WO2021113736A1 - Single-domain antibody to chloramphenicol - Google Patents

Abstract

Disclosed are single variable domain antibodies (VHH) that specifically bind to chloramphenicol and/or a chloramphenicol analog and nucleic acids encoding the same, devices including such VHHs for determining the presence or absence of chloramphenicol or a chloramphenicol analog, and methods of using the same.

Description

SINGLE-DOMAIN ANTIBODY TO CHLORAMPHENICOL

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 62/944,150, filed December 5, 2019, the entire disclosure of which is hereby incorporated herein by reference.

FIELD OF THE INVENTION

[0002] Die present invention is in the field of contaminant detection in food, feed, environmental samples, and medical samples. More particularly, the invention relates to small molecule detection with single domain antibodies and other immunoglobulins.

BACKGROUND OF THE INVENTION

[0003] Antibodies have long been used in diagnostic and therapeutic applications as highly selective binders to a target of interest, but their large size and multiple domains make library screening difficult and limit their binding capabilities in challenging environments such as high temperatures or after long-term storage. See, e.g., Ingram et al., Annu. Rev. Immunol (2018) 36:695-715

[0004] Smaller naturally occurring antibodies, such as single-chain antibodies found in camelids, and antibody fragments such as Fab, Fab’, F(ab’)2, and Fv fragments, scFv’s, and single domain variable antibodies (VHHs) have been used for specific binding to targeted larger molecules such as proteins, but few have been isolated or engineered to detect smaller molecules. For example, single domain antibodies specific for mycotoxins, insecticides, biomarkers, and flame retardants have been characterized, but their affinities for these targets have been moderate and have not led to any real-world applications.

[0005] Thus, what is needed are improved VHHs with high specificity and selectivity tor small molecules

SUMMARY OF THE INVENTION

[0006] Die present disclosure provides single variable domain antibodies or VHHs that specifically bind to chloramphenicol or a chloramphenicol analog, where the VHHs comprise favorable properties for biotechnological applications.

[0007] In one aspect, the present disclosure is directed to a single variable domain antibody (VHH) that specifically binds to chloramphenicol and/or a chloramphenicol analog, comprising: (a) a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the ammo acid sequence GRX3FSX4X5AMG (SEQ ID NQ:1), wherein: X3 is S or T; X4 is S, T, or N; and X5 is F or Y; (b) a CDR2 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the ammo acid sequence L ! S \ V X ~ X X .· X : : X : ; V (SEQ ID NO:2), wherein: X₇ is S or N; X₈ is H, G, P, or is absent; X₉ is G or I; Xjo is I, 8, or R; and Xu is T or 8; and (c) a CDR3 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to tire amino acid sequence ADSIPYGXisXieXnYRNPGY (SEQ ID NO:3), wherein: X₁₅ is D or S; Xi_{6 i}s S, A, or V; and Xn is R or S.

[0008] in an exemplar}' embodiment, the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence:

QX1QLVEX2GGGLVQAGGSLRLSCAASGRX3FSX4X5AMGWFRQAPGKEREX6VAAIS

WX₇X8X9XioXiiYXi2DSVKGRmSRDXi3AKNTVYLXi4MNSLKPEDTAVYYCAADSIP

YGX 15X10X57YRNPG YWGQGTQ VTV 8 S (SEQ ID NO:4), wherein: X, is V orL; X₂ is 8 or T; X3 is S or T; X4 is S, T, or N; X5 is F or Y ; X_¾ is W or F; X_? is S or N; Xs is H, G, P, or is absent; X9 is G or I; X10 is I, 8, or R; X11 is T or S; X12 is T or A; X13 is N or S; X» is Q or E; Xi5 is D or S; Xjg is S, A, or V; and Xn is R or S.

[0009] In an exemplar}- embodiment, XV, is W

[0010] in an exemplary embodiment, the CDR1 comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to an ammo acid sequence selected from SEQ ID NOs:5, 6, 7, or 8. In an exemplar}- embodiment, the CDR2 comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs:9, 10, 11, or 12. In an exemplary embodiment, tire CDR3 comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs:13, 14, or 15.

[0011] in an exemplary embodiment, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO:5, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0012] in an exemplary embodiment, the VHH comprises tire amino acid sequence set forth in SEQ ID NO: 16. In an exemplar}- embodiment, the VHH comprises the amino acid sequence set forth in SEQ ID NO: 17. In an exemplary embodiment, the VHH comprises the am o acid sequence set forth in SEQ ID NO:23.

[0013] In an exemplary embodiment, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO:6, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, and the CDR3 comprises the ammo acid sequence set forth in SEQ ID NO: 14.

[0014] In an exemplar}- embodiment, the VHH comprises the amino acid sequence set forth in SEQ ID NO: 18. In an exemplary embodiment, the VHH comprises the amino acid sequence set forth in SEQ ID NO:22.

[0015] In an exemplary embodiment, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO:7, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15.

[0016] In an exemplar}' embodiment, the VHH comprises the amino acid sequence set forth in SEQ ID NO: 19.

[0017] In an exemplary embodiment, the CDR1 comprises the amino acid sequence set forth in SEQ ID NQ:8, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, and the CDR3 comprises the amino acid sequence set forth in SE1Q ID NO: 13.

[0018] In an exemplar} embodiment, the VHH comprises the amino acid sequence set forth in SEQ ID NO:2(). In an exemplary' embodiment, the VHH comprises the amino acid sequence set forth in SEQ ID NO:21. [0019] In an exemplar}- embodiment, the VHH is derived from the coordinates set forth in

Table 8.

[0020] in any one of the above aspects and embodiments, the VHH comprises two constant domai s.

[0021] In any one of the above aspects and embodiments, the chloramphenicol analog is selected from the group consisting of florfenicoi, thiamphenicol, azidamphenicol, and an amphenicol antibiotic comprising a phenylpropanoid structure.

[0022] In any one of the above aspects and embodiments, the VHH further comprises an affinity tag. In an exemplary embodiment, the affinity tag is a polynucleotide or a polypeptide. In an exemplary embodiment, the affinity tag is selected from the group consisting of a FI AG -tag, a 6xHis-tag, and biotin.

[0023] In any one of the above aspects and embodiments, the VHH is immobilized on a solid substrate. In an exemplary embodiment, the solid substrate comprises a carbon surface, a glass surface, a silica surface, a plastic surface, a metal surface, a surface comprising a metallic coating, a surface comprising a chemical coating, a microbead, a porous polymer matrix, a ceiluiosie fiber, or any combination thereof in an exemplary embodiment, the solid substrate is a single walled carbon nanotube (SWCNT).

[0024] In another aspect, the present disclosure is directed to a nucleic acid encoding the VHH of any one of the above aspects and embodiments, optionally wherein the nucleic acid is comprised within a vector or host ceil.

[0025] In another aspect, the present disclosure is directed to a method for detecting the presence or absence of chloramphenicol or a chloramphenicol analog in a sample, comprising: contacting the sample with the VHH of any one of the above aspects and embodiments further comprising an affinity tag: and detecting the presence or absence of the affinity tag, thereby detecting the presence or absence of chloramphenicol or a chloramphenicol analog in the sample.

[0026] In an exemplary' embodiment, the sample comprises a food substance. In an exemplar}- embodiment, the sample comprises a drug substance.

[0Q27] In an exemplary embodiment, the affinity tag is a polynucleotide or a polypeptide in an exemplary embodiment, the affini ty tag is selected from the group consisting of a FLAG-tag, a 6xHis-tag, and biotin.

[0028] In an exemplar} embodiment, the VHH is immobilized on a solid substrate. In an exemplary embodiment, the solid substrate comprises a carbon surface, a glass surface, a silica surface, a plastic surface, a metal surface, a surface comprising a metallic coating, a surface comprising a chem ical coating, a microbead, a porous polymer matrix, a cellulosic fiber, or any combination thereof.

[0Q29] In an exemplary embodiment, the step of detecting the presence or absence of the affinity tag comprises contacting the sample containing the VHH with a detection reagent comprising a detectable moiety and a ligand that specifically binds to the affinity tag.

[0030] In an exemplar}' embodiment, the ligand that specifically binds to the affinity tag is a receptor for the affinity tag. In an exemplary embodiment, the ligand that specifically binds to the affinity tag is an antibody.

[0031] In an exemplary embodiment, the detectable moiety is selected from the group consisting of a fluorescent substance, a chemiluminescent substance, a colored substance, a magnetic substance, a radioactive substance, an enzyme, and a metal particle. In an exemplary embodiment, the detectable moiety produces a signal selected from the group consisting of fluorescence, chemiluminescence, radiation, color, and a magnetic property. [0032] in an exemplary embodiment, the chloramphenicol analog is selected from the group consisting of florfenicoL thiamphenicol, azidamphenicol, and an amphenicol antibiotic comprising a phenylpropanoid stracture.

[0033] in another aspect, the present disclosure is directed to a method for detecting the presence or absence of chloramphenicol or a chloramphenicol analog in a sample, comprising: contacting the sample with the VHH of any one of the above aspects and embodiments, wherein the VHH is immobilized on a single walled carbon nanotube (SWCNT); and detecting a signal output emitted by the 8WCNT, thereby detecting the presence or absence of chloramphenicol or a chloramphenicol analog in the sample.

[0034] In an exemplary embodiment, if the sample contains chloramphenicol, the SWCNT emits a first signal output. In an exemplary_'· embodiment, if tire sample does not contain chloramphenicol, the SWCNT emits a second signal output.

[0035] In an exemplary- embodiment, the sample comprises a food substance. In an exemplary embodiment, the sample comprises a drug substance.

[0036] In an exemplary embodiment, the chloramphenicol analog is selected from the group consisting of florfenicoL thiamphenicol, azidamphenicol, and an amphenicol antibiotic comprising a phenylpropanoid structure.

[0037] In another aspect, the present disclosure is directed to a device tor determining the presence of absence of chloramphenicol or a chloramphenicol analog in a sample, comprising: (a) a sample receiving member; (b) a carrier in fluid communication with the sample receiving member, wherein the carrier comprises a detection zone comprising immobilized chloramphenicol or a chloramphenicol analog; (c) the VHH of any one of the above aspects and embodiments further comprising an affinity- tag, which is mobile in the carrier in the presence of the sample; and (d) a detection reagent comprising a detectable moiety and a ligand that specifically binds to the affinity tag; wherein when the sample is applied to the sample receiving member, the VHH is mobilized such that the sample and the VHH is transported along the length of the carrier to the detection zone.

[0038] In an exemplary embodiment, if the sample contains chloramphenicol, the detectable moiety will not be detected in the detection zone. In an exemplary embodiment, if the sample does not contain chloramphenicol, the detectable moiety will be detected in the detection zone.

