阅读说明：本技术 用于帽柱木碱的免疫测定 (Immunoassay for quebracho ) 是由 徐国平 I·纳沙希比 蔡蓬 C·拉曼 林英奇 L·安尼 A·普雷斯蒂贾科莫 于 2020-03-12 设计创作，主要内容包括：提供或引入帽柱木碱的衍生物尤其是作为半抗原和免疫原的组合物、方法、测定和试剂盒。(Compositions, methods, assays and kits are provided or introduced for derivatives of quebracho, particularly as haptens and immunogens.)

1. A quebracho derivative having the general structure:

wherein:

x is CH₂CO or CS;

y is an organic spacer-K-Q-, wherein K ═ N, NH, O, or C comprising a substituted or unsubstituted linear or branched, saturated or unsaturated alkylene moiety_(1-10)(ii) a And Q ═ comprises carboxylic acids or esters, carbonyl, aldehydes, amines or amino, amides, maleimides, halogenated carboxylic acids or esters, diThiopyridyl moiety, vinylsulfone moiety, and/or C of thiocarboxylic acid or ester thereof_(1-10)(ii) a And is

Z is a handle group selected from the group consisting of: a detectable label, an antigen carrier, a coupling agent, a terminal group, a protein, a lipoprotein, a glycoprotein, a polypeptide, a polysaccharide, a nucleic acid, a polynucleotide, a teichoic acid, a radioisotope, an enzyme fragment, an enzyme donor fragment, an enzyme acceptor fragment, an enzyme substrate, an enzyme inhibitor, a coenzyme, a fluorescent moiety, a phosphorescent moiety, an anti-stokes upregulating moiety, a chemiluminescent moiety, a luminescent moiety, a dye, a sensitizer, a particle, a microparticle, a magnetic particle, a solid support, a liposome, a ligand, an acceptor, and combinations thereof.

2. The derivative of claim 1, wherein Z is an antigen carrier selected from the group consisting of: bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, bovine gamma globulin, and combinations thereof.

3. The derivative of claim 1, wherein:

x is CO;

k is N or NH; and is

Q is C_(2-10)A carboxylic acid or ester thereof.

4. The derivative of claim 3, wherein Z is selected from the group consisting of: a protein, a lipoprotein, a glycoprotein, a polypeptide, an enzyme fragment, an enzyme donor fragment, an enzyme acceptor fragment, an enzyme substrate, a coenzyme, an antigen carrier selected from the group consisting of: bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, and bovine gamma globulin.

5. The derivative of claim 1, wherein:

x is CO;

k is N or NH; and is

Q is selected from the group consisting of: carboxylic acids or esters thereof, carbonyl, amine or amino, amides, maleimides, and combinations thereof.

6. An antibody produced by or against the derivative of claim 1, wherein the antibody has 100% cross-reactivity with cuprammine.

7. An immunoassay for quebracho comprising:

the antibody of claim 6; and

a conjugate formed from a mercaptoethylamine-HCl adduct of quebracho, the conjugate adapted to produce a measurable signal to indicate the presence of quebracho in a fluid sample.

8. A method of producing an antibody having 100% cross-reactivity with cuprammine, comprising injecting the derivative of claim 1 into a suitable antibody-producing animal.

9. A method of detecting the presence of a quebracho compound in a biological sample, the method comprising:

contacting a biological sample with the derivative of claim 1; and

contacting the biological sample with a second reagent comprising:

a second operative member configured to cooperate with the operative member to produce a detectable signal; and

an antibody raised against the derivative of claim 1, wherein the antibody is (i) conjugated to a second operational member and (ii) 100% cross-reactive with cuprammine,

wherein the presence of the quebracho compound in the biological sample is confirmed by detecting a difference between the measured amount of the detectable signal and the expected amount of the detectable signal, the quebracho compound competing with the quebracho derivative for binding to the antibody.

10. A kit for detecting the presence of a quebracho compound in a biological sample, the kit comprising:

(i) a first detection reagent and a second detection reagent, the first reagent comprising:

a first fluid comprising a first buffer; and

the derivative of claim 1 disposed in the first fluid,

the second detection reagent comprises:

a second fluid comprising a second buffer;

a second operative member configured to cooperate with the operative member to produce a detectable signal; and

(ii) an antibody raised against the derivative of claim 1, wherein the antibody is (i) conjugated to a second operational member and (ii) cross-reactive with cephalothin.

11. An antibody produced by or against an artificial derivative of cuprammonium having the general structure:

wherein:

x is CH₂CO or CS;

y is an organic spacer-K-Q-, wherein K ═ N, NH, O, or C comprising a substituted or unsubstituted linear or branched, saturated or unsaturated alkylene moiety_(1-10)(ii) a And Q ═ C comprising carboxylic acid or ester thereof, carbonyl, aldehyde, amine or amino, amide, maleimide, halocarboxylic acid or ester thereof, dithiopyridyl moiety, vinylsulfone moiety, and/or thiocarboxylic acid or ester thereof_(1-10)(ii) a And is

Z is an antigen carrier selected from the group consisting of: bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, bovine gamma globulin, and combinations thereof; and is

Wherein the antibody has 100% cross-reactivity with quebracho.

12. The antibody of claim 12, wherein:

x is CO;

k is N or NH; and is

Q is selected from the group consisting of: carboxylic acids or esters thereof, carbonyl, amine or amino, amides, maleimides, and combinations thereof.

13. An immunoassay for quebracho comprising:

(i) an antibody produced by or directed against an artificial derivative of cuprammine, said artificial derivative of cuprammine having the general structure:

wherein:

x is CH₂CO or CS;

y is an organic spacer-K-Q-, wherein K ═ N, NH, O, or C comprising a substituted or unsubstituted linear or branched, saturated or unsaturated alkylene moiety_(1-10)(ii) a And Q ═ C comprising carboxylic acid or ester thereof, carbonyl, aldehyde, amine or amino, amide, maleimide, halocarboxylic acid or ester thereof, dithiopyridyl moiety, vinylsulfone moiety, and/or thiocarboxylic acid or ester thereof_(1-10)(ii) a And is

Z is an antigen carrier selected from the group consisting of: bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, bovine gamma globulin, and combinations thereof, and

wherein the antibody has 100% cross-reactivity with quebracho; and

(ii) a conjugate formed from a mercaptoethylamine-HCl adduct of cyprocombine and capable of producing a measurable signal to indicate the presence of cyprocombine in a fluid sample.

