at any other site. Therefore, there is a linear relationship between the total amount of glycated hemoglobin (GHb) and any particular form of glycated hemoglobin (e.g., HbA1c).
A determination of the level of GHb in an individual can serve as an index of the blood glucose levels in the blood over time. The measurement of GHb) in diabetics is a useful indicator of disease state. Because the average blood cell has a lifespan of 120 days, (GHb levels reflect the blood glucose history of an individual over several months (Singer,
Ann. Intern. Med
., 1989, 110:125-137), with most recent blood glucose levels being most strongly represented. It is recommended that diabetes be monitored by determining levels of GHb every 2-3 months to determine the progress of disease, the efficacy of control of the disease, or both.
Many methods for assessing GHb concentrations require separation of GHb from Hb. One type of separation method is based on the charge difference between GHb and Hb. This can be done, for example, by electrophoresis, high performance liquid chromatography (HPLC), or conventional ion exchange chromatography. Structural differences have also been exploited in assays of GHb, using methods such as affinity binding, chromatography, and immunoassay (Goldstein et al.,
Clin. Chem
., 1996, 32/10B, B64-B70).
Antibodies that have limited specificity for native HbA1c have been obtained by using glycosylated synthetic peptide sequences from the N-terminal region of the Hb &bgr;-chain (Knowles, U.S. Pat. Nos. 4,647,654 and 4,727,036; Mezei, U.S. Pat. No. 4,478,744). These antibodies have significantly less affinity for native HbA1c compared to the peptides against which they were raised. Because the antigenic site is difficult to access in native HbA1c, methods to denature HbA1c (Lewis, EP 401 860 A2; Knowles U.S. Pat. No. 4,727,036) have been used to develop immunoassays for HbA1c. Another approach is to degrade the Hb molecule with an enzyme, e.g., pepsin, to expose the N-terminal &bgr;-chain site. Methods for immunoassay of GHb generally employ denaturation or degradation pretreatment steps for antibody and/or assay development.
SUMMARY OF THE INVENTION
In general, the invention features a device and assay method for determining GHb in a biological fluid, antibodies useful in the device and assay method, and a method for producing such antibodies. The device and assay method are useful for the evaluation of GHb in disease states such as diabetes.
The invention features a device or kit for assaying GHb in a biological fluid. The device or kit includes a solid support member, which comprises a predetermined amount of an immobilized GHb/Hb capture molecule. Optionally, the device or kit includes a means of detecting a glycation probe that is specific for GHb, or an Hb/Hp-specific probe that is used to detect Hb bound to haptoglobin (Hp). The glycation probe can be, e.g., an antibody or a boronate derivative. The Hb/Hp-specific probe can be an antibody. The capture molecule can be haptoglobin or an antibody. The means for detecting the glycation probe can involve any suitable method, e.g., chemiluminescence, electrosensing, fluorimetry, fluorescence quenching, enzymatic immunocytochemistry, or visualization of particles.
The invention also features a method for detecting glycated hemoglobin in a biological fluid. The method includes the steps of: (a) providing a solid support member, which includes a predetermined amount of a GHb/Hb capture molecule, (b) contacting the biological fluid with the support member, thereby forming a GHb/Hb-saturated support member, (c) contacting the GHb/Hb-saturated support member with a glycation probe, (d) removing unbound glycation probe, and (e) detecting the glycation probe bound to captured GHb.
Glycated hemoglobin can also be assayed in a biological fluid by a method that includes the steps of: (a) providing a solid support member, which includes a predetermined amount of a GHb/Hb capture molecule, (b) contacting the biological fluid with the support member, thereby forming a GHb/Hb-saturated support member, contacting the GHb/Hb-saturated support member with an Hb/Hp-specific probe, (d) removing unbound Hb/Hp-specific probe, and (e) determining the amount of the Hb/Hb-specific probe bound to captured Hb, and calculating how much GHb is in the biological fluid, if present.
The glycation probe or Hb/Hp-specific probe can be labelled directly or detected indirectly, e.g., with an antibody. The glycation probe can specifically bind nondenatured GHb or GHb conformer H. The Hb/Hp-specific probe can specifically bind Hb or Hb conformer H. Preferably, the glycation probe specifically binds GHb conformer H. It is preferred that an Hb/Hp-specific probe bind Hb conformer H.
Preferably, the capture molecule is haptoglobin. Alternatively, it can be an antibody. The biological fluid is from a human or a nonhuman mammal. typically, the biological fluid is blood.
The invention also features a method for producing nondenatured GHb-specific or GHb conformer H-specific antibodies. The method includes the steps of: (a) binding GHb to haptoglobin (Hp), thereby producing a GHb/Hp complex, (b) placing the GHb/Hp complex into an antibody production system, (c) obtaining an antibody-containing product from the production system, and (d) detecting GHb or GHb conformer H-specific antibodies in the product.
The invention also features a method for producing Hb conformer H-specific antibodies. The method includes the steps of: (a) binding Hb to haptoglobin (Hp), thereby producing a Hb/Hp complex, (b) placing the Hb/Hp complex into an antibody production system, (c) obtaining an antibody-containing product from the production system, and (d) detecting Hb conformer H-specific antibodies in the product. The binding between GHb or Hb and Hp can be covalent or non-covalent. Hp may be from any mammal, preferably a human or a sheep. The antibody production system can be any nonhuman mammal, preferably a rabbit, a mouse, a rat, a goat, a chicken, a donkey, or a sheep. Alternatively, the antibody production system can be an in vitro system.
The invention also features an antibody produced by the methods described above, wherein the antibody selectively binds to nondenatured GHb, GHb conformer H, or Hb conformer H in the presence of Hb. For example, the antibody can display any of the following binding specificities: (a) nondenatured GHb, (b) GHb conformer H, (c) nondenatured GHb and GHb conformer H, (d) nondenatured Hb, (e) Hb conformer H, and (f) nondenatured Hb and Hb conformer H. Preferably the antibody specifically binds nondenatured GHb, GHb conformer H, or both.
As used herein, “hemoglobin” (Hb) means all naturally occurring forms of non-glycated hemoglobin, including tetrameric forms of &agr;&agr;&bgr;&bgr; top and dimeric forms, e.g., &agr;&bgr;, and disease variants that bind to capture molecules.
As used herein, “glycated hemoglobin” (GHb) means hemoglobin having covalently attached sugar moieties (e.g., HbA1c) and GHb formed through a non-enzymatic reaction via an Amadori rearrangement.
As used herein, “hemoglobin conformer H” (Hb conformer H) means a hemoglobin in a non-native conformation inducible by a haptoglobin binding event.
As used herein, “glycated hemoglobin conformer H” (GHb conformer H) means a g
Ito Ralph K.
Sanghera Gordon
Abbott Laboratories
Chin Christopher L.
Pope Lawrence S.
Weinstein David L.
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