[0039] In an exemplary' embodiment, the affinity tag is a polynucleotide or a polypeptide. In an exemplary- embodiment, the affinity tag is selected from the group consisting of a FLAG-tag, a 6xHis-tag, and biotin. [0040] in an exemplary embodiment, the ligand that specifically binds to the affinity tag is a receptor for the affinity tag. in an exemplary embodiment, the ligand that specifically binds to the affinity tag is an antibody.

[0041] in an exemplary embodiment, tire detectable moiety is selected from the group consisting of a fluorescent substance, a chemiluminescent substance, a colored substance, a magnetic substance, a radioactive substance, an enzyme, and a metal particle. In an exemplary' embodiment, the detectable moiety produces a signal selected from the group consisting of fluorescence, chemiluminescence, radiation, color, and a magnetic property. [0Q42] in an exemplary embodiment, the sample comprises a food substance in an exemplary_' embodiment, the sample comprises a drug substance.

[0043] in an exemplary' embodiment, the chloramphenicol analog is selected from the group consisting of florfenicol, thiamphenicol, azidamphenicol, and an amphenicol antibiotic comprising a phenylpropanoid structure.

[0Q44] In another aspect, the present disclosure is directed to a device for determining the presence or absence of chloramphenicol or a chloramphenicol analog in a sample, comprising the VI II I of any one of the above aspects and embodiments, wherein the VHH is immobilized on a single walled carbon nanotube (SWCNT).

[0045] in an exemplary' embodiment, the sample comprises a food substance in an exemplary embodiment, the sample comprises a drug substance.

[0046] In an exemplary- embodiment, the chloramphenicol analog is selected from the group consisting of florfenicol, thiamphenicol, azidamphenicol, and an amphenicol antibiotic comprising a phenylpropanoid structure.

[0047] In another aspect, the present disclosure is directed to a device for determining the presence or absence of one or more analytes in a sample, comprising a plurality of single walled carbon nanotube (SWCNT) sensors configured to detect the one or more anal vies, wherein at least one SWCNT sensor is configured to detect chloramphenicol or a chloramphenicol analog, and comprises the VHH of any one of the above aspects and embodiments, wherein the VHH is immobilized on a single walled carbon nanotube (SWCNT).

[0048] In an exemplary embodiment, the sample comprises a food substance. In an exemplary embodiment, the sample comprises a drug substance.

[0049] in an exemplary' embodiment, the chloramphenicol analog is selected from the group consisting of florfenicol, thiamphenicol, azidamphenicol, and an amphenicol antibiotic comprising a phenylpropanoid structure. [0050] in another aspect, the present disclosure is directed to the use of the VHH of any one of the above aspects and embodiments, in a method for detecting chloramphenicol or a chloramphenicol analog in a sample

[0051] in an exemplary embodiment, the sample comprises a food substance in an exemplary embodiment, the sample comprises a drug substance.

[0052] In an exemplar}- embodiment, the chloramphenicol analog is selected from the group consisting of f!orfenicol, thiamphenicol, azidamphenicol, and an amphenicol antibiotic comprising a phenylpropanoid structure.

DESCRIPTION OF THE DRAWINGS

[0053] The foregoing and other objects of the present disclosure, the various features thereof as well as the disclosure itself may be more fully understood from the following description, when read together with the accompanying drawings in which:

[0054] FIG. 1 is a schematic representation of a sequence logo plot that shows the amino acid variation between the top 10 VHH hits that bind to chloramphenicol, where the CDR regions are indicated below the corresponding amino acids.

DESCRIPTION

[0055] Hie disclosures of these patents, patent applications, and publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein. The instant disclosure will govern in the instance that there is any inconsistency between the patents, patent applications, and publications and this disclosure.

[0056] Unless defined otheiwise, all 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. The initial definition provided for a group or term herein applies to that group or term throughout the present specification individually or as part of another group, unless otherwise indicated.

[0057] As used herein, the articles “a ’ and ‘"an” refer to one or to more than one (/. ? , to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the temi “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0058] As used herein, the term “about” will be understood by persons of ordinal} skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or ±10%, including ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0059] By the term “specifically binds,” as used herein with respect to a VHH, is meant a VI II I which recognizes a specific target, but does not substantially recognize or bind other molecules in a sample. For example, a VHH of the present disclosure specifically binds to chloramphenicol or a chloramphenicol analog.

[0060J As used herein, the term “chloramphenicol analog” refers to, for example, fiorfenicol, thiamphenicol, azidamphen col, and any amphenicol antibiotic comprising a phenylpropanoid structure. As such, in some examples, a VHH of the present disclosure specifically binds chloramphenicol, fiorfenicol, thiamphenicol, azidamphenicol, and any amphenicol antibiotic comprising a phenylpropanoid structure.

[0Q61] Amounts throughout this disclosure, various aspects of the disclosure can he presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should he considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range

[0062] As used herein, the term “antibody fragment” refers to a portion of an intact antibody of any type and refers to the antigenic determining and binding variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments.

[0063] As used herein, the term “VHH”, also known as single domain antibody fragments, VHH antibody fragments, VHH domains, and VHH antibodies, refers to the antigen binding fragment of heavy chain only antibodies that do not comprise a light chain.

[0064] The term “expression” or “expresses” are used herein to refer to transcription and translation occurring within a host cell, e.g., the expression of a single domain antibody that binds chloramphenicol . The level of expression of a product gene in a host cell can be determined on the basis of either the amount of corresponding mRNA that is present in the ceil or the amount of the protein encoded by the product gene that is produced by the cell. [0065] Food substance includes any ingestible, nutritious, animal, vegetable or other digestible material.

[0066] Drag substance includes any medicament in any form that provides a benefit or treatment to a live organism such as a mammal that may is ingestible, inhalable, or otherwise tolerable to a live organism.

Antibodies that Bind Chloramphenicol

[0067] The present disclosure provides single variable domain antibodies or VHHs comprising favorable properties for biotechnological applications.

[0068] Antibodies are useful in diagnostic and therapeutic applications as highly selective binders to a target of interest. However, their large size and having multiple domains make library screening difficult and limit their binding capabilities in challenging environments such as high temperatures or after long-term storage. See, e.g., Ingram et al., Anmii. Rev. Immunol (2.018) 36:695-715. Heavy chain-only antibodies, as they occur in camelids. can be minimized into a single variable domain called a VHH (marketed as a Nanobody® by Ablynx N.V.) with favorable properties for biotechnological applications.

[0069] Hie VHHs of the present disclosure comprises favorable properties for biotechnological applications VHHs are distinguished from conventional antibodies which consist of two heavy chains and two light chains. These heavy chain-only antibodies contain a single variable domain (VHH) and two constant domains (CH2, CHS). VHHs are further characterized by the presence of one or more distinguishing residues (‘"hallmark residues”) in one or more of the framework sequences. Generally, a VHH can be described as an amino acid sequence comprising a general structure of: FRl - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4, wherein FRl, FR2, FR3, and FR4 refer to framework regions 1 to 4, respectively, and CDR1, CDR2, and CDR3 refer to complementarity determining regions 1 to 3, respectively. Such hallmark residues are described in PCT Patent Publication No. WO 08/020079. VHHs of the present disclosure contain one or more of the hallmark residues described in WO 08/020079, or other hallmark residues of VHHs known in the art.

[0070] Hie cloned and isolated VHHs possess full antigen binding capacity and stability. Due to their small size and unique structure, VHHs find use in a variety of applications, including, but not limited to, those that are employed in fusion construct format, multivalent construct format, and those having various other modifications, e.g., half-life extending modifications. Examples of the applications and modifications of VHHs can he found in PCX Publication Nos. WO 08/101985 and WO 08/142164.

[0071] VHHs according to the disclosure can be stored at 4^CC and can tolerate incubation at 37°C for several months, thus having an improved shelf life for various applications, e.g., in a diagnostic platform. See, e.g., Anderson et al., Anal. Chem. (2008) 80:9604-9611. As further described herein, the WHs according to the disclosure can be employed as selective binding agents for a target small molecule (e.g. , chloramphenicol) to be used as a sensor for detecting the same in samples, such as food or drug samples.

[0Q72] in one example, the VHHs according to the disclosure specifically bind to chloramphenicol or a chloramphenicol analog. It will readily be recognized to those of skill in the art that use of the term “chloramphenicol,” as used herein, is expressly intended to refer to chloramphenicol and analogs of chloramphenicol that are known in the art.

Examples of chloramphenicol analogs i nclude, but are not limited to, florfenicoi, thiamphenicol, azidamphemcol, and any amphenicol antibiotic comprising a phenylpropanoid structure. Other examples of chloramphenicol are known in the art, and are described in, e.g., Dinos et al ., Antibiotics (2016) 5(2): 20.

[0073] VHHs of the present disclosure comprise die general structure: FRl - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4, wherein FRl, FR2, FR3, and FR4 refer to framework regions 1 to 4, respectively, and CDR1, CDR2, and CDR3 refer to complementarity determining regions 1 to 3, respectively. As known in the art, the CDR regions of antibodies determine antigen binding specificity.

[0074] Amino acid sequences making up the VHHs according to the disclosure are presented herein using the single letter code known to the skilled artisan, and are shown below in Table 1 with their three latter codes. Table 1:

[0075] A chloramphenicol-specific VHH of the disclosure may comprise a COR1 comprising an ammo acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence GRX3FSX4X5AMG (SEQ ID NO: I), wherein: X3 is S or T; X4 is S, T, or N; and X5 is F or Y. For example, a chloramphenicol specific VHH of the disclosure may comprise a CDR1 comprising an ammo acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs:5, 6, 7, or 8. A chloramphenicol specific VHH of the disclosure may comprise a CDR2 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence AiSWXrXgXgXioXnY (SEQ ID NO:2), wherein: X_{? i}s S or N; Xs is H, G, P, or is absent; Xg is G or I; X o is I, S, or R; and Xu is T or S. in one example, a chloramphenicol specific VHH of the disclosure may comprise a CDR2 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence AISWXrXsXsXioXiiY (SEQ ID NQ:2), wherein: X? is S or N; Xs is H, G, or P; X9 is G or I; Xio is I, S, or R; and Xu is T or S. In another example, a chloramphenicol specific VHH of the disclosure may comprise a CDR2 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence AISWX₇X₉X₁₀X₁₁ Y (SEQ ID NO:24), wherein: X? is S or N; Xg is G or 1; Xio is I, S, or R; and Xu is T or S. For example, a chloramphenicol specific VHH of the disclosure may comprise a CDR2 comprising an ammo acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs:9, 10, 11, or 12. A chloramphenicol specific VHH of the disclosure may comprise a CDR3 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence ADSIPYGXisXieXnYRNPGY (SEQ ID NQ:3), wherein: Xis is D or S; Xie is S,

A, or V; and X17 is R or S. For example, a chloramphenicol specific VHH of the disclosure may comprise a CDR3 comprising an ammo acid sequence having at least 80%, 5%, 90%, 95%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 13, 14, or 15.

