WO2008076382A2 - Bacteriophage particle agglutination - Google Patents

Abstract

A composition for detecting one or more species, classes, and/or types of bacteriophage present in a sample. In some embodiments, the disclosed composition includes a plurality of detection particles, wherein the plurality of detection particles includes one or more binding molecules complexed therewith, each of which specifically binds to one or more species of bacteriophage. Also provided are methods for employing the disclosed compositions for testing of and/or for direct or indirect identification of indicators of the presence of bacteria, viruses, or other microbes in any sample for which such testing and/or identification is desired.

Description

DESCRIPTION BACTERIOPHAGE PARTICLE AGGLUTINATION

CROSS REFERENCE TO RELATED APPLICATION The presently disclosed subject matter claims the benefit of U.S. Provisional Patent Application Serial No.60/875,128, filed December 15, 2006; the disclosure of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The presently disclosed subject matter relates to the field of public health and safety, more particularly to methods and compositions for the rapid testing of aqueous and other samples that might come into direct or indirect contact with members of the public for microbial contamination.

BACKGROUND Water quality is a global public health concern. In developing countries, there is inadequate access to safe drinking water and its sources. Unsafe water, sanitation, and hygiene cause around 1.7 million deaths each year worldwide, mostly from infectious diarrhea in children in developing countries (55). Microbial pathogens causing gastrointestinal, dermal, and respiratory infections can be spread by drinking, bathing, or cleaning with water polluted with feces (56). In developed countries, waterbome disease outbreaks and discrete disease cases continue to occur despite government regulations on wastewater and drinking water quality, treatment, and monitoring-based warning systems for wastewater effluents, recreational waters, and shellfish- growing waters (11 , 27, 36). Fecal indicator microorganisms, such as fecal coliforms, Escherichia coli, and enterococci, are used to measure the efficacy of water and wastewater treatment, drinking water quality, and the sanitary quality of bathing and shellfishing waters (32). However, current microbial indicators are bacteria, and many waterborne pathogens are enteric viruses, for which bacterial indicators are inadequate or unreliable in certain circumstances. For example, bacterial indicators can be inadequate due to greater virus and bacteriophage resistance to water and wastewater treatment processes (21 , 26) and greater virus and bacteriophage persistence in freshwater and seawater (10, 14, 31). Hence, there is a need for quick, easy-to-use, reliable viral indicators and effective methods to detect and assay them.

U.S. ambient water monitoring programs are just one example of the need for improved fecal indicator detection. Bacterial indicator assays used by regulators to monitor ambient water quality require 18 to 96 hours for results, which causes water quality decisions and warnings/advisories to be posted days after contamination events occur (32). Fecal pollution events in water are intermittent and often return to below threshold levels in 24 hours (5, 28). The same bacterial indicator assays cannot differentiate human and nonhuman fecal wastes for tracking and controlling their sources without extra and advanced steps, and they have a lack of predictability for enteric virus contamination (12). In 2005, regulators issued around 20,400 days of closures or advisories at U.S. beaches and lakes due to exceedances of bacterial fecal indicators (33). About 75% of those 20,400 exceedances were caused by unknown sources of fecal pollution that could not be tracked, treated, or managed (33).

Coliphages are alternatives to bacterial indicators. Coliphages are bacterial viruses that reside in the guts of animals, sometimes at titers similar to those of bacterial gut flora (1). Coliphages are obligate intracellular parasitic microorganisms that generally do not replicate in environments outside the gut, where host bacterial levels are >10⁴ CFU/ml (50, 54), or in nutrient-poor environments that do not support host growth (54). In addition, coliphage lysis of bacteria only occurs in bacterial cultures undergoing exponential (logarithmic-phase) growth (37). F+ coliphages infect the F pili of coliform bacteria, which stop forming below 25⁰C (34, 53), further constraining the natural conditions needed for coliphage replication. Coliphages are useful at indicating public health risks for water users and shellfish consumers, as in some studies the presence of coliphages was correlated with the presence of pathogenic human viruses in water and shellfish and with the risk of viral illness (9, 12, 13, 25, 48). F+ coliphages can be divided into two families, namely, the Leviviridae, containing RNA genomes (F+ RNA coliphages), and the Inoviridae, containing DNA genomes (F+ DNA coliphages) (46). F+ RNA coliphages can be serotyped into distinct groups present in human fecal waste (groups Il and III) or animal fecal waste (groups I and IV) (8, 15, 23). Microbial source tracking with F+ RNA coliphages has been used to identify and control human and animal sources of fecal pollution in surface waters (3, 17, 42). Current coliphage recovery and detection assays are as time consuming as culture-based bacterial indicator methods, taking 1 to 3 days for coliphage culture and plating methods (44, 45), 1 to 2 days for coliphage serotyping methods (23), and 2 days for molecular coliphage methods, including reverse transcriptase PCR (RT-PCR) and probe hybridization (47). What is needed, then, are rapid, easily portable systems that can be employed for measuring contamination of water samples and other samples suspected of being contaminated with microbes. This and other needs in the art are addressed by the presently disclosed subject matter.

SUMMARY

This Summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features. The presently disclosed subject matter provides compositions for detecting one or more species, classes, and/or types of bacteriophage present in a sample. In some embodiments, the compositions comprise a plurality of detection particles, wherein (i) the plurality of detection particles comprises one or more binding molecules complexed therewith; and (ii) the one or more binding molecules each specifically binds to one or more species of bacteriophage. In some embodiments, at least one of the one or more species of bacteriophage is a male-specific (i.e., an F⁺) coliphage. In some embodiments, the one or more species of bacteriophage includes F⁺ RNA- containing coliphage. In some embodiments, the F⁺ RNA-containing coliphage include Group I {e.g., MS2-like), Il (e.g., GA-like), III (e.g., QB-like), IV (e.g.,

SP- and Fl-like), and M11-like coliphage. In some embodiments, the one or more species of bacteriophage includes F⁺ DNA-containing coliphage. In some embodiments, the F⁺ DNA-containing coliphage include Fd, F1 , M13, Φ15,

Φ16, Φ18, and taxonomicaliy, genetically, and antigenically related coliphage.

In some embodiments, the plurality of detection particles comprises beads coated with the one or more binding molecules. In some embodiments, the beads are polystyrene beads. In some embodiments, the plurality of detection particles are present in the composition at about a 1 % w/v suspension in a neutral buffer, and the 1% w/v suspension further comprises about 0.01 % w/v of a blocking agent. In some embodiments, the blocking agent comprises bovine serum albumin (BSA).

In some embodiments of the presently disclosed subject matter, the one or more binding molecules comprise a plurality of antibodies, fragments thereof, derivatives thereof, or combinations thereof. In some embodiments, at least one of the plurality of antibodies, fragments thereof, derivatives thereof, or combinations thereof is selected from the group consisting of a whole immunoglobulin molecule, an scFv antibody, a chimeric antibody, an Fab fragment, an Fab' fragment, an F(ab')₂ fragment, and combinations thereof. In some embodiments, at least one of the plurality of antibodies, fragments thereof, derivatives thereof, or combinations thereof binds to at least two different epitopes present on a single species or particle of bacteriophage. In some embodiments, the plurality of antibodies, fragments thereof, derivatives thereof, or combinations thereof binds to at least two different epitopes present on different species or particles of bacteriophage. In some embodiments, each member of the plurality of detection particles is complexed with a single species of binding molecule that binds a single ligand. In some embodiments, the single species of binding molecule comprises a monoclonal antibody, or a fragment or derivative thereof, which binds to a single epitope present on at least one of the one or more species of bacteriophage.

In some embodiments of the presently disclosed subject matter the plurality of detection particles comprises a mixture of detection particles comprising a plurality of species of binding molecules. In some embodiments, the plurality of species of binding molecules comprises a polyclonal antiserum, a mixture of monoclonal antibodies raised against different bacteriophage epitopes, fragments or derivatives thereof, or combinations thereof. In some embodiments of the presently disclosed subject matter, one or more members of the plurality of detection particles are complexed with a mixture of at least two different species of binding molecules.

The compositions provided herein are in some embodiments stable for extended periods. In some embodiments, the composition is stable at about 25°C for at least 1 , 2, 3, 4, 5, or 6 months or more, or at about 4°C for at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months or more.

The presently disclosed subject matter also provides methods for detecting the presence of one or more species of bacteriophage present in a sample, the methods comprising contacting the sample with one or more of the disclosed compositions under conditions sufficient to specifically bind at least one of the one or more binding molecules to at least one of the one or more species of bacteriophage, if present, to form a complex; and detecting the complex formed, whereby the presence of at least one of the one or more species of bacteriophage present in the sample is detected. In some embodiments, the complex formed is visible to the naked eye.

In some embodiments, the presently disclosed methods for detecting the presence of one or more species of bacteriophage present in a sample comprise contacting a sample with a composition comprising a plurality of detection particles, wherein the plurality of detection particles comprises one or more binding molecules complexed therewith, the one or more binding molecules each specifically binds to one or more species of bacteriophage, and the contacting is under conditions sufficient to bind at least one of the binding molecules to at least one of the one or more species of bacteriophage if present in the sample; and identifying a complex comprising one or more binding molecules and the at least one of the one or more species of bacteriophage, whereby the presence of one or more species of bacteriophage in the sample is detected. In some embodiments of the presently disclosed methods, at least one of the one or more species of bacteriophage is an F⁺ coliphage. In some embodiments, the F⁺ RNA-containing coliphage is selected from the group consisting of Group I (MS2-like), Group Il (GA-like), Group III ((QB-like), Group IV (SP- and Fl-like), and M11-like coliphage. In some embodiments, at least one of the one or more species of bacteriophage is an F⁺ DNA-containing coliphage. In some embodiments, the F⁺ DNA-containing coliphage is selected from the group consisting of Fd, F1 , M13, Φ15, Φ16, Φ18, and taxonomically, genetically, and antigenically similar coliphage.