14. The immunoassay of claim 13, wherein:

x is CO;

k is N or NH; and is

Q is selected from the group consisting of: carboxylic acids or esters thereof, carbonyl, amine or amino, amides, maleimides, and combinations thereof.

15. A method of detecting the presence of a quebracho compound in a biological sample, the method comprising:

contacting the biological sample with a first detection reagent and a second detection reagent, the first reagent comprising a quebracho derivative having the general structure:

wherein:

x is CH₂CO or CS;

y is an organic spacer-K-Q-, wherein K ═ N, NH, O, or C comprising a substituted or unsubstituted linear or branched, saturated or unsaturated alkylene moiety_(1-10)(ii) a And Q ═ C comprising carboxylic acid or ester thereof, carbonyl, aldehyde, amine or amino, amide, maleimide, halocarboxylic acid or ester thereof, dithiopyridyl moiety, vinylsulfone moiety, and/or thiocarboxylic acid or ester thereof_(1-10)(ii) a And is

Z is a member of the first operation,

the second reagent comprises:

a second operative member configured to cooperate with the first operative member to produce a detectable signal; and

an antibody raised against said cephalomannine derivative, wherein said antibody is (i) conjugated to said second manipulation member and (ii) has 100% cross-reactivity with cephalomannine,

wherein the presence of the quebracho compound in the biological sample is confirmed by detecting a difference between the measured amount of the detectable signal and the expected amount of the detectable signal, the quebracho compound competing with the quebracho derivative for binding to the antibody.

16. The method of claim 15, wherein:

x is CO;

k is N or NH; and is

Q is selected from the group consisting of: carboxylic acids or esters thereof, carbonyl, amine or amino, amides, maleimides, and combinations thereof.

17. A kit for detecting the presence of a quebracho compound in a biological sample, the kit comprising:

a first detection reagent and a second detection reagent, the first reagent comprising:

a first fluid comprising a first buffer; and

a cap post lignan derivative disposed in the first fluid, the cap post lignan derivative having the following general structure:

wherein:

x is CH₂CO or CS;

y is an organic spacer-K-Q-, wherein K ═ N, NH, O, or C comprising a substituted or unsubstituted linear or branched, saturated or unsaturated alkylene moiety_(1-10)(ii) a And Q ═ C comprising carboxylic acid or ester thereof, carbonyl, aldehyde, amine or amino, amide, maleimide, halocarboxylic acid or ester thereof, dithiopyridyl moiety, vinylsulfone moiety, and/or thiocarboxylic acid or ester thereof_(1-10)(ii) a And is

Z is a member of the first operation,

the second reagent comprises:

a second fluid comprising a second buffer;

a second operative member configured to cooperate with the first operative member to produce a detectable signal; and

an antibody raised against said cephalomannine derivative, wherein said antibody is (i) conjugated to said second manipulation member and (ii) cross-reactive with cephalomannine.

18. The kit of claim 12, wherein:

x is CO;

k is N or NH; and is

Q is selected from the group consisting of: carboxylic acids or esters thereof, carbonyl, amine or amino, amides, maleimides, and combinations thereof.

19. An immunogenic precursor derived from cephalomannine, said immunogenic precursor having the following structure:

20. an immunogenic precursor derived from cephalomannine, said immunogenic precursor having the following structure:

21. an immunogenic precursor derived from cephalomannine, said immunogenic precursor having the following structure:

I. Field of the invention

The present disclosure relates generally to compositions, kits, and methods for measuring the concentration of quebracho in human biological material samples, and particularly in fluid samples, including oral fluid, urine, serum, plasma, or whole blood. In particular, the present disclosure relates to the preparation and use of haptens (haptenes) (immunogen precursors) and immunogens (immunogenes) derived from cephalomannine and useful for producing antibodies specific to cephalomannine and/or metabolites of cephalomannine, as well as labeled conjugates and immunoassays (immunoassay) for measuring the concentration of cephalomannine or metabolites of cephalomannine, and methods of making and using the same.

Related Art

The major constituent indole alkaloids of the species corynespora pseudocerana (cortinanthe), trichophylline (mitragynine) (IUPAC name (E) -2- [ (2S,3S,12bS) -3-ethyl-8-methoxy-1, 2,3,4,6,7,12,12 b-octahydroindolo [2,3-a ] quinolizin-2-yl ] -3-methoxyprop-2-enoic acid methyl ester), is the plant species meilitha pseudocapila (mitrayna specosa, commonly known as "carpain (kratom)" or "biak-biak"). Although katain leaves have a medicinal tradition in some countries, recreational use and abuse of katain has led to increased addiction and death to cephalothin. Recent increases in the mental stimulation caused by recreational drugs, and the accompanying increases in the types and amounts of substances ingested to achieve this effect, including the use of pain. Cardamine is said to have pharmaceutical properties and although illegal in some countries it is still legal in the uk and in europe and most parts of the us, although it is listed as a substance of interest by the us drug administration (u.s.drug Enforcement Agency) and banned in some states.

There have been many reports of deaths related to their intake recently, and there is increasing interest in detecting katain for toxicological and research purposes by means of cuphealine and other alkaloids of katain including, but not limited to, paynanthine (paynantheine), beauty-cuphealine (speciogynine), speekin (specociliatine), 7-hydroxy-cuphealine (7-hydroxy-mitragine). Analytical methods that have been described for the detection and quantification of hattacrine and other katain alkaloids use relatively expensive, professional-operator-dependent High Performance Liquid Chromatography (HPLC) techniques or mass spectrometry techniques in conjunction with gas chromatography (GC-MS) or liquid chromatography (LC-MS) (e.g., Kaewklum, 2005; janchaweee, 2007; Le, 2012). These techniques can be more complex due to the need to pre-treat the sample prior to analysis. Therefore, there is a need for rapid and economical detection and quantification of patient samples and of cephalothin and its metabolites suspected of being incorporated into carpain substances in clinical and forensic settings.

