Lateral flow assay device and method for rapid detection of fentanyl and fentanyl analogs

Abstract

The invention concerns uniquely designed lateral flow assay devices for detection of a target analyte. Particularly, the invention concerns the detection of fentanyl and fentanyl analogues such as carfentanil using specific anti-fentanyl / carfentanil monoclonal antibodies.

Description

[0001] LATERAL FLOW ASSAY DEVICE AND METHOD FOR

[0002] RAPID DETECTION OF FENTANYL AND FENTANYL

[0003] ANALOGS

[0004] TECHNOLOGICAL FIELD

[0005] The present invention generally relates to antibodies directed against fentanyl and fentanyl analogs and to methods and uses thereof in detection of these substances in a sample.

[0006] BACKGROUND ART

[0007] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0008] 1 Angelini, D.J. et al. The use of lateral flow immunoassays for the detection of fentanyl in seized drug samples and postmortem urine. J Forensic Sci. 66: 758- 765. doi: 10.1111 / 1556-4029.14631.(2021).

[0009] 2 Baehr, C. et al. Monoclonal Antibodies Counteract Opioid-Induced Behavioral and Toxic Effects in Mice and Rats. J Pharmacol Exp Ther 375:469-477. DOI: https: / / doi.org / 10.1124 / jpet.120.000124 (2020).

[0010] 3 Ban, B. et al. Novel chimeric monoclonal antibodies that block fentanyl effects and alter fentanyl biodistribution in mice. MABS 13, el991552, doi: 10.1080 / 19420862.2021.1991552 (2021).

[0011] 4 France, C.P. et al. Countermeasures for Preventing and Treating Opioid Overdose. Clin Pharmacol Ther 109: 578-590, doi: 10.1002 / cpt.2098 (2021).

[0012] 5 Frens, G. Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions. Nat. Phys. Sci., 241, 20-22 (1973).

[0013] 6 Guo, L. et al, Gold Nanoparticle-Based Paper Sensor for Simultaneous Detection of 11 Benzimidazoles by One Monoclonal Antibody. Small, 14, 1701782 (2018). 7 Noy-Porat, T. et al. Isolation of Anti-Ricin Protective Antibodies Exhibiting High .Affinity from Immunized Non-Human Primates. Toxins 8, Doi: 10.3390 / toxins8030064 (2016) .

[0014] 8 Noy-Porat, T. et al. A panel of human neutrali zing mAbs targeting SARS-CoV-2 spike at multiple epitopes. Nat. Commun. 11, 4303. Doi: 10.1038 / s41467-020- 18159-4 (2020).

[0015] 9 Rosenfeld, R. et al. Isolation and chimerization of a highly neutralizing antibody conferring passive protection against lethal Bacillus anthracis infection. PLoS One 4, e6351. Doi: 10.1371 / journal. pone.0006351 (2009).

[0016] 10 Smith, L.C. et al. Monoclonal Antibodies for Combating Synthetic Opioid Intoxication. J Am Chem Soc 141 : 10489-10503, doi: 10.1021 / jacs.9b04872 (2019).

[0017] Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

[0018] BACKGROUND

[0019] Opioids are a diverse class of drugs, used to relieve pain for over 200 years. However, since the late 1990s, a widespread overuse of opioid medications, both from prescriptions and illegal sale, is recorded, causing an exponential rise in the number of individuals suffering from opioid dependence and addiction, presently referred to as opioid use disorder (OUD). Currently, synthetic opioids such as fentanyl and its analogs, are the main cause of opioid involved deaths, accounting for approximately 88.5% of all opioid-related overdose deaths and 66.6% of all drug-related overdose deaths in 2021, in the United States alone. Synthetic opioids readily cross the blood-brain barrier stimulating p-opioid receptors (MORs) in the brain, thereby eliciting pain relieving effects. However, MOR binding also induces addiction and depressive effects of the central nervous system, which can lead to lethal respiratory depression, the primary cause of drug overdose deaths. Fentanyl is estimated to be 100 times more potent than morphine, and 10 times more powerful than heroin. Carfentanil, a structural analog of fentanyl, is the most potent synthetic opioid detected, with an estimated potency of 100 times greater than fentanyl. The very high potency, ease of synthesis and widespread availability of these synthetic opioids, pose a significant public health risk, to civilians as well as law enforcement personnel, first responders and the military since these chemicals have the potential to be weaponized.

[0020] US2015 / 0268257 discloses a method and a device for detecting illegal drugs, whereby the detecting moiety is the ligand-binding domain of an opioid-binding receptor molecule.

[0021] Angelini et al (2021) describe lateral flow immunoassays (LFIs) for detecting fentanyl in biofluid matrices. These assays contain antibodies designed to react with fentanyl and are based on a conventional competition design whereby a positive result is manifested by the disappearance of a test band.

[0022] GENERAL DESCRIPTION

[0023] In a first of its aspects, the present invention provides, a lateral flow assay device for detection of a target analyte, comprising: a cassette, wherein said cassette comprises a sample well for receiving a sample, wherein said cassette further comprises a test result window, wherein a result of an assay and a validity of an assay are individually displayable in said test result window; a conjugate pad positioned in said cassette, wherein said conjugate pad comprises a plurality of soluble test labeling probes and a plurality of soluble control labeling probes, wherein said test labeling probes are conjugated with:

[0024] (i) antibodies directed to the target analyte, and

[0025] (ii) an additional antigen (A), and wherein said control labeling probes are conjugated with a polypeptide (P) being other than said additional antigen (A); a membrane positioned in said cassette having a preceding area (area #1) comprising a plurality of target analyte-protein complexes immobilized to said membrane, a test area (area #2) comprising a plurality of antibodies directed to the antigen (A) immobilized to said membrane, and a control area (area #3) comprising a plurality of binding partners directed to the control polypeptide (P) immobilized to said membrane.

[0026] In another aspect, the present inventio provides, a method for detecting a target analyte in a sample, comprising the steps of: providing a lateral flow assay device comprising a cassette, wherein said cassette comprises a sample well and a test result window, wherein a conjugate pad is positioned in said cassette, wherein said conjugate pad comprises a plurality of soluble test labeling probes and a plurality of soluble control labeling probes, wherein said test labeling probes are conjugated with: i. antibodies directed to the target analyte, and ii. an additional antigen (A), and wherein said control labeling probes are conjugated with a polypeptide (P) being other than said additional antigen (A); wherein a membrane is positioned in said cassette having a preceding area (area #1) comprising a plurality of target analyte-protein complexes immobilized to said membrane, a test area (area #2) comprising a plurality of antibodies directed to the antigen (A) immobilized to said membrane, and a control area (area #3) comprising a plurality of binding partners directed to the control polypeptide (P) immobilized to said membrane; introducing the sample to said sample well, and determining the presence of the target analyte.

[0027] In one embodiment, said cassette further comprises a sample pad.

[0028] In one embodiment, said sample pad is composed of cellulose fibers or woven meshes (e.g., glass fiber).

[0029] In one embodiment, said cassette further comprises an absorbance pad.

[0030] In one embodiment, said absorbance pad is composed of cellulose fibers.

[0031] In one embodiment, said labeling probes are selected from a group consisting of gold nanoparticles, latex microparticles, magnetic nanoparticles, carbon nanoparticles, silica nanoparticles, and quantum dots.

[0032] In one embodiment, said membrane is a nitrocellulose membrane.

[0033] In one embodiment, said target analyte is at least one of fentanyl or a fentanyl analog.

[0034] In one embodiment, said fentanyl analog is one or more of carfentanil, sufentanil, or remifentanil.

[0035] In one embodiment, said additional antigen (A) is a non-specific antibody designated herein antibody II, and wherein said plurality of antibodies directed to the antigen (A) are anti-antibody II antibodies designated herein antibody III. In one embodiment, said cassette is contained in a housing.

[0036] In one embodiment, said sample is a body fluid obtained from a subject.

[0037] In one embodiment, said body fluid is selected from a group consisting of blood, urine, saliva, sweat.

[0038] In one embodiment, said sample is taken from the surface of an object.

[0039] In one embodiment, said object is selected from a group consisting of glass, tiles, wood, concrete and metal surfaces.

[0040] In one embodiment, said sample is a solid in a particulate form, such as powder, granules, pellets and the like.

[0041] In another aspect, the present inventio provides, an isolated monoclonal antibody or antigen-binding fragments thereof which binds to fentanyl and / or a fentanyl analog, wherein said isolated monoclonal antibody comprises complementarity determining regions selected from a group consisting of: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 145, CDRH2 denoted by SEQ ID NO. 146, CDRH3 denoted by SEQ ID NO. 147, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 148, a CDRL2 denoted by SEQ ID NO. 149, and a CDRL3 denoted by SEQ ID NO. 150 (designated HP 15); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 115, CDRH2 denoted by SEQ ID NO. 116, CDRH3 denoted by SEQ ID NO. 117, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 118, a CDRL2 denoted by SEQ ID NO. 119, and a CDRL3 denoted by SEQ ID NO. 120 (designated HP9); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 127, CDRH2 denoted by SEQ ID NO. 128, CDRH3 denoted by SEQ ID NO. 129, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 130, a CDRL2 denoted by SEQ ID NO. 131, and a CDRL3 denoted by SEQ ID NO. 132 (designated HP11); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 151, CDRH2 denoted by SEQ ID NO. 152, CDRH3 denoted by SEQ ID NO. 153, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 154, a CDRL2 denoted by SEQ ID NO. 155, and a CDRL3 denoted by SEQ ID NO. 156 (designated HP 16); e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 103, CDRH2 denoted by SEQ ID NO. 104, CDRH3 denoted by SEQ ID NO. 105, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 106, a CDRL2 denoted by SEQ ID NO. 107, and a CDRL3 denoted by SEQ ID NO. 108 (designated 7); f. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 109, CDRH2 denoted by SEQ ID NO. 110, CDRH3 denoted by SEQ ID NO. I l l, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 112, a CDRL2 denoted by SEQ ID NO. 113, and a CDRL3 denoted by SEQ ID NO. 114 (designated HP8); g. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 121, CDRH2 denoted by SEQ ID NO. 122, CDRH3 denoted by SEQ ID NO. 123, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 124, a CDRL2 denoted by SEQ ID NO. 125, and a CDRL3 denoted by SEQ ID NO. 126 (designated HP 10); h. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 133, CDRH2 denoted by SEQ ID NO. 134, CDRH3 denoted by SEQ ID NO. 135, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 136, a CDRL2 denoted by SEQ ID NO. 137, and a CDRL3 denoted by SEQ ID NO. 138 (designated HP 13); i. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 139, CDRH2 denoted by SEQ ID NO. 140, CDRH3 denoted by SEQ ID NO. 141, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 142, a CDRL2 denoted by SEQ ID NO. 143, and a CDRL3 denoted by SEQ ID NO. 144 (designated HP 14); j . a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 157, CDRH2 denoted by SEQ ID NO. 158, CDRH3 denoted by SEQ ID NO. 159, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 160, a CDRL2 denoted by SEQ ID NO. 161, and a CDRL3 denoted by SEQ ID NO. 162 (designated HP 18); k. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 163, CDRH2 denoted by SEQ ID NO. 164, CDRH3 denoted by SEQ ID NO. 165, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 166, a CDRL2 denoted by SEQ ID NO. 167, and a CDRL3 denoted by SEQ ID NO. 168 (designated HP 19); l. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 169, CDRH2 denoted by SEQ ID NO. 170, CDRH3 denoted by SEQ ID NO. 171, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 172, a CDRL2 denoted by SEQ ID NO. 173, and a CDRL3 denoted by SEQ ID NO. 174 (designated HP20); m. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 175, CDRH2 denoted by SEQ ID NO. 176, CDRH3 denoted by SEQ ID NO. 177, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 178, a CDRL2 denoted by SEQ ID NO. 179, and a CDRL3 denoted by SEQ ID NO. 180 (designated HP21); n. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 33, CDRH2 denoted by SEQ ID NO. 34, CDRH3 denoted by SEQ ID NO. 35, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 36, a CDRL2 denoted by SEQ ID NO. 37, and a CDRL3 denoted by SEQ ID NO. 38 (designated HP3); o. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 21, CDRH2 denoted by SEQ ID NO. 22, CDRH3 denoted by SEQ ID NO. 23, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 24, a CDRL2 denoted by SEQ ID NO. 25, and a CDRL3 denoted by SEQ ID NO. 26 (designated HP1); p. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 27, CDRH2 denoted by SEQ ID NO. 28, CDRH3 denoted by SEQ ID NO. 29, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 30, a CDRL2 denoted by SEQ ID NO. 31, and a CDRL3 denoted by SEQ ID NO. 32 (designated HP2); q. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 39, CDRH2 denoted by SEQ ID NO. 40, CDRH3 denoted by SEQ ID NO. 41, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 42, a CDRL2 denoted by SEQ ID NO. 43, and a CDRL3 denoted by SEQ ID NO. 44 (designated HP4); r. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 45, CDRH2 denoted by SEQ ID NO. 46, CDRH3 denoted by SEQ ID NO. 47, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 48, a CDRL2 denoted by SEQ ID NO. 49, and a CDRL3 denoted by SEQ ID NO. 50 (designated HP5).