[0Q76] Accordingly, the present disclosure provides a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog, comprising: (a) a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence GRX3FSX4X5AMG (SEQ ID NO: 1), wherein: X₃ is S or T; X is S, T, or N; and X₅ is F or Y; (b) a CDR2 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100%

I I sequence identity to the amino acid sequence AISWX₇X₈X₉X₁₀X₁1 Y (SEQ ID NO:2), wherein: X? is S or N; Xs is H, G, P, or is absent; X9 is G or 1; X10 is I, S, or R; and Xn is T or S; and (c) a CDR3 comprising an amino acid sequence having at {east 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence ADSIPYGXisXieXnYRNPGY (SEQ ID NO:3), wherein: X_{15 i}s D or S; X_i6 is S, A, or V; and X37 is R or S. In one example, the VHH comprises: (a) a complementarity detennining region 1 (CDR1) comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence GRX3FSX4X5AMG (SEQ ID NO: 1), wherein: X3 is S or T; X₄ is S, T, or N; and X5 is F or Y : (b) a CDR2 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to tiie amino acid sequence AlSWX_/XsXyXioXnY (SEQ ID N():2), wherein: X·_/ is S or N; Xg is H, G, or P; X9 is G or I: X o is I, S, or R; and Xu is T or S; and (c) a CDR3 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence ADSIPYGX15X16X17YRNPGY (SEQ ID NO:3), wherein: X15 is D or S; Xie is S, A, or V; and X17 is R or S. in another example, the VHH comprises: (a) a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the ammo acid sequence GRX3FSX4X5AMG (SEQ ID NO: 1), wherein: X_{3 i}s S or T; X_{4 i}s S, T, or N; and Xs is F or Y: (b) a CDR2 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence AISWX7X9X10X1 iY (SEQ ID NG:24), wherein: X_? is S or N; X₉ is G or I; XJO is I, S, or R; and Xu is T or S; and (c) a CDR3 comprising an ammo acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence ADSIPYGXisXieXnYRNPGY (SEQ ID NO:3), wherein: X₁₅ is D or S; X 6 is S, A, or V; and X₁₇ is R or S. As provided herein, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog comprises a CDR1 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs:5, 6, 7, or 8; a CDR2 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NQs:9, 10, 11, or 12; and a CDR3 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 13,

14, or 15. For example, a VHH of the disclosure that specifically binds to chloramphenicol and/or a chloramphenicol analog comprises a CDRi comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5, a CDR2 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NQ:9, and a CDR3 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In another example, a VHH of the disclosure that specifically binds to chloramphenicol and/or a chloramphenicol analog comprises a CDRl comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NQ:6, a CDR2 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In another example, a VHH of the disclosure that specifically binds to chloramphenicol and/or a chloramphenicol analog comprises a CDRl comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In another example, a VHH of die disclosure that specifically binds to chloramphenicol and/or a chloramphenicol analog comprises a CDRl comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the ammo acid sequence set forth in SEQ ID NO: 8, a CDR2 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the ammo acid sequence set forth in SEQ ID NO: 13.

[0077] As will be appreciated by those of skill in the art, outside the CDR sequences are the framework sequences. Hie CDR and framework sequences together make up the variable domain (or the VHH). Hie present disclosure also provides a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog, comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence:

QX1QLVEX2GGGLVQAGGSLRLSCAASGRX3FSX4X5AMGWFRQAPGKEREX6VAAIS

WX7X8X9X10X11YX12DSVKGRFTISRDX13AKNTVYLX14MNSLKPEDTAVYYCAADSIP YGXisXieXnYRNPGYWGQGTQVTVSS (SEQ ID N0:4), wherein: Xi is V or L; X_{2 i}s S or T: X₃ is S or T; X, is S, T, or N; X₅ is F or Y; X_{6 i}s W or F: X_{7 i}s S or N; Xs is H, G, P, or is absent; s is G or I; Xio is I, S, or R; Xn is T or S; Xn is T or A; Xu is N or S; X» is Q or E; Xi5 is D or S; C_½ is S, A. or V; and Xn is R or S. In one example, the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence:

QX1QLVEX2GGGLVQAGGSLRLSCAASGRX3FSX4X5AMGWFRQAPGKEREX6VAAIS WX₇X8X9XioXiiYXi2DSVKGRFnSRDXi₃AKNTVYLXi4MNSLKPEDTAVYYCAADSIP YGXiiXieXnYRNPGYWGQGTQVTVSS (SEQ ID NQ:4), wherein: Xi is V or L; X_{2 i}s S or T; X3 is S or T; X4 is S, T, or N; X5 is F or Y ; X_{¾ i}s W or F; X_? is S or N; Xs is H, G, or P; X9 is G or I; Xio is I, 8, or R; Xu is T or S; X ₂ is T or A; Xi₃ is N or S; X14 is Q or E; Xn is D or S; Xn is S, A, or V; and Xn is R or S. in one example, the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the ammo acid sequence:

QXIQLVEX₂GGGLVQAGGSLRLSCAASGRX₃FSX4X5AMGWFRQAPGKEREX6VAAIS

WX₇X9XioXnYXi2DSVKGRFnSRDXi₃AKNTVYLXi₄MNSLKPEDTAVYYCAADSIPY

GX 5X eX 7YRNPGYWGQGTQ VTV S S (SEQ ID NO:25), wherein: Xi is V or L; X_{2 i}s S or T; X3 is S or T; X4 is S, T, or N; X5 is F or Y; Xe is W or F; X? is S or N; X9 is G or I; Xio is I, S, or R; Xn is T or S; X12 is T or A; X13 is N or S: X14 is Q or E: X15 is D or S; Xig is S, A, or V; and Xi? is R or S For example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to an ammo acid sequence set forth in SEQ ID NQs:I5, 16, 17, 18, 19, 2.0, 21, 22 or 23 In another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may comprise an amino acid sequence having at least 80%, at least 85%, at least 90'%, at least 95%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 15. In another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to an ammo acid sequence set forth in SEQ ID NO: 16 In another example, a VHH that specifically hinds to chloramphenicol and/or a chloramphenicol analog may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 17. In another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may comprise an amino acid sequence having at least 80%, at least 85%, at least 90'%, at least 95%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 18. in another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to an ammo acid sequence set forth in SEQ ID NO: 19. in another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%_>, at least 99%, or 100%₎ sequence identity to an amino acid sequence set forth in SEQ ID NO:20. In another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%₎, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NQ:2I. In another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%o, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NQ:22. In another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%₎ sequence identity to an amino acid sequence set forth in SEQ ID NG:23.

[0078] A VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may consist of an amino acid sequence set forth in SEQ ID NOs: 15, 16, 17, 18, 19,

20, 21, 22, or 23. For example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may consist of an amino acid sequence set forth in SEQ ID NO: 15. In another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may consist of an amino acid sequence set forth in SEQ ID NO: 16. In still another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may consist of an amino acid sequence set forth in SEQ ID NO: 17. In yet another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may consist of an amino acid sequence set forth in SEQ ID NO: 18. In another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may consist of an amino acid sequence set forth in SEQ ID NO: 19. In still another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may consist of an asnino acid sequence set forth in SE1Q ID NO:20. In yet another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may consist of an amino acid sequence se t forth in SEQ ID NQ:21.

In another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may consist of an amino acid sequence set forth in SEQ ID NQ:22

In yet another example, a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog may consist of an amino acid sequence set forth in SEQ ID NO:23. [0079] Certain framework residues were found by the present inventors to contribute to enhanced binding properties. While competitive ELISAs are useful for gaining preliminary data on sensitivity and selectivity of a binder during screening, more robust methods are used to determine quantitative binding constants. Isothermal titration calorimetry (ITC) is a well- established quantitative technique that can determine the binding affinity, enthalpy change, and binding stoichiometry of two interacting molecules in solution. ITC was used to determine the binding behavior of promising binders against both its target of interest and structural analogs to more accurately assess selectivity. In addition to ITC, a crystal structure of a potentially useful VHH was obtained both bound and unbound to its target small molecule. Using this structure, the binding pocket within the protein and what residues bind directly to the small molecule have been determined. This structural information was used to guide site-directed mutagenesis of the VHH to improve its binding affinity

[0080] in one example, the binding affinity of a chloramphenicol-specific VHH was improved through structure-guided site-directed mutagenesis and the use of ITC. ITC results of the top two bits from the preliminary screen against chloramphenicol had dissoc iation constants of 1 iiM (Chl-B2; SEQ ID NO: 16) and lOQnM (Chl-Dl; SEQ ID NO: 18) (see,

Table 2). A crystal structure with a 1.4 A resolution of CM-B2 bound to chloramphenicol was obtained that provided a clear picture of the binding pocket. By comparing the crystal structure and sequence of Chl-B2 with the sequence of Chl-Dl, it was determined that a residue change from phenylalanine to tryptophan at position 47 improved binding affinity. By- introducing tliis one amino acid substitution into the Chl-B2 binder, the binding affinity was improved 19-fold (see, Table 2). Chl-B2F47W (SEQ ID NO:23), has a dissociation constant of 57 nM.

Table 2:

[0081] Accordingly, the present disclosure also provides a VHH that specifically binds to chloramphenicol and/or a chloramphenicol analog, comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence:

QX1QLVEX2GGGLVQAGGSLRLSCAASGRX3FSX4X5AMGWFRQAPGKEREX6VAAIS

WX7X8X9XioXuYXi2DSVKGRFnSRDXi3AKNTVYLX 4MNSLKPEDTAVYYCAADSIP

Y GX₁₅X₁₆X ₁₇YRNPGYW GQGTQVTV SS (SEQ ID NO:4), wherein: X, is V or L; X_{2 i}s S or T; ¾ is S or T; X₄ is S, T, or N; X_{5 i}s F or Y; X_{6 i}s W; X? is S or N; X* is H, G, P, or is absent: X9 is G or I; X₁₀ is I, S, or R; Xn is T or S; X1₂ is T or A; Xn is N or S: X14 is Q or E: Xis is D or S; X e is S, A, or V; and Xn is R or S. In one example, die VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence:

QX1QLVEX2GGGLVQAGGSLRLSCAASGRX3FSX4X5AMGWFRQAPGKEREX6VAAIS WX7XeX9XioXiiYXi2DSVKGRraSRDXi₃AKNTVYLXi4MNSLKPEDTAVYYCAADSIP YGXisXisXnYRNPGYWGQGTQVTVSS (SEQ ID NG:4), wherein: Xi is V or L; X_{2 i}s S or T; X_{3 i}s S or T; X_{4 i}s S, T, or N; X₅ is F or Y; X_{6 i}s W or F; X_{7 i}s S or N; X_s is H, G, or P; X₉ is G or I; X10 is I, S, or R; X is T or S; X ₂ is T or A; Xn is N or S; Xu is Q or E; Xu is D or S; Xi6 is S, A, or V; and Xi? is R or S. In one example, the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence:

QXiQLVEXaGGGLYQAGGSLRLSCAASGRXsFSXiXsAMGWFRQAPGKEREXeVAAIS WX7X9XioXiiYXi2DSVKGRFTlSRDX₁₃AKNlA'YLXi4MNSLKPEDTA\YYCAADSiPY GXisXieXnYRNPGYWGQGTQVTVSS (SEQ ID NG:25), wherein: Xs is V or L; X_{2 i}s S or T; Xi is S or T; X4 is S, T, or N; X5 is F or Y; Xc, is W or F; X? is S or N; X9 is G or I; X o is 1, S, or R; n is T or S; C·₂ is T or A; Xn is N or S; Xu is Q or E; u is D or S; Xu is S, A, or V; and Xs? is R or S. For example, a VHH of the present disclosure comprises a tryptophan (W) at position 47 (i.e., at position Xe of SEQ ID NO:4). For example, a VHH of the present disclosure comprises or consists of the amino acid sequence set forth in SEQ ID NQ:23.