Early methods for the detection and quantification of hattacrine included High Performance Liquid Chromatography (HPLC), and mass spectrometry after separation by gas chromatography (GC-MS) or liquid chromatography (LC-MS).

WO 2013/147586(Tan et al) and US 9,952,206(Benchikh et al) describe immunoassay methods for the detection and measurement of quebracho and other related alkaloids. Specifically, Tan et al describe a method of synthesizing cuphealine-p-aminobenzoic acid (PABA-cuphealine) to produce an immunogen for the production of antibodies. Benchikh et al teach the use of derivatives of 8-demethylidecadrine (8-desmethylnigaryne, also known as 9-hydroxyconicanthine) for the production of polyclonal antibodies that specifically bind to calophylline, 8-demethylidecadrine, 8-sulfonylcalophylline (8-sulphonylmitrayne) and/or 8-glucuronidategaryne (8-glucuronidyrityne).

Limsuwanchote et al (Forensic Science International,244: 70-77; 2014) teach monoclonal antibodies (1A6) produced from immunogenic conjugates of hattacrine (16-carboxy hattacrine) and bovine serum albumin. According to Limsuwanchote et al, a carboxyl group is added to the capping-post lignin to allow conjugation to an antigenic protein (to produce an immunogen). The resulting monoclonal antibody was used in an indirect ELISA assay to measure binding to the coated antigen (mitragynine-glutaraldehyde-ovalbumin) and showed higher affinity for the coated antigen-ovalbumin complex than for free pillarine. Since ELISA immunoassays were developed for the purpose of quantifying the presence of quebracho in samples extracted from katakana leaves, no human samples were detected. The measurement range of the assay developed by Limsuwanchote et al is 32.92-250. mu.g/ml, which is not sensitive enough to detect the presence of capelin in human samples.

Randox Laboratories Limited has commercialized an ELISA immunoassay that quantitatively measures hattacrine and 9-O-desmethyl hattacrine from urine and whole blood, which has a low level of cross-reactivity to 7-alpha-hydroxy hattacrine with a detection limit of about 0.54ng/ml (in whole blood).

Philipp et al (Anal Bioanal Chem,400: 127-. Due to the strong metabolism of katain alkaloids, Philipp et al emphasized the need to include metabolites when screening for katain use, and determined that in addition to quebracho, the metabolites 16-carboxyquebracho (quebracho), 9-O-demethylated quebracho and/or 9-O-demethyl-16-carboxyquebracho are also suitable for inclusion in assays for monitoring katain use.

Problems in the art

At least one antibody specific for a target antigen typically forms the basis of a useful immunoassay, but small molecules such as drugs are typically not immunogenic and therefore do not elicit an immune response when injected into animals, including those animals commonly used for commercial antibody production, such as mice, rats, rabbits, goats, horses, and other mammals known in the art. In addition, some active forms of the target drug may be toxic to the animal recipient, even in small doses. Therefore, it may be desirable to prepare derivatives of the target drug as immunogens for antibody production. However, antibodies raised against the derivatized target must still be able to cross-react with the drug that is expected to be present in the patient sample.

Thus, there are many problems that can be solved in the field of immunoassay generation of small molecules, including immunoassays for the detection of hattacrine, especially when developing antibodies and other reagents.

Background

Disclosure of Invention

The present disclosure relates to pharmaceutical analysis and to the field of detection and quantification of the main component of quebracho (Mitragyna speciosa, commonly known as "carpain") for quebracho (systematic name: (E) -2- [ (25,35,12b5) -3-ethyl-8-methoxy-1, 2,3,4,6,7,12,12 b-octahydroindolo [2,3-a ] quinolizin-2-yl ] -3-methoxyacrylate), with low, if any, cross-reactivity with major metabolites including 16-carboxyquebracho (hattaconic acid) and 9-hydroxycicotinine, also known as 8-demethylidenemine (systematic name: (E) -2- [ (25,35,12b5) -3-ethyl-8-hydroxy-1, methyl 2,3,4,6,7,12,12 b-octahydroindolo [2,3-a ] quinolizin-2-yl ] -3-methoxyacrylate).

The present disclosure solves one or more of the above or other problems in the art by compositions, kits, and methods for detecting cephalosporins in a biological sample, such as a body fluid. Embodiments may include immunogenic compounds or immunogens derived from cephalosporine. In some embodiments, the immunogen comprises a hapten-carrier complex wherein the hapten moiety is comprised of a small molecule derived from a pillarine structure. The pillarine-derived hapten (or immunogenic precursor) can be conjugated to an antigenic biomolecule (e.g., an antigenic protein, polypeptide, etc.) to produce an immunogenic hapten-carrier complex or immunogen. The immunogen derived from and/or comprising a cephalomannine-derived moiety is capable of stimulating the production of antibodies that (i) specifically interact with and have high affinity for at least a portion of the hapten and/or immunogenic hapten-carrier complex, and (ii) are specific for or cross-reactive with at least a portion of the target drug, cephalomannine and/or cephalomannine metabolites. The immunogen comprising the hattacrine-derived moiety is operable to trigger a robust immune response in a suitable animal to which the immunogen is administered and results in the production of antibodies that are capable of recognizing, binding and/or having (strong) affinity and specificity for the immunogen and/or the hattacrine-derived hapten. Preferably, the immunogen is operable to trigger an antibody-mediated immune response that results in the production of antibodies that are specific for and/or cross-reactive with cuprammonium and/or cuprammonium metabolites.

Embodiments of the present disclosure may additionally relate to compositions, products, and kits comprising one or more antibodies raised against and/or specific for (or with binding specificity for) cupdrine and/or a cupdrine derivative. The compositions, products, and kits can additionally include cupnoline and/or one or more derivatives of cupnoline, including cupnoline haptens and/or cupnoline derivative precursors. As one non-limiting example, the disclosed products or kits can include one or more immunodiagnostic assays for detecting the presence and/or concentration of cephalothin and/or active metabolites thereof.

