Propofol immunoassay

Abstract

An antibody for binding propofol is provided. Also provided is an immunoassay for assaying propofol and an immunobiosensor for rapid, point-of-care of propofol.

Description

Technical Field

[0001] The present invention generally relates to apparatus, methods, antigens and antibodies for performing immunoassays, and particularly to, but not limited to, immunobiosensors for propofol. Background Technology

[0002] An immunobiosensor is a biosensor that uses antibodies or antigens as biological receptors to detect the formation of immune complexes.

[0003] Propofol (2,6-diisopropylphenol, Formula I) is a hypnotic alkylphenol derivative and a parenteral anesthetic commonly used to produce sedation and general anesthesia.

[0004]

[0005] Propofol: C 12 H 18 O Molecular weight 178 In general anesthesia practice, propofol can be used for both induction and maintenance phases, a process known as total intravenous anesthesia (TIVA). Growing evidence suggests that TIVA offers advantages over more traditional volatile anesthesia, including: reduced short-term side effects such as nausea and vomiting, reduced cognitive impact, potential for improved long-term survival in cancer patients, and significantly reduced environmental impact.

[0006] A major obstacle to the widespread adoption of TIVA is the lack of suitable methods for continuous, real-time monitoring of propofol concentrations in the blood of anesthetized patients.

[0007] Similarly, the optimization of propofol sedation is limited by the lack of suitable methods for real-time monitoring of propofol concentrations in the blood of patients receiving sedation.

[0008] The goal of achieving real-time monitoring of blood propofol concentrations during general anesthesia or sedation places several requirements on any potential propofol sensing technology. For example, any method must be able to return results within a sufficiently narrow time window to provide information of practical use to anesthesiologists or other medical professionals.

[0009] For use in patient monitoring within a surgical setting, any sensor system needs to produce stable results throughout the surgical procedure, potentially lasting 8 hours or longer. Furthermore, propofol has been shown to redistribute slowly over time between plasma and blood cell membranes, meaning the time between sample collection and measurement needs to be strictly controlled. Therefore, any technology used for real-time propofol monitoring should be suitable for automation and require minimal sample handling.

[0010] Propofol is likely to be administered co-administered with other drugs, such as lidocaine, opioid analgesics like fentanyl or morphine, competitive neuromuscular blocking agents like atracurium or rocuronium, antibiotics, and anti-inflammatory drugs; therefore, any propofol assay must have sufficient specificity. Immunosensors, due to their high selectivity and sensitivity resulting from the specific binding between antibodies and corresponding antigens, are a suitable platform for applications in the medical field.

[0011] When antibodies are produced using traditional methods, the antigen is injected into the animal, and then the antibodies produced as part of the immune response are extracted.

[0012] Propofol is a small molecule: a small molecule is an organic compound with a low molecular weight (≤ 1000 Da).

[0013] Small molecules are non-immunogenic due to their size, meaning they do not elicit an immune response and therefore do not produce antibodies.

[0014] This problem can be overcome by conjugating small molecules to carrier proteins, in which case it is called a hapten (half antigen). Carrier proteins, such as keyhole limpethemocyanin (KLH) and bovine serum albumin (BSA), are conjugated to multiple small molecules. The presence of the larger protein molecule elicits an immune response in the injected animal, and a portion of the antibodies produced are specific to the hapten.

[0015] Some of these antibodies can bind to protein-coupled and uncoupled small molecules, but most only bind to the coupled form, thus requiring screening. Once screened, some of these antibodies exhibit good affinity for their small molecule targets, with reported KD values ​​in the nanomolar range; however, this is usually only achievable after extensive and rigorous in vitro selection and affinity maturation from an immune library.

[0016] Despite these successes, the failure rate in anti-hapten antibody development remains high. It is estimated that 50-75% of all anti-hapten antibody development projects fail to deliver reagents with sufficient affinity or specificity. Unfortunately, the use of carrier molecules often results in developed antibodies that are specific to the hapten-carrier molecule linker region, rather than to the desired hapten. Conjugating small molecules to carrier proteins also limits the epitopes available for antibody binding, leading to differences in antibody performance when binding to the conjugated target versus when free in solution, with significantly lower affinity for free molecules in solution. Summary of the Invention

[0017] One aspect of the present invention provides an antibody generated by immunizing a non-human animal with propofol-4-carboxylic acid conjugated to a carrier protein.

[0018] One aspect of the present invention provides antibodies generated by immunizing non-human animals with an antigen consisting of propofol-4-carboxylic acid coupled to a carrier protein.

[0019] Another aspect of the present invention provides an antibody generated by immunizing a non-human animal with propofol-4-carboxylic acid conjugated to a carrier protein.

[0020] Another aspect of the present invention provides antibodies generated by immunizing non-human animals with propofol-4-carboxylic acid or its analogues or functional equivalents conjugated to a carrier protein.

[0021] Another aspect of the invention provides antibodies generated by immunizing non-human animals with propofol-4-carboxylic acid (including its salts and esters) coupled to a carrier protein.

[0022] On the other hand, antibodies were provided that were produced by immunizing non-human animals with HS357 conjugated to a carrier protein.

[0023] On the other hand, antibodies that specifically bind to propofol are provided, which are generated against an antigen containing propofol-4-carboxylic acid coupled to a carrier protein.

[0024] On the other hand, antibodies that specifically bind to propofol are provided, said antibodies being generated against an antigen containing HS357 coupled to a carrier protein.

[0025] In some aspects and implementations, the carrier is KLH.

[0026] In some aspects and implementations, the carrier is BSA.

[0027] The antibodies formed according to the present invention can be monoclonal or polyclonal.

[0028] In some aspects and implementation methods, antibodies that are specific to haptens are selected.

[0029] The present invention also provides an antibody that binds to propofol, comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56.

[0030] The present invention also provides an antibody that binds to propofol, comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64.

[0031] The present invention also provides an antibody that binds to propofol, comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72.

[0032] The present invention also provides an antibody that binds to propofol, comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 79 and SEQ ID NO: 80.

[0033] The present invention also provides a monoclonal antibody for binding propofol, said antibody comprising one or more of SEQ ID NO: 1 to SEQ ID NO: 80.

[0034] On the other hand, an immunoassay for measuring propofol in a sample is provided, comprising an antibody as defined herein.

[0035] Immunoassays are available in the form of plate ELISA.

[0036] On the other hand, it provides a rapid, immediate care immune biosensor for propofol in plasma based on antibodies defined in this paper.