[0082] Exemplary VHHs of the present disclosure are described in Table 3. Table 3:

jOOOl j Determination of a CDR and identification of residues comprising the binding site of an antibody (e.g, a VHH that specifically binds to chloramphenicol) is accomplished by- solving the stracture of the antibody and/or sol ving the structure of the anti body-ligand complex (e.g., a VHH-chloramphenicol complex). A crystal structure was obtained with a resolution <1.4/1 of Chl-B2 bound to chloramphenicol. The coordinates for the crystal structure discussed above are presented in Table 8. By comparing the crystal structure and sequence of Cbl-B2 with the sequence of Chl-Dl, an important residue change from phenylalanine to tryptophan was determined to improve the binding affinity. By introducing this one phenylalanine to tryptophan substitution at position 47 into the Chi-B2 VHH, the binding affinity of the VHH was improved 19-fold (see, Table 2) It will be readily appreciated by those of skill m the art that the binding affinity-improving phenylalanine to tryptophan substitution at position 47 of Chl-B2 can be used to identify a corresponding position for introducing a similar substitution in any VHH provided herein.

[0002] It should be noted that the present disclosure is not limited to as to the origin of the amino acid sequence or nucleic acid sequence encoding the same, nor as to the method in which the amino acid sequence of nucleic acid sequence encoding the same has been obtained or generated. For example, the present disclosure also includes, without limitation, humanized VHH sequences (e.g., a partially or fully humanized VHH sequence), VHH sequences that have been obtained by standard techniques known in the art such as affinity- maturation, CDR grafting, veneering, combining fragments derived from different VHHs, PCR assembly using overlapping primers, and similar techniques for engineering antibody- sequences well known to the skilled artisan. For example, humanization is generally known to those of skill in the art to involve replacing one or more amino acid residues in the sequence of a naturally occurring VHH with the am o acid residues that occur at the same position in a human heavy chain variable domain. Standard techniques are available and readily accessible to those of skill in the art, for example, in standard handbooks.

VHH Conjugates

[0003] The VHHs described herein can be a part of a conjugate. For example, A VHH of the present disclosure can further comprise two constant domains. Such a construct is known in the art as a heavy chain only antibody.

[0004] The present disclosure also encompasses constructs and polypeptides that comprise a VHH described herein. For example, a VHH described herein can comprise other functional moieties that find various uses in the industry. Such moieties include, without limitation, chemical groups, modified residues. The functional moieties may be functional on their own (e.g., independently possessing biological and/or pharmacological properties), or are functional in the context of the rest of the construct or polypeptide that comprises a VHH of the disclosure. It will be appreciated to those of skill in the art that a functional moiety can be linked to a VHH of the disclosure directly or indirectly. For example, a VHH described herein can be linked to a functional moiety directly, e.g., via covalent means or other means known the art. Alternatively, a VHH described herein can be linked to a functional moiety indirectly, e.g. , via a linker moleeuie. A linker molecule can be a variety of linkers known to those of skill in the art, e.g., a peptide or nucleic acid linker.

[0005] A functional moiety includes an affinity tag. An affinity tag refers to a moleeuie incorporated to either the N- or C -terminal end of a recombinant protein (e.g., a VHH of the present disclosure). In some cases, the affinity tag may be a molecule that is incorporated within the sequence of a recombinant protein (e.g., a VHH of the present disclosure). In such cases, it is typically desired that the affinity tag docs not interfere with physiological aspects of the recombinant protein (e.g. , a VHH of the present disclosure). For example, an affinity tag may be incorporated within the sequence of a recombinant protein by appending the tag to a residue of the recombinant protein in a manner that does not interfere with the folding and/or function of the recombinant protein. Affinity tags arc used in the art to facilitate purification of the tagged protein and in other applications such as facilitating the detection of the tagged protein, and/or improving the solubility of the tagged protein. A VHH of the present disclosure can be linked to any affinity tag known in the art. For example, an affinity tag may be a polynucleotide or a polypeptide. Examples of affinity tags include, without limitation, a polyhistidine tag (several consecutive histidine residues, e.g., a 6xHis tag), a polyarginine tag (several consecutive arginine residues), a glutathione-S-transferase (GST) tag (having affinity for glutathione), a FLAG teg, a streptavidin-binding peptide, a calmodulin-binding peptide, a chitin-binding tag, a maltose-binding teg, a cellulose-binding tag, and a biotin tag. The uses and advantages of each affinity tag will be clear to the skilled artisan, who will readily be able to select the appropriate affinity tag for an intended purpose. [0006] Other functional moieties include, for example, detectable moiety and/or signal generating moieties. The skilled artisan will readily be able to select the appropriate detectable moiety and/or signal-generating moiety for an intended purpose. Detectable moieties can be any moiety that produces or can be induced to produce a signal. Examples of such include, without limitation, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrm, phycocyamn, aliophycocyanin, o-phthaldehyde, and fluoreseamine and fluorescent metals such as 152Eu or others metals from the lanthanide series), phosphorescent labels, chemiluminescent labels or biolumine scent labels (such as luminal, isolumino!, theromatic aeridinium ester, imidazole, aeridinium salts, oxalate ester, dioxetane or GFP and its analogs), radio-isotopes (such as 3FI 1251, 32P, 35S, 14C, 51Cr, 36C1, 57Co, 58Co, 59Fe, and 75Se), metals, metals chelates or metallic cations (for example metallic cations such as 99mTc, 1231, l l lln, 1311, 97Ru, 67€u, 67Ga, 68Ga, !57Gd, 55Mn, 162Dy, 52Cr, and 36Fe)), colored moieties (e.g., latex or colloidal gold), as well as chfomophores and enzymes (such as rnalate dehydrogenase, staphylococcal nuclease, delte- V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, b-galactosidase, ribonuclease, urease, catalase, glucose- Vl-phosphate dehydrogenase, glucoamyiase and acetylcholine esterase).

Accordingly, the detectable moiety can be any detectable moiety selected from, without limitation, a fluorescent substance, a chemiluminescent substance, a colored substance, a magnetic substance, a radioactive substance, an enzyme, or a metal particle.

[0007] The detectable moiety can be directly or indirectly coupled to a VHH of the present disclosure. For example, where the detectable moiety is a metal or metallic cation, the VHH may include a chelating group. Suitable chelating groups include, without limitation, diethyl-enetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA). Detectable moieties may be attached to a VHH of the present disclosure using conventional chemistry' and means known in the art.

[0008] Such detectable moieties may be linked to a ligand that specifically binds to an affinity tag, e.g., a VHH described herein that further comprises an affinity tag. The ligand can be any ligand known in the art that comprises specificity for the affinity tag, e.g., a receptor that specifically binds the affinity tag, or an antibody that specifically binds the affinity tag.

[0009] The detectable moiety can produce a detectable signal, for example, without limitation, a fluorescent signal, a chemiluminescent signal, color, radiation, a magnetic property, or a paramagnetic property . Other suitable detectable moieties will be clear to the skilled artisan. The detectable moiety may be detected by a detector that is configured to detect the signal produced by the detectable moiety. Examples of suitable detectors include without limitation, those that detect signals using NMR or E8R spectroscopy, detectors configured to detect fluorescence or luminescence, color, radioactivity, enzyme activity, or changes in magnetic field. Further examples of detectors are those that can detect a detectable moiety via optical interrogation, e.g, via light absorption, light scattering, light diffraction, light refraction, light reflection, fluorescence, or luminescence.

[0010] Other suitable functional moieties are those that are part of a specific binding pair, such as the biotin~(strept)avidiii binding pair. Such a functional moiety may be used to link a VHH of the present disclosure to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair. For example, a VHH of the present disclosure may be conjugated to biotin, and linked to another protein, polypeptide, compound or earner conjugated to avidin or streptavidin. For example, such a conjugated construct or polypeptide of the invention may be used as a reporter, for example in a diagnostic system where a detectable signal -producing agent is conjugated to avidin or streptavidin.

[0011] Other suitable functional moieties will he recognized by those of skill in the art, for example, without limitation, toxins, or therapeutics. VHHs according to the disclosure can be immobilized (e.g., adhered or conjugated to) on a substrate

[0012] Hie substrate may be a solid substrate. Examples of suitable substrates include, without limitation, those that comprise a carbon surface, a glass surface, a silica surface, a plastic surface, a metal surface, a surface comprising a metallic coating, a surface comprising a chemical coating, a microbead, a porous polymer matrix, a cellulosic fiber, or any combination thereof. Other substrates will be known to those of skill in the art and selected for an intended purpose in one example, the solid substrate is a single walled carbon nanotube (SWCNT).

[0013] Such VHH conjugates described herein may, for example, be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other “sandwich assays”, etc., and lateral flow assays, e.g., lateral flow immunoassays). Conjugation of VHHs to SWNTs for Optical Detection

[0Q14| Single walled carbon nanotubes (SWCNTs) are nanomaterials that when singly dispersed in solution phase via non-covalent conjugation exhibit near infrared (iiiR) fluorescence upon visible light excitation. Non-covalent wrapping in some cases imparts varying degrees of optical signal modulation in response to changes of the local environment or the presence of specific molecules. In addition to using the nanotube corona itself, additional techniques have been developed to allow for the tethering of molecular recognition elements such that a binding event can be translated to a subsequent optical response. Further description of SWCNTs can be found in, e.g., U.S. Patent No. 9,901,295.