Embodiments of the present disclosure may include methods for synthesizing a hattacrine derivative and/or an immunogen comprising a hattacrine derivative. The disclosed methods can be used to produce one or more elements (elements) of the disclosed compositions, products, and kits. Additionally or alternatively, elements of the disclosed compositions, products, and kits can be used in the various methods disclosed herein. For example, one or more of the immunodiagnostic assays disclosed herein may be used in the disclosed methods for detecting the presence or concentration of cephalosporine and/or an active metabolite thereof. Accordingly, embodiments of the present disclosure may include methods of detecting the presence or concentration of cephalosporins and/or active metabolites thereof, for example, in a body fluid.

Embodiments of the present disclosure additionally include methods for synthesizing a pillarine derivative, which may include an operational group, such as an antigenic moiety that can be used as a hapten in an immunogenic hapten-carrier complex to make anti-pillarine antibodies, an antigenic moiety that can be used in an immunodiagnostic assay for pillarine, or a tracer moiety that can be used in an immunodiagnostic assay. In some embodiments, the cephalomannine derivatives are provided in the disclosed products and/or kits, e.g., in a competitive immunodiagnostic assay, to compete with the presence of cephalomannine and/or cephalomannine metabolites in a sample for binding to primary anti-cephalomannine antibodies.

In one aspect, the invention comprises haptens and derivatives produced by derivatizing the nitrogen atom at position 12 of a fused heterocyclic ring of a cuprammine. The hapten produced can be conjugated to: (i) a carrier polypeptide to produce an immunogen for antibody production; and/or (ii) an enzyme, particle, radioisotope, fluorescent molecule, dye or other directly or indirectly detectable label to produce a labeled conjugate for use as a detection reagent in an immunoassay for cephalomannine and/or a metabolite of cephalomannine. In addition, primary anti-hatscheline antibodies, haptens or derivatives/analogs of hatscheline and/or anti-hatscheline antibodies can be attached to solid supports, including particles, multi-well plates, chips, and the like, for use in immunoassays.

The immunogen(s) (or hapten component of the hapten-carrier immunogenic complex comprising derivatives of cuprammonium) of the present disclosure has the following general structure (I):

wherein:

x is C, CO or CS;

y is an organic spacer-K-Q-, wherein K ═ N, O or a linear or branched chain comprising substituents or unsubstitutedC of a saturated or unsaturated alkylene moiety_(1-10)(ii) a And Q ═ C comprising a carboxylic acid or ester thereof, an aldehyde, an amino, a maleimide, a halogenated carboxylic acid or ester thereof, a dithiopyridyl moiety, a vinylsulfone moiety, or a thiocarboxylic acid or ester thereof_(1-10)(ii) a And is

Z is an operable group comprising one or more of: a detectable label, an antigen carrier, a coupling agent, an end group, a protein, a lipoprotein, a glycoprotein, a polypeptide, a polysaccharide, a nucleic acid, a polynucleotide, a teichoic acid, a radioisotope, an enzyme fragment, an enzyme donor fragment, an enzyme acceptor fragment, an enzyme substrate, an enzyme inhibitor, a coenzyme, a fluorescent moiety, a phosphorescent moiety, an anti-stokes upregulating moiety, a chemiluminescent moiety, a luminescent moiety, a dye, a sensitizer, a particle, a microparticle, a magnetic particle, a solid support, a liposome, a ligand, an acceptor, or a combination thereof.

In one embodiment, Z is an antigen carrier comprising one or more of: a protein, a polypeptide, a glycoprotein, a polysaccharide, a particle, a microparticle, a nucleic acid, a polynucleotide, or a combination thereof. In another embodiment, the antigen carrier is a protein comprising: bovine serum albumin ("BSA"), keyhole limpet hemocyanin ("KLH"), ovalbumin, bovine gamma globulin ("BGG"), or a combination thereof.

In another aspect, the invention comprises an antibody produced by inoculating a mammal with an immunogen. As used herein, the term "antibody" refers to polyclonal and monoclonal antibodies as well as recombinant antibodies, including functional fragments and derivatives thereof. One exemplary method of producing antibodies is to administer an immunogen, usually in combination with an adjuvant such as Freund's adjuvant, to a host animal (e.g., a mammal, such as a mouse, rat, rabbit, etc.) in a series of injections for the purpose of inducing an immune response. Such methods are well known to those skilled in the art. Methods for producing monoclonal antibodies were first described by Kohler and Milstein (Nature, Vol 256, pp 495-497, 1975; which is incorporated herein by reference in its entirety) and have been subject to numerous modifications since the advent of this publication. Hybridoma technology has been described in U.S. Pat. Nos. 4,491,632, 4,472,500 and 4,444,887, and Methods in Enzymology,73B:3 (1981); each of which is incorporated herein by reference in its entirety. Since the particular method of producing antibodies to the immunogen is not critical, any suitable, acceptable and/or proven method known in the art can be used to produce polyclonal or monoclonal antibodies using the immunogens described herein.

These and other aspects, features, embodiments and/or embodiments of the present disclosure and inventions disclosed and described herein will be more fully apparent from the following description and appended claims, or may be learned by the practice of the embodiments and/or inventions as set forth hereinafter.

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description and appended claims, which form a part of the present disclosure. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

Brief description of the drawings

Various embodiments of the present disclosure will now be discussed with reference to the figures. It is appreciated that these drawings depict only typical embodiments of the disclosure and are therefore not to be considered limiting of its scope.

Figure 1 shows a compound having the general structure (I) comprising an immunogen (or a hapten-carrier immunogenic complex comprising a hapten component comprising a pillarine derivative) according to one embodiment of the present disclosure;

figure 2 illustrates the production of an ester (compound 3) from compound 1 according to one embodiment of the present disclosure;

figure 3 shows the generation of a pillarine (derivatized) hapten (compound 4) from compound 3 according to another embodiment of the disclosure;

figure 4 shows the generation of an activated ester of a quebracho hapten (compound 4) (compound 5) according to another embodiment of the present disclosure;

figure 5 shows the generation of a pillarine (derived) immunogen (compound 6) from compound 5 according to another embodiment of the present disclosure;

figure 6 illustrates the production of a hattacrine MEA adduct (compound 7) from compound 5 according to another embodiment of the present disclosure;

figure 7 shows the generation of an enzyme donor conjugate (compound 8) from compound 7 according to another embodiment of the present disclosure; and

figure 8 shows an alternative hapten derived from cyprodinil according to another embodiment of the present disclosure.