[0037] sequence list SEQ ID NO: 1 CE8_HSnaa Cloning the CE8 heavy chain signal peptide protein sequence MGWSWIFLFLLSGTAGVHS SEQ ID NO: 2 CE8_HCSnuc Cloning the DNA sequence of the CE8 heavy chain signal peptide ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCT SEQ ID NO: 3 CE8_HCVaa Cloning the protein sequence of the CE8 heavy chain variable region EVQLQQSGPELVKPGASMKISCKASGYSFTDYTMNWVNWVKQSHGKNLEWIGHINPYNGGSSYNQKFRGKATLTVDKSSTTAYMELLSLTSEDSAVYYCAVIYYDYDGDIFAYWGQGTLVTVSA SEQ ID NO: 4 CE8_HCVnuc Cloning the CE8 heavy chain variable region DNA sequence GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACACCATGAACTGGGTGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACATATTAATCCTTACAATGGTGGTTCTAGC TACAACCAGAAGTTCAGGGGCAAGGCCACATTAACTGTAGACAAGTCATCCACCACAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCTGTAATCTACTATGATTACGACGGGGATATTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA SEQ ID NO: 5 CE8_HCCaa Cloning the protein sequence of the CE8 heavy chain constant region AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 6 CE8_HCCnuc Clone the DNA sequence of the CE8 heavy chain constant region GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA SEQ ID NO: 7 CE8_LCSaa Cloning the CE8 light chain signal peptide protein sequence MSVPTQVLGLLLLWLTGARC SEQ ID NO: 8 CE8_LCSnuc Cloning the DNA sequence of the CE8 light chain signal peptide ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGT SEQ ID NO: 9 CE8_LCVaa Cloning the protein sequence of the CE8 light chain variable region DIQMTQSPASSLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNSKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPFTFGSGTKLEIK SEQ ID NO: 10 CE8_LCVnuc Cloning the CE8 light chain variable region DNA sequence GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAACTCCTGGTCTATAATTCAAAAACCT TAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTACTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAAATAAAA SEQ ID NO: 11 CE8_LCCaa Cloning the protein sequence of the CE8 light chain constant region RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 12 CE8_LCCnuc Cloning the DNA sequence of the CE8 light chain constant region CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAACCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGA ACAGTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT SEQ ID NO: 13 CE7_HCSaa Cloning the CE7 heavy chain signal peptide protein sequence MRVLILLCLFTAFPGILS SEQ ID NO: 14 CE7_HCSnuc Cloning the DNA sequence of the CE7 heavy chain signal peptide ATGAGAGTGCTGATTCTTTTGGTGCCTGTTCACAGCCTTTCCTGGTATCCTGTCT SEQ ID NO: 15 CE7_HCVaa Cloning the protein sequence of the CE7 heavy chain variable region DVQLQESGPDLVKPSQSLSLTCTVTGYSLTSGFTWHWIRQFPGNKLEWMGYLHYSGDTNYNPSLRSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGGITSALWGQGTLVTVSA SEQ ID NO: 16 CE7_HCVnuc Cloning the CE7 heavy chain variable region DNA sequence GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCCCTCACCTGCACTGTCACTGGCTACTCCCTCACCAGTGGTTTTACCTGGCACTGGATCCGGCAGTTTCCAGGGAACAAACTGGAGTGGATGGGCTACCTACACTACAGTGGTGACACTAACTACAACCCATCTCTCAGAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGAGGCGGGATTACGTCGGCTCTCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA SEQ ID NO: 17 CE7_HCCaa Clone CE7 heavy chain constant region protein sequence AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 18 CE7_HCCnuc Clone CE7 heavy chain constant region DNA sequence GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA SEQ ID NO: 19 CE7_LCSaa Cloning the CE7 light chain signal peptide protein sequence MSVPTQVLGLLLLWLTDARC SEQ ID NO: 20 CE7_LCSnuc Cloning the DNA sequence of the CE7 light chain signal peptide ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGATGCCAGATGT SEQ ID NO: 21 CE7_LCVaa Cloning the protein sequence of the CE7 light chain variable region DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYGATNLADGVPSRFSGSGSGTQFSLKINRLQSEDFGIYYCHHLWGIPYTFGGGTKLEIK SEQ ID NO: 22 CE7_LCVnuc Cloning the CE7 light chain variable region DNA sequence GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGTATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTAATTTAGCATGGTATCAGCAGAAGCAGGGAAAATCTCCTCAGCTCCTGGTCTATGGTGCTACAAACT TAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCCCTCAAGATCAACAGGTTGCAGTCTGAAGATTTTGGGATTTACTACTGTCACCATTTATGGGGTATACCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA SEQ ID NO: 23 CE7_LCCaa Cloning the protein sequence of the CE7 light chain constant region RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 24 CE7_LCCnuc Cloning the protein sequence of the CE7 light chain constant region CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAACCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGA ACAGTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT SEQ ID NO: 25 FH10_HCSaa Cloning the FH10 heavy chain signal peptide protein sequence MRVLILLWLFTAFPGILS SEQ ID NO: 26 FH10_HCSnuc Cloning the FH10 heavy chain signal peptide DNA sequence ATGAGAGTGCTGATTCTTTTGGTGGCTGTTCACAGCCTTTCCTGGTATCCTGTCT SEQ ID NO: 27 FH10_HCVaa Cloning the FH10 heavy chain variable region protein sequence DVQVQESGPGLVKPSQSLSLTCTVTGYSITSNYAWNWIRQFPGDKLEWMGFITYSGSSTYNPSLKSRISITRDTSKNQFFLQLNSVTSEDTATYYCASVYYDYDAWFAYWGQGTLVTVSA SEQ ID NO: 28 FH10_HCVnuc Cloning the FH10 heavy chain variable region DNA sequence GATGTGCAGGTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTAACTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAGACAAACTGGAGTGGATGGGCTTCATAACCTACAGTGGTAGTTCTACCTACAACCCCTCTCTCAAGAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTTCAATTGAATTCTGTGACTTCTGAGGACACAGCCACTTATTACTGTGCAAGTGTCTATTATGATTACGACGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA SEQ ID NO: 29 FH10_HCCaa Cloning of the protein sequence of the constant region of the FH10 heavy chain AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 30 FH10_HCCnuc Cloning of the DNA sequence of the constant region of the FH10 heavy chain GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA SEQ ID NO: 31 FH10_LCSaa Cloning the FH10 light chain signal peptide protein sequence MSVPTQVLGLLLLWLTGARC SEQ ID NO: 32 FH10_LCSnuc Cloning the FH10 light chain signal peptide DNA sequence ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTACTGTGGCTTACAGGTGCCAGATGT SEQ ID NO: 33 FH10_LCVaa Cloning the FH10 light chain variable region protein sequence DIQMTQSPASSLSASVGETVTITCRASENIYTYLAWYQLKQGKSPQLLVYNAKTLAGGVPSRFSASGSGTQFSLKINSLQPEDFGSFYCHHHYHTPFTFGSGTRLEIN SEQ ID NO: 34 FH10_LCVnuc Cloning the FH10 light chain variable region DNA sequence GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACACTTATTTAGCATGGTATCAGCTGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCT TAGCGGAGGTGTGCCATCAAGGTTCAGTGCCAGTGGATCAGGCACACAATTTTCTCTGAAGATCAACAGCCTGCAGCCTGAGGATTTTGGGAGTTTCTACTGTCACCATCATTATCATACTCCTTTCACGTTCGGCTCGGGGACAAGGTTGGAGATAAAC SEQ ID NO: 35 FH10_LCCaa Cloning the protein sequence of the FH10 light chain constant region RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 36 FH10_LCCnuc Cloning the FH10 light chain constant region DNA sequence CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAACCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGA ACAGTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT SEQ ID NO: 37 GH5_HCSaa Cloning the GH5 heavy chain signal peptide protein sequence MGWSWIFLFLLSGTAGVHS SEQ ID NO: 38 GH5_HCSnuc Cloning the GH5 heavy chain signal peptide DNA sequence ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCT SEQ ID NO: 39 GH5_HCVaa Cloning the GH5 heavy chain variable region protein sequence EVQLQQSGPELVKPGASMKISCKASDYSFTDYTMTWINWVKQSHEKNLEWIGHINPYNGGTSYNQKFRGKATLTVDKSSSAAYMELLSLTSEDSAVYYCAVVYYDYGGDIFAYWGQGTLVTVSA SEQ ID NO: 40 GH5_HCVnuc Cloning the GH5 heavy chain variable region DNA sequence GAGGTCCAACTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGATTACTCATTCACTGACTACACCATGACCTGGATCAACTGGGTGAAGCAGAGCCATGAAAAGAACCTTGAGTGGATTGGACATATTAATCCTTACAATGGTGGTACTAGTTACAACCAGAAGTTCAGGGGCAAGGCCACATTAACTGTAGACAAGTCATCCAGCGCAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCTGTAGTCTACTATGATTACGGCGGGGATATTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA SEQ ID NO: 41 GH5_HCCaa Clone GH5 heavy chain constant region protein sequence AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 42 GH5_HCCnuc Clone GH5 heavy chain constant region DNA sequence GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA SEQ ID NO: 43 GH5_LCSaa Cloning the GH5 light chain signal peptide protein sequence MSVPTQVLGLLLLWLTGARC SEQ ID NO: 44 GH5_LCSnuc Cloning the DNA sequence of the GH5 light chain signal peptide ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGT SEQ ID NO: 45 GH5_LCVaa Cloning the protein sequence of the GH5 light chain variable region DIQMTQSPASSLSASVGETVTITCRASENIYNYLAWYQQRQGKSPQLLVYNSKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGNYFCQHHSGTPFTFGSGTKLDLK SEQ ID NO: 46 GH5_LCVnuc Cloning the GH5 light chain variable region DNA sequence GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACCGTCACCATCACATGTCGAGCAAGTGAAAATATTTACAATTATTTAGCATGGTATCAACAGAGACAGGGAAAATCTCCTCAACTCCTGGTCTATAATTCAAAAACCT TAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAATTATTTCTGTCAACATCATTCTGGTACTCCATTCACGTTCGGCTCGGGGACAAAGTTGGATTTAAAA SEQ ID NO: 47 GH5_LCCaa Cloning the protein sequence of the GH5 light chain constant region RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 48 GH5_LCCnuc Cloning the GH5 light chain constant region DNA sequence CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAACCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGA ACAGTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT SEQ ID NO: 49 CE8_VHaa Cloning the CE8 primary VH sequence EVQLQQSGPELVKPGASMKISCKASGYSFTDYTMNWVNWVKQSHGKNLEWIGHINPYNGGSSYNQKFRGKATLTVDKSSTTAYMELLSLTSEDSAVYYCAVIYYDYDGDIFAYWGQGTLVTVSA SEQ ID NO: 50 CDR-H1aa Cloning the CE8 CDR-H1 sequence DYTMN SEQ ID NO: 51 CDR-H2aa Cloning the CE8 CDR-H2 sequence WIGHINPYNGGSSYNQKFRG SEQ ID NO: 52 CDR-H3aa Cloning the CE8 CDR-H3 sequence IYYDYDGDIFAY SEQ ID NO: 53 CE8_VLaa Cloning CE8 primary VL sequence DIQMTQSPASSLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNSKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPFTFGSGTKLEIK SEQ ID NO: 54 CDR-L1aa Cloning the CE8 CDR-L1 sequence RASENIYSYLA SEQ ID NO: 55 CDR-L2aa Cloning the CE8 CDR-L2 sequence NSKTLAE SEQ ID NO: 56 CDR-L3aa Cloning the CE8 CDR-L2 sequence QHHYGTPFT SEQ ID NO: 57 CE7_VHaa Cloning the CE7 primary VH sequence DVQLQESGPDLVKPSQSLSLTCTVTGYSLTSGFTWHWIRQFPGNKLEWMGYLHYSGDTNYNPSLRSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGGITSALWGQGTLVTVSA SEQ ID NO: 58 CDR-H1aa Cloning CE7 CDR-H1 SGFTWH SEQ ID NO: 59 CDR-H2aa Cloning CE7 CDR-H2 YLHYSGDTNYNPSLRS SEQ ID NO: 60 CDR-H3aa Cloning CE7 CDR-H3 GGITSAL SEQ ID NO: 61 CE7_VLaa Cloning the CE7 primary VL sequence DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYGATNLADGVPSRFSGSGSGTQFSLKINRLQSEDFGIYYCHHLWGIPYTFGGGTKLEIK SEQ ID NO: 62 CDR-L1aa Cloning CE7 CDR-L1 RASENIYSNLA SEQ ID NO: 63 CDR-L2aa Cloning CE7 CDR-L2 GATNLAD SEQ ID NO: 64 CDR-L3aa Cloning CE7 CDR-L3 HHLWGIPYT SEQ ID NO: 65 FH10_VHaa Cloning the FH10 primary VH sequence DVQVQESGPGLVKPSQSLSLTCTVTGYSITSNYAWNWIRQFPGDKLEWMGFITYSGSSTYNPSLKSRISITRDTSKNQFFLQLNSVTSEDTATYYCASVYYDYDAWFAYWGQGTLVTVSA SEQ ID NO: 66 CDR-H1aa Cloning FH10 CDR-H1 SNYAWN SEQ ID NO: 67 CDR-H2aa Cloning FH10 CDR-H2 FITYSGSSTYNPSLKS SEQ ID NO: 68 CDR-H3aa Cloning FH10 CDR-H3 VYYDYDAWFAY SEQ ID NO: 69 FH10_VLaa Cloning FH10 primary VL sequence DIQMTQSPASSLSASVGETVTITCRASENIYTYLAWYQLKQGKSPQLLVYNAKTLAGGVPSRFSASGSGTQFSLKINSLQPEDFGSFYCHHHYHTPFTFGSGTRLEIN SEQ ID NO: 70 CDR-L1aa Cloning FH10 CDR-L1 RASENIYTYLA SEQ ID NO: 71 CDR-L2aa Clone FH10 CDR-L2 NAKTLAG SEQ ID NO: 72 CDR-L3aa Clone FH10 CDR-L3 HHHYHTPFT SEQ ID NO: 73 GH5_VHaa Clone GH5 primary VH sequence EVQLQQSGPELVKPGASMKISCKASDYSFTDYTMTWINWVKQSHEKNLEWIGHINPYNGGTSYNQKFRGKATLTVDKSSSAAYMELLSLTSEDSAVYYCAVVYYDYGGDIFAYWGQGTLVTVSA SEQ ID NO: 74 CDR-H1aa Clone GH5 CDR-H1aa DYTMT SEQ ID NO: 75 CDR-H2aa Clone GH5 CDR-H2 WIGHINPYNGGTSYNQKFRG SEQ ID NO: 76 CDR-H3aa Clone GH5 CDR-H3 VYYDYGGDIFAY SEQ ID NO: 77 GH5_VLaa Clone GH5 primary VL sequence DIQMTQSPASLSASVGETVTITCRASENIYNYLAWYQQRQGKSPQLLVYNSKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGNYFCQHHSGTPFTFGSGTKLDLK SEQ ID NO: 78 CDR-L1aa Clone GH5 CDR-L1 RASENIYNYLA SEQ ID NO: 79 CDR-L2aa Clone GH5 CDR-L2 NSKTLAE SEQ ID NO: 80 CDR-L3aa Clone GH5 CDR-L3 QHHSGTPFT Detailed Implementation