In some embodiments of the presently disclosed methods, the plurality of detection particles comprises beads coated with the one or more binding molecules. In some embodiments, the beads are polystyrene beads.

In some embodiments of the presently disclosed methods, the one or more binding molecules comprise a plurality of antibodies, fragments thereof, derivatives thereof, or combinations thereof. In some embodiments, at least one of the members of the antibodies, fragments thereof, derivatives thereof, or combinations thereof is selected from the group consisting of a whole immunoglobulin molecule, an scFv antibody, a chimeric antibody, an Fab fragment, an Fab¹ fragment, an F(ab')₂ fragment, and combinations thereof. In some embodiments, at least one of the plurality of antibodies, fragments thereof, derivatives thereof, or combinations thereof binds to at least two different epitopes present on a single species or particle of bacteriophage. In some embodiments, at least one the plurality of antibodies, fragments thereof, derivatives thereof, or combinations thereof binds to at least two different epitopes present on different species or particles of bacteriophage. In some embodiments, at least one member of the plurality of detection particles is complexed with a single species of binding molecule that binds a single ligand. In some embodiments, the single species of binding molecule comprises a monoclonal antibody, or a fragment or derivative thereof.

In some embodiments of the presently disclosed methods, at least one of the plurality of detection particles comprises a mixture of detection particles comprising a plurality of species of binding molecules. In some embodiments, the plurality of species of binding molecules comprises a polyclonal antiserum, a mixture of monoclonal antibodies raised against different coliphage epitopes, or combinations thereof. In some embodiments, at least one members of the plurality of detection particles are complexed with a mixture of at least two different species of binding molecules.

In some embodiments of the presently disclosed methods, the complex formed is visible to the naked eye. In some embodiments, the complex that is visible to the naked eye forms in less than 60 seconds at 25⁰C.

The presently disclosed compositions and methods can be employed for testing of and/or for direct or indirect identification of indicators of the presence of bacteria, viruses, or other microbes in any sample for which such testing and/or identification is desired. In some embodiments, a sample is selected from the group consisting of a drinking water sample, an ambient water sample (e.g., surface water or seawater), a groundwater sample, an irrigation water sample, a sewage sample, a biosolid sample, a manure sample, a soil or sediment sample, a produce sample, a meat sample, a shellfish sample, one or more treated samples thereof, combinations thereof, and a culture of one or more of the these samples, wherein the culture comprises some or all of one or more of the enumerated samples grown under conditions sufficient to support the growth of a bacteriophage, if present, in the sample.

In some embodiments, the presently disclosed methods further comprise culturing the sample, or a portion thereof, under conditions sufficient to amplify the one or more species of bacteriophage if present in the sample to produce a culture, and employing the culture as the sample in the contacting step. In some embodiments, the culturing is performed for a short period of time {e.g., for not more than about two to three hours).

In some embodiments of the presently disclosed subject matter, methods are provided of serotyping one or more species of bacteriophage present in a sample, comprising, contacting the sample containing the one or more species of bacteriophage with a plurality of detection particles comprising an antibody complexed therewith, which is specific for an antigen on one or more of the species of bacteriophage, under conditions sufficient to allow for a binding complex to occur between the bacteriophage and the antibody; detecting the complex formed; and assigning a serotype to the one or more species of bacteriophage based on the detection of the complex between the bacteriophage and the bacteriophage antigen-specific antibody. In some embodiments, the detecting the complex formed is through visualization by the naked eye. In some embodiments, the complex that is visible to the naked eye forms in less than 60 seconds at 25°C. In some embodiments, the sample, or a portion thereof, is first cultured, preferably for no more than about two to three hours, under conditions sufficient to amplify the one or more species of bacteriophage to produce a culture, and the culture is employed as the sample in the contacting step. In some embodiments, the one or more species of bacteriophage are F+ coliphage.

In some embodiments, the antibody is a polyclonal antiserum against F+ coliphage. In some embodiments, the antiserum is rabbit antiserum. In some embodiments, the F+ coliphage is F+ RNA coliphage including Group I coliphage, preferably MS2-like coliphage; Group Il coliphage, preferably GA- like coliphage; Group III coliphage, preferably Qβ-like coliphage; Group IV coliphage, preferably SP-like or Fl-like coliphage; and M11-like coliphage. In some embodiments, the F+ coliphage is F+ DNA coliphage including Fd, F1 ,

M13, Φ15, Φ16, and Φ18, and coliphage that are taxonomically, genetically, and/or antigenically related to one or more of Fd, F1 , M13, Φ15, Φ16, and Φ18.

In some embodiments, kits which comprise the detection particles of the presently disclosed subject matter are provided for detecting the presence of and/or serotyping the one or more species of bacteriophage present in a sample.

It is an object of the presently disclosed subject matter to provide a composition for testing of and/or for direct or indirect identification of indicators of the presence of bacteria, viruses, or other microbes in any sample for which such testing and/or identification is desired.

An object of the presently disclosed subject matter having been stated hereinabove, and which is achieved in whole or in part by the presently disclosed subject matter, other objects will become evident as the description proceeds when taken in connection with the accompanying drawings as best described hereinbelow. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a diagram outlining an exemplary approach to coliphage agglutination. Positive coliphage agglutination can be visualized 30-60 seconds after mixing equal volumes of coliphage enrichments with particles (e.g., polystyrene beads) comprising molecules that bind to domains and/or epitopes that are present on the coliphage (e.g., anti-coliphage antibodies, including fragments or derivatives thereof).

Figures 2A-2D are graphs showing rapid culture enrichment of F+ RNA coliphage prototype strains (squares) with host E. coli F_amp (circles). 2A) F+ RNA coliphage prototype strain MS2. 2B) F+ RNA coliphage prototype strain

Qβ. 2C) F+ RNA coliphage prototype strain Sp. 2D) F+ RNA coliphage prototype strain Fi. The error bars show standard deviations for coliphages (n

= 3) are obscured by some square data points. The preculture level of E. coli

F_amp was 1 X 10⁷ CFU/ml. E. coli levels during the experiment were measured by spectrophotometric absorbance at 520 nm.

DETAILED DESCRIPTION

Simple, rapid, and reliable fecal indicator tests are needed to better monitor and manage the sanitary quality of ambient waters and treated waters and wastes. Immunological particle agglutination assays are potentially rapid, simple, specific, and inexpensive and their reagents can be stored at ambient temperatures for months. The presently disclosed subject matter provides same-day optimized and validated microbial monitoring assays using immunological agglutination of viral indicators of various samples. Rapid, simple and inexpensive methods for detecting bacteriophages are provided by the presently disclosed subject matter. For example, phages in aquatic and terrestrial environments are not well characterized because often <1 % of their natural hosts are culturable, resulting in the "great plaque count anomaly" (49, 51 ). One advantage of the presently disclosed subject matter is that it enables detection of bacteriophage strains that infect bacterial hosts but do not form plaques, thereby obviating or circumventing the need for conventional serotyping methods based on neutralization of virus infectivity. Accordingly, in some embodiments, the presently disclosed compositions and methods are useful for detecting marine bacteriophages and providing useful information about bacteriophage occurrence, ecology, properties, and public health risks. In some embodiments, the bacteriophages being detected are coliphages. In some embodiments, the bacteriophages being detected include other fecal indicator viruses, such as Bacteroides fragilis phages, Salmonella phages, and somatic coliphages as well as phages in terrestrial and marine environments.

In some embodiments rapid microbial detection assays provided herein are based on an antibody immunological approach. For example, disclosed herein are optimized and validated rapid microbial monitoring assays using immunological agglutination of viral indicators of various samples. The presently disclosed immunoassays are simple to perform on a cardboard card by mixing a drop of virus enrichment culture with a drop of detection reagent. Visual agglutination or clumping of positive samples can occur in less than 60 seconds. The assay has a sensitivity over 95% and a specificity higher than 97% for two families of fecal indicator viruses. The assays successfully detected and identified viruses in similar proportions as a gold standard nucleic hybridization assay.

This successful development and evaluation of a new immunological agglutination technique for rapid and simple detection of fecal indicator viruses provides an improved tool to monitor the microbiological quality of drinking, recreational, shellfishing, and other waters, as well as other samples for which monitoring would be desirable.

L General Considerations

F⁺ coliphages are viruses of E. coli bacteria and can be used to determine the sanitary quality of water (drinking water, surface water, etc), foods, and other environmental media to protect people from exposure to harmful enteric microbes. Disclosed herein are optimized and validated F⁺ coliphage detection assays based on agglutination of F⁺ coliphage groups with antibody-coated particles for rapid microbial water quality monitoring. The F⁺ Coliphage Agglutination and Typing (CLAT) assay was performed on a cardboard card by mixing a drop of coliphage enrichment culture with a drop of antibody-coated polymeric beads as the detection reagent. Visual agglutination or clumping of positive samples occurred in less than 60 seconds. This method could be produced as a kit for water quality monitoring in developed and developing countries. All references listed in the instant disclosure, including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and GENBANK® database entries (including all annotations available therein) are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for or teach methodology, techniques and/or compositions employed herein.

!L Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are now described.

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

Following long-standing patent law convention, the terms "a", "an", and

"the" refer to "one or more" when used in this application, including the claims.

Thus, for example, reference to "a phage" includes a plurality of such phage, and so forth.

Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term

"about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter. As used herein, the term "about," when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, in some embodiments ±0.1 %, and in some embodiments ±0.1 % from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

The terms "CAT" and "CLAT" are herein used interchangeably.

