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Improved immunoassay methods

A technology of subjects and antibodies, applied in the field of detection of antibodies and biomarkers, can solve problems such as intolerant changes in the amount of antibodies

Active Publication Date: 2008-06-18
福瑞姆有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, these methods do not allow for variation in the amount of antibody detected between individuals, leading to the occurrence of said false positives and false negatives

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0136] Example 1 - General Protocol for Titrating Antigens in Autoantibody Assays

[0137] Samples of (biotinylated) tumor marker antigens can be prepared by recombinant expression, following a method similar to that described in WO99 / 58978.

[0138] Briefly, the cDNA encoding the marker antigen of interest was cloned into the pET21 vector (Invitrogen) that had been modified to encode a biotin tag and a 6x histidine tag to aid in the purification of the expressed protein. The resulting clones were cultured (in inclusion bodies) in suitable bacterial host cells, the bacteria were lysed and denatured, and the expressed antigen was recovered by passage through a nickel chelate affinity column (Hi-trap, available from Amersham, following the manufacturer's protocol). The expressed antigen was refolded by dialysis in an appropriate buffer and the yield of expressed antigen was assessed by SDS-PAGE, western blot and ELISA and quantified before storage.

[0139] Negative control VO...

Embodiment 2

[0162] Example 2, detection of autoantibodies in primary breast cancer

[0163] The following data were obtained from a pilot study evaluating the sensitivity and reproducibility of a titrated autoantibody assay in primary breast cancer (PBC). The study included serum samples from 17 women with no evidence of cancer as well as preoperative serum samples from 20 women with primary breast cancer. Normal and cancer samples were age-matched. One normal sample and three cancer samples could only be removed because they showed evidence of an anti-biotin antibody response and therefore could not be assessed using the current assay format. Approximately 10% of the population is thought to develop an immune response against biotin.

[0164] The assay was carried out according to the procedure given in Example 1 using the antigens p53, c-myc, NY-ESO-1 and BRCA2.

[0165] Figure 1 gives an example of the curve obtained when this antigen titration assay was used to measure p53 autoan...

Embodiment 3

[0173] Example 3, Determination of Autoantibodies in Lung Cancer

[0174] Analysis of autoantibody responses to 2 antigens (p53 and NY-ESO) in a pilot lung cancer study (10 normal and 9 lung cancer plasma) showed a detection rate of 78% (Figure 3).

[0175] The assay was performed according to the general protocol in Example 1 except that plasma samples were used instead of serum.

[0176] Positive patient samples exhibited a reverse sigmoid titration curve similar to that shown in Figure 1. Figure 3 shows a comparison of p53 and NY-ESO autoantibody levels in normal individuals and lung cancer patients measured using an antigen titration assay. The normal cutoff was calculated as the mean plus 2 standard deviations for the normal population.

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Abstract

The invention relates to a method of detecting a disease state or disease susceptibility in a mammalian subject which comprises detecting an antibody in a test sample comprising a bodily fluid from said mammalian subject wherein said antibody is a biological marker of a disease state or disease susceptibility, the method comprising: (a) contacting said test sample with a plurality of different amounts of an antigen specific for said antibody, (b) detecting the amount of specific binding between said antibody and said antigen, (c) plotting or calculating a curve of the amount of said specific binding versus the amount of antigen for each amount of antigen used in step (a) and (d) determining the presence or absence of said disease state or disease susceptibility based upon the amount of specific binding between said antibody and said antigen at each different antigen concentration used.

Description

technical field [0001] The present invention relates generally to the field of diagnostic or prognostic assays, and in particular to assays optimized for the detection of antibodies in samples comprising bodily fluids of a patient, where such antibodies are used as biomarkers of a disease state or susceptibility to disease. Background technique [0002] Many diagnostic, prognostic, and / or monitoring assays rely on the detection of biomarkers of a particular disease state or disease susceptibility. Such biomarkers are typically proteins or polypeptides that are characteristic of a particular disease or associated with disease susceptibility. [0003] It has become increasingly apparent in recent years that antibodies, especially autoantibodies, can also serve as biomarkers of disease or susceptibility to disease. Autoantibodies are naturally occurring antibodies directed against an antigen that is recognized by an individual's immune system as foreign, even though the antige...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/564G01N33/574
CPCG01N2800/102G01N33/564G01N2800/52G01N33/574G01N33/57484G01N33/53
Inventor 约翰·福赛思·鲁塞尔·罗伯特森托尼·巴尔内斯安德烈亚·默里卡罗琳·查普曼
Owner 福瑞姆有限公司
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