[0038] Different aspects and embodiments of the present invention may be used individually or in combination.

[0039] Other specific and preferred aspects of the invention are set forth in the appended independent and dependent claims. Features of the dependent claims may be suitably combined with features of the independent claims, and in any manner not expressly set forth in the claims. Each aspect may be practiced independently of the other aspects or in combination with one or more other aspects.

[0040] This is not intended to limit the specific forms disclosed. Rather, it should include all modifications, equivalents, and alternatives that fall within the scope of the appended claims.

[0041] Unless otherwise defined, all terms used herein (including technical and scientific terms) shall be interpreted in accordance with the conventions of the field. It should also be understood that commonly used terms shall be interpreted in accordance with the conventions of the relevant field, rather than in an idealized or overly formal sense, unless explicitly defined herein.

[0042] Phase 1 – Development of propofol-specific monoclonal antibodies The size of propofol severely limits the successful production of high-quality antibodies.

[0043] Propofol will be conjugated with KLH and BSA carrier proteins for use in immunization and screening.

[0044] Target Propofol was conjugated with carrier proteins (KLH and BSA) and used as an immunogen and screening material for any subsequently generated antibodies.

[0045] Four mice were immunized with KLH-conjugated propofol, and then BSA-conjugated materials were used in the screening phase to select antibodies specific to propofol.

[0046] Once candidates are selected, the chosen clonal cell lines are amplified to confirm stable antibody expression, and antibody production at a scale of 10 mg is carried out after successful clonal confirmation.

[0047] Report – Preparation of propofol conjugates and mouse immunization Material Propofol – purchased from Sigma Aldrich P1643-1ML, lot number LRAC4221, pharmaceutical grade secondary standard reference material.

[0048] KLH Imject KLH #77653 Thermo Fisher Lot No. 1902219 cBSA Imject cBSA - cBSA #77150 Thermo Fisher Lot No. VH312389 Formaldehyde solution Sigma Aldrich F8776, batch number MKPC0535 MES Sigma Aldrich M8250 Lot No. SLBQ4750V Anhydrous ethanol Fisher E / 0650DF / 17, batch number 2189134 NaCl Fisher S / 3160 / 63 Lot No. 2033832 Sodium hydroxide Acros 308215000, batch number A0384089 HCl SevernBio 20-555-50, Lot No. 21331 DPBS GIBCO 14190136 Lot No. 2234628 PD10 column GE17-0851-01, batch number 17179054 method The initial attempt was made according to the Mannich condensation reaction described in Hermanson Bioconjugation Techniques (Second Edition), as listed on pages 776-779. This method is not detailed in the Third Edition, likely because it is considered unreliable. It is noted that the recommended buffer solution of 0.1M MES / 0.15M NaCl did not provide significant buffering capacity at the desired pH of 4.50.

[0049] Initial method Dissolve BSA and KLH in MES buffer to a concentration of 10 mg / ml.

[0050] Dissolve propofol in ethanol to a concentration of 10 mg / ml. The reaction mixture was added to the glass vial containing cBSA and KLH as follows: cBSA – 1000μL, 10mg / ml +250μL ETOH +1000 μL propofol / ETOH solution, 10 mg / ml +250μL 37% formaldehyde KLH – 2 x 200μL, 10mg / ml (separate vials) +50μL ETOH / bottle +200 μL propofol / ETOH solution, 10 mg / ml +50μL formaldehyde Mix the components gently and thoroughly, shaking and stirring, and incubate overnight at 37°C. Note that the mixture is slightly milky white at the start of the reaction, but has aggregated and precipitated by the next morning.