As used herein, the phrase "indicator(s) of the presence of bacteria, viruses, or other microbes" refers to any material that can be assayed and is indicative of the presence in a sample of a bacterium, a virus, or any other microbe or microorganism for which knowledge of its presence in a sample would be desirable. Representative, non-limiting indicators of the presence of a bacterium, a virus, or another microbe include the physical bacterium, virus, or microbe itself and a fragment of a bacterium, virus, or microbe (e.g., an immunogenic fragment such as an epitope, which in some embodiments can be specific for a particular species, class, and/or type of bacterium, virus, or microbe). Other indicators include, but are not limited to entities that are typically found in the presence of a bacterium, virus, or microbe, such as a bacteriophage that is generally not detectable in a sample if the bacterium that serves as its host is not also present in the sample. As such, in some embodiments a bacteriophage can be used as a proxy for its host bacterium, and detection of the bacteriophage in a sample serves as a positive indicator of the presence of the host bacterium in the sample. As used herein, the phrase "species, classes, and/ortypes" refers to any classification scheme and is not to be interpreted narrowly as referring to a taxonomic meaning for these terms. Accordingly, a "species" of a bacteriophage, a binding molecule, an antibody, etc. refers to any such bacteriophage, binding molecule, antibody, etc., classified in whatever grouping is convenient.

The term "antibody" includes both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass or combination thereof, including human (including CDR-grafted antibodies), humanized, chimeric, multi-specific, monoclonal, polyclonal, and oligomers thereof, irrespective of whether such antibodies are produced, in whole or in part, via immunization, through recombinant technology, by way of in vitro synthetic means, or otherwise. Thus, the term "antibody" includes those that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transfected to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA sequences. Such antibodies have variable and constant regions derived from germline immunoglobulin sequences of two distinct species of animals. In certain embodiments, however, such antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human immunoglobulin sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and V_L regions of the antibodies are sequences that, while derived from and related to the germline V_H and VL sequences of a particular species (e.g., human), may not naturally exist within that species' antibody germline repertoire in vivo.

In some embodiments, a whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding region thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V_H) and a heavy chain constant region, comprised of three domains (abbreviated herein as CH 1 , CH2, and CH3). Each light chain is comprised of a light chain variable region (abbreviated herein as V_L) and a light chain constant region, comprised of one domain (abbreviated herein as C_L). The V_H and V_L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_H and V_L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system {e.g., effector cells) and the first component (C 1q) of the classical complement system. An amino acid sequence which is substantially the same as a heavy or light chain CDR exhibits a considerable amount or extent of sequence identity when compared to a reference sequence and contributes favorably to specific binding of an antigen bound specifically by an antibody having the reference sequence. Such identity is definitively known or recognizable as representing the amino acid sequence of the particular human monoclonal antibody. Substantially the same heavy and light chain CDR amino acid sequence can have, for example, minor modifications or conservative substitutions of amino acids so long as the ability to bind a particular antigen is maintained. The term "human monoclonal antibody" is intended to include a monoclonal antibody with substantially human CDR amino acid sequences produced, for example, by recombinant methods, by lymphocytes or by hybridoma cells.

The term "antigen-binding region" of an antibody means one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a phage antigen) that is specifically bound by a reference antibody, as disclosed herein. An "antigen-binding regions" of an antibody can include, for example, polypeptides comprising individual heavy or light chains and fragments thereof, such as V_Ll V_H, and Fd regions; monovalent fragments, such as Fv, Fab, and Fab¹ regions; bivalent fragments such as F(ab')₂; single chain antibodies, such as single chain Fv (scFv) regions; Fc fragments; diabodies; Fd (including the V_H and CH1 domains), maxibodies (bivalent scFV fused to the amino terminus of the Fc (CH2-CH3 domains) of IgGI) and complementarity determining region (CDR) domains. Such terms are described, for example, in Harlow & Lane, 1988; Myers, 1993; Pluckthun & Skerra, 1989; and in Day, 1990, each of which is incorporated herein by reference.

The term "antigen-binding region" also includes, for example, fragments produced by protease digestion or reduction of a human monoclonal antibody and by recombinant DNA methods known to those skilled in the art. One skilled in the art knows that the exact boundaries of a fragment of a human monoclonal antibody can be variable, so long as the fragment maintains a functional activity. Using well-known recombinant methods, one skilled in the art can engineer a nucleic acid to express a functional fragment with any endpoints desired for a particular application.

Furthermore, although the two domains of the Fv fragment, V_L and V_H, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V_L and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988; and Huston et al., 1988). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding region" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Such fragments include those obtained by amino-terminal and/or carboxy-terminal deletions, but where the remaining amino acid sequence is substantially identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full-length cDNA sequence.

Antigen binding regions also include fragments of an antibody which retain at least one (e.g. , 1 , 2, 3 or more) heavy chain sequences and/or at least one (e.g., 1 , 2, 3 or more) light chain sequences for a particular complementarity determining region (CDR) (i.e., at least one or more of CDR1 , CDR2, and/or CDR3 from the heavy and/or light chain). Fusions of CDR containing sequences to an Fc region (or a constant heavy 2 (CH2) or constant heavy 3 (CH3) containing region thereof) are included within the scope of this definition including, for example, scFV fused, directly or indirectly, to an Fc are included herein. An antigen binding region is inclusive of, but not limited to, those derived from an antibody or fragment thereof (e.g., by enzymatic digestion or reduction of disulfide bonds), produced synthetically using recombinant methods (e.g., transfectomas), created via in vitro synthetic means (e.g., Merrifield resins), combinations thereof, or through other methods. Antigen-binding regions may also comprise multiple fragments, such as CDR fragments, linked together synthetically, chemically, or otherwise, in the form of oligomers.

The term "V_L fragment" refers to a fragment of the light chain of a monoclonal antibody that includes all or part of the light chain variable region, including the CDRs. A VL fragment can further include light chain constant region sequences.

The term "Fd fragment" refers to a fragment of the heavy chain of a monoclonal antibody which includes all or part of the V_H heavy chain variable region, including the CDRS. An Fd fragment can further include CH1 heavy chain constant region sequences.

The term "Fv fragment" refers to a monovalent antigen-binding fragment of a monoclonal antibody, including all or part of the variable regions of the heavy and light chains, and absent of the constant regions of the heavy and light chains. The variable regions of the heavy and light chains include, for example, the CDRs. For example, an Fv fragment includes all or part of the amino terminal variable region of about 1 10 amino acids of both the heavy and light chains.

The term "Fab fragment" refers to a monovalent antigen-binding fragment of an antibody consisting of the V_L, V_H, C_L, and CH1 domains, which is larger than an Fv fragment. For example, an Fab fragment includes the variable regions, and all or part of the first constant domain of the heavy and light chains. Thus, a Fab fragment additionally includes, for example, amino acid residues from about 1 10 to about 220 of the heavy and light chains. The term "Fab¹ fragment" refers to a monovalent antigen-binding fragment of a monoclonal antibody that is larger than a Fab fragment. For example, a Fab¹ fragment includes all of the light chain, all of the variable region of the heavy chain, and all or part of the first and second constant domains of the heavy chain. For example, a Fab' fragment can additionally include some or all of amino acid residues 220 to 330 of the heavy chain.

The term "F(ab')₂ fragment" refers to a bivalent antigen-binding fragment of a monoclonal antibody comprising two Fab fragments linked by a disulfide bridge at the hinge region. An F(ab')₂ fragment includes, for example, all or part of the variable regions of two heavy chains and two light chains, and can further include all or part of the first constant domains of two heavy chains and two light chains.

The term "dAb fragment" refers to a fragment consisting of the VH domain, as described by Ward et al., 1989.

The term "CDR" refers to the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat ef a/., 1983; Chothia et al., 1987; and by MacCallum et al., 1996, each of which is incorporated herein by reference, where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or functional fragment thereof is intended to be within the scope of the term as defined and used herein. The exact amino acid residue numbers which encompass a particular CDR will vary depending on the structure of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody. Those skilled in the art can compare two or more antibody sequences by defining regions or individual amino acid positions of the respective sequences with the same CDR definition. The term "isolated" in the context of an antibody refers to separated from one or more compound that is found with the antibody or polypeptide in nature or in a synthetic reaction used to produce the antibody including, for example, a reagent, precursor or other reaction product, and preferably substantially free from any other contaminating mammalian polypeptides that would interfere with its therapeutic or diagnostic use. An isolated agent also includes a substantially pure agent. The term can include naturally occurring molecules such as products of biosynthetic reactions or synthetic molecules. An antibody is also considered "isolated", for example, when it is substantially free of other antibodies having different antigenic specificities. Also, a substance is "isolated" if it is bound or conjugated to a polypeptide or other substance to which it is not bound in nature.

The term "substantially pure" refers to a substance that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition) and comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, or alternatively more than about 85%, 90%, 95%, and 99%. A substance is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Similarly, a substance is "isolated" if in the course of manufacture or formulation it is "isolated" or "substantially pure" as described above, and then combined with other agents in a well-defined composition, notwithstanding the substance in the well-defined composition is not the predominant species present.

As used herein, the terms "specifically binds" and "specific binding" mean that a compound preferentially or selectively recognizes and binds to a mature, full-length or partial-length epitope of a bacteriophage, or an ortholog thereof, such that its affinity (as determined by, e.g., Affinity ELISA, BIAcore, or other assays) or its neutralization capability (as determined by e.g., Neutralization ELISA assays or similar assays) is in some embodiments at least 10 times as great, in some embodiments 50 times as great, in some embodiments 100 as great, in some embodiments 250 as great, in some embodiments 500 times as great, or in some embodiments at least 1000 times as great as the affinity or neutralization capability of the same for any other polypeptide, wherein the peptide portion of the peptibody is first fused to a human Fc moiety for evaluation in such assay. Typically, the antibody binds with an affinity of at least about 1 X 10⁷M^"1, and binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. As used herein, an antibody "recognizing" or "specific for" an antigen is considered equivalent to "binding specifically" to an antigen.