Detailed Description

Example embodiments are described below. Many different forms and embodiments are possible without departing from the spirit and teachings of the present disclosure, and therefore the present disclosure should not be construed as limited to the example embodiments set forth herein. Rather, these example embodiments are provided so that this disclosure will be thorough and complete, and will convey the scope of the disclosure to those skilled in the art.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of this application and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. In case of conflict in terminology, the present specification will control.

The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Although a variety of methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, only certain exemplary materials and methods are described herein.

All publications, patent applications, patents, or other references mentioned herein are incorporated by reference in their entirety.

Various aspects of the disclosure, including devices, systems, methods, etc., may be described with reference to one or more exemplary embodiments. As used herein, the terms "exemplary" and "illustrative" mean "serving as an example, instance, or illustration," and should not necessarily be construed as preferred or advantageous over other embodiments disclosed herein. Furthermore, references to "an embodiment" or "an embodiment" of the present disclosure or invention include specific references to one or more embodiments thereof, and vice versa, and are intended to provide illustrative examples without limiting the scope of the invention, which is indicated by the appended claims rather than by the following description.

It will be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a tile" includes one, two or more tiles. Similarly, reference to multiple referenced objects should be understood to include a single referenced object and/or multiple referenced objects unless the content and/or context clearly dictates otherwise. Thus, reference to "tiles" does not necessarily require a plurality of such tiles. In contrast, it will be understood that the font change (concatenation) is independent; one or more tiles are contemplated herein.

As used throughout this application, the words "can" and "may" are used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). Furthermore, as used herein (including the claims), the terms "comprising", "having", "involving", "containing", "characterized by", variants thereof (e.g., "comprises", "having", "has", "involves", "contains", etc.), and like terms, shall be inclusive and/or open-ended, shall have the same meaning as the word "comprising" and variants thereof (e.g., "comprises" and "contains"), and are illustratively not exclusive of additional, unrecited elements or method steps.

It will be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. Thus, a "first" element may be termed a "second" element without departing from the teachings of embodiments of the present invention.

It should also be understood that various embodiments described herein may be used in combination with any other embodiment described or disclosed without departing from the scope of the present disclosure. Thus, products, members, elements, devices, systems, methods, processes, compositions, and/or kits according to certain embodiments of the present disclosure may include, incorporate, or otherwise contain the properties, features, components, members, elements, and/or steps, etc., described in other embodiments (including systems, methods, and/or devices, etc.) disclosed herein without departing from the scope of the present disclosure. Thus, references to a particular feature associated with one embodiment should not be construed as limited to use with that embodiment.

The headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claims. To facilitate understanding, like reference numerals have been used, where possible, to designate like elements that are common to the figures. Moreover, where possible, like element numbers are used in the various figures. Further, alternative configurations of specific elements may each include a separate letter appended to the element number.

As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list.

In developing the various embodiments of the present disclosure, including immunoassays for detecting target drugs such as hattacrine and/or its metabolites that lack inherent immunogenicity, an immunogenic compound or immunogen comprising a hapten derived from hattacrine is prepared. Briefly, haptens (structures derived from cephalothin) are conjugated to larger antigenic proteins, polypeptides, or other antigenic biomolecules. The resulting immunogen is capable of stimulating the production in a mammal of antibodies that specifically interact with, and in some cases have high affinity for, at least the hapten portion of the immunogen, and not only the hapten-carrier complex comprising the immunogen. The antibody may additionally or alternatively be specific for and/or cross-reactive with cephalomannine or an active metabolite thereof.

In addition, detection of cephalosporins in immunoassays typically requires the use of detectable components or labels, including but not limited to radioisotopes, byproducts of enzymatic reactions (e.g., byproducts of horseradish peroxidase, alkaline phosphatase, glucose oxidase, or beta galactosidase), fluorescent molecules, and particles. The detectable component may be conjugated to a secondary antibody specific for a primary antibody capable of binding to the cephalothin or an active metabolite thereof.

In embodiments comprising an immunoassay, the target or primary antibody may be conjugated to a reporter (e.g., a detectable component or label) or immobilized to a substrate, as is known in the art. The antibody-bound antigen can be detected directly (in the case of a reporter-labeled antibody) or by a reporter-conjugated secondary antibody. Accordingly, aspects of the present disclosure include immunoassays with a primary antibody specific for a target molecule (e.g., a cuprammine or an active metabolite thereof). An assay may be performed on a sample that may contain the target molecule, and the presence and/or level of a detection signal may be determined therefrom, as is known in the art (e.g., enzymatic reaction products, changes in fluorescence signal, etc.).

In some embodiments, a competitive immunoassay is provided. Competitive immunoassays may include labeled versions of target molecules (e.g., cuprammine or its active metabolites) that compete for binding to a limited number of target antibodies. Thus, when the target molecule is present in a sample, such as a sample obtained from a diagnostic or drug test detection kit (e.g., for judicial identification, legal and/or criminal judicial purposes), the target molecule competes with labeled cephaeline (or an active metabolite thereof) for binding to the target antibody, producing a detectable (e.g., quantifiable or quantitative) signal. In some embodiments, the detectable signal comprises a difference between the measured amount of the detection signal and the expected amount of the detection signal. In some embodiments, the detectable signal is indirectly related to the concentration of the target molecule in the sample (e.g., less label measured in a competitive immunoassay means more (unlabeled) target molecule is present in the sample).

The optimal immunogen may comprise a drug derivative comprising an antigenic biomolecule and a hapten (structures derived from calophylline) which combine to form an immunogen configured to elicit a robust immune response in a suitable animal into which the immunogen is introduced and capable of raising antibodies to the immunogen. In particular, the structure of the immunogen of the present disclosure is used to trigger a robust immune response, which results in the production of antibodies that recognize, bind and/or have (strong) specificity and/or affinity for the hapten and/or hattacrine and/or its metabolites. In certain embodiments, antibodies raised against a hapten form of quebracho can have up to 100% cross-reactivity with quebracho and/or quebracho metabolites. In some embodiments, the antibody can have at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% cross-reactivity with cephalomannine and/or cephalomannine metabolites.