[0042] In some embodiments, said isolated monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein: a. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 65 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 66; or b. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 55 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 56; or c. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 59 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 60; or d. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 67 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 68; or e. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 51 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 52; or f. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 53 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 54; or g. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 57 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 58; or h. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 61 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 62; or i. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 63 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 64; or j . said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 69 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 70; or k. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 71 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 72; or l. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 73 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 74; or m. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 75 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 76; or n. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 5 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 6; or o. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 1 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 2; or p. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 3 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 4; or q. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 7 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 8; or r. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 9 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 10.

[0043] In some embodiments, said isolated monoclonal antibody comprises: a. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 91 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 92, or a variant thereof; or b. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 81 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 82, or a variant thereof; or c. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 85 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 86, or a variant thereof; or d. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 93 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 94, or a variant thereof; or e. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 77 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 78, or a variant thereof; or f. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 79 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 80, or a variant thereof; or g. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 83 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 84, or a variant thereof; or h. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 87 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 88, or a variant thereof; or i. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 89 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 90, or a variant thereof; or j. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 95 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 96, or a variant thereof; or k. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 97 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 98, or a variant thereof; or l. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 99 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 100, or a variant thereof; or m. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 101 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 102, or a variant thereof; or n. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 15 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 16, or a variant thereof; or o. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 11 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 12, or a variant thereof; or p. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 13 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 14, or a variant thereof; or q. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 17 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 18, or a variant thereof; or r. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 19 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 20, or a variant thereof.

[0044] In one embodiment, said antibody or antigen-binding fragment thereof binds fentanyl and / or a fentanyl analog with a KD of about 2nM to about IpM.

[0045] In one embodiment, said antibody or antigen-binding fragment thereof is a singlechain Fv-Fc (scFv-Fc) molecule, single-chain Fv (scFv), Fv, heavy chain variable region, light chain variable region, Fab, F(ab)2(or any combination thereof.

[0046] In one embodiment, said antibody or antigen-binding fragment thereof is a scFv- Fc molecule. In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or any antigen-binding fragment thereof according to the invention.

[0047] The present invention also provides an expression vector comprising the isolated nucleic acid molecule of the invention, and a host cell transfected with the expression vector of the invention.

[0048] In a specific embodiment, the present invention provides the lateral flow assay device according to the invention, or the method according to the invention, wherein said antibodies directed to the target analyte are the antibodies of the invention as described herein above.

[0049] In a specific embodiment, the present invention provides a lateral flow assay device for detection of fentanyl and / or a fentanyl analog, comprising: a cassette, wherein said cassette comprises a sample well for receiving a sample, wherein said cassette further comprises a test result window, wherein a result of an assay and a validity of an assay are individually displayable in said test result window; a conjugate pad positioned in said cassette, wherein said conjugate pad comprises a plurality of soluble test labeling probes and a plurality of soluble control labeling probes, wherein said test labeling probes are conjugated with:

[0050] (iii) antibodies directed to fentanyl and / or a fentanyl analog, and

[0051] (iv) an additional antigen (A), and wherein said control labeling probes are conjugated with a polypeptide (P) being other than said additional antigen (A); a membrane positioned in said cassette having a preceding area (area #1) comprising a plurality of fentanyl-protein complexes immobilized to said membrane, a test area (area #2) comprising a plurality of antibodies directed to the antigen (A) immobilized to said membrane, and a control area (area #3) comprising a plurality of binding partners directed to the control polypeptide (P) immobilized to said membrane, wherein said antibodies directed to fentanyl or a fentanyl analog comprise complementarity determining regions selected from a group consisting of: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 145, CDRH2 denoted by SEQ ID NO. 146, CDRH3 denoted by SEQ ID NO. 147, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 148, a CDRL2 denoted by SEQ ID NO. 149, and a CDRL3 denoted by SEQ ID NO. 150 (designated HP15); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 115, CDRH2 denoted by SEQ ID NO. 116, CDRH3 denoted by SEQ ID NO. 117, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 118, a CDRL2 denoted by SEQ ID NO. 119, and a CDRL3 denoted by SEQ ID NO. 120 (designated HP9); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 127, CDRH2 denoted by SEQ ID NO. 128, CDRH3 denoted by SEQ ID NO. 129, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 130, a CDRL2 denoted by SEQ ID NO. 131, and a CDRL3 denoted by SEQ ID NO. 132 (designated HP11); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 151, CDRH2 denoted by SEQ ID NO. 152, CDRH3 denoted by SEQ ID NO. 153, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 154, a CDRL2 denoted by SEQ ID NO. 155, and a CDRL3 denoted by SEQ ID NO. 156 (designated HP 16); e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 103, CDRH2 denoted by SEQ ID NO. 104, CDRH3 denoted by SEQ ID NO. 105, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 106, a CDRL2 denoted by SEQ ID NO. 107, and a CDRL3 denoted by SEQ ID NO. 108 (designated 7); f. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 109, CDRH2 denoted by SEQ ID NO. 110, CDRH3 denoted by SEQ ID NO. I l l, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 112, a CDRL2 denoted by SEQ ID NO. 113, and a CDRL3 denoted by SEQ ID NO. 114 (designated HP8); g. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 121, CDRH2 denoted by SEQ ID NO. 122, CDRH3 denoted by SEQ ID NO. 123, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 124, a CDRL2 denoted by SEQ ID NO. 125, and a CDRL3 denoted by SEQ ID NO. 126 (designated HP 10); h. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 133, CDRH2 denoted by SEQ ID NO. 134, CDRH3 denoted by SEQ ID NO. 135, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 136, a CDRL2 denoted by SEQ ID NO. 137, and a CDRL3 denoted by SEQ ID NO. 138 (designated HP13); i. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 139, CDRH2 denoted by SEQ ID NO. 140, CDRH3 denoted by SEQ ID NO. 141, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 142, a CDRL2 denoted by SEQ ID NO. 143, and a CDRL3 denoted by SEQ ID NO. 144 (designated HP14); j . a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 157, CDRH2 denoted by SEQ ID NO. 158, CDRH3 denoted by SEQ ID NO. 159, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 160, a CDRL2 denoted by SEQ ID NO. 161, and a CDRL3 denoted by SEQ ID NO. 162 (designated HP18); k. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 163, CDRH2 denoted by SEQ ID NO. 164, CDRH3 denoted by SEQ ID NO. 165, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 166, a CDRL2 denoted by SEQ ID NO. 167, and a CDRL3 denoted by SEQ ID NO. 168 (designated HP 19); l. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 169, CDRH2 denoted by SEQ ID NO. 170, CDRH3 denoted by SEQ ID NO. 171, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 172, a CDRL2 denoted by SEQ ID NO. 173, and a CDRL3 denoted by SEQ ID NO. 174 (designated HP20); m. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 175, CDRH2 denoted by SEQ ID NO. 176, CDRH3 denoted by SEQ ID NO. 177, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 178, a CDRL2 denoted by SEQ ID NO. 179, and a CDRL3 denoted by SEQ ID NO. 180 (designated HP21); n. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 21, CDRH2 denoted by SEQ ID NO. 22, CDRH3 denoted by SEQ ID NO. 23, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 24, a CDRL2 denoted by SEQ ID NO. 25, and a CDRL3 denoted by SEQ ID NO. 26 (designated HP1); o. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 27, CDRH2 denoted by SEQ ID NO. 28, CDRH3 denoted by SEQ ID NO. 29, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 30, a CDRL2 denoted by SEQ ID NO. 31, and a CDRL3 denoted by SEQ ID NO. 32 (designated HP2); p. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 33, CDRH2 denoted by SEQ ID NO. 34, CDRH3 denoted by SEQ ID NO. 35, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 36, a CDRL2 denoted by SEQ ID NO. 37, and a CDRL3 denoted by SEQ ID NO. 38 (designated HP3); q. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 39, CDRH2 denoted by SEQ ID NO. 40, CDRH3 denoted by SEQ ID NO. 41, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 42, a CDRL2 denoted by SEQ ID NO. 43, and a CDRL3 denoted by SEQ ID NO. 44 (designated HP4); r. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 45, CDRH2 denoted by SEQ ID NO. 46, CDRH3 denoted by SEQ ID NO. 47, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 48, a CDRL2 denoted by SEQ ID NO. 49, and a CDRL3 denoted by SEQ ID NO. 50 (designated HP5). In another specific embodiment, the present invention provides a method for detecting fentanyl and / or a fentanyl analog in a sample, comprising the steps of providing a lateral flow assay device comprising a cassette, wherein said cassette comprises a sample well and a test result window, wherein a conjugate pad is positioned in said cassette, wherein said conjugate pad comprises a plurality of soluble test labeling probes and a plurality of soluble control labeling probes, wherein said test labeling probes are conjugated with:

[0052] (v) antibodies directed to fentanyl and / or a fentanyl analog, and

[0053] (vi) an additional antigen (A), and wherein said control labeling probes are conjugated with a polypeptide (P) being other than said additional antigen (A); wherein a membrane is positioned in said cassette having a preceding area (area #1) comprising a plurality of fentanyl-protein complexes immobilized to said membrane, a test area (area #2) comprising a plurality of antibodies directed to the antigen (A) immobilized to said membrane, and a control area (area #3) comprising a plurality of binding partners directed to the control polypeptide (P) immobilized to said membrane; introducing the sample to said sample well, and determining the presence of the target analyte, wherein said antibodies directed to fentanyl or a fentanyl analog comprise complementarity determining regions selected from a group consisting of a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 145, CDRH2 denoted by SEQ ID NO. 146, CDRH3 denoted by SEQ ID NO. 147, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 148, a CDRL2 denoted by SEQ ID NO. 149, and a CDRL3 denoted by SEQ ID NO. 150 (designated HP15); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 115, CDRH2 denoted by SEQ ID NO. 116, CDRH3 denoted by SEQ ID NO. 117, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 118, a CDRL2 denoted by SEQ ID NO. 119, and a CDRL3 denoted by SEQ ID NO. 120 (designated HP9); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 127, CDRH2 denoted by SEQ ID NO. 128, CDRH3 denoted by SEQ ID NO. 129, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 130, a CDRL2 denoted by SEQ ID NO. 131, and a CDRL3 denoted by SEQ ID NO. 132 (designated HP11); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 151, CDRH2 denoted by SEQ ID NO. 152, CDRH3 denoted by SEQ ID NO. 153, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 154, a CDRL2 denoted by SEQ ID NO. 155, and a CDRL3 denoted by SEQ ID NO. 156 (designated HP 16); e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 103, CDRH2 denoted by SEQ ID NO. 104, CDRH3 denoted by SEQ ID NO. 105, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 106, a CDRL2 denoted by SEQ ID NO. 107, and a CDRL3 denoted by SEQ ID NO. 108 (designated 7); f. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 109, CDRH2 denoted by SEQ ID NO. 110, CDRH3 denoted by SEQ ID NO. I l l, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 112, a CDRL2 denoted by SEQ ID NO. 113, and a CDRL3 denoted by SEQ ID NO. 114 (designated HP8); g. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 121, CDRH2 denoted by SEQ ID NO. 122, CDRH3 denoted by SEQ ID NO. 123, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 124, a CDRL2 denoted by SEQ ID NO. 125, and a CDRL3 denoted by SEQ ID NO. 126 (designated HP 10); h. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 133, CDRH2 denoted by SEQ ID NO. 134, CDRH3 denoted by SEQ ID NO. 135, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 136, a CDRL2 denoted by SEQ ID NO. 137, and a CDRL3 denoted by SEQ ID NO. 138 (designated HP13); i. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 139, CDRH2 denoted by SEQ ID NO. 140, CDRH3 denoted by SEQ ID NO. 141, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 142, a CDRL2 denoted by SEQ ID NO. 143, and a CDRL3 denoted by SEQ ID NO. 144 (designated HP14); j . a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 157, CDRH2 denoted by SEQ ID NO. 158, CDRH3 denoted by SEQ ID NO. 159, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 160, a CDRL2 denoted by SEQ ID NO. 161, and a CDRL3 denoted by SEQ ID NO. 162 (designated HP18); k. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 163, CDRH2 denoted by SEQ ID NO. 164, CDRH3 denoted by SEQ ID NO. 165, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 166, a CDRL2 denoted by SEQ ID NO. 167, and a CDRL3 denoted by SEQ ID NO. 168 (designated HP 19); l. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 169, CDRH2 denoted by SEQ ID NO. 170, CDRH3 denoted by SEQ ID NO. 171, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 172, a CDRL2 denoted by SEQ ID NO. 173, and a CDRL3 denoted by SEQ ID NO. 174 (designated HP20); m. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 175, CDRH2 denoted by SEQ ID NO. 176, CDRH3 denoted by SEQ ID NO. 177, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 178, a CDRL2 denoted by SEQ ID NO. 179, and a CDRL3 denoted by SEQ ID NO. 180 (designated HP21); n. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 33, CDRH2 denoted by SEQ ID NO. 34, CDRH3 denoted by SEQ ID NO. 35, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 36, a CDRL2 denoted by SEQ ID NO. 37, and a CDRL3 denoted by SEQ ID NO. 38 (designated HP3) o. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 21, CDRH2 denoted by SEQ ID NO. 22, CDRH3 denoted by SEQ ID NO. 23, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 24, a CDRL2 denoted by SEQ ID NO. 25, and a CDRL3 denoted by SEQ ID NO. 26 (designated HP1); p. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 27, CDRH2 denoted by SEQ ID NO. 28, CDRH3 denoted by SEQ ID NO. 29, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 30, a CDRL2 denoted by SEQ ID NO. 31, and a CDRL3 denoted by SEQ ID NO. 32 (designated HP2); q. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 39, CDRH2 denoted by SEQ ID NO. 40, CDRH3 denoted by SEQ ID NO. 41, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 42, a CDRL2 denoted by SEQ ID NO. 43, and a CDRL3 denoted by SEQ ID NO. 44 (designated HP4); r. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 45, CDRH2 denoted by SEQ ID NO. 46, CDRH3 denoted by SEQ ID NO. 47, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 48, a CDRL2 denoted by SEQ ID NO. 49, and a CDRL3 denoted by SEQ ID NO. 50 (designated HP5).