The term "epitope" refers to that portion of any molecule capable of being recognized by and bound by a specific binding agent, e.g., an antibody, at one or more of the binding agent's antigen binding regions. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An antibody is considered to specifically bind an antigen when the dissociation constant is in some embodiments less than or equal to about 1 μM, in some embodiments less than or equal to about 100 nM, and in some embodiments less than 10 nM. The antibodies and antigen-binding regions of such antibodies of the presently disclosed subject matter include antibodies and antigen-binding regions thereof that are generated using the epitopic determinants defined herein, or that are generated using epitopic determinants having substantial identity to the epitopic determinants defined herein. In this context, the term "substantial identity" means that the sequences share sufficient identity that an antibody that binds to the modified epitopic determinant competitively inhibits binding of an antibody to the epitopic determinants described herein.

The antibodies encompassed by the presently disclosed subject matter include, but are not limited to IgG, IgA, IgGi_-4, IgE, IgM, and IgD antibodies, e.g., IgG_1K, or lgG_u isotypes, or lgG_4κ or lgG_u isotypes. Methods of using the antibodies of the presently disclosed subject matter to detect the presence of a target microbe (e.g., a bacteriophage), either in vitro or in vivo, are also encompassed by the presently disclosed subject matter.

The antibodies and antigen binding regions of the presently disclosed subject matter can be constructed by any number of different methods, including but not limited to via immunization of animals (e.g., with a microbial antigen that elicits the production of antibodies that specifically bind to a microbe that expresses the antigen); via hybridomas (e.g., employing B-cells from transgenic or non-transgenic animals); via recombinant methods (e.g., CHO transfectomas; see Morrison, 1985), or via in vitro synthetic means (e.g., solid-phase polypeptide synthesis).

Mouse splenocytes can be isolated and fused with PEG to a mouse myeloma cell line based upon standard protocols. The resulting hybridomas are then screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice are fused to one-sixth the number of P3x63-Ag8.653 non-secreting mouse myeloma cells (Catalogue No. CRL 1580 from the American Type Culture Collection (ATCC®)) with 50% PEG. Cells are plated at approximately 2 X 10⁵ in flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and 1X HAT (Sigma Chemical Co., St. Louis, Missouri, United States of America); the HAT is added 24 hours after the fusion). After two weeks, cells are cultured in medium in which the HAT is replaced with HT. Individual wells are then screened by ELISA using relevant antigens to identify appropriate monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, medium is observed usually after 10-14 days. The antibody secreting hybhdomas are replated, screened again, and if still positive for desired monoclonal antibodies, can be subcloned at least twice by limiting dilution. The stable subclones are then cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.

IiL Representative Embodiments

In some embodiments, the presently disclosed subject matter provides compositions that can be employed for testing of and/or for direct or indirect identification of indicators of the presence of bacteria, viruses, or other microbes in any sample for which such testing and/or identification is desired. In some embodiments, the compositions comprise two primary components: a detection particle and one or more binding molecules complexed thereto.

With respect to the detection particle, any solid support can be employed. In some embodiments, a detection particle is one that is small enough that it can be carried conveniently and can be employed without the need for complex laboratory equipment. In some embodiments, a detection particle is a bead or other small item to which binding molecules can be attached. An exemplary bead is a polystyrene bead such as the 0.29 μm diameter polystyrene particles sold under the registered trademark OPTIBIND® by Seradyn Inc., of Indianapolis, Indiana, United States of America.

The second component of the composition is a binding molecule that is complexed to the detection particle. Binding molecules can be any type of molecule that can bind specifically to a particular target. Thus, the term "binding molecule" as used herein refers to a molecule or other chemical entity having a capacity for binding to a target. The binding molecules of the presently disclosed subject matter include ligands that comprise, for example, a peptide, an oligomer, a nucleic acid (e.g., an aptamer), a small molecule (e.g., a chemical compound), an antibody or fragment or derivative thereof, a nucleic acid-protein fusion, and/or any other affinity agent or combination thereof.

In some embodiments, a binding molecule is an antibody or a fragment or derivative thereof. Fragments and derivatives of antibodies that retain the ability to bind to targets are known, and include, but are not limited to whole immunoglobulin molecules, scFv antibodies, chimeric antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, and combinations thereof. Methods for producing these binding molecules are also known in the art (see e.g., Harlow & Lane, 1988; Harlow & Lane, 1999).

The choice of binding molecules can be made based on the nature of the molecule to which the binding molecules are to be bound (e.g., the nature of the indicators of the presence of bacteria, viruses, or other microbes in a sample). In some embodiments, the compositions of the presently disclosed subject matter are designed to bind to a specific indicator (e.g., a specific species, class, and/or type of bacteriophage), and the binding molecules are identified by assaying one or more potential binding molecules for specific binding to the indicator.

In some embodiments, a binding molecule is identified from a library of potential binding molecules. Representative libraries include but are not limited to a peptide library (U.S. Patent Nos. 6,156,511 , 6,107,059, 5,922,545, and 5,223,409), an oligomer library (U.S. Patent Nos. 5,650,489 and 5,858,670), an aptamer library (U.S. Patent No. 6,180,348 and 5,756,291), a small molecule library (U.S. Patent Nos. 6,168,912 and 5,738,996), a library of antibodies or antibody fragments (for example, an scFv library or an Fab antibody library; U.S. Patent Nos. 6,174,708, 6,057,098, 5,922,254, 5,840,479, 5,780,225, 5,702,892, and 5,667988), a library of nucleic acid-protein fusions (U.S. Patent No. 6,214,553), and a library of any other affinity agent that can potentially bind to an indicator of the presence of bacteria, viruses, or other microbes in a sample (e.g., U.S. Patent Nos. 5,948,635, 5,747,334, and 5,498,538). In one embodiment, a library is a phage-displayed antibody library. In another embodiment, a library is a phage-displayed scFv library. In another embodiment, a library is a phage-displayed Fab library. In still another embodiment, a library is a soluble scFv antibody library. The molecules of a library can be produced in vitro, or they can be synthesized in vivo, for example by expression of a molecule in vivo. Also, the molecules of a library can be displayed on any relevant support, for example, on bacterial pili (Lu et a!., 1995) or on phage (Smith, 1985).

Once identified, one or more of the binding molecules are complexed with the detection particles. This can be accomplished by any suitable technique. The choice of how to complex the binding molecules to the detection particles can consider inter alia the nature of the detection particles and of the binding molecules themselves. An exemplary technique for which is disclosed in EXAMPLE 3. Additionally, the nature of the complexes between the detection particles and the binding molecules can be varied as desired. For example, in some embodiments only a single specificity of binding molecule (i.e., a single antibody or fragment or derivative thereof, which binds to a single epitope) is bound to each unit of the detection particle. For example, a single specificity of a binding molecule such as an antibody or fragment or derivative thereof can be bound to a plurality of polystyrene beads, although each bead would be expected have more than one copies of the antibody or the fragment or derivative thereof bound to it. The plurality of beads with the single specificity of binding molecule could then be employed in the methods of the presently disclosed subject matter as is, or they could be mixed with one or more other pluralities of beads with different specificities of binding molecules for use in the disclosed methods. Alternatively or in addition, more than one specificity of binding molecule can be mixed prior to complexing the binding molecules with the detection particles such that each individual detection particle (e.g., each bead) comprises more than one specificity of binding molecule. In particular, in some embodiments of the presently disclosed subject matter the methods employ compositions with more than one specificity of binding molecule either in order to create crosslinking of the detection particle units in the event that different detection particle units bind to different targets on the same indicator individual (see Figure 1 for a depiction of the crosslinking). In some embodiments, compositions are provided for detecting one or more species, classes, and/or types of bacteriophage present in a sample, wherein the composition comprises a plurality of detection particles, and further wherein the plurality of detection particles comprise one or more binding molecules complexed therewith; and the one or more binding molecules each specifically binds to one or more species of bacteriophage. In some embodiments, the one or more species of bacteriophage includes an F⁺ coliphage, preferably an F⁺ RNA-containing coliphage or an F⁺ DNA-containing coliphage. In some embodiments, the F⁺ RNA-containing coliphage include

Group I coliphage, preferably MS2-like coliphage; Group Il coliphage, preferably GA-like coliphage; Group III coliphage, preferably QB-like coliphage; Group IV coliphage, preferably SP-like or Fl-like coliphage; and M11-like coliphage. In some embodiments, the F⁺ DNA-containing coliphage include Fd, F1 , M13, Φ15, Φ16, and Φ18, and coliphage that are taxonomically, genetically, and/or antigenically related to one or more of Fd, F1 , M13, Φ15, Φ16, and Φ18.

In some embodiments, the presently disclosed subject matter provide methods for testing of and/or for direct or indirect identification of indicators of the presence of bacteria, viruses, or other microbes in any sample for which such testing and/or identification is desired. In some embodiments, the methods comprise contacting a sample, or a portion thereof or a culture thereof, with a composition of the presently disclosed subject matter under conditions sufficient to specifically bind at least one of the one or more binding molecules to at least one of the one or more species, classes, and/or types of bacteriophage, if present, to form a complex. The complex is then detected, and the presence of the complex is indicative of the presence of species, classes, and/or types of bacteriophage or other indicator.