The hapten portion of the immunogen (or derivative) and/or the antibody specific for the hapten and/or cross-reactive with the cephalomannine and/or active cephalomannine metabolites may be conjugated to one or more detectable entities, such as labels, markers, enzymes, and the like, as known in the art. The conjugates are useful as reagents for developing and performing immunoassays to detect quebracho and/or quebracho metabolites. Such immunoassays include, for example, enzyme-linked immunoassays (ELISA), Fluorescence Polarization Immunoassays (FPIA), immunoturbidimetry assays, and clonase-donor immunoassays (CEDIA), among others.

In particular, the CEDIA technology has proven to be a highly accurate method for the quantification of therapeutic and abuse drugs. The CEDIA technology is the subject of several patents, including U.S. Pat. No. 4,708,929 (incorporated herein by reference in its entirety) which claims competitive homogeneous assay methods, U.S. Pat. No. 5,120,653 (incorporated herein by reference in its entirety) which claims recombinant DNA sequences encoding enzyme donor compound fragments and hosts for such vectors, U.S. Pat. No. 5,604,091 (incorporated herein by reference in its entirety) which claims amino acid sequences of enzyme donor fragments, and teaching and claims for CEDIA^TMU.S. patent No. 5,643,734 (incorporated herein by reference in its entirety) for a kit of assays. Competitive homogeneous assays are preferred over heterogeneous assays, including ELISA assays, because there is no need to separate unbound labeled conjugate from bound labeled conjugate, which requires time consuming washing steps.

Further details regarding the use and conjugation of immunogens (or derivatives) and/or antibodies, detectable entities such as labels, markers, enzymes, etc., and the development and performance of (reagents for) immunoassays, including the CEDIA assay, can be found in U.S. patent No. 7,138,504 (incorporated herein by reference in its entirety).

Preparation of haptens, immunogens and detection agents

While haptens provide defined structural epitopes, they are generally not immunogenic in themselves, and are therefore often conjugated to carrier materials to produce immunogens which, when administered to a host animal, will elicit an antibody-mediated immune response. Suitable carrier materials typically contain poly (amino acid) segments and include polypeptides, proteins, and protein fragments. Illustrative examples of useful carrier materials include bovine serum albumin, ovalbumin, bovine gamma globulin, bovine thyroglobulin, keyhole limpet hemocyanin, and the like. Alternatively, synthetic poly (amino acids) having a sufficient number of available amino groups (e.g., lysine), as well as other synthetic or natural polymeric materials with reactive functional groups, can be used. In addition, carbohydrates, polysaccharides, or microbial components may be conjugated to haptens to produce immunogens. The formation of immunogens for use in the invention described herein involves conventional conjugation chemistry. To confirm that sufficient conjugation of the hapten to the carrier material has been achieved prior to immunization, each immunogen can be evaluated using matrix assisted UV laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS).

The hapten can also be coupled to a detectable label to form a conjugate. The detectable label may be an enzyme (e.g., horseradish peroxidase or other enzymes described above) used to prepare a detection reagent for use in an immunoassay, a substance with fluorescent properties, or a radioactive label. The fluorescent substance may be, for example, a monovalent residue of fluorescein or a derivative thereof.

The process of developing immunoassays is well known to those skilled in the art. Briefly, for a competitive immunoassay in which the target is a non-immunogenic molecule, such as a hapten, the following process can be performed: a detection agent (e.g., a labeled conjugated target) is added to a sample containing the target and a target-specific (or cross-reactive) antibody, and the detection agent and target compete for binding to the antibody. The process may include immobilizing the antibody to a support matrix (backing), such as a polystyrene solid support or a ceramic chip. The antibody may be a polyclonal or monoclonal antibody obtained using standard techniques. The signal emitted in an immunoassay is directly proportional to the amount of detector bound to the antibody, which in turn is inversely proportional to the analyte concentration. The signal may be detected or quantified by comparison with a reporter specific measurement device or calibrator as known in the art.

Further details regarding haptens, immunogens and detection agents, as well as compositions, products, kits, assays and methods comprising the same, as well as cephalothin and metabolites and derivatives thereof, can be found in U.S. patent No. 9,952,206 (incorporated herein by reference in its entirety). Each of U.S. publication No. US 2005/0176080 and WIPO publication No. WO 2013/147586 is also incorporated herein by reference in its entirety.

Examples

The following examples further illustrate the invention. It is to be understood, however, that the embodiments are set forth by way of illustration, not of limitation, and that various modifications may be made by one of ordinary skill in the art.

Example 1: preparation of ester (Compound 3)

A solution containing 50.0mg (0.125mmol) of pillarine (compound 1) in 5.0ml of Dimethylformamide (DMF) is prepared at Room Temperature (RT). NaH (60% in oil, 50.0mg (1.25mmol)) was added with stirring and stirred at RT under nitrogen for about 25-30 min. Then 162.0mg (1.25mmol) of ethyl 2-isocyanatoacetate (compound 2) are added slowly at RT. The resulting mixture was stirred at RT for about 2.0 h. HPLC and/or LC-MS analysis showed that more than 95% of the original capped pillared xyline was converted to compound 3. Dichloromethane (DCM) (25mL) was added, and saturated NH added₄The mixture was washed with Cl (25 mL). Separating the organic layer over Na₂SO₄Dried, filtered and concentrated. The crude product was purified by silica gel column chromatography (3% to 5% MeOH in DCM) to give 42.4mg (-70% purity) of the ester (compound 3) as a light yellow solid.

Example 2: preparation of Douglas hapten (Compound 4)

To a solution containing 42.4mg (0.054mmol) of the ester (compound 3) in 1.0ml Tetrahydrofuran (THF) and 0.5ml methanol at RT was added dropwise an aqueous solution of LiOH (9.0mg in o.5ml distilled water, 0.21mmol) at RT. Mixing the mixture at RT and N₂Stirring for about 1.0 h. The conversion of compound 3 to compound 4 was monitored by LC-MS. When about 100% of compound 3 is converted to compound 4, it is passed through a rotary evaporatorTHF and MeOH were removed, and the residue was diluted with water (1.5 mL). The aqueous solution was extracted with DCM (2X2.0mL), cooled to 0 ℃ and acidified to pH3 with 1N HCl (aq). The aqueous mixture was extracted with DCM (3X3.0 mL). The combined organic layers were washed with Na₂SO₄Drying, filtration and concentration gave 25.2mg (40.4%) of the quebracho hapten (compound 4) as a pale yellow solid.