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

[0055] To better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0056] Fig. 1A-B is a representative example of an LFIA cassette for detection of macromolecules as known in the art (LFIA sandwich-based method). Fig. 1 A is a cassette representing a negative test showing one band at the C (control) area. Fig. IB is a cassette representing a positive test showing two bands, one band at the C (control) area and one band at the T (test) area. S is the sample placement area.

[0057] Fig. 2A-B is a representative example of an LFIA cassette for detection of small molecules as known in the art (LFIA competitive-based method). Fig. 2A is a cassette representing a negative test showing two bands at the C (control) and T (test) areas. Fig. 2B is a cassette representing a positive test showing one band only at the C (control) area. S is the sample placement area.

[0058] Fig- 3 is a graphic representation of an example of a cassette of the invention.

[0059] Fig. 4A-B is a graphic representation of an example of a negative test. Fig. 4A shows test labeling probes (referred to in the figure as “labelling particle”) comprising two components: (a) antibody I - which is the anti-target molecule and (b) the antigen of antibody II (which is a non-specific antibody, attached to the nitrocellulose (NC) membrane in area #2 - Test) flow through the cassette. Since there is no target molecule in the sample, antibody I remains free (“without target molecules”) and thus when passing through NC membrane / area #1 it binds to the protein-target molecule complex attached to the NC membrane and remains stuck in area #1. Accordingly, area #2 (Test) comprising antibody II remains void and no signal is presented. Fig. 4B is a cassette representing the negative test result showing the sample well, area #1 (which is not externally visible) and the test result window with a single band at the C (control area #3) and no band at the T (test area #2).

[0060] Fig. 5A-B is a graphic representation of an example of a positive test. Fig. 5A shows test labeling probes (referred to in the figure as “labelling particle”) comprising two components: (a) antibody I - which is the anti-target molecule and (b) the antigen of antibody II (which is a non-specific antibody, attached to the nitrocellulose (NC) membrane in area #2 - Test) flow through the cassette. The target molecules in the sample (“with target molecules”) bind to antibody I thus when passing through area #1 the test labeling probes do not bind to the protein-target molecule complex, attached to the NC membrane, and flow towards area #2 where they become attached to antibody II in area #2 - Test. Fig. 5B is a cassette representing the positive test result showing the sample well, area #1 (which is not externally visible) and the test result window with two bands: one band at the C (control area #3) and one band at the T (test area #2).

[0061] Fig. 6A-D are graphs showing results of a binding assay (ELISA) of the various antibodies. Results are presented as O.D. (405nm) as a function of antibody concentration shown in ng / ml. Fig. 6A shows binding results of the antibodies HP1, HP2, HP3, HP4 and HP5 to BSA-fentanyl hapten. Fig. 6B shows binding results of the antibodies HP7, HP8, HP9, HP 10 and HP11 to BSA-carf entanil hapten. Fig. 6C shows binding results of the antibodies HP 16, HP 18, HP 19, HP20 and HP21 to BSA-carfentanil hapten. Fig. 6D shows binding results of the antibodies HP 14 and HP 15 to BSA-carfentanil hapten.

[0062] Fig. 7A-D are graphs showing results of a competitive binding assay of various antibodies to fentanyl and its analogs: carfentanil (Car-F), sufentanil (Su-F), and remifentanil (Remi-F). Results are presented as % of maximal binding as a function of the competitors’ concentration (pM). A. HP1 (10 pg / ml). B. HP2 (4 pg / ml). C. HP3 (1 pg / ml). D. HP4 (4 pg / ml).

[0063] Fig. 8A-F are graphs showing results of a competitive binding assay of various antibodies to carfentanil (Figs. A-C) and fentanyl (Figs. D-F). Results are presented as % of maximal binding as a function of the competitors’ concentration (pM). A. HP7, HP8, HP9, HP10, HP11, HP13 (10 pg / ml). B. HP16, HP18, HP19, HP20, HP21 (10 pg / ml). C. HP14, HP15 (10 pg / ml). D. HP7, HP8, HP9, HP10, HP11, HP13 (10 pg / ml). E. HP16, HP18, HP19, HP20, HP21 (10 pg / ml). F. HP14, HP15 (10 pg / ml).

[0064] Fig. 9A-D are graphs showing results of competitive binding assay of the antibodies: HP9 (Fig. 9A), HP11 (Fig. 9B), HP15 (Fig. 9C) and HP16 (Fig. 9D) against fentanyl and the fentanyl analogs Car-F, Nor-F, Nor-Car, Su-F, Remi-F and Al-F. Results are presented as % of maximal binding, as a function of the competitors’ concentration (pM). All antibodies were used at a concentration of 1 pg / ml.

[0065] Fig. 10A-B is a photograph of an exemplary cassette of the invention. Fig. 10A shows the sample well and the test result window showing results for negative control and three amounts of fentanyl: lOOpg, Ipg, and 0.1 pg. Fig. 10B shows the cassette without the lids thus revealing the internal compartments of the strip.

[0066] DETAILED DESCRIPTION OF EMBODIMENTS

[0067] The present invention concerns an effective system for immunological detection of fentanyl and its analogs. The system is based on antibody-antigen recognition and is hence highly specific and sensitive. The invention also concerns an immunodetection device based on a chromatographic immunological assay, such as a Lateral flow Immunoassay (LFIA). The development of LFIA kits for the detection and identification of biological macromolecules is based usually on “Sandwich” assays, namely, employing two different antibodies directed to two different epitopes on the macromolecule. In kits for detection of such compounds the presence of the compound is indicated by the appearance of a band in the test area (see for example Fig. 1).

[0068] The detection and identification of small chemical molecules (also referred to herein as “target analytes”) is more challenging since it is difficult to produce antibodies to two separate epitopes on the small molecule. Most commercial kits for detection and identification of small molecules are based on the disappearance of the test band (see for example Fig. 2).

[0069] In contrast, the method and device provided by the present invention is based on a novel configuration of lateral flow assay device, as is described below, whereby the presence of the target analyte (e.g., fentanyl and / or an analog thereof) in a sample (i.e., a positive test) is indicated by appearance of an optically detectable band.

[0070] The general structure of the LFIA cassette of the invention is depicted in Figure 3. The cassette is preferably comprised in a housing (e.g., a plastic housing) and comprises four components: a sample pad preferably composed of cellulose fibers or woven meshes (e.g., glass fiber), a conjugate pad preferably composed of cellulose or glass fiber, a nitrocellulose membrane, and an absorbance pad preferably composed of cellulose fibers. The housing has two openings at the upper surface, one serves for application of the sample (also referred to as the “sample well”) and the second serves for reading the results (also referred to herein as the “test result window”) and is positioned to allow optical detection of the target analyte.

[0071] The sample well is configured to receive one or more drops of the sample.

[0072] The conjugate pad comprises test labeling probes (also referred to as test labeling particles) and control labeling probes (also referred to as control labeling particles). The labeling probes may be gold nanoparticles (also referred to as Au nanoparticles, e.g., colloidal Au nanoparticles), latex microparticles, magnetic nanoparticles, carbon nanoparticles, silica nanoparticles, or quantum dots. The test labeling probes comprise two types of components fixed on their surface, the first component being an antibody (I) which recognizes and binds the target molecules, and the second component being a member of a specific recognition couple (e.g., antibody-antigen, receptor-ligand) for example an antigen (designated herein as “A”) of antibody II which is a non-specific antibody. The control labeling probes comprise a polypeptide (P) fixed thereon.

[0073] The NC membrane (shown in Fig. 3 as the nitrocellulose membrane) comprises three areas: the preceding area (area #1 in Fig. 3) which comprises complexes of the target molecules covalently bound to a protein, such as BSA, immobilized on the membrane. These complexes serve to capture the first antibodies (I) which are bound to the test labeling probes. Area #2 (Fig. 3) comprises immobilized antibody II which recognizes and binds the antigen of antibody II (A) which is attached to the test labeling probes together with antibody I. Area #3 (Fig. 3) is a control area comprising immobilized capture molecules which selectively bind the control protein anchored on the surface of the control labeling probes.

[0074] Fig. 4 depicts a negative test outcome, namely an exemplary case in which the sample does not comprise the target molecules. In such case the anti-target molecule antibodies (I) on the test labeling probes remain free of target molecules and available for binding to the target molecules attached to the protein in preceding area #1. In the absence of target molecules in the sample, the test labeling probes remain stuck in area #1 and fail to reach the test area (area #2). Accordingly, the test area will not be marked by the test labeling probes and no band will be optically detected. In parallel a reaction will be monitored (namely, a band will appear) in control area #3 since the control labeling probes will not be retained in the preceding area, thus attesting to the validity of the test.

[0075] Fig. 5 depicts a positive test outcome, namely an exemplary case in which the sample comprises the target molecules. In such case the anti-target molecule antibodies (I) on the test labeling probes bind the target molecules in the sample and hence they are not available for binding to the target molecules attached to the protein in preceding area #1. Therefore, in the presence of target molecules in the sample, the test labeling probes flow through area #1, reach test area #2, where antibody II captures the particles via binding to antigen of antibody II (A). Accordingly, the test area will be marked by the test labeling probes and a band will be optically detected. In parallel a reaction will be monitored (namely, a band will appear) in control area #3 since the control labeling probes will not be retained in the preceding area, nor on the test area #2, thus attesting to the validity of the test.

[0076] Labeling probes can be prepared using any method known in the art, for example using the Frens method (Frens, 1973), or as described in the examples below. The attachment of antibodies and proteins to the probes can be performed using any method known in the art, for example it may be based on electrostatic interaction (e.g., as described in Guo et al. 2018), or as described in the examples below.

[0077] Immobilization of materials on the NC membrane surface can be performed for example by spraying using e.g., a lateral flow dispenser.

[0078] The invention also provides a method for detecting a target analyte in a sample, comprising the steps of: providing a lateral flow assay device comprising a cassette, wherein said cassette comprises a sample well and a test result window, wherein a conjugate pad is positioned in said cassette, wherein said conjugate pad comprises a plurality of soluble test labeling probes and a plurality of soluble control labeling probes, wherein said test labeling probes are conjugated with:

[0079] (i) antibodies directed to the target analyte, and

[0080] (ii) an additional antigen (A), and wherein said control labeling probes are conjugated with a polypeptide (P) being other than said additional antigen (A); wherein a membrane is positioned in said cassette having a preceding area (area #1) comprising a plurality of target analyte-protein complexes immobilized to said membrane, a test area (area #2) comprising a plurality of antibodies directed to the antigen (A) immobilized to said membrane, and a control area (area #3) comprising a plurality of binding partners directed to the control polypeptide (P) immobilized to said membrane; introducing the sample to said sample well, and determining the presence of the target analyte.

[0081] The sample may be a body fluid. The sample may be administered as such or may be diluted in a physiological solution or buffer prior to administration into the cassette.

[0082] In another embodiment, the sample may be obtained from a surface, e.g., by wiping the surface with a swab.

[0083] After sampling (e.g., by wiping a surface), the swab is inserted into a buffer solution for extraction and then several drops are added onto the cassette. In an embodiment, the target analyte is fentanyl and / or one or more fentanyl analogs, and antibody (I) is an anti-fentanyl antibody. The invention thus provides novel antibodies selective for fentanyl and its analogs. To generate anti fentanyl antibodies, appropriate haptens were synthesized and fixated on a carrier protein, e.g., Keyhole limpet hemocyanin (KLH), which was injected into rabbits. Polyclonal and subsequently monoclonal antibodies were generated.

[0084] These specific antibodies were used in the lateral flow assay device of the invention.

[0085] Specific and high-affinity antibodies were prepared from spleen, bone marrow and peripheral blood of rabbits immunized with a KLH-Fentanyl or KLH-Carfentanil haptens, by efficient screening methods using phage-display libraries.

[0086] Based on the construction of a combinatorial single-chain (scFv) library, and selection based on binding to BSA-Fentanyl hapten, 5 high-affinity antibodies were identified, and constructed to full length antibodies, termed herein HP1, HP2, HP3, HP4 and HP5. The anti-fentanyl antibodies described herein were capable of binding fentanyl as well as the fentanyl analogs carfentanil (Car-F) and sufentanil (Su-F), albeit with a different sensitivity.