As used herein, the phrase "conditions sufficient to specifically bind at least one of the one or more binding molecules to at least one of the one or more species, classes, and/or types of microbes, if present, to form a complex" refers to any conditions that allow specific binding of one or more of the binding molecules to an indicator of the presence of bacteria, viruses, or other microbes in a sample to an extent that the complex can be detected. Additionally, any suitable detection method can be employed. In some embodiments, the detection method is a visual detection of an agglutination or clumping of antibody-coated beads that can occur when a target indicator is present in the sample. This visual detection can be facilitated using any available method including, but not limited to shining light through a transparent or translucent vessel in which the method is carried out or placing a drop of the sample or culture on a cardboard card and adding a drop of the binding molecule/detection particle complex.

In some embodiments of the presently disclosed subject matter, methods are provided of serotyping one or more species of bacteriophage present in a sample, comprising, contacting the sample containing the one or more species of bacteriophage with a plurality of detection particles comprising an antibody complexed therewith, which is specific for an antigen on one or more of the species of bacteriophage, under conditions sufficient to allow for a binding complex to occur between the bacteriophage and the antibody; detecting the complex formed; and assigning a serotype to the one or more species of bacteriophage based on the detection of the complex between the bacteriophage and the bacteriophage antigen-specific antibody.

In some embodiments, the detecting the complex formed is through visualization by the naked eye. In some embodiments, the complex that is visible to the naked eye forms in less than 60 seconds at 25°C. In some embodiments, the sample, or a portion thereof, is first cultured, preferably for no more than about two to three hours, under conditions sufficient to amplify the one or more species of bacteriophage to produce a culture, and the culture is employed as the sample in the contacting step. In some embodiments, the one or more species of bacteriophage are F+ coliphage.

In some embodiments, the antibody is a polyclonal antiserum against F+ coliphage. In some embodiments, the antiserum is rabbit antiserum. In some embodiments, the F+ coliphage is F+ RNA coliphage selected from the group consisting of Group I coliphage, preferably MS2-like coliphage; Group Il coliphage, preferably GA-like coliphage; Group III coliphage, preferably Qβ-like coliphage; Group IV coliphage, preferably SP-like or Fl-like coliphage; and M11-like coliphage. In some embodiments, the F+ coliphage is F+ DNA coliphage selected from the group consisting of Fd, F1 , M13, Φ15, Φ16, and Φ18, and coliphage that are taxonomically, genetically, and/or antigenically related to one or more of Fd, F1 , M13, Φ15, Φ16, and Φ18.

The presently disclosed subject matter relates to the testing of samples for the presence of microbes and/or microorganisms. The nature of the sample to be tested is not limiting to the practice of the presently disclosed subject matter. Representative samples include, but are not limited to drinking water samples, ambient water samples, groundwater samples, irrigation water samples, sewage samples, biosolid samples, manure samples, soil or sediment samples, produce samples, meat samples, shellfish samples, treated samples thereof, combinations thereof, and cultures of one or more of the these samples, wherein the culture comprises some or all of one or more of the enumerated samples grown under conditions sufficient to support the growth of a bacteriophage, if present, in the sample.

In some embodiments, the presently disclosed methods further comprise culturing the sample, or a portion thereof, under conditions sufficient to amplify the one or more species of bacteriophage if present in the sample to produce a culture, and employing the culture as the sample in the contacting step. EXAMPLE 1 at the section titled "Rapid F+ coliphage culture" and EXAMPLE 2 for representative embodiments of culturing methods. In some embodiments, the culturing is performed for a short period of time (e.g., for not more than about two to three hours).

In some embodiments, kits which comprise the detection particles of the presently disclosed subject matter are provided for detecting the presence of and/or serotyping the one or more species of bacteriophage present in a sample. In some embodiments, the kits are field-portable. The field-portable applications necessitate the use of simple methods, robust but nonsterile techniques, and inexpensive and stable detection materials. In some embodiments the culturing of the sample, or a portion thereof, under conditions sufficient to amplify the one or more species of bacteriophage is performed in a field-portable water bath. In some embodiments, the field-portable water bath comprises an insulated cooler, an aquarium heater, and a deep-cycle marine battery to create an inexpensive 35°C water bath that can run for more than 5 hours.

EXAMPLES

The following Examples have been included to provide illustrations of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the spirit and scope of the presently disclosed subject matter.

EXAMPLE 1

Materials and Methods

Virus strains, bacterial hosts, and environmental F+ coliphage isolates. F⁺ RNA coliphage prototype strains MS2 (serogroup I), GA (serogroup II), Qβ (serogroup III), M11 (serogroup III), SP (serogroup IV), and Fl (serogroup IV) and F⁺ DNA coliphage prototype strains Fd, F1 , and M13 were used as positive controls. F⁺ coliphage field isolates were recovered from samples of shellfish tissue, water, and bird feces at estuaries in Florida, North Carolina, Delaware, New Hampshire, Massachusetts, Rhode Island, and California by previously described methods (see Sobsey era/., 1990; U.S. EPA, 2001a, b), using a permissive E. coli F_amp host (American Type Culture Collection (ATCC®) Catalogue No. 700891). F⁺ coliphage isolates were enriched under conditions described in EPA Method 1601 (U.S. EPA, 2001a), using liquid culture to promote high phage titers. Enriched material was clarified by centrifugation at 1 ,200 x g for 15 minutes, and the resulting supernatant was frozen at -80⁰C in tryptic soy broth (TSB).

Rapid F+ coliphage culture. A 180 minute F⁺ coliphage culture enrichment was developed as a modified version of the 16 to 24 hour culture step of EPA method 1601 (U.S. EPA, 2001a). Rapid F⁺ coliphage culture conditions differed from those in EPA method 1601 by the use of an optimized initial log-phase host concentration of 1 x 10⁷ CFU E. coli F_amp per ml of culture and lasted 2 to 3 hours in a 35 to 37°C water bath, at which time host bacteria entered stationary-phase growth. Rapid F⁺ coliphage enrichments were compared for prototype F⁺ RNA coliphages (MS2, Qβ, SP, and Fl) by inoculating 1 to 3 PFU into 333-ml broth cultures and tracking bacterial and coliphage levels at times throughout the culture period (0, 30, 60, 90, 120, 180, and 360 min). F⁺ RNA coliphages were quantified on tryptic soy agar (TSA) spot plates containing host E. coli F_amp lawns, and E. coli was quantified before and after log-phase growth on TSA plates and during log-phase growth by measuring the optical density at 520 nm in a spectrophotometer (Spectronic

1201 ; Milton Roy Company, Ivyland, Pennsylvania, United States of America).

RNase test for detecting F+ RNA and F+ DNA coliphages. F+ coliphage field isolates were replated with and without RNase (RNase A; Sigma-Aldrich, St. Louis, Missouri, United States of America) to distinguish viral nucleic acid content as DNA or RNA (23). RNase infectivity neutralization tests were performed on spot plates of 0.75% TSA containing the log-phase E. coli

Famp host, streptomycin, ampicillin (each at 15 μg/ml), and RNase (100 μg/ml).

F+ coliphage genogrouping. F+ coliphage isolates were also subjected to molecular typing to distinguish the four groups of F+ RNA coliphages (groups I, II, III, and IV) by RT-PCR of the replicase gene and to F+ DNA coliphage analysis by PCR (47). A reverse line blot (RLB) hybridization assay was performed as previously described (47) to confirm the RT-PCR-amplified products. A new RT-PCR assay was developed to amplify the levivirus capsid region. Capsid region amplification used previously reported reaction conditions (47), with the modification of the RT and annealing steps being increased to a temperature of 50⁰C. F+ RNA and F+ DNA coliphage field isolates were also genogrouped by nucleic acid sequencing (UNC Nucleic Acids Core Facility, Chapel Hill, North Carolina, United States of America). Sequences were aligned using free software (Bioedit and Chromas Lite v. 2.0) (18), and phylogenetic trees were created using Jukes and Cantor distance estimation and 100 bootstrap values (TreeCon v. 1.3b) (58). Rabbit antiserum production and collection. To generate polyclonal antibodies against F⁺ coliphages, New Zealand White rabbits were given intradermal inoculations with each F⁺ RNA coliphage group (group I, MS2; group II, GA; group III, Qβ; and group IV, SP and Fl) and with F⁺ DNA coliphages (Fd, F1 , M13, Φ15, Φ16, and Φ18). Initial virus inocula and a 1- month booster had titers of 10¹⁰ to 10¹¹ PFU/ml and were partially purified and suspended in Freund's complete adjuvant. Antisera were collected from rabbits at 30, 45, 60, and/or 90 days post-immunization to obtain polyclonal rabbit immunoglobulins against coliphage antigens and were stored at -20⁰C. No purification was performed to separate immunoglobulin G (IgG) or other immunoglobulin classes or other serum constituents. Anti-MS2 serum had a protein concentration of about 3 mg/ml, while other serum protein levels were not measured.