[0015] The near infrared region of the electromagnetic spectrum has advantages tor in vivo fluorescence imaging, due to minimal auto fluorescence and absorption of blood and tissue. See, Frangiom, Curr Opin. Chem. Biol 7, 626-634 (2003), and Wray et al, Biochim. et Biophys. Acta 933, 184-192 (1988). Examples of common nlR fluorescent agents include organic nlRf!uorophores, such as Indocyanine green (ICG), semiconductor qua tum dots (Qdots), and single-walled carbon nanotubes (SWNT). The ICG dye has previously been utilized for real time detection of liver cancer, and sentinel lymph node mapping in breast cancer patient. Biofunctionalized CdSe/ZnS Qdots and InAs/InP/ZnSe Qdots has previously been used for tumor targeting and fluorescent imaging in mice. In another example, nlR fluorescent phosphine coated CdTe/CdSe Qdots have been utilized for sentinel lymph node mapping.

[0016] Several different conjugation strategies can be used for VHH attachment to the nlR sensor. One molecular recognition scheme utilizes a known recognition element (e.g., antibody, receptor, affinity peptide) containing a hexahistidine tag. Chitosan-wrapped SWCNTs are embedded a hydrogel and functionalized with nitrilotriacetic acid (NTA) chelating groups to tether Cu(II) ions close to the nanotube surface. In one example, the Cu(II) ions function as proximity quenchers of SWCNT fluorescence and bind to the hexahistidine-tag of the recognition element. Subsequent binding of the analyte protein changes the position of the Cu(ll) ions, resulting in a fluorescence response. This recognition scheme has been employed in a variety of studies including the detection of glycoproteins, protein A and human immunoglobulin G.

[0017] Another approach for conjugation involves the attachment of a DNA oligo to the VHH and using a complementary DNA sequence wrapped around the SWCNT. Previous studies have attached DNA oligos of various lengths to the C terminus of VHHs via SMCC conjugation. See, e.g., Mann et al., Angew. Chemie - Int. Ed. (2019) 58:11469-11473, and W u eta!., (2019) Angew. Chernie - Ini. Ed. 58: 14224-14228. In brief, 3 "-amine-modified DMA oligos are reacted with the bifunetional crosslinker sulfosuecimmidyi 4-(N-maleimido- methyl)cyclohexane-l-carboxylate (Sulfo-SMCC) to generate cysteine -reactive DMA oligonucleotides. These modified DMA sequences can be used to noneovalently wrap the hydrophobic SWCNT surface via ultrasomcation and can then react to an exposed cysteine on the VHH. Since the position of the cysteine in the binder may affect both the binding pocket to the target (e.g., chloramphenicol), as well as the signal transduction to the SWCNT, various strategies are employed in determining the ideal cysteine location on the binder in one example, a cysteine is attached to the C terminus of the binder (e.g., a VHH of the present disclosure) where it is least likely to affect the residues that contribute to the binding pocket of the binder. While this location can be consistent across all binders, its location may affect the ability of the SWCNT to sense a binding event far from the linker. In another example, surface-exposed positions on the binder where a cysteine could be inserted that are dynamically correlated to binding pocket residues, can be modeled using computational methods. These sites sense a binding event on the binder, and if a linker is atached at this position, will transduce an optical signal to the SWCNT.

[0018] Additionally, the DNA sequence employed to w rap around the SWCNT may affect sensor output. Adsorption of the DNA on SWCNT can be modulated by base pair choice.

See, e.g., Shankar et ah, Langmuir (2014) 30:3176-3183, and Salem et ah, J Am. Chem. Soc. (2017) 139: 16791-16802. Those of skill in the art will be able to test and select a suitable linker from those of varying lengths of differently adsorbing sequences to tether the binder at a distance such that the binding event can be optimally transduced.

[0019] Accordingly, a VHH-SWCNT conjugate has a characteristic baseline fluorescence signal. When the target of the VHH (e.g., chloramphenicol) binds to the VHH-SWCNT, it will cause either an intensity or wavelength shift in the nIR optical signal as compared to the baseline. The magnitude of this deviation can be correlated to the amount of chloramphenicol present in the sample. The skilled artisan would understand that the deviation may be dependent on the sensitivity of the detection equipment employed. Each VHH-SWCNT may have a unique response that would have different sensitivities. In one example, a VHH-SWCNT shows a decrease in nIR signal when bound to the target ligand (e.g., chloramphenicol). In another example, a VHH-SWCNT show's an increase nIR signal when bound to the target ligand (e.g., chloramphenicol). In addition, the baseline signal can depend on the pH and osmolarity of the sample solution. [0020] As would be appreciated by those of skill in the art, in order to perform such measurements relating to the amount of an analyte (e.g., chloramphenicol) present in a sample, basic components needed include, e.g., a visible light source, optics to direct the light, and a detector to measure the nIR signal in one example, a laser can be used as a light source. In another example, a light emitting diode (LED) can be used as a light source. Further, VHH-SWCNTs provided by the present disclosure can be configured in a variety of form factors, including in a solution, embedded in a hydrogel, or deposited on a surface such as a glass slide or paper format.

VT1H Production

[0021] The present disclosure also provides methods for preparing the VHHs described herein. VHHs of the present disclosure can be prepared by any method known to those in the art for the preparation of antibodies and/or the preparation of antibody fragments. For example, methods for producing VHHs generally include recombinant expression of the VHH in a suitable host ceil or host organism, or other suitable expression systems for the expression of a nucleic acid that encodes the VHH. In particular, expression of the VHH in a suitable host cell or host organism includes cultivating and/or maintaining the host cell or organism under conditions such that the host cell or organism expresses the VHH. Following expression of the VHH, isolation and/or purification of the VHH is performed, thereby obtaining an isolated VHH of the present disclosure.

[0022] Accordingly, the present disclosure also provides nucleic acid sequences that encode a VHH as described herein. A nucleotide sequence encoding a VHH described herein can be present in an expression vector and/or a cloning vector. An expression vector can include a selectable marker, an origin of replication, and other features that provide tor replication and/or maintenance of the vector.

[0023] Large numbers of suitable vectors with appropriate promoters for various expression hosts are known to those of skill in the art; many are commercially available for generating a VHH described herein. Exemplary bacterial expression vectors include, for example, the pET vector series from Miilipore Sigma.

[0024] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g. , Li et a!.. Invest Opthalmol Vis Set 35:2543 2549, 1994; Borras eta!., Gene B ' ier 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et ah, H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno- associated vims (see, e.g., Ali et ah, Hum Gene Ther 9:81 86, 1998, Flannery et ai. , PNAS 94:6916 6921, 1997; Bennett et ai, Invest Opthalmol Vis Sci 38:28572863, 1997; Jomaty et ah, Gene Ther 4:683 690, 1997, Rolling et ah, Hum Gene Ther 10:641 648, 1999; Ali et ah. Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski etah,J Vir. (1989) 63:3822-3828; Mendelson etal, Virol (1988) 166:154-165; and Flotte et ah, PNAS (1993) 90:10613-10617); SV40; herpes simplex vims; human immunodeficiency vims (see, e.g., Miyoshi et ah, PNAS 94:10319 23, 1997; Takahashi etal.,J. Virol. 73:78127816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency vims, myeloproliferative sarcoma vims, and mammary tumor virus); and the like.

[0025] Suitable promoter and enhancer elements are known in the art. For expression in a bacterial cell, suitable promoters include, but are not limited to, lad, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, light and/or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters.

[0Q26] For expression in a yeast ceil, a suitable promoter is, e.g. , a constitutive promoter such as an ADH1 promoter, a PGKI promoter, an ENQ promoter, a PYKI promoter and the like; or a regulatable promoter such as a GALI promoter, a GAL 10 promoter, an ADH2 promoter, a PHOS promoter, a CUP1 promoter, a GALT promoter, a MET25 promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1 promoter, and AOX1 (e.g., for use in Piehia). Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. [0027] Suitable promoters for use in prokaryotic host cells include, but are not limited to, a bacteriophage T7 RNA polymerase promoter; atrp promoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/Iae promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as an ssaG promoter or a related promoter (see, e.g., U.S. Patent Publication No. 20040131637), a pagC promoter (Pulkkinen and Miller, J. Bacterial., 1991: 173(1): 86-93; Alpuche -Aranda et al, PNAS, 1992; 89(21): 10079-83), a mrB promoter (Harhome et al., (1992) Mol. Micro. 6:2805-2813), and the like (see, e.g., Dunstan etal, (1999) Infect. Immun. 67:5133-5141; McKelvie et al, (2004) Vaccine 22:3243-3255; and Chatfield ei ai.., (1992) Biotechnoi. 10:888-892); a sigma70 promoter, e.g., a consensus sigma?0 promoter (see, e.g., GenBank Accession Nos. AX798980, .4X798961, and AX798183); a stationary phase promoter, e.g., a dps promoter, a spy promoter, and the like; a promoter derived from the pathogenicity island SPI-2 (see, e.g., W096/17951); an aetA promoter (see, e.g., Shetron-Rama et al, (2002) Infect. Immun. 70: 1087-1096); an rpsM promoter (see, e.g, Valdivia and Falkow (1996). Mol. Microbiol. 22:367); a let promoter (see, e.g., Hillen, et al, (1989) In Saenger, W. and Heinematm, U. (eds), Topics in Molecular and Structural Biology, Protein— Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); an SP6 promoter (see, e.g, Melton et al, (1984) Nucl. Acids Res. 12:7035); and the like. Suitable strong promoters for use in prokaryotes such as Escherichia coli include, but are not limited to Tre, Tac, T5, T7, and pLambda. Non-limiting examples of operators for use in bacterial host cells include a lactose promoter operator (Lad repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), a tryptophan promoter operator (when complexed with tryptophan, TrpR repressor protein has a conformation that binds tire operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and a tac promoter operator (see, for example, deBoer et al, (1983 ) Proc. Natl. Acad. Sci. U.S.A. 80:21-25).

[0028] The present disclosure also provides isolated genetically modified host cells that are genetically modified with a nucleic acid described herein. Isolated genetically modified host cells described herein am produce a VHH or VHH conjugate of the present disclosure. [0029] Suitable host cells include eukaryotic host cells, such as a mammalian cell, an insect host cell, a yeast cell; and prokaryotic cells, such as a bacterial cell. Introduction of a nucleic acid into the host cell can be achieved, for example, by calcium phosphate precipitation, DEAL dextran mediated transfection, liposome -mediated transfection, electroporation, or other known method.

[0030] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate ceil lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL6L CRL9096), 293 cells (e.g, ATCC No. CRL- 1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g, ATCC No CCL10), PC 12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.