[0051] An attempt was made to separate the coupled protein from the reaction mixture using a PD10 column, but the protein could not be recovered. The material was discarded and new cBSA and KLH were purchased.

[0052] Sedimentation survey 1) Buffer Concentration - Further conjugates were prepared using standard components V BSA, varying the MES / NaCl buffer concentration, the amount of ethanol added, and the relative concentration of formaldehyde. Precipitation occurred in all cases, but the standard method tried was the least bad.

[0053] 2) Buffer Types – Buffers were prepared using acetate, citrate, and citrate-phosphate formulations at a concentration of 0.1 M and a pH of approximately 4.50. Various concentrations of BSA, formaldehyde, and ETOH were evaluated. All buffers showed precipitation after incubation at 37°C, but less than the MES buffer system.

[0054] 3) Buffer Type – Re-evaluate using variations of the previous three buffers, changing the required volume / concentration, and using plastic reaction vessels instead of glass reaction vessels. It was found that precipitation was slower under some of these changes compared to others.

[0055] 4) Based on previous work, the changes in buffer type and volume / concentration / temperature were evaluated using cBSA and KLH. Citrate-phosphate buffer showed minimal precipitation under various conditions.

[0056] 5) Final execution: 0.1M citrate-phosphate buffer: 39.45 ml 0.1 M citric acid (21 g / L) + 44.1 ml 0.2 M Na₂HPO₄ (32 g / L) + 0.165 M NaCl. pH 4.48 On a roller, cBSA and KLH were dissolved in CP buffer (citrate-phosphate buffer) for 30 min. The reaction was carried out in polypropylene tubes.

[0057] 200 μL KLH tubes, 10 mg / ml + 50 μL formaldehyde + 25 μL ethanol containing 80 mg / ml propofol. Mix.

[0058] 200 μL cBSA tubes, 10 mg / ml + 200 μL CP buffer + 50 μL formaldehyde + 75 μL ethanol containing 80 mg / ml propofol.

[0059] The reaction was carried out overnight at 37°C. It was found that almost all materials precipitated and could not pass through a PD10 column.

[0060] Protein-propofol material was collected by centrifugation at 17,000g for 10 minutes. The precipitate was resuspended in DPBS and frozen.

[0061] Thaw the precipitate, resuspend it, and disperse the aggregates using an ultrasonic water bath. Wash twice more with DPBS to remove any residual formaldehyde and uncoupled propofol. Finally, resuspend the aggregates in a water bath and open the tube cap to allow any residual formaldehyde to evaporate.

[0062] Final estimated protein recovery: KLH 2.8mg, 1.68mg / ml, combined with two coupling reactions.

[0063] cBSA 3.2mg, 1.86mg / ml, combined with two coupling reactions.

[0064] The material was sent to be used as an immunogen. Soluble conjugates could not be produced using this method. However, aggregated proteins could also be used for this purpose.

[0065] Screening of blood samples after immunization

[0066] Among them, 1 / 50 is 1 in 50; 1 / 500 is 1 in 500; 1 / 5000 is 1 in 5000; and 1 / 50K is 1 in 50K.

[0067]

[0068]

[0069] result A weak but clear positive reaction was observed in mice 4, indicating that propofol was successfully conjugated, but it failed to elicit a strong antibody response. This was a small, measurable response, but it is likely 100 times weaker than what we expected to require to advance the project.

[0070] There were no problems with the antigen preparation or immunization process; all mice were correctly immunized and showed no obvious signs of disease.

[0071] Other mice from a similar cohort (M-NAC-25 mice) showed strong responses as expected, so we believe there is no problem with the animals themselves or the immune process.

[0072] Regarding the assay, these same reagents were used to analyze other test sera, which also showed strong reactions, indicating that there was no problem with the assay reagents used.

[0073] The conclusion is that small-molecule propofol either has poor immunogenicity or produces antibodies that can only bind weakly to small targets.

[0074] discuss Methods for the synthesis of coupling compounds:

[0075] This route did not produce sensitive or specific antibodies, possibly due to steric hindrance.

[0076] No linker was used. Consider using a 6-carbon linker between propofol and the protein conjugate to obtain a better antigen response.

[0077] Consider using propofol analogues, such as: 2-(3-Ethyl-4-hydroxy-5-isopropyl-phenyl)-3,3,3-trifluoro-2-hydroxypropionamide; HS357.5

[0078] Compound structures and design. Propofol, HS245, and HS357. HS245 contains a hydroxyamide moiety, while HS357 contains a six-carbon linker between the phenyl and hydroxyamide moieties.

[0079] Using propofol-BSA for screening may be problematic because serum albumin contains two pocket binding sites for propofol. Would this be beneficial and generate two sets of antibodies specific to free propofol and bound propofol, respectively? For the same reason, propofol-BSA cannot be truly used for assay development; it will nonspecifically bind propofol in the sample and introduce bias in measurements of low concentrations of propofol.

[0080] Recommendation: Propofol analogues, such as 2-(3-ethyl-4-hydroxy-5-isopropyl-phenyl)-3,3,3-trifluoro-2-hydroxypropamide.

[0081] Phase 2 – Conjugation of propofol derivatives and generation of mouse anti-propofol monoclonal antibodies A working program was initiated to generate a monoclonal antibody against the target biomarker propofol (CAS:2078-54-8). For conjugation purposes, an analogue with a suitable functional handle, such as propofol-4-carboxylic acid, was proposed to obtain "propofol" conjugated to the carrier protein.

[0082]

[0083] propofol

[0084] Propofol-4-carboxylic acid Stage 1. Preparation of protein-propofol-4-carboxylic acid conjugate Starting at a 10 mg protein scale, propofol-4-carboxylic acid 2 (TRC, P829760-1G) was conjugated with KLH (Sigma, H7017) and BSA (Sigma, A7030) using EDC / NHS conjugation chemistry to generate propofol immunogen and screening conjugates, respectively. Following conjugation, the products were purified by gel filtration (e.g., Sephadex G25) followed by elution with PBS pH 6.7 to remove unreacted hapten and conjugating agents. Protein concentration was determined by UV-Vis spectrophotometry. Hapten incorporation was qualitatively assessed by TNBS assay and UV-Vis spectrophotometry. The products were normalized to ≥1 mg / ml and finally filtered to 0.2 μm.

