Detection of PSA-α2-macroglobulin complex in a biological fluid
AssigneeBayer Aktiengesellschaft
InventorPrakash Tewari
Life of this patent
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Abstract
The present invention provides methods for detecting and quantitating PSA-α2-macroglobulin complexes in the sera of patients at risk for prostate cancer. Methods for detecting and monitoring prostate cancer in a patient by determining the serum level of the complex also are described, as are kits for performing the methods.
Claims13
A method for detecting the presence or amount of PSA-α2-macroglobulin complex in blood drawn from an individual, said method comprising the steps of:
- a) processing said blood to separate serum or plasma therefrom,
- b) exposing, in vitro, said serum or plasma of said individual to a pH pre-treatment that initially adjusts the pH to 3 or less, after which said serum or plasma is neutralized using an alkaline solution, whereby, after such exposure, a PSA epitope previously entrapped becomes available for specific binding,
- c) contacting said serum or plasma sample with an antibody or antibody fragment to PSA that binds PSA in PSA-α2-macroglobulin complex, said antibody or antibody fragment being immobilized on a solid support to form a PSA-α2-macroglobulin—solid phase conjugate;
- d) isolating from said serum or plasma sample, said PSA-α2-macroglobulin—solid phase conjugate;
- e) contacting said isolated PSA-α2-macroglobulin—solid phase conjugate with a labeling antibody or antibody fragment that binds α2-macroglobulin in said complex to form a labeled complexed antibody or antibody fragment thereof;
- and
- f) measuring the amount of said labeled antibody or antibody fragment bound to said complex to determine the amount of PSA-α2-macroglobulin complex.
A method for pretreating a serum sample to be assayed for concentration of PSA-α2-M complex formed in vivo, wherein said method does not lead to the dissociation of said complex, said method comprising:
- a) adjusting the pH of said sample to approximately pH 1.5 to 3.0, by the addition of acid,
- b) allowing said sample with adjusted pH to remain at room temperature for about one hour, and
- c) after said one hour, adjusting the pH of said sample to approximately 5.8 to 7.0, by the addition of base.
A method for detecting the presence or amount of PSA-alpha2-macroglobulin complex in blood drawn from an individual, said method comprising the steps of:
- a) processing said blood to separate serum or plasma therefrom,
- b) treating said serum or plasma by
- (1) adjusting the pH of said sample to approximately pH 1.5 to 3.0, by the addition of acid,
- (2) allowing said sample with adjusted pH to remain at room temperature for about one hour, and
- (3) after said one hour, adjusting the pH said sample to approximately 5.8 to 7.0, by the addition of base,
- c) combining a sample of said serum or plasma from step (a) with a first conjugate comprising (i) a PSA-specific antibody or a PSA-specific antibody fragment thereof and (ii) a solid phase, under conditions sufficient to induce specific binding between said first conjugate and PSA in said sample to form a PSA-antibody solid phase conjugate;
- d) contacting said PSA-antibody solid phase conjugate formed in (b) with a second conjugate comprising (i) an α2-macroglobulin-specific antibody or an α2-macroglobulin-specific antibody fragment thereof and (ii) a detectable label, under conditions sufficient to induce specific binding between the second conjugate and α2-macroglobulin in said PSA-antibody solid phase conjugate in said serum or plasma to form an α2-macroglobulin-label complex;
- e) separating said solid phase conjugate from said sample;
- and
- f) detecting presence or amount of said label attached to said separated solid phase conjugate,
- wherein the presence or amount of PSA-α2-macroglobulin complex present is determined by correlating the results from step (f) to a calibration curve generated by the use of known amounts of PSA-α2-macroglobulin.
Dependencies are inferred from the claim text.