Example 3: preparation of activated ester of quebracho hapten (Compound 5)

To a stirred solution containing 9.0mg (0.018mmol) of the pillarine hapten (compound 4) in 1.2ml of DMF were added 8.0mg (0.045mmol) of endo-N-hydroxy-5-norbornene-2, 3-dicarboximide (NHDC) and 8.6mg (0.045mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCI). The resulting mixture was stirred at RT for about 16h and the crude reaction mixture of activated ester (compound 5) was used directly in the next step.

Example 4: preparation of quebracho immunogen

About 53.2mg BSA was added to about 3.0ml Na under stirring at RT₂CO₃/NaHCO₃Buffer (pH 9). The resulting clear solution was cooled to about 0 ℃ for about 20 minutes. A solution of the activated ester (compound 5) in 1.5ml DMF (0.018mmol) was added dropwise at 0 ℃ and the resulting mixture was stirred at RT for 16 h. By adding phosphate buffer (10mM Na)₂HPO₄-NaH₂PO₄pH 7.0) was dialyzed 3 times to purify the resulting crude immunogen (compound 6). The concentration of the pillarine-BSA immunogen (compound 6) was measured using a bicinchoninic acid (BCA) protein concentration assay. The immunogen (compound 6) was at a concentration of 6.0mg/mL and a hapten count of 9.8 and was available for immunization.

Example 5: preparation of the hatschel-wood-alkali MEA adduct (Compound 7)

To a stirred solution containing 10.6mg of activated ester (Compound 5) in DMF was added 8.4mg of mercaptoethylamine-HCl (MEA-HCl) and 18.6mg of diisopropylethylamine. The resulting mixture was stirred at RT overnight (about 16 h). The mixture was directly purified by HPLC to give about 5.9mg of the adduct (compound 7) as a white solid.

Example 6: preparation of enzyme Donor conjugate (Compound 8)

About 0.9mg of MEA adduct (compound 7) in 0.5ml DMF was added dropwise to a stirred solution containing 2.0mg of desalted cloned enzyme donor ED28 in about 0.91ml of phosphate buffer (pH 7.0) at 0 ℃. The resulting mixture was stirred at 0 ℃ for about 20 min. The crude mixture was purified by HPLC to give about 0.63mg of the pillarine-ED 28 conjugate (compound 8) as a colorless liquid in about 10.0mL of a mixture of acetonitrile and water containing 0.1% TFA. The concentration of compound 8 was measured using absorbance at 280nm and determined to be about 0.062 mg/ml.

Example 7: production of monoclonal antibodies against cephalomannine and/or cephalomannine metabolites

In a specific embodiment of the invention, the BSA-containing immunogen of example 4 (compound 6) was administered to Balb/c mice in a series of injections as is conventional in the art. Alternatively, other mammals are suitable for antibody production. Antisera samples taken from immunized mice were screened to assess antibody titers and to evaluate the ability of the antibodies in the samples to bind to the enzyme-donor conjugates described in example 6.

Example 8: by usingDevelopment of CEDIA assay for Cylindrine and/or Cylindrine metabolites

A preferred form of immunoassay is the cloned enzyme donor immunoassay or CEDIA assay, which is based on the reassociation (re-association) of an enzymatically inactive polypeptide fragment of β -galactosidase. Specifically, a β -galactosidase donor polypeptide fragment is combined with a β -galactosidase receptor fragment to form an active β -galactosidase. The active enzyme complex is capable of converting a substrate to a differentially detectable product. Typically, the product is a different color than the substrate and is quantified using spectrophotometry. Conjugating haptens or other small analytes or analyte analogues to the enzyme donor fragment at certain sites does not affect the ability to form active enzymes by complementary reactions and does not affect the rate of enzyme activity in the presence of a substrate for β -galactosidase. However, when the enzyme donor-hapten conjugate is bound by an anti-analyte antibody, e.g., when little or no analyte is present in the sample being tested, the complementary reaction is inhibited, reducing the amount of active enzyme present in the reaction mixture. Thus, the rate of the enzyme-catalyzed reaction is reduced under such conditions. In contrast, when the test sample contains a significant concentration of the target analyte, it competes with the enzyme donor-hapten for binding sites on the anti-analyte antibody, thereby increasing the amount of active enzyme formed by the complementary reaction. Thus, the rate of the enzyme-catalyzed reaction is directly proportional to the concentration of the target analyte present in the test sample. The preferred β -galactosidase donor is ED28, a polypeptide containing residues 6-45 of β -galactosidase, which have cysteines at positions 1 and 46 (numbering relative to the original β -galactosidase fragment).

As one example, the CEDIA kit for measuring the concentration of cephalothin in a biological fluid contains a β -galactosidase receptor (EA) reagent comprising EA lyophilized in a buffered saline solution, preferably at a concentration of about 0.118 grams of EA per liter of buffered saline solution prior to lyophilization. Preservatives such as sodium azide are advantageous for extending shelf life (shelf life). Also included is EA reconstitution buffer, which may include an antibody capable of specifically binding to cephalothin as described herein. Preferred buffers include PIPES, MOPS, HEPES, TES or Tris.

The Enzyme Donor (ED) fragment conjugated to the pillarine MEA adduct prepared as described in example 6 can be provided in a kit as a separate reagent lyophilized together with the substrate. Chlorophenol red-beta-D-galactopyranoside at a concentration of about 10nM (about 3.0g/L) is the preferred substrate. In addition, stabilizers such as bovine serum albumin fragments and preservatives such as sodium azide are advantageous for extending shelf life. The ED reagent was reconstituted with ED reconstitution buffer containing potassium phosphate along with surfactants and preservatives. Other components of the kit include instructions for performing the assay. Optionally, the kit may include calibrators, e.g., at least one calibrator without hattacrine (0ng/mL hattacrine) and one with a higher concentration range (. gtoreq.200 ng/mL) and a control containing a known drug concentration. The calibrator and/or control may be included in the kit or provided as a separate component.