[0087] In addition, based on the construction of a combinatorial single-chain (scFv) library, and selection based on binding to BSA-Carfentanil haptens, 13 additional high- affinity antibodies were identified, and constructed to full length antibodies, termed herein HP7, HP8, HP9, HP10, HP I 1, HP13, HP14, HP15, HP16, HP18, HP19, HP20 and HP21. The anti-carfentanil antibodies described herein were capable of binding carfentanil as well as fentanyl, with similar sensitivity.

[0088] An efficient antibody-based detection system should preferably contain antibodies that recognize at least some of the clinically relevant fentanyl analogs.

[0089] The antibodies of the present invention can be used inter alia in detection assays for fentanyl and its analogs, for example in the unique method and cassette described in the invention.

[0090] Therefore, in one of its aspects, the present invention provides isolated monoclonal antibodies or antigen-binding fragments thereof which bind to fentanyl wherein said isolated monoclonal antibodies are selected from a group consisting of the antibodies denoted HPI, HP2, HP3, HP4 and HP5.

[0091] The present invention also provides isolated monoclonal antibodies or antigen- binding fragments thereof which bind to carfentanil wherein said isolated monoclonal antibodies are selected from a group consisting of the antibodies denoted HP7, HP8, HP9, HP10, HP11, HP13, HP14, HP15, HP16, HP18, HP19, HP20 and HP2 I .

[0092] As used herein the term “fentanyl” encompasses fentanyl and its analogs which are members of the class of drugs known as rapid-acting synthetic opioids used to reduce pain. The term “fentanyl analogs” includes by is not limited to acetylfentanyl, butyrfentanyl, norfentanyl (Nor-F), alfentanil (Al-F), carfentanil (Car-F), norcarfentanil (Nor-Car), sufentanil (Su-F), and remifentanil (Remi-F).

[0093] Fentanyl (N-phenyl-N-[l-(2-phenylethyl)piperidin-4-yl]propanamide) is represented by the compound of formula I:

[0094] Carfentanil (methyl l-(2-phenylethyl)-4-(N-propanoylanilino)piperidine-4- carboxylate) is represented by the compound of formula II:

[0095]

[0096] Sufentanil (N-[4-(methoxymethyl)-l-(2-thiophen-2-ylethyl)piperidin-4-yl]-N- phenylpropanamide) is represented by the compound of formula III:

[0097] Remifentanil (methyl 1 -(3 -methoxy-3 -oxopropyl)-4-(N- propanoylanilino)piperidine-4-carboxylate) is represented by the compound of formula IV:

[0098]

[0099] Specifically, the present invention provides isolated monoclonal antibodies or antigen-binding fragments thereof which bind to fentanyl, wherein said isolated monoclonal antibodies comprise complementarity determining regions selected from a group consisting of a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 33, CDRH2 denoted by SEQ ID NO. 34, CDRH3 denoted by SEQ ID NO. 35, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 36, a CDRL2 denoted by SEQ ID NO. 37, and a CDRL3 denoted by SEQ ID NO. 38 (designated HP3); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 21, CDRH2 denoted by SEQ ID NO. 22, CDRH3 denoted by SEQ ID NO. 23, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 24, a CDRL2 denoted by SEQ ID NO. 25, and a CDRL3 denoted by SEQ ID NO. 26 (designated HP1); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 27, CDRH2 denoted by SEQ ID NO. 28, CDRH3 denoted by SEQ ID NO. 29, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 30, a CDRL2 denoted by SEQ ID NO. 31, and a CDRL3 denoted by SEQ ID NO. 32 (designated HP2); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 39, CDRH2 denoted by SEQ ID NO. 40, CDRH3 denoted by SEQ ID NO. 41, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 42, a CDRL2 denoted by SEQ ID NO. 43, and a CDRL3 denoted by SEQ ID NO. 44 (designated HP4); and e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 45, CDRH2 denoted by SEQ ID NO. 46, CDRH3 denoted by SEQ ID NO. 47, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 48, a CDRL2 denoted by SEQ ID NO. 49, and a CDRL3 denoted by SEQ ID NO. 50 (designated HP5);

[0100] The present invention also provides isolated monoclonal antibodies or antigenbinding fragments thereof which bind to carfentanil, wherein said isolated monoclonal antibodies comprise complementarity determining regions selected from a group consisting of: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 145, CDRH2 denoted by SEQ ID NO. 146, CDRH3 denoted by SEQ ID NO. 147, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 148, a CDRL2 denoted by SEQ ID NO. 149, and a CDRL3 denoted by SEQ ID NO. 150 (designated HP15); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 115, CDRH2 denoted by SEQ ID NO. 116, CDRH3 denoted by SEQ ID NO. 117, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 118, a CDRL2 denoted by SEQ ID NO. 119, and a CDRL3 denoted by SEQ ID NO. 120 (designated HP9); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 127, CDRH2 denoted by SEQ ID NO. 128, CDRH3 denoted by SEQ ID NO. 129, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 130, a CDRL2 denoted by SEQ ID NO. 131, and a CDRL3 denoted by SEQ ID NO. 132 (designated HP11); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 151, CDRH2 denoted by SEQ ID NO. 152, CDRH3 denoted by SEQ ID NO. 153, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 154, a CDRL2 denoted by SEQ ID NO. 155, and a CDRL3 denoted by SEQ ID NO. 156 (designated HP 16); e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 103, CDRH2 denoted by SEQ ID NO. 104, CDRH3 denoted by SEQ ID NO. 105, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 106, a CDRL2 denoted by SEQ ID NO. 107, and a CDRL3 denoted by SEQ ID NO. 108 (designated 7); f. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 109, CDRH2 denoted by SEQ ID NO. 110, CDRH3 denoted by SEQ ID NO. I l l, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 112, a CDRL2 denoted by SEQ ID NO. 113, and a CDRL3 denoted by SEQ ID NO. 114 (designated HP8); g. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 121, CDRH2 denoted by SEQ ID NO. 122, CDRH3 denoted by SEQ ID NO. 123, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 124, a CDRL2 denoted by SEQ ID NO. 125, and a CDRL3 denoted by SEQ ID NO. 126 (designated HP 10); h. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 133, CDRH2 denoted by SEQ ID NO. 134, CDRH3 denoted by SEQ ID NO. 135, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 136, a CDRL2 denoted by SEQ ID NO. 137, and a CDRL3 denoted by SEQ ID NO. 138 (designated HP13); i. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 139, CDRH2 denoted by SEQ ID NO. 140, CDRH3 denoted by SEQ ID NO. 141, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 142, a CDRL2 denoted by SEQ ID NO. 143, and a CDRL3 denoted by SEQ ID NO. 144 (designated HP14); j . a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 157, CDRH2 denoted by SEQ ID NO. 158, CDRH3 denoted by SEQ ID NO. 159, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 160, a CDRL2 denoted by SEQ ID NO. 161, and a CDRL3 denoted by SEQ ID NO. 162 (designated HP18); k. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 163, CDRH2 denoted by SEQ ID NO. 164, CDRH3 denoted by SEQ ID NO. 165, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 166, a CDRL2 denoted by SEQ ID NO. 167, and a CDRL3 denoted by SEQ ID NO. 168 (designated HP 19); l. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 169, CDRH2 denoted by SEQ ID NO. 170, CDRH3 denoted by SEQ ID NO. 171, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 172, a CDRL2 denoted by SEQ ID NO. 173, and a CDRL3 denoted by SEQ ID NO. 174 (designated HP20); and m. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 175, CDRH2 denoted by SEQ ID NO. 176, CDRH3 denoted by SEQ ID NO. 177, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 178, a CDRL2 denoted by SEQ ID NO. 179, and a CDRL3 denoted by SEQ ID NO. 180 (designated HP21).

[0101] The KD values of the anti-fentanyl / anti-carfentanil antibodies were calculated using biolayer interferometry (BLI). The KD values as measured by BLI are presented in Tables 5 and 6. The antibodies had a broad affinity range, with HP9 showing the highest affinity.

[0102] Determination of the dissociation constant KD is well known to those of skill in the art and may be performed for example as described in the Examples below.

[0103] Any of the isolated antibodies of the invention may be used for detection as a single agent or in combination with another antibody of the invention. As used herein, the term “antibody” refers to a polypeptide encoded by an immunoglobulin gene that specifically binds and recognizes an antigen, in the present case fentanyl and fentanyl analogs.

[0104] The term “monoclonal antibody”, “monoclonal antibodies” or m. \b as herein defined refers to a population of substantially homogenous antibodies, i.e., the individual antibodies comprising the population are identical except for possibly naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are directed against a single antigenic site (epitope).

[0105] Monoclonal antibodies may be prepared and purified by any method known in the art. For example, monoclonal antibodies may be prepared from B cells taken from the spleen, bone marrow, peripheral blood, or lymph nodes of immunized animals (e.g., rabbits, rats, mice, or monkeys). mRNA is extracted from these cells, reverse-transcribed into cDNA and the heavy and light chain variable regions are amplified and a phage display library of combinatorial single chain (scFv) antibodies is constructed.

[0106] Full length antibodies are then produced from selected scFv, as known in the art, and as described below.

[0107] Purification of monoclonal antibodies may be performed using any method known in the art, for example by affinity chromatography, namely, by using an affinity column to which a specific epitope (or antigen) is conjugated. Alternatively, purification of antibodies may be based on using protein A and protein G column chromatography.

[0108] An exemplary antibody structural unit comprises a tetramer, as known in the art. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light chain” and one “heavy chain” . The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen (or epitope) recognition.

[0109] Thus, the terms “heavy chain variable region” (VH) and “light chain variable region” (VL) refer to these heavy and light chains, respectively. More specifically, the variable region is subdivided into hypervariable and framework (FR) regions. Hypervariable regions have a high ratio of different amino acids in each position, relative to the most common amino acid in that position. Four FR regions which have more stable amino acids sequences are positioned between the hypervariable regions. The hypervariable regions directly contact a portion of the antigen's surface. For this reason, hypervariable regions are referred to as "complementarity determining regions " . or "CDRs", the CDRs are positioned either at the heavy chain of the antibody (a "heavy chain complementarity determining region") or at the light chain of the antibody (a "light chain complementarity determining region").

[0110] From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the CDR is located.

[0111] Thus, the complementarity determining regions CDRH1, CDRH2 and CDRH3 refer to the three complementarity determining regions starting from the N-terminus of the antibody’s heavy chain (also referred to herein as heavy chain complementarity determining region) and the complementarity determining regions CDRL1, CDRL2 and CDRL3 refer to the three complementarity determining regions starting from the N- terminus of the antibody’s light chain (also referred to herein as light chain complementarity determining region).

[0112] The present invention encompasses antigen-binding fragments of the isolated anti fentanyl monoclonal antibodies of the invention.

[0113] As used herein the term "antigen binding fragment" relates to a fragment of the full length antibody which retains the antibody's specificity of binding to fentanyl. An antigen binding fragment encompasses but is not limited to Fv, single chain Fv (scFv), single chain Fv-Fc (scFv-Fc), Fab’, Fab, F(ab’)2 and F(ab)2.

[0114] Such fragments may be produced by any method known in the art, for example by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).

[0115] Thus, in some embodiments the antibody according to the invention is wherein said antibody is an antibody fragment selected from the group consisting of a single-chain Fv-Fc (scFv-Fc) molecule, single chain Fv (scFv), Fv, Fab’, Fab, F(ab’)2, F(ab)2 and any combination thereof.

[0116] In some specific embodiments, the antibody or antigen-binding fragment thereof is a single chain variable fragment-Fc fragment (scFv-Fc) molecule. In other specific embodiments, the antibody or antigen-binding fragment thereof is a single chain variable fragment (scFv) molecule. In some specific embodiments, scFv-Fc molecule is converted to a full-length IgG antibody using methods known in the art.

[0117] In some embodiments, the present invention provides an anti-fentanyl / carfentanil isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 145, CDRH2 denoted by SEQ ID NO. 146, CDRH3 denoted by SEQ ID NO. 147, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 148, a CDRL2 denoted by SEQ ID NO. 149, and a CDRL3 denoted by SEQ ID NO. 150 (designated HP15).

[0118] In some embodiments, the present invention provides an anti-fentanyl / carfentanil isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 115, CDRH2 denoted by SEQ ID NO. 116, CDRH3 denoted by SEQ ID NO. 117, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 118, a CDRL2 denoted by SEQ ID NO. 119, and a CDRL3 denoted by SEQ ID NO. 120 (designated HP9).

[0119] In some embodiments, the present invention provides an anti-fentanyl / carfentanil isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 127, CDRH2 denoted by SEQ ID NO. 128, CDRH3 denoted by SEQ ID NO. 129, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 130, a CDRL2 denoted by SEQ ID NO. 131, and a CDRL3 denoted by SEQ ID NO. 132 (designated HP11).

[0120] In some embodiments, the present invention provides an anti-fentanyl / carfentanil isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 151, CDRH2 denoted by SEQ ID NO. 152, CDRH3 denoted by SEQ ID NO. 153, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 154, a CDRL2 denoted by SEQ ID NO. 155, and a CDRL3 denoted by SEQ ID NO. 156 (designated HP16).

[0121] In some embodiments, the present invention provides an anti-fentanyl / carfentanil isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 103, CDRH2 denoted by SEQ ID NO. 104, CDRH3 denoted by SEQ ID NO. 105, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 106, a CDRL2 denoted by SEQ ID NO. 107, and a CDRL3 denoted by SEQ ID NO. 108 (designated 7).