Antiserum labeling of agglutinable particles. For the F+ coliphage latex agglutination and typing (CLAT) assay, polystyrene particles were first labeled with F+ coliphage antisera. A 1 % suspension of 0.29-μm-diameter polystyrene particles (OptiBind particles; Seradyn Inc., Indianapolis, Indiana, United States of America) was made from the commercial 10% stock solution of particles by dilution in either phosphatebuffered saline (PBS; 0.136 M sodium chloride, 2.68 mM potassium chloride, 0.88 mM monobasic potassium phosphate, 3.4 mM dibasic sodium phosphate, pH 7.2 and 8.2) or citrate phosphate (CP) buffer (1.36 mM citric acid, 7.28 mM dibasic sodium phosphate, pH 6.2). Rabbit antisera against F+ coliphages or PBS (negative control) was added in an equal volume to that of the polystyrene particles to the 1% polystyrene particle-buffer solution. The antibody-particlebuffer mixtures were agitated by being pipetted up and down for several seconds (not vortexed) and then rocked at 150 rpm on a rotary platform (Orbit shaker; Lab- Line Instruments, Melrose Park, Illinois, United States of America) for 1 hour at room temperature to facilitate hydrophobic adsorption of antibodies to particles. Samples were then microcentrifuged at 14,000 rpm (Eppendorf 5415C centrifuge; Brinkman Instruments, Westbury, New York, United States of America) for 5 minutes, and the unbound antiserum in the supernatant was decanted from the antiserum-labeled particles in the pellet. The pellet was resuspended by pipetting (not vortexing) to give a 1 % particle solution in either PBS-0.01 % bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, Missouri, United States of America) (pH 7.2 or 8.2) or CP-0.01 % BSA (pH 6.2) to match the original buffer. BSA was used to block unbound particle binding sites and to create a more stable solution for long-term storage. Labeled particles were stored at 4°C or used directly. Five F+ RNA coliphage antiserum-labeled particle suspensions (anti-MS2, anti-GA, anti-QB, anti-SP, and anti-Fi) and six F+ DNA coliphage antiserum-labeled particle suspensions (anti-Fd, anti-F1 , anti-M13, anti-Φ15, anti-Φ16, and anti-Φ18) were prepared.

F+ coliphage agglutination assay and optimization. Equal 2.5-μl volumes of antibody-labeled particles and coliphage enrichment cultures (or controls) were mixed on a black cardboard card (agglutination cards; Pro-Lab Diagnostics, Austin, Texas, United States of America) with a toothpick and then rocked by hand for 30 seconds. Coliphage positive samples showed agglutination within 30 to 60 seconds, as visualized by the naked eye, with particles clumping together due to antibodies on different particle-binding coliphages (Figure 1). Negative samples where no coliphages were detected appeared as a cloudy or "milky" liquid suspension of particles with no visible clumping. To determine the appropriate types and concentrations of antisera for F+ coliphage typing and detection, a diverse panel of 32 F+ RNA and F+ DNA coliphage field isolates (confirmed by nucleic acid sequencing of the replicase gene of F+ RNA coliphages and gene IV of F+ DNA coliphages) (47), prototype strains (F+ RNA coliphages MS2, GA, Qβ, SP, and Fi and F+ DNA coliphages F1 , Fd, and M13), and negative controls (unlabeled particles and bacterial host cultures in TSB) was tested. Optimization experiments used a "checkerboard" titration system with combinations of various amounts of antigen and antisera (serial twofold dilutions of antisera from 1 :4 to 1 :128) per sample. The lower detection limit of the CLAT assay was determined using 5-μl volumes of enriched F+ RNA and F+ DNA coliphage prototype strains and fivefold serial dilutions thereof.

Protein assay for antisera. A protein detection assay (BCA protein assay kit; Pierce, Rockford, Illinois, United States of America) was used according to the manufacturer's instructions to determine the levels of antisera adsorbed to polystyrene particles and in stocks of antisera. Briefly, the absorbance at 562 nm (by spectrophotometry) was used to generate an albumin standard curve, which was confirmed to have an r² value of 99% and then used for comparison to unknown samples. The amount of antiserum labeling particles was taken to be the initial amount of protein added to particles minus the amount of unbound protein in the supernatant after centhfugation.

Statistical methods. Proportions of coliphages detected by CLAT and

RLB hybridization assays were compared using a two-sided Ztestwith a preset significance level of 0.05, and P values are reported. The Kruskal-Wallis test, a nonparametric analysis of variance, and Dunn's multiple comparison test were used to compare more than two variables, including different buffers for antibody binding efficiency and antibody dilutions. Statistics were calculated in Excel and InStat (version 3.06; GraphPad Software Inc., San Diego, California United States of America).

EXAMPLE 2

Rapid F+ Coliphage Culture

A modified version of EPA method 1601 was used to rapidly enrich F+ RNA coliphage prototype strains in culture broths of host E. coli F_amp initially inoculated with 1 to 3 PFU of F+ RNA coliphage (MS2, Qβ, Sp, or Fi) and incubated at 35 to 37°C. Enrichment of these low levels of coliphage produced progeny coliphage at levels of 1.2 X 10⁵ to 5.3 X 10⁶ PFU/ml in 120 min and 4.3 X 10⁶ to 5.5 X 10⁸ PFU/ml in 180 min (Fig. 2). Rapid coliphage culture was achieved by increasing the concentration of the log-phase E. coli F_amp host in broth cultures from about 10⁴ CFU/ml in the overnight culture approach to as much as 10⁷ CFU/ml in the new rapid approach. E. coli F_am_p reached stationary-phase growth in 180 min, with levels of 7.7 X 10⁸ to 4.4 X 10⁹ CFU/ml (Figure 2). EXAMPLE 3

Efficiency of Adsorption of Antisera to Polystyrene Particles

Because the adsorption of immunoglobulins varies with the isoelectric points of the antibodies in sera, the pH of the adsorption buffer and the electrolyte content were varied by employing three buffer pH levels, 6.2, 7.2, and 8.2, at six antiserum dilutions (1 :4 to 1 :128) to examine their effects on anti-MS2 serum binding to polystyrene particles. Binding of antiserum to polystyrene particles was measured by a spectrophotometry protein detection assay. The saturation point for polystyrene particles with anti-MS2 serum was the 1 :32 dilution, with decreased binding efficiency both above and below this saturation point (Table 1). The highest binding efficiencies were in PBS at pH

7.2, with 106%±1 % and 100%±2% binding of antisera at 1 :64 and 1 :32 antiserum dilutions, respectively (Table 1). PBS at pH 7.2 was significantly better than CP buffer at pH 6.2 or PBS at pH 8.2 at promoting adsorption of anti- MS2 serum to particles, and significant differences were seen among the three pH buffers at antiserum dilutions of 1 :16 (P = 0.0265), 1 :32 (P = 0.0036), and 1 :64 (P = 0.0379) (Table 1). Subsequent antiserum binding assays used

PBS at pH 7.2 as the optimized adsorption buffer.

TABLE 1. Binding efficiency of diluted MS2 antiserum to polystyrene particles

Buffei % Binding efficiency foi antiseium dilution (mean ± SDf solution L :4 1 :8 I: :16* 1: 1:64* 1: 128

CP (pH 6.2) 35 ± 6 50 ± 4 76 H- 4* 90 ± 6 71 ± 3* 49 ± 9 PBS (pH 7.2) 40 ± 2 50 ± 3 71 ± 0.1 100 ± 2* 106 ± 1* PBS (pH 8.2) 38 ± 3 46 ± 4 54 ± 2* 74 ± 3* 78 ± 4 50 ± 2

" Data aic means of thiec replicates. *, statistically significant difference among all three variables in a column (foi asterisks in column headings) or between two variables within a column (for asterisks on individual values). Significance was set at an α level ol 0.05. b Avei age of two ieplicates with no standard deviation. EXAMPLE 4

Optimizing particle agglutination by F+ coliphages

A series of experiments explored and optimized CLAT with various types and concentrations of antisera in "checkerboard" assays, based on true- and false-positive and true- and false-negative agglutination with a diverse panel of

32 nucleic acid-sequenced F+ coliphage field isolates, F+ coliphage prototype strains, and negative controls. Optimal concentrations of antisera were selected to detect truly positive reactions in the F+ coliphage panel while minimizing nonspecific agglutination and falsepositive reactions. As shown in Table 2, CLAT detected and typed F+ RNA coliphage prototype strains into each of four serogroups and gave true negative results for other F+ RNA and F+ DNA coliphage prototype strains, with the exceptions of anti-Fi sera cross-reacting with F+ RNA coliphage strain Sp (Table 2). No agglutination occurred when CLAT was performed with negative controls of TSB alone and stationaryphase E. coli cultures in TSB (Table 2). For F+ DNA coliphage detection, CLAT could detect all F+ DNA coliphage reference strains but could not serotype F+ DNA coliphage field strains. Anti-Fd, anti-F1 , anti-M13, and anti-Φ16 sera reacted with the three F+ DNA coliphage prototype strains, while no F+ DNA coliphage antisera reacted with F+ RNA coliphages or negative controls (Table 2). Anti- M13 serum at the 1 :8 dilution was the most reactive antiserum and was the only antiserum to detect all 16 F+ DNA coliphage field strains. Anti-Φ15 and anti-Φ18 sera gave only weakly positive agglutination at the 1 :4 dilution and therefore were not pursued further.

TABLE 2. Reaction matrix for testing agglutination of antiserum-coated particles with F+ coliphage antigens

Presence of F+ coliphage anlisci luin-hbelcd pai tick's⁰

F+ colrphnge F+ RNA coliphage {antiserum dilution) F+ DNA < roliphage (i antiserum dilution) μrotot>pc strain υi cυntiυt MS2 GA 0|3 SV Fi Fd Fl MU ΦI5 Ψ16 Φ1S ( 1: 16) (1:32) (1:8) (1 : 16) (1: 16) (1:16) ( 1: 16) ( 1:8) ( 1:4) ( 1:1ft) ( 1:4)

F+ RNA coliplmgcs MS2 GA Oβ SP - Fi I ^{I I I} +

F+ DNΛ coliplmςcs Fd - _ + + + + + Fl - + + + +

M13 ^{I I I I} + + + + + + +

Negative contiols E. coli F₁₁-, in TSB - - ¹¹¹ - - - TSB -¹ +_ _ _ _ _ a Checkerboaid titration of nntiseimn-labeled paiticles (antiserum dilutions of 1:4. 1:8. 1:16, l:M. 1:64. and 1: 128) to empiiicattv deteimine optimi dilutions for detecting and typing a diverse panef of M nucleic aoid-scqucnccd F+ RNA and F-H DNA cotiphage field isolates, S reference sπains, and ncgai The table gives a summary of agglutinat ion results with F+ coliphage reference strains and controls ai optimum antibody dilutions. +, arMtserum-labeied p detected; ~, no particles were detected.