Example 9: development of homogeneous microparticle immunoassays

Homogeneous microparticle immunoassay ("HMI") technology, which may be referred to as immunoturbidimetry, is based on the agglutination of particles and compounds in solution. Immunoturbidimetry assay techniques are described in U.S. patent nos. 5,571,728, 4,847,209, 6,514,770 and 6,248,597, which are incorporated herein by reference. Briefly, in the homogeneous assay method, light attenuation, nephelometry (nephelometric method) or turbidimetry (turbidimetric method) is mainly used. When particles and/or chemical compounds agglutinate, the turbidity of the solution increases. HMI assays can be configured to be performed using hatscheline and/or hatscheline haptens (analogs) loaded onto the microparticles, or using anti-hatscheline antibodies loaded onto the microparticles. The use of microparticles loaded with an analog may be particularly advantageous because of the ability to efficiently load the microparticles. In any case, HMI or immunoturbidimetric assays are well known in the art methods for measuring drug concentration in a sample.

Example 10: fluorescence polarization immunoassay for pillared xylosidosines

The Fluorescence Polarization Immunoassay (FPIA) technique is based on competitive binding between the antigen/drug in the sample and a known concentration of labeled antigen/drug. FPIA technology is described in U.S. patent nos. 4,593,089, 4,492,762, 4,668,640, and 4,751,190, which are incorporated herein by reference. Thus, the FPIA reagents, systems and devices described in the incorporated references can be used with anti-pillarine antibodies.

Example 11: chemiluminescent microparticle immunoassay for pillarine

Competitive assays using chemiluminescent microparticle immunoassay ("CMIA") technology can also be used to assess the presence or absence of pillarine in a sample. Various types of CMIA techniques are well known in the art of heterogeneous immunoassays for determining the presence and/or amount of a chemical entity in a sample. Some CMIA technologies may be exemplified by U.S. patent nos. 6,448,091, 5,798,083, and 5,834,206, which are incorporated herein by reference. CMIA assays may include the use of anti-pillarine antibodies, which are capable of binding to pillarine and/or its metabolites or analogs, coupled to particles such as magnetic particles or particles suitable for separation by filtration, sedimentation and/or other means. In addition, a tracer may be used which may include a pillarine analogue linked to a suitable chemiluminescent moiety to compete with free pillarine in the patient sample for a limited amount of anti-pillarine antibody on the particle. After the sample, tracer and antibody particles interact and a conventional washing step has removed unbound tracer, the amount of tracer bound to the antibody particles can be measured by chemiluminescence, where chemiluminescence is expressed in relative light units (RULE). The amount of chemiluminescence is inversely related to the amount of free drug in the patient sample, and the concentration is determined by constructing a standard curve using known values of the drug.

The pillarine haptens, derivatives, analogs, conjugates, antibodies, immunogens and/or other conjugates described herein are also suitable for use in any of a variety of other types of immunoassays with a range of detection systems, including but not limited to ELISA assays, rapid lateral flow assays, and antibody arrays or chips in multi-well plates, as well as formats yet to be developed.

Example 12: specificity and Cross-reactivity

The cross-reactivity of anti-hattacrine antibodies derived from the above immunogens was tested against certain katain alkaloids and hattacrine metabolites shown below. The antibody was 100% cross-reactive with cephalomannine tested at a concentration of 50ng/ml and showed only low levels of cross-reactivity with the metabolites 16-carboxy cephalomannine (cephalomannine acid) and 9-hydroxyconiphylline. The antibodies have a cross-reactivity of 0.2% or less with the alkaloids 7-OH quebrachuinine, phoenix and speepttin tested at higher concentrations as shown in Table 1 below.

TABLE 1

Example 13: accuracy of CEDIA hatscherin assay

The CEDIA cap column lignan assay was compared to liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a qualitative and semi-quantitative mode in 100 human urine samples using a 50ng/mL cutoff calibrator. The CEDIA assay showed 100% agreement with the LC-MS/MS method, as shown in Table 2 below.

TABLE 2

	LC-MS/MS positive	LC-MS/MS negative
			Positive assay for CEDIA	50	0
CEDIA assay negative	0	50

Example 14: accuracy of CEDIA hatscherin assay

A 20 day accuracy was performed by testing urine samples spiked with cuprammonium in 25% increments or decrements from a 50ng/mL cutoff using a Beckman Coulter AU680 analyzer. Samples were tested in duplicate (n-2) twice daily for 20 days in both qualitative and semi-quantitative modes (total n-80 per concentration level). The accuracy in the qualitative mode was < 2% CV, and the accuracy in the semi-quantitative mode was < 10% CV.

Example 15: substitutional haptens derived from quebracho

The following haptens are also derived from cephalosporins.

Conclusion

Although the foregoing detailed description has referred to specific exemplary embodiments, the disclosure may be embodied or carried out in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments, aspects and/or features are, therefore, to be considered in all respects as illustrative and/or exemplary and not restrictive. For example, various substitutions, alterations, and/or modifications of the inventive features described and/or illustrated herein, and other applications of the principles described and/or illustrated herein, which would occur to one skilled in the relevant art and having possession of this disclosure, may be made to the described and/or illustrated embodiments without departing from the spirit and scope of the invention as defined by the appended claims.

It should also be understood that various features of certain embodiments may be compatible with, combined with, included in, and/or incorporated into other embodiments of the present disclosure. For example, systems, methods, and/or articles of manufacture according to certain embodiments of the present disclosure may include, incorporate, or otherwise contain features described in other embodiments disclosed and/or described herein. Thus, the disclosure of certain features relative to particular embodiments of the present disclosure should not be construed as limiting the use or inclusion of such features to the particular embodiments. In addition, unless a feature is described as required in a particular embodiment, the feature described in various embodiments may be optional and may not be included in other embodiments of the disclosure. In addition, any feature herein may be combined with any other feature of the same or different embodiments disclosed herein, unless one feature is described as requiring combination with another feature.

The scope of any invention disclosed and/or described herein is indicated by the appended claims rather than by the foregoing description. The limitations described in the claims are to be interpreted broadly based on the language employed in the claims and not limited to specific embodiments described in the foregoing detailed description, which embodiments are to be construed as non-exclusive and non-exhaustive. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.