[0122] In some embodiments, the present invention provides an anti-fentanyl / carfentanil isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 109, CDRH2 denoted by SEQ ID NO. 110, CDRH3 denoted by SEQ ID NO. I l l, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 112, a CDRL2 denoted by SEQ ID NO. 113, and a CDRL3 denoted by SEQ ID NO. 114 (designated HP8).

[0123] In some embodiments, the present invention provides an anti-fentanyl / carfentanil isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 121, CDRH2 denoted by SEQ ID NO. 122, CDRH3 denoted by SEQ ID NO. 123, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 124, a CDRL2 denoted by SEQ ID NO. 125, and a CDRL3 denoted by SEQ ID NO. 126 (designated HP10).

[0124] In some embodiments, the present invention provides an anti-fentanyl / carfentanil isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 133, CDRH2 denoted by SEQ ID NO. 134, CDRH3 denoted by SEQ ID NO. 135, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 136, a CDRL2 denoted by SEQ ID NO. 137, and a CDRL3 denoted by SEQ ID NO. 138 (designated HP13).

[0125] In some embodiments, the present invention provides an anti-fentanyl / carfentanil isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 139, CDRH2 denoted by SEQ ID NO. 140, CDRH3 denoted by SEQ ID NO. 141, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 142, a CDRL2 denoted by SEQ ID NO. 143, and a CDRL3 denoted by SEQ ID NO. 144 (designated HP14). In some embodiments, the present invention provides an anti-fentanyl / carfentanil isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 157, CDRH2 denoted by SEQ ID NO. 158, CDRH3 denoted by SEQ ID NO. 159, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 160, a CDRL2 denoted by SEQ ID NO. 161, and a CDRL3 denoted by SEQ ID NO. 162 (designated HP 18).

[0126] In some embodiments, the present invention provides an anti-fentanyl / carfentanil isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 163, CDRH2 denoted by SEQ ID NO. 164, CDRH3 denoted by SEQ ID NO. 165, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 166, a CDRL2 denoted by SEQ ID NO. 167, and a CDRL3 denoted by SEQ ID NO. 168 (designated HP 19).

[0127] In some embodiments, the present invention provides an anti-fentanyl / carfentanil isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 169, CDRH2 denoted by SEQ ID NO. 170, CDRH3 denoted by SEQ ID NO. 171, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 172, a CDRL2 denoted by SEQ ID NO. 173, and a CDRL3 denoted by SEQ ID NO. 174 (designated HP20).

[0128] In some embodiments, the present invention provides an anti-fentanyl / carfentanil isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 175, CDRH2 denoted by SEQ ID NO. 176, CDRH3 denoted by SEQ ID NO. 177, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 178, a CDRL2 denoted by SEQ ID NO. 179, and a CDRL3 denoted by SEQ ID NO. 180 (designated HP21).

[0129] In some embodiments, the present invention provides an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 33, CDRH2 denoted by SEQ ID NO. 34, CDRH3 denoted by SEQ ID NO. 35, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 36, a CDRL2 denoted by SEQ ID NO. 37, and a CDRL3 denoted by SEQ ID NO. 38 (designated HP3).

[0130] In some embodiments, the present invention provides an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 21, CDRH2 denoted by SEQ ID NO. 22, CDRH3 denoted by SEQ ID NO. 23, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 24, a CDRL2 denoted by SEQ ID NO. 25, and a CDRL3 denoted by SEQ ID NO. 26 (designated HP1).

[0131] In some embodiments, the present invention provides an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 27, CDRH2 denoted by SEQ ID NO. 28, CDRH3 denoted by SEQ ID NO. 29, and a light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 30, a CDRL2 denoted by SEQ ID NO. 31, and a CDRL3 denoted by SEQ ID NO. 32 (designated HP2).

[0132] In some embodiments, the present invention provides an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 39, CDRH2 denoted by SEQ ID NO. 40, CDRH3 denoted by SEQ ID NO. 41, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 42, a CDRL2 denoted by SEQ ID NO. 43, and a CDRL3 denoted by SEQ ID NO. 44 (designated HP4).

[0133] In certain embodiments, the present invention provides an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 45, CDRH2 denoted by SEQ ID NO. 46, CDRH3 denoted by SEQ ID NO. 47, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 48, a CDRL2 denoted by SEQ ID NO. 49, and a CDRL3 denoted by SEQ ID NO. 50 (designated HP5).

[0134] In other embodiments, the present invention provides an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 5 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 6.

[0135] In other embodiments, the present invention provides an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 1 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 2.

[0136] In certain embodiments, the present invention provides an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 3 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 4.

[0137] In some embodiments, the present invention provides an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 7 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 8. In some embodiments, the present invention provides an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 9 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70%, or 75%, or 80%, or 85%, or 90% or more identical to the nucleic acid sequence denoted by SEQ ID NO. 10.

[0138] In other embodiments, the present invention provides an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 15 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 16, or a variant thereof.

[0139] In one embodiment, the present invention provides an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 11 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 12, or a variant thereof.

[0140] In one embodiment, the present invention provides an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 13 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 14, or a variant thereof.

[0141] In one embodiment, the present invention provides an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 17 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 18, or a variant thereof. In one embodiment, the present invention provides an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 19 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 20, or a variant thereof.

[0142] In other embodiments the isolated antibody according to the invention is wherein said antibody is an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 33-38, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16.

[0143] In other embodiments the isolated antibody according to the invention is wherein said antibody is an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos 21-26, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 11 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 12.

[0144] In various other embodiments the isolated antibody according to the invention is wherein said antibody is an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 27-32, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 13 and a light chain variable region comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO. 14.

[0145] In various embodiments the isolated antibody according to the invention is wherein said antibody is an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 39-44, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 17 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 18.

[0146] In other embodiments the isolated antibody according to the invention is wherein said antibody is an anti-fentanyl isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody comprises six CDR sequences as denoted by SEQ ID Nos. 45-50, and a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 19 and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 20.

[0147] As detailed above, in specific embodiments the isolated antibody according to the invention is an scFv-Fc antibody, namely an antibody comprising a single chain variable fragment comprising both the heavy and light chain CDRs fused to an Fc fragment.

[0148] The nucleic acid sequences encoding the heavy and light chains of the antibodies termed HP1, HP2, HP3, HP4, HP5, HP7, HP8, HP9, HP10, HP11, HP13, HP14, HP15, HP 16, HP 18, HP 19, HP20, andHP21 are detailed in Table 1 below. In addition, the amino acid sequences of the heavy and light chains of the antibodies described herein are detailed in Table 2 below. Furthermore, the sequences of the CDRs of the above antibodies are displayed in Table 3 below.

[0149] Table 1: Nucleic acid sequences of the light and heavy chains of the antibodies HP1, HP2, HP3, HP4, HP5, HP7, HP8, HP9, HP10, HP11, HP13, HP14, HP15, HP16, HP18, HP19, HP20, and HP21

[0150] Table 2: Amino acid sequences of the light and heavy chains of the antibodies HP1, HP2, HP3, HP4, HP5, HP7, HP8, HP9, HP10, HP11, HP13, HP14, HP15, HP16,

[0151] HP18, HP19, HP20, and HP21

[0152] The CDR sequences are highlighted within the heavy and the light chain amino acid sequences. Table 3: CDR amino acid sequences of the antibodies HP1, HP2, HP3, HP4, HP5, HP7, HP8, HP9, HP10, HP11, HP13, HP14, HP15, HP16, HP18, HP19, HP20, and HP21

[0153] The present invention also encompasses variants of the heavy and light chain variable regions. The variants may include mutations in the complementarity determining regions of the heavy and light chains which do not alter the binding activity of the antibodies herein described, or in the framework region.

[0154] The term “variant” refers to sequences of amino acids or nucleotides different from the sequences specifically identified herein, in which one or more amino acid residues or nucleotides are deleted, substituted, or added.

[0155] It should be appreciated that by the term "added ", as used herein it is meant any addition of amino acid residues to the sequences described herein.

[0156] Variants encompass various amino acid substitutions. An amino acid “substitution” is the result of replacing one amino acid with another amino acid which has similar or different structural and / or chemical properties. Amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0157] Typically, variants encompass conservative amino acid substitutions. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0158] Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another:

[0159] 1) Alanine (A), Glycine (G);

[0160] 2) Aspartic acid (D), Glutamic acid (E);

[0161] 3) Asparagine (N), Glutamine (Q);

[0162] 4) Arginine (R), Lysine (K);

[0163] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0164] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0165] 7) Serine (S), Threonine (T); and

[0166] 8) Cysteine (C), Methionine (M).

[0167] Conservative nucleic acid substitutions are nucleic acid substitutions resulting in conservative amino acid substitutions as defined above.

[0168] Variants in accordance with the invention also encompass non-polar to polar amino acid substitutions and vice-versa.

[0169] As used herein, the term “ amino acid" or “ amino acid residue refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function like the naturally occurring amino acids.

[0170] Variant sequences refer to amino acid or nucleic acids sequences that may be characterized by the percentage of the identity of their amino acid or nucleotide sequences with the amino acid or nucleotide sequences described herein (for example, the amino acid or nucleotide sequences of the heavy and light chains of the antibodies herein described).

[0171] In some embodiments, variant sequences as herein defined refer to nucleic acid sequences that encode the heavy and light chain variable regions, each having a sequence of nucleotides with at least 70% or 75% of sequence identity, around 80% or 85% of sequence identity, around 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity when compared to the sequences of the heavy and light chain variable regions described herein.

[0172] The term “binding activity of the antibodies means the ability of the antibodies to bind fentanyl. The binding activity of the antibodies can be measured using methods known in the art e.g., as described in the Examples below.

[0173] The binding of the antibody of the invention to fentanyl may be measured for example using ELISA, biolayer interferometry (BLI), Western blot or immunofluorescence assays.

[0174] In another one of its aspects the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof according to the invention.

[0175] The term “nucleic acid" or “nucleic acid molecule as herein defined refers to a polymer of nucleotides, which may be either single- or double-stranded, which is a polynucleotide such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides. The term DNA used herein also encompasses cDNA, i.e., complementary DNA produced from an RNA template by the action of reverse transcriptase (RNA-dependent DNA polymerase).

[0176] The invention further provides an expression vector comprising the isolated nucleic acid molecule as herein defined.

[0177] “Expression vector sometimes referred to as “expression vehicle or “expression construct, as used herein, encompasses vectors such as plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles, which enable the integration of DNA fragments into the genome of the host. Expression vectors are typically self-replicating DNA or RNA constructs containing the desired gene or its fragments, and operably linked genetic control elements that are recognized in a suitable host cell and effect expression of the desired genes. These control elements are capable of effecting expression within a suitable host. The expression vector in accordance with the invention may be competent with expression in bacterial, yeast, or mammalian host cells, to name but few.

[0178] In yet another one of its aspects the present invention provides a host cell transfected with the isolated nucleic acid molecule according to the invention or with the expression vector according to the invention.

[0179] The term "host cells as used herein refers to cells which are susceptible to the introduction of the isolated nucleic acid molecule according to the invention or with the expression vector according to the invention. Preferably, said cells are mammalian cells, for example CHO cells. Transfection of the isolated nucleic acid molecule or the expression vector according to the invention to the host cell may be performed by any method known in the art.

[0180] The antibodies of the invention can bind fentanyl and detect fentanyl in a sample.

[0181] As used herein the term “sample” comprises a body fluid obtained from a subject (e.g., a human subject) for example, but not limited to blood, plasma, urine, saliva, and sweat. The term also encompasses a sample taken from the surface of an object, for example, but not limited to, glass, tiles, wood, concrete, metal surfaces. The term encompasses the surface of any object that is found in the tested environment. The term also encompasses samples of solids in a particulate form, such as powder, granules, pellets and the like, including samples of encapsulated powder (i.e., powder contained within capsules).

[0182] It is appreciated that the term “purified" or "isolated" refers to molecules, such as amino acid or nucleic acid sequences, peptides, polypeptides, or antibodies that are removed from their natural environment, isolated, or separated. An “isolated antibody” is therefore a purified antibody. As used herein, the term “purified" or “to purify” also refers to the removal of contaminants from a sample.

[0183] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. Disclosed and described, it is to be understood that this invention is not limited to the examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing specific embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

[0184] It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[0185] Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word comprise , and variations such as comprises and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0186] EXAMPLES

[0187] Methods

[0188] Immunization

[0189] Four rabbits were immunized with 170 pg of KLH-Fentanyl hapten (2 rabbits with Alum adjuvant and two with CFA), followed by three monthly booster injections (final booster dose - 280 pg of KLH-Fentanyl hapten). Eight additional rabbits were immunized with KLH-Carfentanil hapten, in the same amounts and immunization regimes. 10 days after the last boost, the animals were sacrificed, and samples were taken from the blood, spleen, and bone marrow to isolate mRNA that was subsequently used for VH / VL amplification.