[0031] Suitable yeast cells include, but are not limited to, Pichia pasterns, Pichia finlandica , Pichia trehalophila, Pichia koclamae, Pichia memhranaefaciem , Pichia opuntiae , Pichia thermotolerans , Pichia salictaria , Pichia guercuum, Pichia pi j peri, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polyrnorpha , Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowen.se, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonas remhardtii, and the like

[0032] Suitable prokaryotic cells include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Lactobacillus sp., Salmonella sp., Shigella sp., and the like. See, e.g.. Carrier etal., (1992) J Immunol. 148: 1176-1181; U S. Pat No. 6,447,784; and Sizemore et ah, (1995) Science 270:299-302. Examples of Salmonella strains which can be employed in the present invention include, but are not limited to. Salmonella typhi and S. typhimurium . Suitable Shigella strains include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella disenteriae. Typically, the laboratory_' strain is one that is non- pathogenic. Non-limiting examples of other suitable bacteria include, but are not limited to, Bacillus subtilis. Pseudomonas pudita, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroid.es, Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus sp., and the like. A useful host cell is Escherichia coli.

[0033] The VHH of the present disclosure may then be isolated from the host cell or host organism and/or from the medium in which the host cell or organism was cultivated.

Methods for the isolation and/or purification of proteins from cells and media are know n to those of skill in the art, for example, chromatography and electrophoresis techniques, differential precipitation techniques, affinity techniques (e.g., using affinity tags/labels), and or immunological techniques (e.g., immunoaffmity techniques).

Applications for Chlora phenicol VHHs

[0034] Testing for the presence of small molecule contaminants in food processing is important for the health of consumers. Current testing of products in the food supply chain is slow and expensive. Most samples are sent to third-party labs which have slow turn-around time, requires the use of expensive equipment such as GC-MS, LC-MS, etc., requires sample preparation prior to measurement, and requires a different test for each analyte that must be tested in a sample. Current field tests lack quantitative measurements and one test that can test for a variety of analytes.

[0035] Because these food products are passing quickly through many hands along the supply chain, current testing methods are either too slow, expensive, or inadequate to keep up. Field tests do not provide quantitative measurements, but only indicate whether a contaminant is present or absent. Quantitative measurements are important since low-level amoun ts of certain contaminants are allowed at low doses. In addition, there is a separate kit for every analyte, making it difficult to test each potential contaminant in a rapid manner. Because of this, many samples are sent to third party labs for testing in order to pro vide quantitative data. Unfortunately, this usually requires expensive equipment and the turn around time is long, on the order of days. This is inefficient and is difficult for perishable food items that need results quickly.

[0036] Seafood is one of the largest traded commodities in the world today and over half originates in developing countries. Because fish grown in confined aquaculture areas can have high rates of bacterial infections, farmers may treat them with drugs, such as antibiotics and antifungal agents, to increase their survival rates. Once farmers introduce drugs, either m feed or water, drag residues can remain in the fish through harvesting, processing, and consumption. The use of antibiotics or chemicals, such as malachite green, nitrofurans, and fluoroquinolones during the various stages of aquaculture can result in the presence of residues of the parent compound or its metabolites in the edible portion of the seafood. The presence of antibiotic residues may contribute to an increase of antimicrobial resistance in human pathogens. Moreover, prolonged exposure to nitrofurans, malachite green, and gentian violet has been shown to have a carcinogenic effect. See, e.g., FDA. Import Alert (2019) 16- 131. (2039).

[0037] It is difficult for inspection agencies to increase the volume and frequency of safety tests due to the cost and time required to do the rigorous analytical experiments using HPLC and mass spectrometry to determine all the potential adulterants in a food product. On the other hand, current rapid test kits for these adulterants have low accuracy, low throughput, and an inability to test for multiple adul terants.

[0038] Accordingly, the VHHs and VHH conjugates of the present disclosure can be used in the detection of contaminants {e.g., chloramphenicol and chloramphenicol analogs) in food, feed, environmental samples, and medical samples. In one example, the VHHs and VHH conjugates of the present disclosure can be used to detect the presence or absence of chloramphenicol and chloramphenicol analogs in plants, feed, and food of non-animal origin, including, e.g., without limitation, various herbs (e.g., Thalictrum, Artemisia, Thermopsis species), various grasses, straw (i.e., in animal feed), wheat, and maize. In another example, the VHHs and VHH conjugates of the present disclosure can be used to detect the presence or absence of chloramphenicol and chloramphenicol analogs in food of animal origin, including, e.g., without limitation, honey, royal jelly, milk, milk products (e.g., powdered milk), seafood (e.g., fish, shrimp, farmed and wild seafoods), seafood products, meat (e.g, poultry', bovine, pig, sheep, goat, horse, rabbit, farmed game, and wild game meat), and meat products (e.g., eggs). In another example, the VHHs and VHH conjugates of the present disclosure can be used to detect the presence or absence of chloramphenicol and chloramphenicol analogs in medical samples, including, e.g., without limitation, a sample obtained from medical equipment, drug formulations (e.g., ophthalmic solutions), drug substances.

[0039] The chloramphenicol VHHs and VHH conjugates disclosed here can be used in various technology platforms that provides quantitative measurement of chloramphenicol and its analogs on-site. These analogs include florfenicol, thiamphenicoi, azidamphenicol, and other amphenicol antibiotics with a phenylpropanoid structure.

Assay Methods Using VHHs

[0040] The present disclosure provides assay methods for the quantitative determination of an analyte (e.g., chloramphenicol) in a sample. Various assays, e.g., immunoassays are known in the art and can employ the VHH and VHH conjugates of the present disclosure.

An immunoassay is a biochemical method that identifies and quantifies an analyte in a sample, typically using antibody reactions. A variety of methods are known to those of skill in the art for visualizing a primary antibody-antigen reaction, e.g. , a VHH-chloramphenicol reaction. Precipi tation of large cross linked VHH-chloramphenicol complexes can be visible to the naked eyes or detection can be enhanced with the use of labeled reagents.

[0041] immunoassays generally employ a variety of different labels or moieties to allow the detection of antibodies and antigens, e.g., VHHs or VHH conjugates of the present disclosure and chloramphenicol. In one example, a radioimmunoassay (RIA) is provided that employs the use of radioactive isotopes to label the VHH. In an RIA, the radioactivity is detected to measure the VHH-antigen complex with high sensitivity. In another example, an enzyme immunoassay (El A) is provided that employs enzymes (e.g, HRP, AP) as probes. Hie enzymes allow detection in the form of an observable color change in the presence of certain substrate and chromogen reagents based on enzymatic reactions in another example, a fluoroimmunoassay (FIA) is provided that is similar to El A but employs the use of f!uorophores (e.g, FITC, phycoerythrin) instead of a radioisotope. The resulting fluorescence signal can be measured directly by a detecting instrument configured to do the same. In another example, a chemiluminescence immunoassay (CLTA) is provided that determines the concentration of an analyte in a sample according to the intensity of the luminescence that results from a chemical reaction. Various chemiluminescent labels and substrates are known in the ait and suitable for use, for example, lumino!, isohiminol, acridinium ester derivatives, and peroxidase.

[0042] in another example, nanocomposite immunosensors can employ the VHH and VHH conjugates of the present disclosure. For example, the electrochemical properties of Prussian blue-functionalized graphene oxide can be used in conjunction with a VHH of the present disclosure in order to develop a highly selective and sensitive immunosensor for the detection of a chloramphenicol. Functionalized graphene oxide is further described in Pan et ui . Chem. Ear. J (2018) 24:9869-9876.

[0043] Also described herein are applications using a VHH and/or VHH conjugate of the present disclosure as optical sensors, in the context of single w alled carbon nanotube (SWCNT). Optical sensors based on the properties of SWC Ts are known in the art and employed by those of skill in the art for converting biological cues into detectable signals. Such devices are of high interest by the analytical community due to their high sensitivity, rapid detection rate, non-invasive nature, and non-destructive mode of action. SWCNT sensors are further described in Hofferber etai, J Electrochem. Society (2020) 167:037530.

Kits and Devices

[0044] The present disclosure also provides kits and de vices comprising a VHH described herein. In one example, the kits and devices of the present disclosure find use in situations where the determination of the presence or absence of an analyte (e.g., chloramphenicol and/or analogs thereof) is desired.

[0045] Certain devices include SWCNT sensors, such as those that can be configured and integrated into a portable assay kit. In one example, one or more SWCNT sensors can be incorporated into the same device, e.g., a multiplexed array in a paper-based format for the development of a portable assay kit. The portable setup consists of a simple optical setup with LED excitation and an NIR photo detector. The SWCNT -VHH complex can be deposited and stored on a nitrocellulose barcode (see, e.g. , Salem ei a!.. Anal. Chem. (2020) 92:916-923) read out by translating the optical beam across the barcode regions. Using multiple sensors on a singl e test solution, an array of targets can be differentiated and measured in parallel.

[0046] in one example, the present disclosure provides a device for determining the presence or absence of chloramphenicol or a chloramphenicol analog in a sample, comprising a VHH as described herein, wherein the VHH is immobilized on a single walled carbon nanotube (SWCNT). Regarding a multiplexed array, in another example, provided herein is a device for determining the presence or absence of one or more analytes in a sample, comprising a plurality of single walled carbon nanotube (SWCNT) sensors configured to detect the one or more analytes, wherein at least one SWCNT sensor is configured to detect chloramphenicol or a chloramphenicol analog, and comprises a VHH as described herein, wherein the VHH is immobilized on a single walled carbon nanotube (S WCNT).

[0047] Other devices provided herein are configured to perform in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other “sandwich assays”, etc., and lateral flow assays, e.g., lateral flow immunoassays).

[0048] In one example, a lateral flow immunoassay device is provided. For example, provided herein is a device for determining the presence of absence of chloramphenicol or a chloramphenicol analog in a sample, comprising: (a) a sample receiving member: (b) a carrier in fluid communication with the sample receiving member, wherein the carrier comprises a detection zone comprising immobilized chloramphenicol or a chloramphenicol analog; (c) a VHH as described herein further comprising an affinity tag, which is mobile in the carrier in the presence of the sample; and (d) a detection reagent comprising a detectable moiety and a ligand that specifically binds to the affinity tag; wherein when the sample is applied to the sample receiving member, the VHH is mobilized such that the sample and the VHH is transported along the length of the carrier to the detection zone. In such a device, when employed, if the sample contains chloramphenicol, the detectable moiety is not detected in the detection zone. When employed, if the sample does not contain chloramphenicol, the detectable moiety is detected in the detection zone.

[0049] In one example, the affinity tag is a polynucleotide or a polypeptide. In one example, the affinity tag is selected from the group consisting of a FLAG -tag, a 6xHis-tag, and biotin. In one example, the ligand that specifically binds to the affinity tag is a receptor for the affinity tag. In one example, the ligand that specifically binds to the affinity tag is an antibody. In one example, the detectable moiety is selected from the group consisting of a fluorescent substance, a chemiluminescent substance, a colored substance, a magnetic substance, a radioactive substance, an enzyme, and a metal particle. In one example, the detectable moiety produces a signal selected from the group consisting of fluorescence, chemiluminescence, radiation, color, and a magnetic property in one example, the sample is obtained from food, feed, the environment, or a medical sample. In one example, the chloramphenicol analog is selected from the group consisting of fiorfenieol, thiamphenicol, azidamphenico!, and an amphenico! antibiotic comprising a phenyl propan oid structure.