EXAMPLE 5

Lower detection limit of CLAT for F+ coliphaqes The detection limit of the CLAT assay was determined using F+ coliphage prototype strains cultured overnight by EPA method 1601. Samples were scored as positive or negative by the presence of agglutination. The detection limit for F+ RNA coliphages ranged from 5 X 10³ to ^{I I I I I} 1 X 10⁵ PFU depending upon the antiserum used (Table 3). The detection limit for F+ DNA

^{I I I I !} coliphages was 1 X 10⁶ to 5 X 10⁶ PFU (Table 3). I ^{I 1 I I} TABLE 3. Lower detection limits for F+ coliphage prototype strains determined using antiserum-labeled polystyrene particles

Detection of V-i- coliphage antiseiuni-labelcϋ particles*

F+ coliphuge prototype

F+ RNΛ coliphage (antiscuim F+ DNA col strain count (I¹FU)" dilution) (antiserum di

MS2 ( 1: 16) CA ( 1:32) OU (1:8) SP ( 1:16) Fi ( 1:16) MU ( 1:8)

₅ X 10⁷ ND ND ND ND ND +

1 X 10⁷ ND NO ND ND ND

5 X 10* ND ND ND + + +

1 X 10« ND ND + + + _

5 X 10' + ND + + + __

I x K)⁵ + ND + + + __

5 X 10⁴ — + — _ + _

1 X IO⁴ — + _ _ _

5 X IO³ _ +

1 X 10^} — _ — _ _

5 X IO² - - - - - -

* F F coliphage piototype stiains (MS2. GA, Oβ. SP. Fi. M 13, and Fd) weic tested against lhcii coπespunding nntiscra. > +, antiserum-labeied particles were detected; — , no particles were detected; ND, not done. EXAMPLE 6

Application of CLAT to serotype F+ RNA coliphaqe field isolates

A diverse panel of F+ RNA and DNA colfphage field isolates were recovered from shellfish and water at 10 estuaries on the East, West, and Gulf Coasts of the United States by lysis zone isolation and overnight reenrichment culture by EPA method 1601. These coliphage isolates were assayed by both

CLAT and (for F+ RNA coliphages) RT-PCR amplification with RLB hybridization genotyping (47). Of the 192 F+ RNA coliphage field isolates tested, CLAT correctly serotyped 185 and RLB hybridization correctly genotyped 177. CLAT and RLB hybridization typed the same number of group I isolates, but CLAT typed significantly more group Il isolates than did RLB hybridization (P = 0.006) (Table 4). RLB hybridization typed 15 more F+ RNA coliphage group III isolates and 4 more F+ RNA coliphage group IV isolates than did CLAT, which were statistically significant differences (P < 0.0002). The false-negative rates were 4% for CLAT and 8% for RLB hybridization, which is a statistically significant difference (P < 0.0002). Both typing methods gave no false-positive results when challenged with 34 known F+ DNA coliphage field isolates (Table 4). Because CLAT serogrouping and RLB hybridization genogrouping provided different results for a small percentage of isolates, these differences were further explored by nucleic acid sequencing.

In this study, CLAT results were scored as positive or negative, while quantification was performed by most-probable-number culture enrichment where replicate volumes in dilution are scored as positive or negative. The presence of host bacteria in enrichment cultures did not adversely affect the detectability of F+ coliphages by CLAT. Coliphage-enriched water samples were analyzed by CLAT, which accurately detected and subtyped prototype F+ RNA coliphage strains into serogroups I, II, III, and IV and did not react with F+ DNA coliphage prototype strains or controls. Subgrouping of F+ RNA coliphages is useful for microbial source tracking (15, 17, 23, 35) but is not used routinely because it is time-consuming and more expensive than bacteriological analysis and requires scientific knowledge and technical skill. The presently disclosed subject matter improves access to F+ RNA coliphage detection and source tracking by making it easier, as affordable as bacteriological analysis, and rapid. In validation studies of the use of the F+ RNA coliphage CLAT assay for serotyping a large panel of F+ coliphage field strains, CLAT sensitivity was 96.4%, and its specificity was 100%. These findings are similar to those of previous F+ RNA coliphage characterization studies, where serotyping classified 99.5% of isolates (23), genotyping by probe hybridization classified 96.6% of isolates (23), and RT-PCR followed by probe hybridization correctly classified 97.8% of isolates (47). The presently disclosed CLAT assay had a similar performance and typing ability as an RT- PCR-probe hybridization assay (47), compared using the same panel of F+ coliphage field strains. Hsu et al. (23) also compared genotyping and serotyping outcomes by using a common isolate panel and arrived at a similar grouping outcome performance to that reported in this study.

EXAMPLE 7 Capsid nucleic acid sequencing of discordantly typed F+ RNA leviviruses

The observed inconsistencies between serogrouping and genogrouping results for 24 of 192 F+ RNA coliphage field strains shown in Table 4 were further analyzed by nucleotide sequencing of the capsid genomic region. The capsid regions of the 24 problematic F+ RNA coliphage field strains were RT- PCR amplified, sequenced, and arranged in a phylogenetic tree alongside the CLAT and RLB hybridization grouping results. Capsid sequence analysis showed 19 isolates clustered with F+ RNA coliphage group I at 90% sequence similarity, 17 of which were classified by CLAT into serogroups I and II. Five isolates clustered as F+ RNA coliphage group II, with slightly less than 90% sequence similarity, and CLAT serogrouping was in agreement for all five of these isolates.

TABLE 4. CLAT Assay Detection and Serotyping of F⁺ RNA Coliphage Field Isolates Compared to a Gold Standard Method

a CLAT assay uses: anti-MS2 sera at 1 :16 dilution for group I; anti-GA sera at 1 :32 dilution for group II; anti-Qβ sera at 1 :8 dilution for group III; and anti-Sp and anti- Fi sera both at 1 :16 dilution for group IV. Optimal antibody dilutions for the particle agglutination assay were determined empirically by checkerboard titration. b RLB = Reverse Line Blot hybridization (Vinje et al., 2004).

EXAMPLE 8

Application of CLAT to detect and type F+ DNA coliphage field isolates

A diverse panel of 164 F+ DNA coliphage field isolates and 132 F+ RNA coliphage field isolates were recovered from shellfish and water at 10 estuaries on the East, West, and Gulf Coasts of the United States by lysis zone isolation and overnight reenrichment culture by EPA method 1601. Subsequently, these coliphage isolates were assayed by both the CLAT assay and an RNase infectivity neutralization assay for F+ DNA coliphages. The RNase infectivity neutralization assay scored coliphages as having either RNA or DNA nucleic acid and was used as a standard for comparing CLAT results. The CLAT assay detected 161 of 164 F+ DNA coliphage field isolates (98%), which was not statistically different from the 164 detections (100%) of the RNase assay (P = 0.82) (Table 5). The CLAT assay failed to detect 3 of 164 F+ DNA coliphage isolates and gave false-positive detection of 3 of 132 F+ RNA coliphage field isolates (2%) (Table 5). The detection rate for F+ DNA coliphages with M13 antiserum-coated particles was 83%, which was improved to 98% detection by including a second level of screening of all negative samples with Fd antiserum particles. TABLE 5. CLAT Assay Detection of F⁺ DNA Coliphage Field Isolates Compared to a Gold Standard Method

a CLAT Assay for F+ DNA coliphage uses M13 antisera (1 :8 antibody dilution) and Fd antisera (1 :16 antibody dilution). b No significant difference exists between the proportion of true positive F+ DNA detected by the 2 methods (P value = 0.82).

The presently disclosed F+ DNA coliphage CLAT assay provides a simple, robust, rapid, and affordable means to facilitate detection of all F+ coliphages, regardless of whether or not F+ RNA coliphages are present. F+ DNA coliphages as fecal indicator viruses have been isolated from wastewater treatment plants and from swine, gull, and cattle waste (8). They have been found in higher proportions than F+ RNA coliphages in surface waters impacted by humans and animals during storm events than during background flows, in warmer waters (8), and in epidemiological-microbiological studies of illness risks from recreational use of water contaminated by nonpoint fecal sources (9). The CLAT detection limit was sufficiently low for both F+ RNA and DNA coliphages that they can be detected readily after enrichment of water samples for 2 to 3 hours. While the lower detection limit was lower for F+ RNA coliphages than for F+ DNA coliphages, this difference does not pose a problem for F+ DNA coliphage CLAT detection, because these coliphages enrich to 2- to 3-log10 higher levels than do F+ RNA coliphages. In the study disclosed herein, all but 3 of 164 F+ DNA coliphage field isolates were detected by the CLAT assay. No difference was observed in the speed or strength of agglutination for the small, icosahedral RNA coliphages compared to the long, rodshaped DNA coliphages tested, suggesting that virus morphology has little influence on CLAT detection.

REFERENCES The references listed below as well as all references cited in the specification are incorporated herein by reference to the extent that they supplement, explain, provide a background for or teach methodology, techniques and/or compounds employed herein.

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Claims

CLAIMSWhat is claimed is:

1 . A composition for detecting one or more species, classes, and/or types of bacteriophage present in a sample, wherein the composition comprises a plurality of detection particles, and further wherein:

(i) the plurality of detection particles comprises one or more binding molecules complexed therewith; and (ii) the one or more binding molecules each specifically binds to one or more species of bacteriophage.

2. The composition of claim 1 , wherein at least one of the one or more species of bacteriophage is an F⁺ coliphage, preferably an F⁺ RNA- containing coliphage or an F⁺ DNA-containing coliphage.