[0190] Library construction mRNA was extracted from spleen, bone marrow and peripheral blood of the immunized rabbits, and was reverse transcribed into cDNA. The Heavy and light chains variable regions (VH and VL, respectively) were amplified from the cDNA, using a set of primers designed to cover all known antibody families of rabbits. VH and VL were randomly assembled by PCR to construct a combinatorial single-chain (scFv) library (Noy-Porat T. et al. 2016), which was then cloned into phagemid pCC16 plasmid (Rosenfeld R. et al. 2009) and transformed into E. coli TGI electro-competent cells (Lucigen, Middleton, WI, USA). The transformed bacteria, containing the final scFv library, were plated on YPD agar (BD, Franklin Lakes, NJ, USA) supplemented with 100 pg / mL ampicillin and 100 mM glucose and, after an overnight culture at 30°C, were harvested, aliquoted and stored at -80°C. Library Screening

[0191] For library packaging, 200 mL Yeast Extract-Peptone-Dextrose (YPD) medium, containing 100 pg / mL ampicillin and 100 mM glucose, were inoculated with 0.5 mL of the harvested bacteria containing the scFv library. Bacteria were grown in a shaker incubator (New Brunswick Scientific, Enfield, CT, USA ) at 37°C, 220 rpm to an O.D. 600 of 0.7. Bacteria (10 mL) were then infected with 700 pL of M13KO7 helper phage (New England Biolabs, Ipswich, MA, USA) by incubating at 37°C for 60 minutes (min) without shaking, followed by 60 min at 120 rpm. Infected cells were then harvested (5 min, 4000 rpm) and re-suspended in 50 mL YPD with 100 pg / mL ampicillin and 50 pg / mL kanamycin. After an overnight culture at 30°C at 200 rpm, cells were pelleted by centrifugation for 10 min, 4000 rpm at 4°C, and the supernatant containing the phages was filtered through a 0.45-pm filter and then precipitated with 1 / 5 volume of 20% PEG6000 (polyethylene glycol) / 2.5 M NaCl solution for 2 hours on ice. The phages were pelleted by centrifugation of 1 hour at 9000 x g, 4°C, and re-suspended in 5 mL phosphate buffered saline (PBS).

[0192] Library panning was performed against BSA-Fentanyl or BSA-carf entanil hapten, directly absorbed to polystyrene plates (Maxisorb 96-well microtiter plates; Nunc, Denmark).

[0193] Phages were incubated with the antigen coated plates for 60 min, followed by 6- 10 washings with phosphate buffered saline Tween-20 (PBST). Phages were eluted by incubation with 1ml of 100 mM Triethylamine (Sigma- Aldrich, St. Louis, MO, USA) for 30 min following neutralization (in 200 pl IM Tris-HCl, pH 7.4). The resulting phages were used to infect TGI cells and grown overnight for phage enrichment. The culture was collected and lOOpl were used for phage rescue, to prepare the input for the next round of panning. In total, three rounds of selection were used to isolate the anti-fentanyl antibodies.

[0194] Single colonies were randomly picked from the third panning output, and phages were rescued and tested for their ability to bind BSA-fentanyl or BSA-carf entanil hapten. ELISA

[0195] Maxisorp 96-well microtiter plates (Nunc, Sigma-Aldrich, St. Louis, MO, USA) were coated overnight with BSA-fentanyl or BSA-carf entanil hapten (50pl / well) in carbonate-bicarbonate (CBC) buffer (50mM, pH 9.6), washed and blocked with PBST buffer (0.05% Tween 20, 2% BSA in PBS) at room temperature for one hour. Individual phage clones or antibodies were added to the plates for a one-hour incubation; the plates were then washed with PBST and incubated with the detecting antibody: horseradish peroxidase (HRP)-conjugated anti-M13 antibody (GE healthcare, Little Chalfont, UK) for phage clones or anti-human IgG conjugated to alkaline phosphatase (Jackson immunoresearch, West Grove, PA, USA) for full antibodies. Detection of HRP conjugates was achieved with 3,3',5,5'-tetramethybenzidine (TMB / E, Millipore, - 89 - Billerica, MA, USA) while detection of alkaline phosphatase conjugates was achieved with SIGMAFAST p-nitrophenyl phosphate tablets (Sigma-Aldrich, St. Louis, MO, USA).

[0196] Nucleic Acid Analysis

[0197] Phagemid DNA was isolated using the QIAprep spin Miniprep kit (Qiagen, GmbH, Hilden, Germany), and scFvs were sequenced by the SeqStudio Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using primers TAB-RI and CBD-AS (NoyPorat T. et al. 2016).

[0198] Production of full-length antibodies

[0199] Phagemid DNA of the antibody clones were isolated using QIAprep spin Miniprep kit and scFv sequences were cloned into a mammalian scFv-Fc expression vector (Noy-Porat et al. 2020). Antibodies were expressed using ExpiCHO™ Expression system (Thermo Scientific, Waltham, MA, USA) and purified on HiTrap Protein-A column (GE healthcare, Little Chalfont, UK).

[0200] Biolayer interferometry for affinity measurements.

[0201] Binding studies were carried out using the Octet system (ForteBio, USA, Version 8.1, 2015) that measures biolayer interferometry (BLI). All steps were performed at 30°C with shaking at 1500 rpm in a black 96-well plate containing 200pl solution in each well. Streptavidin-coated biosensors were loaded with biotinylated-fentanyl or carfentanil hapten (50nM) to reach 0.7-1 nm wavelength shift followed by a wash. The sensors were then reacted for 300 s with increasing concentrations of Antibody (association phase) and then transferred to buffer-containing wells for another 600 s (dissociation phase). Binding and dissociation were measured as changes over time in light interference after subtraction of parallel measurements from unloaded biosensors. Sensorgrams were fitted with a 1 : 1 binding model using the Octet data analysis software 8.1 (Fortebio, USA, 2015), and the presented values are an average of several repeated measurements. Competition assay

[0202] The ability of each antibody to bind fentanyl and its analogs was tested using competitive-ELISA. Microtiter plates were coated over night with BSA-fentanyl hapten in CBC buffer, washed and blocked with PBST. The antibody was pre-incubated with increasing amounts of the relevant compound and the mixture was then added to the plates for a one-hour incubation, the plates were then washed with PBST and incubated with the detecting antibody: anti-human IgG conjugated to alkaline phosphatase. Detection was performed using SIGMAFAST p-nitrophenyl phosphate tablets. Lack of binding to the plate suggests antibody binding to fentanyl or analogs.

[0203] Example 1: Preparation of KLH-fentanyl hapten vaccine

[0204] Hapten-KLH coupling: The corresponding hapten (1 equivalent) was dissolved in dimethyl formamide (DMF) and a small amount of water (10% v / v) was added. A- hydroxy succinimi de (NHS) and 1 -( 3 -di methylamin opropyl)-3 -ethy 1 carbodi imide hydrochloride (EDC)*HC1 were added to the solution. The reaction was stirred at room temperature (RT) for 1.5 hours. An additional amount of EDCºHCl was added, and the reaction was stirred at RT for 2 additional hours. A solution of KLH in PBS was added to the reaction solution. The solution turned turbid with time, and the reaction was left to stir over-night. The whole volume was transferred into a dialysis cassette (MWCO 10,000), and ran against PBS. The buffer was replaced 3 times over 2, 6 and 17 hours. The total mixture volume was kept under refrigerated conditions (~ 14 mL volume).

[0205] Vaccine preparation: Two different adjuvants were used for vaccination: alum and Complete or Incomplete Freund’s Adjuvant (CFA / ICFA). All vaccines were freshly prepared:

[0206] 1. For two Alum injections - Alum (367 pL) was diluted with PBS (1,200 pL) and stirred well. Hapten-KLH (340 pg, 600 pL) was slowly added dropwise and stirred well. Total volume 2.2 mL (1.1 mL for each injection).

[0207] 2. For CFA / ICFA injection - Hapten-KLH (170 pg, 300 pL) was diluted in PBS (700 pL). CFA / ICFA (1000 pL) was added, and the mixture was collected in a 3 mL syringe. The 2-phase mixture was emulsified by "fluid dispensing connector" between 2 syringes at least 10 times. Example 2: Isolation of antibodies from immunized rabbits

[0208] Tissue samples were taken from two rabbits, immunized with KLH-fentanyl hapten and two rabbits immunized with KLH-carfentanil hapten, as described above. Total RNA was extracted, reverse-transcribed into cDNA and further used for construction of phage-display libraries, as detailed above. Individual phage clones were selected as described above.

[0209] After panning and screening of the phage display library, 18 different antibodies, termed HP1, HP2, HP3, HP4, HP5, HP7, HP8, HP9, HP10, HP11, HP13, HP14, HP15, HP 16, HP 18, HP 19, HP20, and HP21, were isolated and sequenced, as described above. The nucleic acid sequences of the heavy and the light chains of the antibodies are shown in Table 1. The amino acid sequences of the heavy and the light chains of the antibodies are shown in Table 2 and the amino acid sequences of the heavy chain CDRs and light chains CDRs of the antibodies are shown in Table 3.

[0210] Example 3: Characterization of anti-fentanyl antibodies’ specificity

[0211] The specific binding of the various antibodies to BSA-f entanyl / carf entanil hapten was determined by ELISA assay. ELISA plates were coated with BSA-fentanyl or BSA- carf entanil hapten and each antibody was tested in serial dilutions starting from 10 pg / ml. As can be seen in Figure 6, all antibodies bind the hapten used for their isolation, fentanyl or carfentanil, with different affinities. Antibodies isolated against fentanyl hapten were then examined for their ability to bind free fentanyl molecules as well as the main fentanyl analogs (Car-F, Su-F and Remi-F), using competition ELISA (Figure 7). ELISA plates were coated with BSA-fentanyl hapten. Each antibody was pre-incubated with increasing amounts of each compound (fentanyl, Car-F, Su-F and Remi-F), used as the competitor, and the mixture was then added to the plates. Antibodies isolated against carfentanil hapten were tested with fentanyl and carfentanil as competitors (Figure 8) and IC50 values were calculated from the binding curves (Table 4). IC50 represents the concentration of the inhibitor (fentanyl or carfentanil) needed to inhibit the binding of the antibody in the competition assay by 50%. Therefore, a lower value indicates stronger recognition of the inhibitor by the antibody. Table 4: IC50 values calculated from the competition assays.

[0212] Four of the antibodies: HP9, HP11, HP15 and HP16 were chosen for additional characterization. These antibodies were further tested with additional fentanyl analogs as competitors (Fig. 9): sufentanil, remifentanil, alfentanil, norfentanyl and norcarfentanil. IC50 values were calculated from the binding curves and are presented in Table 5). Table 5: IC50 values calculated from the competition assays.

[0213] Example 4: Affinity Characterization of the anti-fentanyl antibodies

[0214] The antibodies were analyzed by Biolayer interferometry (BLI) to determine their affinity towards the specific compound. Biotinylated fentanyl or carfentanil hapten was immobilized to streptavidin sensors and interacted with increasing amounts of the relevant antibody. Binding kinetics were fitted using the 1 : 1 binding model. The kinetic parameters obtained are summarized in Tables 6 and 7.

[0215] Table 6: Affinity measurements for antibodies HP1, HP2 and HP3 Table 7: Affinity measurements for antibodies HP7, HP8, HP9, HP10, HP11, HP13,

[0216] HP14, HP15, HP16, HP18, HP19, HP20, and HP21

[0217]

[0218] Example 5: Results obtained with the lateral flow assay device

[0219] For testing the ability of the antibodies of the invention to perform in the LFIA cassette according to the principles of the invention, an LFIA cassette was constructed generally as depicted in Figure 3.

[0220] The conjugate pad comprised test labeling probes and control labeling probes (12 nm colloidal Au nanoparticles prepared as described below). The test labeling probes comprised two types of components fixed on their surface, the first component was the antibody HP3 of the invention, which recognizes and binds fentanyl, and the second component was Mouse Anti-Rat IgG (Jackson ImmunoResearch, code:212-005-168). The control labeling probes comprised Streptavidin from Streptomyces avidinii (SigmaAldrich, S0677) fixed thereon.

[0221] Preparation of the labeling probes: Labeling probes based on 12 nm colloidal Au nanoparticles, were prepared using the Frens method (Frens, 1973), via the addition of 38.8 mM of sodium citrate to a stirred solution of boiling 0.01% w / w HAuCl43H2O. The mixture was boiled for 15 min and then stirred for an additional 10 min as it cooled. The attachment of antibodies and proteins to the probes is based on electrostatic interaction (Guo et al. 2018). For test labeling probes, antibody I (0.1 mg / mL) and antibody II (0.1 mg / mL) were mixed dropwise with 1 mL of 12 nm colloidal Au nanoparticles solution adjusted to pH 8.0 using 0.1 M K2CO3 solution, followed by mild shaking for 1 h. The mixture was centrifuged for 1 h at 7000 rpm. Finally, the bottom colloidal Au nanoparticles conjugated to the antibodies were redissolved in 0.05-0.1 mL of basic buffer (0.02 M PBS, pH 7.4, including 5% sucrose, 0.5% PEG 6000, 0.02% NaNs, and 0.1% Tween) and stored at 4 °C for further use. For control labeling probes, BSA-biotin (0.2 mg / mL) was mixed dropwise with 1 mL of 12 nm colloidal Au nanoparticles solution, followed by mild shaking for 1 h. The mixture was centrifuged for 1 h at 7000 rpm. Finally, the bottom colloidal Au nanoparticles conjugated to the BSA- biotin were redissolved in 0.05-0.1 mL of basic buffer (0.02 M PBS, pH 7.4, including 5% sucrose, 0.5% PEG 6000, 0.02% NaN3, and 0.1% Tween) and stored at 4 °C for further use.