[0050J Reference will now be made to specific examples illustrating the disclosure. It is to be understood that the examples are provided to illustrate exemplary' embodiments and that no limitation to the scope of the disclosure is intended thereby.

EXAMPLES

EXAMPLE I

VHH Expression and Purification

[0051] VHH sequences were cloned into a pHEN perip!asmic expression vector (Hoogenboom et al , (1991) Nucleic Acids Res. 19: 4133-4137) for purification in WK6 E. coli cells (ATCC, Manassas, VA). The pHEN expression plasmid inserts a cleavable pelB peripiasmic secretion signal on the N terminus of the protein and a όc-His tag on the C terminus that is used for nickel affinity purificatio Expression of the VHH is controlled by a lac promoter that responds to IPTG induction. Hie pelB signal is cleaved upon secretion into the periplasm.

[0052] One microliter of pHEN plasmid containing the open reading frame encoding the desired VHH was added to 100 mΐ of chemically competent WK6 cells and incubated on ice for 20 min. Cells were then heat-shocked for 1 :30 min at 42°C, followed by an incubation on ice for 2 min. 0.5 niL of Luria-Bertani (LB) medium was then added to the cells and incubated at 37°C for 30 min. Bacteria were then transferred to 50 mL of LB containing 100 pg/rnL of ampiciiiin (Millipore Sigma, St. Louis, MO) for plasmid retention and grown overnight at 37°C at 200 rpm.

[0053] The next day, 25 mL of the started culture was added to 1 L of Terrific Broth (TB) containing 100 pg/mL ampiciiiin and grown at 37°C until cells reached an ODeoo of approximately 0.6. One mL of 1 M isopropyl b-D-l-thiogalactopyranoside (IPTG, MilliporeSigma) was added to induce protein expression and the culture was grown overnight at 30°C at 200 rpm. [0054] The following day, the bacterial culture was split into two !L centrifugation bottles and spun down at 5000 rpm at 4°C for 20 min. The supernatant was then discarded and each pellet was resuspended in 7 5 mL of TES buffer (200 inM Tris pH 8.0, 0.65 mM EDTA, 0.5 M sucrose). Hie high concentration of sucrose in the TES buffer osmoticaily shocks the cells and releases the VHHs from the periplasmic space into the supernatant lire two pellet suspensions were recombined into one 50mL Falcon centrifuge tube and rocked at 4°C for 4 hr. Afterwards, the suspension was spirt into two 50 mL Falcon centrifuge tubes and 35 mL of 0.25x TES was added to each tube and rocked overnight at 4°C.

[0Q55] The next day, the content of the two tubes was split into three 50 mL Falcon centrifuge tubes to avoid spillage in the centrifuge. Hie tubes were spun at 8000 rpm at 4°C for 20 min and the supernatants are decanted into new 50 mL tubes and the centrifugation step was repeated. Afterwards, these supernatants were collected and used for VHH purification on a nickel affinity column.

[0Q56] To prepare the nickel affinity column, 3 mL of mckel-nitrilotriacetic acid (Ni- NTA) resin was added to a 25 mL gravity flow column (VWR, Radnor, PA) and the flow through was allowed to leave the column. Next, the resin was equil ibrated by adding 25 mL of wash buffer (50 mM Tris pH 7 5, 150 mM NaCl, 10 mM imidazole) through the column and allowed to flow through by gravity. With the column primed, the periplasmic extract w¾s added and allowed to run through by gravity flow. The VHHs contained in the extract bind to the Ni-NTA column with the 6xHxs~tag attached on the C terminus of the protein and the remaining proteins from the extract flow through the column. Afterwards, the column was washed three times with 25 mL of wash buffer. Finally, VHHs were eluted from the column by three successive 2 mL additions of elution buffer (50 mM Tris pH 7.5, 150 mM NaCl, 500 mM imidazole), individually collected iu 2 mL Eppendorf tubes. The high imidazole concentration in the elution buffer displaces the His-tagged VHHs from the Ni- NTA resin to elute the proteins.

[0057] in order to remove the imidazole and exchange the VHH buffer w ith PBS, PD- 10

Columns (Cytiva) were used per manufacturer’s instructions to resuspend the VHHs in 3.5 mL of PBS. The resuspended VHH was then dialyzed, purified by size exclusion, and then concentrated for final use. Final concentration of the VHH was determined via use of a Nanodrop 1000 device (ThermoFisher Scientific, Waltham, MA).

[0058] A typical purification process yielded about 1 mg to 10 mg of VHH in 500 mΐ of PBS EXAMPLE 2

VHHs Against

[0059] Unlike traditional phage display against a protein, it is difficult to immobilize a small molecule during the wash steps necessary to remove unbound phage. In order to solve this problem, three different methods developed by Pirez-Schimier et a!., (Anal. Chem.

(2017) 89:6800-6806) were used to immobilize the small molecule. In all three methods, the small molecule was covalently attached to B8A and coated to the surface of a 96-well plate. The BSA-small molecule conjugate was then probed against the phage library and unbound phage was washed away. In order to select for VHHs that bind specifically to the small molecule, and not the BSA-small molecule conjugate, three methods were performed:

[0060] 1. Selective competition was performed in which small molecule in the well is mixed with the bound phage in order to remove phage that preferentially bound to the small molecule over the BSA-small molecule conjugate.

[0061] 2. Simultaneous competition was performed in which all bound phage was lysed, the phage was mixed with the small molecule alone in a tube, and then the phage was reintroduced to a new well plate with the BSA-srnall molecule conjugated coated to the plate. Any phage that bound to the small molecule have their active site blocked and was removed in the eluate after an incubation period

[0062] 3. Off-rate selection w as performed in which tire small molecule in the well plate was mixed with bound phage and washed off leaving behind phage that bind only to the small molecule. The remaining binders to the plate were then removed by trypsin. Strong binders remain bound to the BSA-small molecule conjugate and the weak binders were removed.

Studies with three structurally distinct small molecules were conducted to determine if a screening platform can be developed: chloramphenicol (a bacteriostatic antibiotic), enrofloxacin (a quinolone antibiotic), and semicarhazide (a metabolite of mtrofuran antibiotics found adulterated food products). These molecules were attached to Keyhole Limpet Hemocyanin (KLH), a carrier protein used to illicit an immune response, and injected into a llama to generate heavy chain-only antibodies against the target molecules. After 50 days, the antibody titer of the llama increased significantly for chloramphenicol and enrofloxacin (see, Table 4) and blood was collected for KNA isolation and construction of a phage library, where each phage was atached to a unique VHH. Table 4:

[0063] Screening against chloramphenicol confirmed that all three methods isolated phage against the small molecule (see, Table 5), with the first two methods performing better at generating a positive hit in an ELISA compared to off-rate selection (see, Table 6). Based on this ELISA, the ten top hits against chloramphenicol were isolated and sequenced (see, Table 3). VHHs were cloned into a periplasmic expression vector rHENό, expressed in WK6 E. cols cells, and purified with the procedure outlined in Example 1. A comparison of these sequences revealed a highly conserved consensus sequence between all 10 top hits (FIG. 1).

Table 5:

Table 6:

[0064] The top hit against chloramphenicol (Chl-B2) was selected and a competitive ELISA was performed to determine selectivity against chloramphenicol, compared to two structurally similar analogs, thiamphenicol and florfenicoi, and a structurally different antibiotic, kanamycin (see, Table 7). Each of the chloramphenicol, florfenicoi, thiamphenicol, and kanamycin experiments were performed in triplicate. The results indicated that the VT1H showed high specificity tor chloramphenicol and florfenicoi, weak affinity to thiamphenicol and showed no affinity for kanamycin. Table 7:

[0065] Isothermal titration calorimetry (ITC) is a well-established quantitative technique that can determine the binding affinity, enthalpy change, and binding stoichiometry of two molecules in solution. ITC was performed to determine the binding kinetics of promising VHHs against both its target of interest and structural analogs to more accurately assess selectivity⁷. In addition to ITC, a crystal structure was obtained of Chl~B2 both bound and unbound to its small molecule. Using this structure, the binding site within the protein was determined and the residues that bind directly to the small molecule. [0066] The binding affinity of a chloramphenicol -specific VI II S was successfully improved through structural analysis and experimental validation by ITC. ITC results of the top two VHH hits from the preliminary screen against chloramphenicol had dissociation constants of ImM (Ch3-B2) and 100 nM (Chl-Dl) (see, Table 2). A crystal structure was obtained with a resolution <1 Ah of Chl-B2 bound to chloramphenicol provided a clear picture of the binding pocket. By comparing the crystal structure and sequence of Chl-B2 with the sequence of Chl-Dl, an important residue change from phenylalanine to tryptophan was determined to improve the binding affinity. By introducing this one substitution into the Chl-B2 VHH, the binding affinity of the VHH was improved 19-fold (see, Table 2). This new VHH, Chi-B2F47W, has a dissociation constant of 57 nM, as determined by ITC analysis, and has the tightest binding to chloramphenicol.

[0067] Hie coordinates for the crystal structure discussed above are presented in Table 8. Table 8:

EXAMPLE 3

Conjugation of VHHs to SWCNTs for Optical Detection

[0068] Single walled carbon nanotubes (SWCNTs) are nanomaterials that when singly dispersed m solution phase via non-covalent conjugation exhibit near infrared (nIR) fluorescence upon visible light excitation. Non-covalent wrapping can impart varying degrees of optical signal modulation in response to changes of the local environment or the presence of specific molecules in addition to using the nanotube corona itself, additional techniques have been developed to allow for the tethering of molecular recognition elements such that a binding event can be translated to a subsequent optical response.

[0069] Several different conjugations strategies can be used for VHH attachment to the nIR sensor. One molecular recognition scheme utilizes a known recognition element (e g., antibody, receptor, affinity' peptide) containing a hexahistidine tag. Cbitosan-wrapped

SWCNTs are embedded in a hydrogel and functionalized with nitrilotriacetic acid (NTA) chelating groups to tether Cu(ii) ions close to the nanotube surface. The Cu(il) ions function as proximity quenchers of SWCNT fluorescence and bind to the hexahistidine-tag of the recognition element. Subsequent binding of the analyte protein changes the position of the Cu(II) ions, resulting in a fluorescence response. To date, we have used this recognition scheme for a variety of studies including tire detection of glycoproteins, protein A and human immunoglobulin G.