3. The composition of claim 2, wherein the F⁺ RNA-containing coliphage are selected from the group consisting of Group I coliphage, preferably

MS2-like coliphage; Group Il coliphage, preferably GA-like coliphage; Group III coliphage, preferably QB-like coliphage; Group IV coliphage, preferably SP-like or Fl-like coliphage; and M1 1-like coliphage.

4. The composition of claim 2, wherein the F⁺ DNA-containing coliphage are selected from the group consisting of Fd, F1 , M13, Φ15, Φ16, and

Φ18, and coliphage that are taxonomically, genetically, and/or antigenically related to one or more of Fd, F1 , M13, Φ15, Φ16, and Φ18.

5. The composition of claim 1 , wherein the plurality of detection particles comprises beads, preferably polystyrene beads, coated with the one or more binding molecules, preferably wherein the plurality of detection particles are present in the composition at about a 1 % w/v suspension in a neutral buffer, and the 1 % w/v suspension further comprises about 0.01 % w/v of a blocking agent, and also preferably wherein the blocking agent comprises bovine serum albumin (BSA).

6. The composition of claim 1 , wherein the one or more binding molecules comprises a plurality of antibodies, fragments thereof, derivatives thereof, or combinations thereof, preferably wherein at least one of the plurality of antibodies, fragments thereof, derivatives thereof, or combinations thereof is selected from the group consisting of a whole immunoglobulin molecule, an scFv antibody, a chimeric antibody, an Fab fragment, an Fab' fragment, an F(ab')₂ fragment, and combinations thereof.

7. The composition of claim 6, wherein at least one of the plurality of antibodies, fragments thereof, derivatives thereof, or combinations thereof binds to at least two different epitopes present on a single species or particle of bacteriophage or to at least two different epitopes present on different species or particles of bacteriophage.

8. The composition of claim 1 , wherein each member of the plurality of detection particles is complexed with a single species of binding molecule that binds a single ligand, preferably wherein the single species of binding molecule comprises a monoclonal antibody, or a fragment or derivative thereof, that binds to a single epitope present on at least one of the one or more species of bacteriophage.

9. The composition of claim 1 , wherein the plurality of detection particles comprises a mixture of detection particles comprising a plurality of species of binding molecules, preferably wherein the plurality of species of binding molecules comprises a polyclonal antiserum, a mixture of monoclonal antibodies raised against different bacteriophage epitopes, fragments or derivatives thereof, or combinations thereof.

10. The composition of claim 1 , wherein one or more members of the plurality of detection particles are complexed with a mixture of at least two different species of binding molecules.

1 1. The composition of claim 1 , wherein the sample is selected from the group consisting of a drinking water sample, an ambient water sample, a groundwater sample, an irrigation water sample, a sewage sample, a biosolid sample, a manure sample, a soil or sediment sample, a produce sample, a meat sample, a shellfish sample, one or more treated samples thereof, combinations thereof, and a culture of one or more of the these samples, wherein the culture comprises some or all of one or more of the enumerated samples grown under conditions sufficient to support the growth of a bacteriophage, if present, in the sample.

12. The composition of claim 1 , wherein the composition is stable at about 25°C for at least 3 months or at about 4°C for at least 6 months.

13. A method of detecting the presence of one or more species of bacteriophage present in a sample, the method comprising:

(a) contacting the sample with a composition comprising a plurality of detection particles comprising one or more binding molecules complexed therewith, each of which specifically binds to one or more species of bacteriophage, under conditions sufficient to specifically bind at least one of the one or more binding molecules to at least one of the one or more species of bacteriophage, if present, to form a complex, preferably a complex that is visible to the naked eye; and

(b) detecting the complex formed, whereby the presence of at least one of the one or more species of bacteriophage present in the sample is detected.

14. The method of claim 13, further comprising cultuhng the sample, or a portion thereof, preferably for no more than about two to three hours, under conditions sufficient to amplify the one or more species of bacteriophage if present in a sample to produce a culture, and employing the culture as the sample in the contacting step.

15. A method for detecting the presence of one or more species of bacteriophage present in a sample, the method comprising:

(a) contacting a sample with a composition comprising a plurality of detection particles, wherein: (i) the plurality of detection particles comprises one or more binding molecules complexed therewith; (ii) the one or more binding molecules each specifically binds to one or more species of bacteriophage; and (iii) the contacting is under conditions sufficient to bind at least one of the binding molecules to at least one of the one or more species of bacteriophage if present in the sample; and

(b) identifying a complex comprising one or more binding molecules and the at least one of the one or more species of bacteriophage, whereby the presence of one or more species of bacteriophage in the sample is detected.

16. The method of claim 15, wherein at least one of the one or more species of bacteriophage is an F⁺ coliphage preferably an F⁺ RNA- containing coliphage or an F⁺ DNA-containing coliphage.

17. The method of claim 16, wherein the F⁺ RNA-containing coliphage are selected from the group consisting of Group I coliphage, preferably MS2-like coliphage; Group Il coliphage, preferably GA-like coliphage; Group III coliphage, preferably Qβ-like coliphage; Group IV coliphage, preferably SP-like or Fl-like coliphage; and M1 1 -like coliphage.

18. The method of claim 16, wherein the F⁺ DNA-containing coliphage are selected from the group consisting of Fd, F1 , M13, Φ15, Φ16, and Φ18, and coliphage that are taxonomically, genetically, and/or antigenically related to one or more of Fd, F1 , M13, Φ15, Φ16, and Φ18.

19. The method of claim 15, wherein the plurality of detection particles comprises beads, preferably polystyrene beads, coated with the one or more binding molecules.

20. The method of claim 15, wherein the one or more binding molecules comprises a plurality of antibodies, fragments thereof, derivatives thereof, or combinations thereof, preferably wherein at least one of the members of the antibodies, fragments thereof, derivatives thereof, or combinations thereof is selected from the group consisting of a whole immunoglobulin molecule, an scFv antibody, a chimeric antibody, an Fab fragment, an Fab' fragment, an F(ab')₂ fragment, and combinations thereof.

21. The method of claim 20, wherein at least one of the plurality of antibodies, fragments thereof, derivatives thereof, or combinations thereof binds to at least two different epitopes present on a single species or particle of bacteriophage or binds to at least two different epitopes present on different species or particles of bacteriophage.

22. The method of claim 15, wherein at least one member of the plurality of detection particles is complexed with a single species of binding molecule that binds a single ligand, preferably a monoclonal antibody or a fragment or derivative thereof.

23. The method of claim 15, wherein at least one of the plurality of detection particle comprises a mixture of detection particles comprising a plurality of species of binding molecules, preferably wherein the plurality of species of binding molecules comprises a polyclonal antiserum, a mixture of monoclonal antibodies raised against different coliphage epitopes, or combinations thereof.

24. The method of claim 15, wherein at least one members of the plurality of detection particles are complexed with a mixture of at least two different species of binding molecules.

25. The method of claim 15, wherein the sample comprises a drinking water sample, an ambient water sample, a groundwater sample, an irrigation water sample, a sewage sample, a biosolid sample, a manure sample, a soil or sediment sample, a produce sample, a meat sample, a shellfish sample, treated samples thereof, combinations thereof, and a culture of one or more of the these samples, wherein the culture comprises the sample grown under conditions sufficient to support the growth of a bacteriophage, if present, in the sample.

26. The method of claim 15, wherein the complex formed is visible to the naked eye.

27. The method of claim 26, wherein the complex that is visible to the naked eye forms in less than 60 seconds at 25°C.

28. The method of claim 15, further comprising culturing the sample, or a portion thereof, preferably for no more than about two to three hours, under conditions sufficient to amplify the one or more species of bacteriophage if present in a sample to produce a culture, and employing the culture as the sample in the contacting step.

29. A method of serotyping one or more species of bacteriophage present in a sample, the method comprising:

(a) contacting the sample containing the one or more species of bacteriophage with a plurality of detection particles comprising an antibody complexed therewith, which is specific for an antigen on one or more of the species of bacteriophage, under conditions sufficient to allow for a binding complex to occur between the bacteriophage and the antibody; (b) detecting the complex formed; and

(c) assigning a serotype to the one or more species of bacteriophage based on the detection of the complex between the bacteriophage and the bacteriophage antigen-specific antibody.

30. The method of claim 29, wherein the detecting the complex formed is through visualization by the naked eye.

31 . The method of claim 30, wherein the complex that is visible to the naked eye forms in less than 60 seconds at 25°C.

32. The method of claim 29, further comprising culturing the sample, or a portion thereof, preferably for no more than about two to three hours, under conditions sufficient to amplify the one or more species of bacteriophage to produce a culture, and employing the culture as the sample in the contacting step.

33. The method of claim 29, wherein the one or more species of bacteriophage are F+ coliphage.

34. The method of claim 29, wherein the antibody is a polyclonal antiserum against F+ coliphage.

35. The method of claim 34, wherein the antiserum is rabbit antiserum.

36. The method of claim 34, wherein the F+ coliphage is F+ RNA coliphage selected from the group consisting of Group I coliphage, preferably MS2-like coliphage; Group Il coliphage, preferably GA-like coliphage; Group III coliphage, preferably Qβ-like coliphage; Group IV coliphage, preferably SP-like or Fl-like coliphage; and M11-like coliphage.

37. The method of claim 34, wherein the F+ coliphage is F+ DNA coliphage selected from the group consisting of Fd, F1 , M13, Φ15, Φ16, and Φ18, and coliphage that are taxonomically, genetically, and/or antigenically related to one or more of Fd, F1 , M13, Φ15, Φ16, and Φ18.

38. A kit for detecting the presence of one or more species of bacteriophage present in a sample, the kit comprising the detection particles of claim 1.