[0222] Immobilization of materials on the NC membrane surface was performed by spraying using a lateral flow dispenser [BioDot].

[0223] The preceding area (area #1 in Fig. 10B) comprised complexes of fentanyl covalently linked to BSA, immobilized on the membrane. Area #2 (Fig. 10B) comprised immobilized Donkey Anti-Mouse IgG (Jackson ImmunoResearch, code: 715-005-150), which recognizes and binds the antigen which is attached to the test labeling probes together with the antibody HP3. Area #3 (Fig. 10B) is a control area comprising immobilized Albumin, Bovine-biotin labeled (Sigma, A-8549) as capture molecules which selectively bind the control protein anchored on the surface of the control labeling probes.

[0224] Three different amounts of fentanyl were tested in the cassettes: lOOpg, Ipg, and 0.1 pg. Fentanyl oxalate stock solutions were prepared in distilled water, and final concentrations of fentanyl were prepared by diluting 20 pl of relevant stock solution in 80 pl of running buffer (1% BSA in PBS with 0.1% Tween 20).

[0225] 100 pl of the mixture were placed in each sample well.

[0226] Fig. 10A and 10B depict the results obtained after 5-10 minutes. As can be seen clearly, the control cassette reveals only one band (the control band) in the test result window, while the cassettes which contained the decreasing amounts of fentanyl show an additional band in the test area (area #2). There is a clear dose response as the cassette with the highest amount of fentanyl shows the strongest test band. In parallel, a band appeared in control area #3 in all the cassettes, attesting to the validity of the test.

Claims

CLAIMS:

1. A lateral flow assay device for detection of a target analyte, comprising: a cassette, wherein said cassette comprises a sample well for receiving a sample, wherein said cassette further comprises a test result window, wherein a result of an assay and a validity of an assay are individually displayable in said test result window; a conjugate pad positioned in said cassette, wherein said conjugate pad comprises a plurality of soluble test labeling probes and a plurality of soluble control labeling probes, wherein said test labeling probes are conjugated with:(i) antibodies directed to the target analyte, and(ii) an additional antigen (A), and wherein said control labeling probes are conjugated with a polypeptide (P) being other than said additional antigen (A); a membrane positioned in said cassette having a preceding area (area #1) comprising a plurality of target analyte-protein complexes immobilized to said membrane, a test area (area #2) comprising a plurality of antibodies directed to the antigen (A) immobilized to said membrane, and a control area (area #3) comprising a plurality of binding partners directed to the control polypeptide (P) immobilized to said membrane.

2. The lateral flow assay device of claim 1, wherein said cassette further comprises a sample pad.

3. The lateral flow assay device of claim 2, wherein said sample pad is composed of cellulose fibers or woven meshes (e.g., glass fiber).

4. The lateral flow assay device of any one of claims 1 to 3, wherein said cassette further comprises an absorbance pad.

5. The lateral flow assay device of claim 4, wherein said absorbance pad is composed of cellulose fibers.

6. The lateral flow assay device of any one of the preceding claims, wherein said labeling probes are selected from a group consisting of gold nanoparticles, latex microparticles, magnetic nanoparticles, carbon nanoparticles, silica nanoparticles, and quantum dots.

7. The lateral flow assay device of any one of the preceding claims, wherein said membrane is a nitrocellulose membrane.

8. The lateral flow assay device of any one of the preceding claims wherein said target analyte is at least one of fentanyl or a fentanyl analog.

9. The lateral flow assay device of claim 8 wherein said fentanyl analog is one or more of carfentanil, sufentanil, or remifentanil.

10. The lateral flow assay device of any one of the preceding claims wherein said additional antigen (A) is a non-specific antibody designated herein antibody II, and wherein said plurality of antibodies directed to the antigen (A) are antiantibody II antibodies designated herein antibody III.

11. The lateral flow assay device of any one of the preceding claims wherein said cassette is contained in a housing.

12. A method for detecting a target analyte in a sample, comprising the steps of: providing a lateral flow assay device comprising a cassette, wherein said cassette comprises a sample well and a test result window, wherein a conjugate pad is positioned in said cassette, wherein said conjugate pad comprises a plurality of soluble test labeling probes and a plurality of soluble control labeling probes, wherein said test labeling probes are conjugated with:(i) antibodies directed to the target analyte, and(ii) an additional antigen (A), and wherein said control labeling probes are conjugated with a polypeptide (P) being other than said additional antigen (A); wherein a membrane is positioned in said cassette having a preceding area (area #1) comprising a plurality of target analyte-protein complexes immobilized to said membrane, a test area (area #2) comprising a plurality of antibodies directed to the antigen (A) immobilized to said membrane, and a control area (area #3) comprising a plurality of binding partners directed to the control polypeptide (P) immobilized to said membrane; introducing the sample to said sample well, and determining the presence of the target analyte.

13. The method of claim 12, wherein said cassette further comprises a sample pad.

14. The method of claim 12 or claim 13, wherein said cassette further comprises an absorbance pad.

15. The method of any one of claims 12 to 14, wherein said labeling probes are selected from a group consisting of gold nanoparticles, latex microparticles, magnetic nanoparticles, carbon nanoparticles, silica nanoparticles, and quantum dots.

16. The method of any one of claims 12-15, wherein said membrane is a nitrocellulose membrane.

17. The method of any one of claims 12 to 16 wherein said target analyte is at least one of fentanyl or a fentanyl analog.

18. The method of claim 17 wherein said fentanyl analog is one or more of carfentanil, sufentanil, or remifentanil.

19. The method of any one of claims 12 to 18 wherein said additional antigen (A) is a non-specific antibody designated herein antibody II, and wherein said plurality of antibodies directed to the antigen (A) are anti-antibody II antibodies designated herein antibody III.

20. The method of any one of claims 12 to 19 wherein said cassette is contained in a housing.

21. The method of any one of claims 12 to 20 wherein said sample is a body fluid obtained from a subject.

22. The method of claim 21 wherein said body fluid is selected from a group consisting of blood, urine, saliva, sweat.

23. The method of any one of claims 12 to 20 wherein said sample is taken from the surface of an object.

24. The method of claim 23 wherein said object is selected from a group consisting of glass, tiles, wood, concrete and metal surfaces.

25. The method of any one of claims 12 to 20 wherein said sample is a solid in a particulate form.

26. The method of claim 25 wherein said solid in a particulate form is selected from a group consisting of powder, granules, and pellets.

27. An isolated monoclonal antibody or antigen-binding fragments thereof which binds to fentanyl and / or a fentanyl analog, wherein said isolated monoclonal antibody comprises complementarity determining regions selected from a group consisting of:a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 145, CDRH2 denoted by SEQ ID NO. 146, CDRH3 denoted by SEQ ID NO. 147, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 148, a CDRL2 denoted by SEQ ID NO. 149, and a CDRL3 denoted by SEQ ID NO. 150 (designated HP15); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 115, CDRH2 denoted by SEQ ID NO. 116, CDRH3 denoted by SEQ ID NO. 117, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 118, a CDRL2 denoted by SEQ ID NO. 119, and a CDRL3 denoted by SEQ ID NO. 120 (designated HP9); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 127, CDRH2 denoted by SEQ ID NO. 128, CDRH3 denoted by SEQ ID NO. 129, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 130, a CDRL2 denoted by SEQ ID NO. 131, and a CDRL3 denoted by SEQ ID NO. 132 (designated HP11); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 151, CDRH2 denoted by SEQ ID NO. 152, CDRH3 denoted by SEQ ID NO. 153, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 154, a CDRL2 denoted by SEQ ID NO. 155, and a CDRL3 denoted by SEQ ID NO. 156 (designated HP 16); e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 103, CDRH2 denoted by SEQ ID NO. 104, CDRH3 denoted by SEQ ID NO. 105, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 106, a CDRL2 denoted by SEQ ID NO. 107, and a CDRL3 denoted by SEQ ID NO. 108 (designated 7); f. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 109, CDRH2 denoted by SEQ ID NO. 110, CDRH3 denoted by SEQ ID NO. 1 l l, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 112, a CDRL2 denoted by SEQ ID NO. 113, and a CDRL3 denoted by SEQ ID NO. 114 (designated HP8);g. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 121, CDRH2 denoted by SEQ ID NO. 122, CDRH3 denoted by SEQ ID NO. 123, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 124, a CDRL2 denoted by SEQ ID NO. 125, and a CDRL3 denoted by SEQ ID NO. 126 (designated HP 10); h. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 133, CDRH2 denoted by SEQ ID NO. 134, CDRH3 denoted by SEQ ID NO. 135, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 136, a CDRL2 denoted by SEQ ID NO. 137, and a CDRL3 denoted by SEQ ID NO. 138 (designated HP13); i. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 139, CDRH2 denoted by SEQ ID NO. 140, CDRH3 denoted by SEQ ID NO. 141, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 142, a CDRL2 denoted by SEQ ID NO. 143, and a CDRL3 denoted by SEQ ID NO. 144 (designated HP14); j . a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 157, CDRH2 denoted by SEQ ID NO. 158, CDRH3 denoted by SEQ ID NO. 159, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 160, a CDRL2 denoted by SEQ ID NO. 161, and a CDRL3 denoted by SEQ ID NO. 162 (designated HP18); k. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 163, CDRH2 denoted by SEQ ID NO. 164, CDRH3 denoted by SEQ ID NO. 165, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 166, a CDRL2 denoted by SEQ ID NO. 167, and a CDRL3 denoted by SEQ ID NO. 168 (designated HP 19); l. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 169, CDRH2 denoted by SEQ ID NO. 170, CDRH3 denoted by SEQ ID NO. 171, and the light chain complementarity determiningregion (CDRL) 1 denoted by SEQ ID NO. 172, a CDRL2 denoted by SEQ ID NO. 173, and a CDRL3 denoted by SEQ ID NO. 174 (designated HP20); m. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 175, CDRH2 denoted by SEQ ID NO. 176, CDRH3 denoted by SEQ ID NO. 177, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 178, a CDRL2 denoted by SEQ ID NO. 179, and a CDRL3 denoted by SEQ ID NO. 180 (designated HP21; n. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 33, CDRH2 denoted by SEQ ID NO. 34, CDRH3 denoted by SEQ ID NO. 35, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 36, a CDRL2 denoted by SEQ ID NO. 37, and a CDRL3 denoted by SEQ ID NO. 38 (designated HP3); o. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 21, CDRH2 denoted by SEQ ID NO. 22, CDRH3 denoted by SEQ ID NO. 23, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 24, a CDRL2 denoted by SEQ ID NO. 25, and a CDRL3 denoted by SEQ ID NO. 26 (designated HP1); p. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 27, CDRH2 denoted by SEQ ID NO. 28, CDRH3 denoted by SEQ ID NO. 29, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 30, a CDRL2 denoted by SEQ ID NO. 31, and a CDRL3 denoted by SEQ ID NO. 32 (designated HP2); q. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 39, CDRH2 denoted by SEQ ID NO. 40, CDRH3 denoted by SEQ ID NO. 41, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 42, a CDRL2 denoted by SEQ ID NO. 43, and a CDRL3 denoted by SEQ ID NO. 44 (designated HP4); r. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 45, CDRH2 denoted by SEQ ID NO. 46, CDRH3 denoted by SEQ ID NO. 47, and the light chain complementarity determiningregion (CDRL) 1 denoted by SEQ ID NO. 48, a CDRL2 denoted by SEQ ID NO. 49, and a CDRL3 denoted by SEQ ID NO. 50 (designated HP5).

28. The isolated monoclonal antibody according to claim 27, wherein said isolated monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein: a. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 65 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 66; or b. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 55 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 56; or c. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 59 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 60; or d. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 67 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 68; or e. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 51 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 52; or f. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 53 and wherein said light chain variable region is encodedby a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 54; or g. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 57 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 58; or h. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 61 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 62; or i. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 63 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 64; or j. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 69 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 70; or k. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 71 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 72; or l. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 73 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO.m. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 75 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 76; or n. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 5 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 6; or o. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 1 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 2; or p. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 3 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 4; or q. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 7 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 8; or r. said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 9 and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 10.

29. The isolated monoclonal antibody according to any one of claims 27 or 28, wherein said isolated monoclonal antibody comprises: a. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 91 or a variant thereof and a light chain variable regioncomprising the amino acid sequence denoted by SEQ ID NO. 92, or a variant thereof; or b. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 81 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 82, or a variant thereof; or c. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 85 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 86, or a variant thereof; or d. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 93 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 94, or a variant thereof; or e. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 77 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 78, or a variant thereof; or f. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 79 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 80, or a variant thereof; or g. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 83 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 84, or a variant thereof; or h. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 87 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 88, or a variant thereof; or i. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 89 or a variant thereof and a light chain variable regioncomprising the amino acid sequence denoted by SEQ ID NO. 90, or a variant thereof; or j . a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 95 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 96, or a variant thereof; or k. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 97 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 98, or a variant thereof; or l. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 99 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 100, or a variant thereof; or m. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 101 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 102, or a variant thereof; or n. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 15 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 16, or a variant thereof; or o. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 11 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 12, or a variant thereof; or p. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 13 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 14, or a variant thereof; or q. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 17 or a variant thereof and a light chain variable regioncomprising the amino acid sequence denoted by SEQ ID NO. 18, or a variant thereof; or r. a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 19 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 20, or a variant thereof.

30. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 27 to 29, wherein said antibody or antigenbinding fragment thereof is a single-chain Fv-Fc (scFv-Fc) molecule, singlechain Fv (scFv), Fv, heavy chain variable region, light chain variable region, Fab, F(ab)2 or any combination thereof.

31. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 27 to 30, wherein said antibody or antigenbinding fragment thereof is a scFv-Fc molecule.

32. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or any antigen-binding fragment thereof according to any one of claims 27 to 31.

33. An expression vector comprising the isolated nucleic acid molecule according to claim 32.

34. A host cell transfected with the expression vector according to claim 33.

35. The lateral flow assay device according to any one of claims 1 to 11, or the method according to any one of claims 12 to 26, wherein said antibodies directed to the target analyte are the antibodies of any one of claims 27 to 31.

36. A lateral flow assay device for detection of fentanyl and / or a fentanyl analog, comprising: a cassette, wherein said cassette comprises a sample well for receiving a sample, wherein said cassette further comprises a test result window, wherein a result of an assay and a validity of an assay are individually displayable in said test result window; a conjugate pad positioned in said cassette, wherein said conjugate pad comprises a plurality of soluble test labeling probes and a plurality of soluble control labeling probes, wherein said test labeling probes are conjugated with:(i) antibodies directed to fentanyl and / or a fentanyl analog, and(ii) an additional antigen (A), and wherein said control labeling probes are conjugated with a polypeptide (P) being other than said additional antigen (A); a membrane positioned in said cassette having a preceding area (area #1) comprising a plurality of fentanyl-protein complexes immobilized to said membrane, a test area (area #2) comprising a plurality of antibodies directed to the antigen (A) immobilized to said membrane, and a control area (area #3) comprising a plurality of binding partners directed to the control polypeptide (P) immobilized to said membrane, wherein said antibodies directed to fentanyl or a fentanyl analog comprise complementarity determining regions selected from a group consisting of: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 145, CDRH2 denoted by SEQ ID NO. 146, CDRH3 denoted by SEQ ID NO. 147, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 148, a CDRL2 denoted by SEQ ID NO. 149, and a CDRL3 denoted by SEQ ID NO. 150 (designated HP15); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 115, CDRH2 denoted by SEQ ID NO. 116, CDRH3 denoted by SEQ ID NO. 117, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 118, a CDRL2 denoted by SEQ ID NO. 119, and a CDRL3 denoted by SEQ ID NO. 120 (designated HP9); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 127, CDRH2 denoted by SEQ ID NO. 128, CDRH3 denoted by SEQ ID NO. 129, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 130, a CDRL2 denoted by SEQ ID NO. 131, and a CDRL3 denoted by SEQ ID NO. 132 (designated HP11); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 151, CDRH2 denoted by SEQ ID NO. 152, CDRH3 denoted by SEQ ID NO. 153, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 154, a CDRL2 denoted by SEQID NO. 155, and a CDRL3 denoted by SEQ ID NO. 156 (designated HP 16); e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 103, CDRH2 denoted by SEQ ID NO. 104, CDRH3 denoted by SEQ ID NO. 105, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 106, a CDRL2 denoted by SEQ ID NO. 107, and a CDRL3 denoted by SEQ ID NO. 108 (designated 7); f. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 109, CDRH2 denoted by SEQ ID NO. 110, CDRH3 denoted by SEQ ID NO. 1 l l, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 112, a CDRL2 denoted by SEQ ID NO. 113, and a CDRL3 denoted by SEQ ID NO. 114 (designated HP8); g. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 121, CDRH2 denoted by SEQ ID NO. 122, CDRH3 denoted by SEQ ID NO. 123, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 124, a CDRL2 denoted by SEQ ID NO. 125, and a CDRL3 denoted by SEQ ID NO. 126 (designated HP 10); h. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 133, CDRH2 denoted by SEQ ID NO. 134, CDRH3 denoted by SEQ ID NO. 135, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 136, a CDRL2 denoted by SEQ ID NO. 137, and a CDRL3 denoted by SEQ ID NO. 138 (designated HP13); i. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 139, CDRH2 denoted by SEQ ID NO. 140, CDRH3 denoted by SEQ ID NO. 141, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 142, a CDRL2 denoted by SEQ ID NO. 143, and a CDRL3 denoted by SEQ ID NO. 144 (designated HP14); j . a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 157, CDRH2 denoted by SEQ ID NO. 158, CDRH3 denoted by SEQ ID NO. 159, and the light chain complementarity determiningregion (CDRL) 1 denoted by SEQ ID NO. 160, a CDRL2 denoted by SEQ ID NO. 161, and a CDRL3 denoted by SEQ ID NO. 162 (designated HP18); k. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 163, CDRH2 denoted by SEQ ID NO. 164, CDRH3 denoted by SEQ ID NO. 165, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 166, a CDRL2 denoted by SEQ ID NO. 167, and a CDRL3 denoted by SEQ ID NO. 168 (designated HP 19); l. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 169, CDRH2 denoted by SEQ ID NO. 170, CDRH3 denoted by SEQ ID NO. 171, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 172, a CDRL2 denoted by SEQ ID NO. 173, and a CDRL3 denoted by SEQ ID NO. 174 (designated HP20); m. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 175, CDRH2 denoted by SEQ ID NO. 176, CDRH3 denoted by SEQ ID NO. 177, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 178, a CDRL2 denoted by SEQ ID NO. 179, and a CDRL3 denoted by SEQ ID NO. 180 (designated HP21 n. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 21, CDRH2 denoted by SEQ ID NO. 22, CDRH3 denoted by SEQ ID NO. 23, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 24, a CDRL2 denoted by SEQ ID NO. 25, and a CDRL3 denoted by SEQ ID NO. 26 (designated HP1); o. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 27, CDRH2 denoted by SEQ ID NO. 28, CDRH3 denoted by SEQ ID NO. 29, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 30, a CDRL2 denoted by SEQ ID NO. 31, and a CDRL3 denoted by SEQ ID NO. 32 (designated HP2); p. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 33, CDRH2 denoted by SEQ ID NO. 34, CDRH3 denoted by SEQ ID NO. 35, and the light chain complementarity determiningregion (CDRL) 1 denoted by SEQ ID NO. 36, a CDRL2 denoted by SEQ ID NO. 37, and a CDRL3 denoted by SEQ ID NO. 38 (designated HP3); q. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 39, CDRH2 denoted by SEQ ID NO. 40, CDRH3 denoted by SEQ ID NO. 41, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 42, a CDRL2 denoted by SEQ ID NO. 43, and a CDRL3 denoted by SEQ ID NO. 44 (designated HP4); r. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 45, CDRH2 denoted by SEQ ID NO. 46, CDRH3 denoted by SEQ ID NO. 47, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 48, a CDRL2 denoted by SEQ ID NO. 49, and a CDRL3 denoted by SEQ ID NO. 50 (designated HP5).

37. A method for detecting fentanyl and / or a fentanyl analog in a sample, comprising the steps of: providing a lateral flow assay device comprising a cassette, wherein said cassette comprises a sample well and a test result window, wherein a conjugate pad is positioned in said cassette, wherein said conjugate pad comprises a plurality of soluble test labeling probes and a plurality of soluble control labeling probes, wherein said test labeling probes are conjugated with:(i) antibodies directed to fentanyl and / or a fentanyl analog, and(ii) an additional antigen (A), and wherein said control labeling probes are conjugated with a polypeptide (P) being other than said additional antigen (A); wherein a membrane is positioned in said cassette having a preceding area (area #1) comprising a plurality of fentanyl-protein complexes immobilized to said membrane, a test area (area #2) comprising a plurality of antibodies directed to the antigen (A) immobilized to said membrane, and a control area (area #3) comprising a plurality of binding partners directed to the control polypeptide (P) immobilized to said membrane; introducing the sample to said sample well, and determining the presence of the target analyte,wherein said antibodies directed to fentanyl or a fentanyl analog comprise complementarity determining regions selected from a group consisting of: a. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 145, CDRH2 denoted by SEQ ID NO. 146, CDRH3 denoted by SEQ ID NO. 147, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 148, a CDRL2 denoted by SEQ ID NO. 149, and a CDRL3 denoted by SEQ ID NO. 150 (designated HP15); b. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 115, CDRH2 denoted by SEQ ID NO. 116, CDRH3 denoted by SEQ ID NO. 117, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 118, a CDRL2 denoted by SEQ ID NO. 119, and a CDRL3 denoted by SEQ ID NO. 120 (designated HP9); c. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 127, CDRH2 denoted by SEQ ID NO. 128, CDRH3 denoted by SEQ ID NO. 129, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 130, a CDRL2 denoted by SEQ ID NO. 131, and a CDRL3 denoted by SEQ ID NO. 132 (designated HP11); d. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 151, CDRH2 denoted by SEQ ID NO. 152, CDRH3 denoted by SEQ ID NO. 153, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 154, a CDRL2 denoted by SEQ ID NO. 155, and a CDRL3 denoted by SEQ ID NO. 156 (designated HP 16); e. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 103, CDRH2 denoted by SEQ ID NO. 104, CDRH3 denoted by SEQ ID NO. 105, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 106, a CDRL2 denoted by SEQ ID NO. 107, and a CDRL3 denoted by SEQ ID NO. 108 (designated 7); f. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 109, CDRH2 denoted by SEQ ID NO. 110, CDRH3 denoted by SEQ ID NO. 1 l l, and the light chain complementarity determiningregion (CDRL) 1 denoted by SEQ ID NO. 112, a CDRL2 denoted by SEQ ID NO. 113, and a CDRL3 denoted by SEQ ID NO. 114 (designated HP8); g. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 121, CDRH2 denoted by SEQ ID NO. 122, CDRH3 denoted by SEQ ID NO. 123, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 124, a CDRL2 denoted by SEQ ID NO. 125, and a CDRL3 denoted by SEQ ID NO. 126 (designated HP 10); h. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 133, CDRH2 denoted by SEQ ID NO. 134, CDRH3 denoted by SEQ ID NO. 135, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 136, a CDRL2 denoted by SEQ ID NO. 137, and a CDRL3 denoted by SEQ ID NO. 138 (designated HP13); i. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 139, CDRH2 denoted by SEQ ID NO. 140, CDRH3 denoted by SEQ ID NO. 141, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 142, a CDRL2 denoted by SEQ ID NO. 143, and a CDRL3 denoted by SEQ ID NO. 144 (designated HP14); j . a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 157, CDRH2 denoted by SEQ ID NO. 158, CDRH3 denoted by SEQ ID NO. 159, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 160, a CDRL2 denoted by SEQ ID NO. 161, and a CDRL3 denoted by SEQ ID NO. 162 (designated HP18); k. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 163, CDRH2 denoted by SEQ ID NO. 164, CDRH3 denoted by SEQ ID NO. 165, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 166, a CDRL2 denoted by SEQ ID NO. 167, and a CDRL3 denoted by SEQ ID NO. 168 (designated HP 19);l. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 169, CDRH2 denoted by SEQ ID NO. 170, CDRH3 denoted by SEQ ID NO. 171, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 172, a CDRL2 denoted by SEQ ID NO. 173, and a CDRL3 denoted by SEQ ID NO. 174 (designated HP20); m. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 175, CDRH2 denoted by SEQ ID NO. 176, CDRH3 denoted by SEQ ID NO. 177, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 178, a CDRL2 denoted by SEQ ID NO. 179, and a CDRL3 denoted by SEQ ID NO. 180 (designated HP21 n. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 33, CDRH2 denoted by SEQ ID NO. 34, CDRH3 denoted by SEQ ID NO. 35, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 36, a CDRL2 denoted by SEQ ID NO. 37, and a CDRL3 denoted by SEQ ID NO. 38 (designated HP3) o. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 21, CDRH2 denoted by SEQ ID NO. 22, CDRH3 denoted by SEQ ID NO. 23, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 24, a CDRL2 denoted by SEQ ID NO. 25, and a CDRL3 denoted by SEQ ID NO. 26 (designated HP1); p. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 27, CDRH2 denoted by SEQ ID NO. 28, CDRH3 denoted by SEQ ID NO. 29, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 30, a CDRL2 denoted by SEQ ID NO. 31, and a CDRL3 denoted by SEQ ID NO. 32 (designated HP2); q. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 39, CDRH2 denoted by SEQ ID NO. 40, CDRH3 denoted by SEQ ID NO. 41, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 42, a CDRL2 denoted by SEQ ID NO. 43, and a CDRL3 denoted by SEQ ID NO. 44 (designated HP4); r. a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 45, CDRH2 denoted by SEQ ID NO. 46, CDRH3 denotedby SEQ ID NO. 47, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 48, a CDRL2 denoted by SEQ ID NO. 49, and a CDRL3 denoted by SEQ ID NO. 50 (designated HP5).

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