[0085] Stage 2. Production of anti-propofol monoclonal antibodies Phase 1 provides an appropriate amount of immunogen and screening conjugates, and performs an immunization procedure to generate monoclonal antibodies against propofol.

[0086] The monoclonal antibody development program is outlined below: Phase 1: Immunization and blood sampling analysis - 4 mice.

[0087] Phase 2: Spleen fusion and screening of two mice.

[0088] Phase 3: Limiting dilution clones, per round, per cell line.

[0089] Phase 4: Final production for each cell line, up to approximately 5 mg. Hybridoma - pilot-scale amplification and purification.

[0090] Phase 3. Evaluate the polyclonal antiserum produced using the immunogen generated in Phase 2 and conduct proof-of-concept development in the form of an immunoassay.

[0091] Development of propofol assay 1. Introduction The objective of this phase is to evaluate the polyclonal antiserum produced using the generated immunogen and to conduct proof-of-concept development in the form of an immunoassay.

[0092] This involves developing a plate-based ELISA format to demonstrate specificity, sensitivity, and precision, with the ultimate goal of developing monoclonal antiserum.

[0093] Polyclonal antiserum has been cultured to be sensitive to propofol (also known as Diprivan), a commonly used anesthetic agent involved in the initiation and maintenance of general anesthesia and other medical applications.

[0094] The development involved optimizing a competitive form of ELISA using a screening antigen (a hapten conjugated to an alternative carrier protein, identical to the immunogen) as the coating, as well as a preliminary investigation of interference and precision in testing several commonly used anesthetics.

[0095] 2. Work Plan Summary The work plan is divided into three objectives: Objective 1 – Initial Optimization Objective 2 – Characterization of antibody reactivity Objective 3 – Matrix tolerance and preliminary determination of precision.

[0096] Objective 1: Prior to assay development, polyclonal antiserum was tested using the propofol-BSA conjugate, with comparable results for both hosts. The anti-rabbit IgG-HRP assay conjugate was then titrated under FBL conditions at a range of concentrations (propofol-BSA: 20–0.156 µg / ml, antiserum: 1 / 250–1 / 512000).

[0097] This form of substitution was then tested with propofol-4-carboxylic acid (a hapten used to generate immunogens) to get a general idea of ​​the assay sensitivity before receiving propofol from Merck.

[0098] Once propofol arrived, it was used as a standard material in all subsequent experiments (after DV305 / 197).

[0099] Standards ranging from 1 µg / ml to 15.625 ng / ml showed a dose-response relationship, but high background was observed. This high background persisted even when tested without propofol-BSA coating, indicating nonspecific binding (DV305 / 195,197,199). We then tested standards with different coatings and antiserum concentrations, as well as uncoated standards, to see if the background could be reduced (DV337 / 01).

[0100] Several different solutions were tested as blocking buffers, followed by a 30-minute blocking step. The solution with the lowest background was found to be 0.1% milk powder (DV337 / 07). This blocking step effectively eliminated the observed nonspecific binding.

[0101] The secondary antibody was then titrated at two different coating concentrations (20 µg / ml and 10 µg / ml) to observe concentrations from 1 / 1000 to 1 / 100,000 (DV337 / 10,12). The resulting ideal conditions were used for all further studies.

[0102] Objective 2: Using the optimized detection conditions described in Objective 1, gradient dilutions of each identified interfering agent were tested. These interfering agents were replicated on each plate with a control. All interfering agents were prepared at the same concentrations as the control series to calculate relative displacement (DV337 / 17,24). This was only necessary for the two interfering agents (propofol-O-glucuronide and propofol-O-sulfate). No antibody displacement was generated for any of the other potential interfering agents tested within the assessed concentration range.

[0103] Objective 3: Serum at different dilutions was tested on propofol curves using the final assay form, and displacement, signal, dynamic range, and nonspecific binding were compared. Minimal differences were found between the control curve and curves in 1 / 5, 1 / 15, and 1 / 20 sera. 1 / 10 sera did not conform to these curves, but were considered abnormal (DV337 / 15) because they did not show the same trend. Therefore, the prototype assay is considered to be tolerable in up to 20% of serum.

[0104] Quality control samples were prepared using mixed human serum at concentrations of 30 pg / ml, 2 pg / ml, and 0.2 pg / ml, and then diluted 1:20 in the test buffer. These were aliquots for single use and stored at <-20°C until testing was required.

[0105] These samples were tested in triplicate in independent runs over 3 days to reach their final form, with a standard curve on each plate. The concentration values ​​for each sample were calculated from these curves using 4PL fitting, and intra- and inter-assay precision was compared (DV337 / 32,34,37).

[0106]

[0107] 3. Materials and Methods 3.1 Reagents: Table 2: Reagent Specifications

[0108] 3.2 Methods Reagent Description: Antipropofol antibody: Antipropofol serum (harvested blood, rabbit 1) was derived from the antibody production program (batch number: 22:05 / 1551-3).

[0109] Anti-rabbit HRP: Anti-rabbit HRP is sourced from Merck (catalog code: batch number: 3920753).

[0110] Analytical standard: Propofol is derived from TCl (catalog code: D0617, batch number: MTBK7900V). Propofol-BSA solution: The propofol-BSA conjugate was prepared by FBL and supplied by SOM / APS (lot number: SOM1 / 2), with a storage concentration of 1.7 mg / ml.

[0111] Buffer 21: See Appendix for formulation.

[0112] Buffer 111: See appendix for formulation.

[0113] Buffer 58: See Appendix for formulation.

[0114] Key equipment: Pipettes: RAININ- P20 (5-20μL, PIP085), P200 (20-200μL, PIP088), P1000 (200-1000μL, PIP090); EPPENDORF- P5000 (1000-5000μL, PIP091) Microcentrifuge tubes: VWR microcentrifuge tubes, 2.0 ml (catalog code: 211-2606) / Microcentrifuge tubes, 0.5 ml (catalog code: 525-1157) Plate: VWR – Perforated Flat Plate (96 Holes) (Catalogue Code: 734-2327) Balances: Sartorius R160D (<100g, BAL 1), Sartorius LP3200D (100-2000g, BAL2), Sartorius ISI 10 (>2000g, BAL3) Plate incubator: Grant Bio PHMP (PINCU2) Plate washer: Biotek ELx50 (WASH2) Board reader: Thermofisher Multiskan FC (READ1) Preparation of working solution: Preparation of propofol-BSA: The optimal assay concentration for propofol-BSA coating was found to be 20 µg / ml.