[0070] Another approach for conjugation involves the attachment of a DN A oligo to the VHH and using a complementary DNA sequence wrapped around the SWCNT. DNA oligos of various lengths have been attached to the C terminus of VHHs via SMCC conjugation (Mann etal., Angew. Chemie - Int. Ed (2019) 58: 11469-11473, and Wu et al., (2019)

Angew. Chemie - Int. Ed. 58:14224-14228). In brief, 3 ’-amine-modified DNA oligos are reacted with the bifunctional crosslinker sulfosuceinimidyl 4-(N-maleimido- methyl)cyclohexane- 1 -carboxylate (Sulfo-SMCC) to generate cysteine-reactive DNA oligonucleotides. These modified DNA sequences are used to noncovalently wrap the hydrophobic SWCNT surface via ultrasonication and can then react to an exposed cysteine on the VHH. Since the position of the cysteine in the VHH may affect both the binding pocket to the adulterant, as well as the signal transduction to the SWCNT, two different strategies is employed in determining the ideal cysteine location on the VHH:

[0071] 1. A cysteine is attached to the C terminus of the protein where it is least likely to affect the residues that contribute to the binding pocket of the VHH. While this location can be consistent across all VHHs, its location may affect the ability of the SWCNT to sense a binding event far from the linker.

[0072] 2. Computational capabilities are used to predict surface-exposed positions on the

VHH where a cysteine could be inserted that are dynamically correlated to binding pocket residues. These sites sense a binding event on the VHH and if a linker is attached at this position, transduce an optical signal to the SWCNT.

[0073] Additionally, the DNA sequence employed to wrap around the SWCNT affects sensor output. Adsorption of the DNA on SWCNT is modulated by base pair choice (Shankar etal., Langmuir (2014) 30:3176-3183, and Salem et al., J. Am. Chem. Soc. (2017)

139: 16791-16802). Part of the nanosensor design involves testing varying lengths of differently adsorbing sequences to tether the VHH at a distance such that the binding event can be optimally transduced.

[0Q74] Successful sensors can be integrated into a multiplexed array in a paper-based format for the development of a postable assay kit. The portable setup consists of a simply optical setup with LED excitation and an NIR photo detector. The SWCNT-NB complex can be deposited and stored on a nitrocellulose barcode (Salem et al., Anal. Chem. (2020) 92:916- 923) read out by translating the optical beam across the barcode regions. Using multiple sensors on a single test solution, an array of targets can be differentiated and measured in parallel.

EXAMPLE 4 Chloramphenicol Lateral Flow Assay

[0075] Chloramphenicol and its analogs can also be tested rapidly using the VHHs disclosed here in a paper-based lateral flow assay, similar to a home pregnancy test kit. The lateral flow assay comprises, in fluid communication, a sample pad, a conjugate pad, a test line, and a control line. A food sample is added to the sample pad and capillary action flows the sample across the conjugate pad, which contains anti -chloramphenicol VHHs that contain a C terminus FLAG-tag (or other affinity tag) as well as an anti-FLAG antibody conjugated to gold nanoparticles or a fluorophore for optical read-out. If chloramphenicol (or other analog) is present in the sample, it binds to the VHH and the anti-FLAG antibody binds to the FLAG-tag on the C terminus. This complex then flows across a test strip that contains BSA protein conjugated with chloramphenicol if chloramphenicol is bound to tire VHH, the complex does not bind to the test strip and no signal is produced, indicating a positive test. If no chloramphenicol is present, the complex binds to the test strip and produces an optical signal which indicates a negative test result. A control strip is added that contains antibodies against the animal that raised the anti-FLAG antibody (e.g . rabbit).

EQUIVALENTS

[0Q76] Those skilled m the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims

What is claimed is:

1. A single variable domain antibody (VHH) that specifically binds to chloramphenicol and/or a chloramphenicol analog, comprising:

(a) a complementarity determining region 1 (CDR!) comprising the amino acid sequence GRX₃FSX₄X₅AMG (SEQ ID NO: 1), wherein:

X is S or T;

X4 is S, T, orN; and X₅ is F orY;

(b) a CDR2 comprising the amino acid sequence AISWX₇X₈X₉X₁₀X₁₁ Y (SEQ ID NO:2) wherein:

X_? is S orN;

Xs is 11 G, P, or is absent;

X9 is G or I;

X10 is I, S, or R; and

Xn is T or S; and

(c) a CDR3 comprising the amino acid sequence ADSIPYGXisXieXnYRNPGY (SEQ ID NO:3), wherein:

Xis is D or S:

Xi6 is S, A, or V; and

Xn is R or S.

2. lire VHH of claim 1, wherein the VHH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence:

QX1QLVEX2GGGLVQAGGSLRLSCAASGRX3FSX4X5AMGWFRQAPGKEREX6VAAIS WX7X8X9X10X11YX12DSVKGRFTISRDX13AKNTVYLX14MNSLKPEDTAVYYCAADSIP YGXisXisXnYRNPGYWGQGTQVTVSS (SEQ ID NO:4), wherein:

Xi is V or L;

X_{2 i}s S ot L

X_{3 i}s S or T;

X4 1S S, T, orN;

X₅ is F orY;

X, is W or F;

X? is S orN; Xg is H, G, P, or is absent;

X is G or I;

Xio is I, S, or R;

Xn is T or S;

Xi2 is T or A;

X i 3 is N or S;

X _{4 i}s Q or E;

Xi5 is D or S;

Xi6 is S, A, or V; and

Xn is R or S.

3. lire VHH of claim 2, wherein X_{6 i}s W.

4. The VHH of claim 1 , wherein the CDRi comprises an amino acid sequence selected from SEQ ID NOs:5, 6, 7, or 8.

5. The VHH of claim 1, wherein the CDR2 comprises an ammo acid sequence selected from SEQ ID NOs:9, 10, 11, or 12.

6. The VHH of claim 1, wherein the CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 13, 14, or 15.

7. The VHH of claim 1, wherein the CDRI comprises the amino acid sequence set forth in SEQ ID NO:5, the CDR2 comprises the amino acid sequence set forth SEQ ID NO:9, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13.

8. The VHH of claim 7, wherein the VHH comprises an amino acid sequence set forth in SEQ ID NO: 16, 17, or 23.

9. The VHH of claim 1, wherein the CDRI comprises the amino acid sequence set forth in SEQ ID NO:6, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 14.

10. Hie VHH of claim 9, wherein the VHH comprises an amino acid sequence set forth in SEQ ID NO: 18 or 22.

11. Hie VHH of claim 1. wherein the CDRi comprises the amino acid sequence set forth in SEQ ID NO: 7, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and the CDR3 comprises the amino acid sequence set forth in S EQ ID NO: 15.

12. Hie VHH of claim 12, wherein the VHH comprises the amino acid sequence set forth in SEQ ID NO: 19.

13. Hie VHH of claim 1, wherein the CDRI comprises the amino acid sequence set forth in SEQ ID NO: 8, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, and the CDR3 comprises the amino acid sequence set forth in S EQ ID NO: 13.

14. Hie VHH of claim 13, wherein the VHH comprises an amino acid sequence set forth in SEQ ID NO:20 or 21.

15. Hie VHH of claim 1, further comprising two constant domains.

16. The VHH of claim 1, wherein the chloramphenicol analog is selected from the group consisting of florfenicol, thiamphenicol, azidarnphenieol, and an amphenicol antibiotic comprising a phenylpropanoid structure.

17. Hie VHH of claim 1, further comprising an affinity tag.

18. The VHH of claim 17, wherein the affinity tag is selected from the group consisting of a polynucleot de, a polypeptide, a FLAG-tag, a 6xHis-tag, and biotin.

19. The VHH of claim 1, immobilized on a solid substrate.

20. The VHH of claim 19, wherein the solid substrate comprises a carbon surface, a glass surface, a silica surface, a plastic surface, a metal surface, a surface comprising a metallic coating, a surface comprising a chemical coating, a microbead, a porous polymer matrix, a cellulosie fiber, or any combination thereof.

21. The VHH of claim 19, wherein the solid substrate is a single walled carbon n anotube ( SWCNT) .

22. A nucleic acid encoding the VHH of claim 1.

23. A vector comprising the nucleic acid of claim 22.

24. A host cell comprising the nucleic acid of claim 22.

25. A method of producing the VHH of claim 1, comprising culturing the host cell of claim 24 under conditions suitable for the expression of the VHH.

26. A method for detecting the presence or absence of chloramphenicol or a chloramphenicol analog in a sample, comprising: contacting the sample with the VHH of claim 1 further comprising an affinity tag; and detecting the presence or absence of the affinity tag, thereby detecting the presence or absence of chloramphenicol or a chloramphenicol analog in the sample.

27. The method of claim 2.6, wherein the sample is obtained from food, feed, the environment, or a medical sample.

28. A method for detecting the presence or absence of chloramphenicol or a chloramphenicol analog in a sample, comprising: contacting the sample with the VHH of claim 1, wherein the VHH is immobilized on a single walled carbon nanotube (SWCNT); and detecting a signal output emitted by the SWCNT, thereby detecting the presence or absence of chloramphenicol or a chloramphenicol analog in the sample.

29. The method of claim 28, wherein if the sample contains chloramphenicol, the SWCNT emits a first signal output, and wherein if the sample does not contain chloramphenicol, the SWCNT emits a second signal output.

30. The method of claim 28, wherein the sample is obtained from food, feed, the environment, or a medical sample.

31. A device for determining the presence of absence of chloramphenicol or a chloramphenicol analog in a sample, comprising:

(a) a sample receiving member;

(b) a carrier in fluid communication with tire sample receiving member, wherein the carrier comprises a detection zone comprising immobilized chloramphenicol or a chloramphenicol analog;

(c) the VHH of claim 1 further comprising an affinity tag, which is mobile in the carrier in the presence of the sample; and

(d) a detection reagent comprising a detectable moiety and a ligand that specifically binds to the affinity tag; wherein when the sample is applied to the sample receiving member, the VHH is mobilized such that the sample and the VHH is transported along the length of the carrier to the detection zone.

32. The device of claim 31, wherein if the sample contains chloramphenicol, the detectable moiety will not be detected in the detection zone, and wherein if the sample does not contain chloramphenicol, the detectable moiety will be detected in the detection zone.

33. A device for determining the presence or absence of chloramphenicol or a chloramphenicol analog in a sample, comprising the VHH of claim 1, wherein the VHH is immobilized on a single walled carbon nanotube (SWCNT).

34. The device of claim 33, wherein the sample is obtained from food, feed, the environment, or a medical sample

35. A device for determining the presence or absence of one or more analytes in a sample, comprising a plurality of single walled carbon nanotube (SWCNT) sensors configured to detect the one or more analytes, wherein at least one SWCNT sensor is configured to detect chloramphenicol or a chloramphenicol analog, and comprises the VHH of claim 1, wherein the VHH is immobilized on a single walled carbon nanotube (SWCNT).

36. The device of claim 35, wherein the sample is obtained from food, feed, the environment, or a medical sample.

37. Use of the VHH of claim 1, in a method for detecting chloramphenicol or a chloramphenicol analog in a sample.

38. The use of claim 37, wherein the sample is obtained from food, feed, the environment, or a medical sample.