Method of releasing solid matrix affinity adsorbed particulates

Details Method of releasing solid matrix affinity adsorbed particulates Method of releasing solid matrix affinity adsorbed particulates

1999-03-22

2001-02-06

Chin, Christopher L. (Department: 1641)

Chemistry: molecular biology and microbiology

Treatment of micro-organisms or enzymes with electrical or...

C435S173900, C435S174000, C435S179000, C435S261000, C435S262000, C435S262500, C435S263000, C435S264000, C435S267000, C435S273000, C435S274000, C435S275000, C435S308100, C435S961000, C436S518000, C436S528000, C436S529000, C436S520000, C436S823000, C436S824000

Reexamination Certificate

active

06184011

ABSTRACT:

FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to a method of releasing solid matrix affinity adsorbed particulates by enzymatically degrading the solid matrix to which the particulates are adsorbed. More particularly, the present invention relates to a method of releasing particulates, such as viruses, cells or fragments thereof, which are affinity adsorbed on a polysaccharide solid matrix, by enzymatically degrading the polysaccharide solid matrix to which the particulates are adsorbed with a polysaccharidase.
The need for efficient affinity adsorption and release methods:
There is a tremendous effort being made to develop rapid cell testing and separation methods to meet the needs of the food, medical, environmental and veterinary industries.
The food industry, for example, needs rapid microbial testing to approve or reject raw materials and determine whether or not to release a held batch of product. Furthermore, with the increasing implementation of hazard analysis and critical control point programs by the food industry, the demand for rapid microbial testing has been steadily on the rise (Rules and Regulations, Department of Agriculture Food Safety and Inspection Service, “Pathogen Reduction; Hazard Analysis and Critical Control Point (HACCP) Systems” (1996) 61 Federal Register 38806).
Rapid microbiological methods such as nucleic acid probe hybridization and immunological assays have advanced dramatically, shortening the time required for the detection of pathogens in meats and other foods.
However, these methods require concentrations of target microorganism of 10
4
to 10
6
cells per ml or more (Blackburn et al., 1994, Lett. Appl. Microbiol. 19:32-36; Swaminathan et al., 1994, Ann. Rev. Microbiol. 48:401-426; and Tian et al., 1996, J. Food Prot. 59:1158-1163). Foods which are contaminated by bacterial pathogens usually have low numbers of bacteria so that an enrichment step is required prior to the application of a rapid detection assay or even a selective culture method.
PCR-based assays have the potential to overcome the need for long selective enrichment steps due to their ability to detect and identify pathogens in the presence of large numbers of background flora (K. Venkateswaran et al., 1997, Appl. Environ. Microbiol. 63:4127-4131).
Nevertheless, when target pathogen concentrations are low, i.e., less than 10
3
CFU/g, current PCR procedures require 6 to 18 hours of enrichment prior to PCR amplification in order to detect such target pathogens. This enrichment step not only brings the target pathogen, which may be present at levels of less than 1 cell per ml or gram of food (Swaminathan et al., 1994, Ann. Rev. Microbiol. 48:401426), to PCR detectable levels but also allows for samples to be diluted or filtered to reduce or partially remove PCR inhibitory food components while bringing the target pathogen to a concentration which is detectable (K. Venkateswaran et al., 1997, Appl. Environ. Microbiol. 63:4127-31).
In culture methods, selective reagents are necessary in order to inhibit the growth of competing microorganisms but they also inhibit target bacteria ultimately increasing the time it requires to achieve detectable levels of the target pathogen. Similarly, many foods contain compounds which are inhibitory to the target bacteria and in many cases the target bacterial cells do not revive or revive very slowly. Furthermore, selection media inhibit the “resuscitation” (reviving the viability) of damaged bacteria present, e.g., in meat samples due to freeze thaw cycles, inhibitory food components, or desiccation during processing.
The possibility of selectively concentrating and thereafter recovering and counting or analyzing target bacteria from a food sample, other than by culture methods, while removing background flora and inhibitory compounds would have a tremendous impact on rapid food testing saving time and increasing sensitivities of existing detection techniques.
Zero tolerance standards require the detection of these damaged pathogens even when they are present at minute levels. On the other hand, selective concentration of target bacteria would enable rapid enrichment to be carried out without the addition of selective reagents which are necessary in order to inhibit the growth of background flora but which also inhibit target bacteria and ultimately increase the time it takes to achieve detectable levels of target pathogen. Removal of competing microorganisms and inhibitory compounds found in food samples to affect resuscitation of target pathogens is best accomplished by selective concentration and subsequent wash steps.
In summary, efficient methods for selective affinity concentration and release of a particular pathogen(s) from foods would not only shorten the overall time required for detection of these pathogens, but would allow for more sensitive detection due to more efficient resuscitation of damaged bacteria.
Furthermore, the ability to collect larger more representative samples while selectively concentrating and recovering a specific pathogen from these samples would greatly increase the probability of detecting a pathogen in foods when they are present at extremely low concentrations.
Methods of efficient affinity adsorption and concentration of a microorganism or microorganisms are disclosed in U.S. patent application Ser. No. 09/175,040, filed Oct. 19, 1998, which is incorporated by reference as if fully set forth herein.
This application teaches methods for concentrating a particular microorganism or microorganisms of interest in a sample. The disclosed methods are generally effected by contacting a sample with a cellulosic or chitin matrix to which is bound a cellulose binding protein (“CBP”)-receptor (i.e., a first member of a binding pair) or cellulose binding domain (“CBD”)-receptor conjugate specific for said microorganism(s).
The methods also include a washing step to remove unbound material of the sample from the matrix. The methods also include an optional step for enriching the concentrated microorganism(s) in situ by addition of a culture medium to the matrix or by enriching the concentrated microorganism(s) in vitro by transferring the microorganism(s) from the matrix to a culture medium. The method also includes an optional step of performing an assay to detect any microorganisms that bind to the CBP- or CBD-receptor conjugate bound to the matrix.
The disclosed methods have utility in concentrating microorganisms in samples, particularly dilute samples, in order to detect the microorganisms by any means known in the art. Such methods of concentration provide improved means of concentrating microorganisms in food, environmental, or biological, such as medical or veterinary, samples.
The disclosed methods have a number of advantages over previously described concentration methods. For example, the use of cellulosic fabric as a matrix allows for larger volumes of liquids (up to 10 liters) to be passed with relatively high flow rates as compared to, for example, the DYNAL® DYNALBEADSO® procedure [Dynal-product Cat. No. 710-03]. The low non-specific binding of the cellulose achieves very low background levels. In certain preferred embodiments, the disclosed methods are able to capture microorganisms present at very low concentrations by use of high surface area cellulosic or chitin matrix, such as, but not limited to gauze. The physical properties of the cellulosic or chitin matrix enable its performance under conditions that Immuno Magnetic Separation (IMS) do not perform effectively, i.e., in the presence of food samples containing milk and food samples containing bacteria at concentrations lower than 10
3
CFU/ml.
However, no efficient method of releasing the captured microorganism is disclosed in U.S. patent application Ser. No. 09/175,040.
There is also a need for efficient adsorption and release of eukaryotic cells. Of particular interest are bone marrow and hematopoietic derived cells. Particular cell types derived from these sources, such as, but not limited to, hematopoietic stem

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