[0115] Prepare from the storage concentration in buffer 21 on the day of use.

[0116] Preparation of anti-propofol antibody: The optimal concentration for assay was found to be a 1 / 500 dilution.

[0117] Prepare in buffer 111 on the day of use.

[0118] Preparation of anti-rabbit HRP antibody: The optimal concentration for assay was found to be a 1 / 5000 dilution.

[0119] Prepare an aliquot of the solution in buffer 111 on the day of use.

[0120] Preparation of propofol standards: The standard series starts at a concentration of 4 µg / ml.

[0121] On the day of use, prepare an initial stock solution by weighing a certain amount of propofol (2-3 mg) and diluting it with methanol to 1 mg / ml.

[0122] Dilute the mixed human serum 1 / 20 in buffer 111.

[0123] Dilute propofol to 4 µg / ml using 1 / 20 of the serum matrix.

[0124] Perform 1 / 4 serial dilutions to produce seven standards ranging from 4 µg / ml to 0.98 ng / ml.

[0125] 1 / 20 of the serum was used as a blank.

[0126] Preparation of blocking buffer: 0.1% milk powder w / w buffer solution was prepared in Buffer 58.

[0127] Measurement Procedure 1. Add 100 µL of propofol-BSA to each well.

[0128] 2. Incubate at 37°C for 1 hour.

[0129] 3. Wash with buffer 58 using the assay procedure 3.

[0130] 4. Add 150 µL of blocking buffer (0.1% milk powder) to each well.

[0131] 5. Incubate at 37°C for 10 minutes.

[0132] 6. Wash with buffer 58 using the assay procedure 3.

[0133] 7. Add 50 µL of propofol standard and sample to the required concentration. 8. Add 50 µL of anti-propofol antibody to each well.

[0134] 9. Incubate at 37°C for 1 hour at 600 rpm.

[0135] 10. Wash with buffer 58 using the assay procedure 3.

[0136] 11. 100 µL of anti-rabbit HRP was added to each well.

[0137] 12. Incubate at 37°C for 1 hour.

[0138] 13. Wash with buffer 58 using the assay procedure 3.

[0139] 14. Add 100 µL of Sureblue stock solution to each well.

[0140] 15. Incubate at room temperature for 10 minutes.

[0141] 16. Add 100 µL of hydrochloric acid to each well to terminate the reaction.

[0142] 17. Read the plate at 450nm and save the results to the project folder.

[0143] Measurement Procedure 3: Extract 400 µL dispensed into each well Repeat steps 1 and 2 three times. Extract result Objective 1 – Initial Optimization Initial coating-antiserum titration – Figure 1 In the initial titrations of the coating and antiserum, coating concentrations ranging from 20–0.156 µg / ml and antiserum concentrations from 1 / 125 to 1 / 512,000 were tested. The tests indicated that this combination could be used to generate a signal, but it has not yet been tested with internal reagents. Anti-rabbit IgG HRP diluted 1 / 1000 was used. Antisera from both hosts were used in this test, showing minimal difference between them. Propofol was not used in this titration to observe the maximum signal for each condition.

[0144] The titers were similar between the two host animals, but rabbit 1 had a slightly higher titer, which was used for subsequent investigations. Then, standards were used to test the optimal three coating conditions and the optimal two antiserum concentrations to examine the substitution.

[0145] A dose-response was observed using a standard curve starting at 2 µg / ml and a 1 / 4 dilution series, but with high background. This was tested using 1 / 125 antiserum and a coating concentration of 20 µg / ml, which was the highest concentration tested.

[0146] Background titration – Figure 2 and 3 To reduce the observed background, different coated and antiserum concentrations were tested again using a standard curve and an uncoated line. Although a slight reduction in background was observed with decreasing concentration, the lowest observed background was 0.65 Abs with a coating concentration of 5 µg / ml and an antiserum concentration of 1 / 2000.

[0147] The blocking step was then introduced, and six different blocking solutions were tested. The 0.1% milk powder blocking solution provided the best background reduction. This was tested using an antiserum concentration of 1 / 500 and a coating concentration of 10 µg / ml. Pierce 'ClearMilk' buffer (Fisher Scientific, 13494209) was diluted 1 / 10 according to the manufacturer's instructions, while Pierce blocking buffer (Fisher Scientific, 37572), 'Superblock' (ThermoScientific, 37515), milk powder, BSA, and Pluronic F-127 solution were prepared at 0.1% in Buffer 58.

[0148] For this assay (DV337 / ) and all subsequent assays, commercially available anti-rabbit HRP (Merck, 12-348) was used.

[0149] Coating-secondary antibody titration – Figure 4 Coating concentrations ranging from 2 to 20 µg / ml and secondary antibody concentrations from 1 / 1000 to 1 / 100,000 were tested to optimize the assay range and maximum signal. 1 / 500 antiserum was used for this purpose, and this was determined as the final antiserum concentration. Relative substitutions for each combination were compared, and the final conditions for 1 / 500 primary antibody, 1 / 5000 secondary antibody, and 20 µg / ml propofol-BSA coating were determined.

[0150] Objective 2 – Characterization of antibody reactivity Figures 5 to 7 To assess the cross-reactivity of the antiserum, standard curves were prepared for 16 common compounds in the same range as the propofol control, including several anesthetics and over-the-counter analgesics. These were prepared as stock solutions in methanol and then diluted in methanol to a 100 µg / ml intermediate. The solutions were then diluted in assay buffer to a 4 µg / ml concentration.

[0151] Several of these are drugs controlled by the Ministry of the Interior and were handled according to relevant procedures. Ketamine, midazolam, morphine, and fentanyl are controlled drugs, classified by the Ministry of the Interior as Schedules 2, 3, 2, and 2, respectively. The use of controlled drugs required in this project was covered by COSHH assessments FBL006, FBL007, and FBL064, while SOP091 details the management instructions for drugs controlled by the Ministry of the Interior's Schedules in accordance with Ministry of the Interior regulations. All were tested on a single-plate, except for thiopental and etomidate, which were delayed due to transportation issues. The etomidate / thiopental result was negative.

[0152] Of the 14 interfering substances tested, two compounds also interacted with the antiserum. These were propofol-O-glucuronide and propofol-O-sulfate, metabolites of propofol with similar molecular structures. Propofol-O-sulfate showed a larger substitution than propofol-O-glucuronide due to its greater structural similarity. These were compared as relative substitutions at the 50% level. The relative substitution calculated for propofol-O-sulfate at the 50% level was 23%.

