Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-05-03
2001-05-01
Wortman, Donna (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C436S066000, C436S518000, C436S813000, C530S412000, C530S422000
Reexamination Certificate
active
06225072
ABSTRACT:
The present invention relates to a process for extracting proteins from gastrointestinal samples and to new and/or improved methods for the determination of said proteins.
The onset and progression of many diseases results in altered patterns of cellular metabolism, antigenicity and changes in the quantitative and qualitative manufacture of cellular products or secretion or release thereof.
One such type of disease is cancer and of particular interest for the purposes of the present invention is cancer of the gastrointestinal (GI) tract. Cancers more remote from the GI tract and other diseases such as inflammatory and infectious diseases are also included within the scope of the invention.
It has been noted by researchers that certain endogenously produced substances are detected in increased or decreased amounts or in variant forms in samples taken from the GI tract of patients with GI or proximate tissue disease. For example, the protein calprotectin, previously referred to as L1 protein, is released in large amounts from leucocytes into the GI tract in response to some diseases, see e.g. U.S. Pat. No. 4,833,074 and U.S. Pat. No. 5,455,160. Calprotectin, a calcium binding heterocomplex protein comprising two heavy chains and one light chain (Fagerhol, et al., 1990), and as another example the protein haemoglobin, have been found in elevated amounts in the faeces of patients suffering from GI cancer (Roseth et al., (1993); Young & St. John, (1992)).
The detection of abnormal amounts or forms of such indicator proteins could be of great predictive and diagnostic value and may be useful in monitoring the progress of disease and also the efficacy of treatment and/or therapeutic procedures. Moreover, although protein profile changes may be detectable in a sample taken from any section of the GI tract, if the sample is a faecal sample then this could represent an entirely non-invasive method by which diagnostic and prognostic appraisals could be made.
Presently, an operator friendly assay that is sufficiently reliable and accurate is not available to measure the titre of such proteins of gastrointestinal origin. This is principally due to the difficulty in extracting the proteins from GI samples, usually faeces, in a form suitable for direct assay by conventional methods e.g. ELISA.
In U.S. Pat. No. 4,833,074, Fagerhol et al. disclose an antibody based detection system for the calprotectin protein and a method for isolation and purification of calprotectin from granulocytes. Such antibody based methods of quantitative detection work well (as will be discussed in greater detail later) when the protein is extracted from more amenable sources than the GI tract. Obviously however it is highly desirable in many respects that the samples from which proteins of interest should be isolated are faecal in origin.
A further immunoassay based method for determining analytes in faecal samples is disclosed in EP-A-0281251 wherein a sample of stool is dispersed in aqueous solution comprising various preservatives, analyte stabilising agents and endogenous interference reducing agents, the solids are allowed to settle and then the liquid phase used for analyte testing.
In U.S. Pat. No. 5,455,160, Fagerhol et al. disclose a kit and a method for the determination of calprotectin in GI samples. This kit and method are presented as a diagnostic tool for use in the detection of cancer and inflammatory bowel diseases. Clearly, these disclosures represent a useful step in the determination and clinical evaluation of altered protein levels, in samples which originate from the GI tract. This method suffers however from severe disadvantages in respect of quantitative accuracy and reproducibility and the ease with which the procedure may be performed by operators in a laboratory.
Firstly, the method of the above mentioned U.S. Pat. No. 5,455,160 describes the use of a sample size of 5 g of faeces which may prove difficult to obtain from elderly or frail subjects. The necessity for such a large amount of faeces creates some storage problems, especially if the samples require freezing.
More importantly, the method entails the process of homogenising the faeces sample in a comparatively small volume (2×) of buffer, in an open tube using a non-disposable rod mixer. Clearly this introduces some health risk to laboratory personnel on account of the aerosol of faeces formed in the course of the procedure. This may result in environmental and operator contamination, possibly cross-contamination of samples, not to mention containment and cleaning problems and a generally unpleasant environment in which to work. These problems may preclude the routine use of such a method as a clinical tool.
The present inventors have established that the extraction steps of the prior art method normally result in the determination of only some 15 to 30% of the total amount of calprotectin present in the faecal sample (Table 1, Example 4 hereinafter). This finding was arrived at by comparing the amount of calprotectin obtained from a single extraction according to the prior art method with the total amount of calprotectin present in a sample. The total amount of calprotectin in a sample was measured by extracting the same sample five times, so that it was essentially depleted of calprotectin, using a method according to the invention, and then summing the five amounts extracted from the sample.
Furthermore, a considerable amount of variability appears to be inherent in the prior art method.
Furthermore, the end product of the prior art extraction procedure is a suspension comprising a large amount of insoluble, particulate detritus. Such a viscous, particulate supernatant may be unsuitable for use with many quantitative determination methods e.g. those using membrane filters loaded with specific antibodies.
In summary therefore, there is a need for a reliable, practicable method by which proteins of interest may be extracted from samples originating from the GI tract, in particular but not exclusively from faeces, preferably in a form which facilitates their analysis and/or quantitation by any method known to one skilled in the art.
It has now surprisingly been found that by using a much smaller sample than in the prior art, for example 50 to 100 mg of faeces (compared to the 5 g of the prior art), a much more accurate and reproducible quantitative determination of the amount of calprotectin or other endogenous protein can be made. Simultaneously, in the same faecal extract as is used for measurement of calprotectin, one may determine the concentration of other endogenous proteins such as haemoglobin. The present inventors have conceived of a new extraction process which ameliorates the problems of the prior art process and provides a practicable, reliable and operator friendly process for the determination of proteins from GI tract samples.
One aspect of the present invention therefore relates to a process for the extraction of proteins from gastrointestinal (GI) tract samples, including faeces, from humans or other mammals, comprising;
a) mixing a small amount of sample (preferably 10 to 500 mg and more preferably 20-150 mg, optionally pre-weighed) with an excess amount of aqueous extraction medium (preferably in the region of 50 fold excess v/w) comprising at least one dissociating, disaggregating and/or chelating agent as described below,
b) homogenising the sample (preferably by vortexing) in a closed tube,
c) separating the solid and liquid material of the dispersion (preferably by centrifugation and additionally or optionally by filtration), and,
d) recovering the clear liquid extract, also referred to hereinafter as the filtrate or the supernatant;
said extract and proteins therein being susceptible of qualitative and/or quantitative determination using any suitable means.
The smaller sample is easier to collect, store and dilute. Collection of the sample is particularly relevant when considering elderly or frail subjects or when the sample of interest is not faeces but a sample which must be obtained by gastroscopy, e
Dale Siri
Fagerhol Magne K
Holtund Jostein
Bacon & Thomas
Fagerhol Magne K.
Wortman Donna
Zeman Robert A.
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