[0153] The substitution of propofol-O-glucuronide could not be calculated at the 50% level, so it was calculated at the 30% substitution level. At 30%, this showed a 2% substitution.

[0154] Table 3 - see attached figure.

[0155] Table 3: Table showing the relative replacement of interfering substances (DV337 / 24) <Results of etomidate / thiopental sodium> Objective 3 - Matrix tolerance and preliminary determination of precision Figure 11 Serum curves were run in 1 / 5, 1 / 10, 1 / 15, and 1 / 20 serum and compared with a control curve in buffer (DV337 / 15). Minimal differences were observed between the serum and control curves, except that a 1 / 10 serum dilution was considered abnormal. Based on these findings, the assay is considered to be tolerable up to 20% serum. However, it should be noted that a shift in the curves occurred compared to buffer only. Therefore, to ensure accuracy, best practice is to prepare calibration curves in diluted matrix. Based on the assay's working range and the target working range, 5% serum is considered optimal for subsequent studies.

[0156] A 1 / 20 dilution was deemed sufficient to minimize any matrix effects; therefore, controls were prepared at 20-fold concentrations, targeting 1500, 200, and 20 ng / ml on the standard curve, representing the high, medium, and low QC ranges, respectively. These were stored as single-use aliquots and frozen. On the day of assay, a 1 / 20 dilution was used with assay buffer and run as a sample.

[0157] To test these, samples were tested in triplicate within independent runs over 3 days, and relative values ​​were calculated from the relevant standard curve. This data was used to compare intra- and inter-assay variability. Figure 12 .

[0158] Table 4-6 - see attached figures.

[0159] Table 4: Summary of optical density values ​​of QC sample tests (DV337 / 32,34,37) Table 5: Summary of signals normalized to average maximum binding (0 standard) (DV337 / 32, 34, 37) Table 6: Back-calculated values ​​(DV337 / 32,34,37) of undiluted QC samples based on 4PL fitting of the standard curve for the corresponding test day.

[0160] in conclusion The objective of this phase of the project is to develop an ELISA-based assay to evaluate anti-propofol antiserum developed in human serum.

[0161] Following our work on antibody development, we went on to develop and successfully produce a prototype assay in the form of a plate-based ELISA suitable for clinical sample analysis.

[0162] The developed assay showed an effective range of 0.02–80 µg / mL, and a 1 / 20 serum dilution met the target range of 0.1–20 µg / mL. The assay appears to tolerate up to 20% serum, but the calibrator should be prepared in a dilution matrix pool. For maximum sensitivity, a 1 / 5 sample dilution provides an effective assay range of 0.005–20 µg / mL.

[0163] No cross-reactivity or interference was observed with the co-administered substances and common interfering agents tested. Cross-reactivity was only observed with structurally similar metabolites. For propofol-glucuronide, this was approximately 2% cross-reactivity, therefore a 50-fold higher concentration of the glucuronide metabolite would be required to produce the same reaction as a given concentration of propofol in the assay. For propofol-sulfate, the cross-reactivity was 23%, therefore a four to five-fold excess of the sulfate metabolite would produce the same assay reaction as a given concentration of propofol. Although relatively low levels of cross-reactivity with metabolites exist, especially considering that glucuronide is the major metabolite, the antiserum is not 100% specific for propofol.

[0164] While it was hoped that the immunogen would be designed to react only to propofol, low levels of cross-reactivity, particularly for glucuronide metabolites, do provide an option. It should be remembered that this is crude polyclonal antiserum, and therefore contains antibodies with a range of different specificities. For the lateral flow assay to be manufactured, monoclonal antibodies would be the preferred implementation. In this case, these data guide the clonal screening method to be used. The same immunogen can be used for immunization, but a stratified approach is needed for screening hits and clones. Initial screening will be done in conjunction with a screening conjugate, followed by subsequent testing to exclude clones that have been replaced by metabolites. This allows clones to be selected for investigation based on minimal cross-reactivity.

[0165] For further assay development using polyclonal antiserum, affinity purification is performed – see Stage 4. The screening conjugates are immobilized on a column, allowing antibody binding. The column is then washed with a solution containing metabolites to remove those cross-reactive antibodies, followed by elution via pH changes to obtain the propofol-specific fraction.

[0166] At this stage of assay development, preliminary assay precision was considered acceptable (<20%). Intra-assay and inter-assay imprecision peaked at high propofol concentrations. The highest level of imprecision observed with calibrators was approximately 16%. While some signal variability existed between plates, this was limited by normalization to the maximum binding signal (0 ng / mL standard). This practice is considered standard for this type of competitive assay. The peak imprecision for back-calculated concentrations of QC samples using 4PL curve fitting was 12%, with a maximum observed deviation from the expected value of 14%.

[0167] Based on the findings of this phase, it was concluded that the proof of concept has been proven.

[0168] Phase 4 - Antigen affinity purification of polyclonal antiserum Following the completion of the plan for cultured polyclonal antisera against propofol and the development of proof-of-concept assays using the antisera, a working plan for antisera affinity purification was initiated.

[0169] During the proof-of-concept phase, the antiserum exhibited some reactivity to propofol sulfate metabolites, but less reactivity to glucuronic acid metabolites. The objective of this phase of the project is to attempt to remove unwanted metabolite reactivity from the polyclonal antiserum while maintaining reactivity with the parent compound.

[0170] Objective 1: Generation and testing of affinity columns The propofol-BSA hapten conjugate will be produced using the method established in Project SOM1. The resulting conjugate will then be immobilized on a Cytiva HiTrap NHS-activated HP 1 mL column. This column can be easily scaled up should the process require future expansion. 1 mL of crude antiserum will be passed through the column and allowed to bind. The captured antibody will be eluted by acidification. This will demonstrate the column's utility and provide a baseline for the proportion of propofol-specific antiserum. The concentration of the eluted antibody will be determined by UV absorbance.

[0171] Objective 2: Reactive removal of metabolites An additional 1 mL aliquot of antiserum will be passed through the column generated in Target 1. After allowing propofol-specific antibody binding, the column will be washed with a solution containing propofol sulfate metabolites. Antibodies reacting with the metabolites will be displaced and collected in the eluent. Remaining non-reactive antibodies from the metabolites will be eluted by acidification.

[0172] The concentration of the eluent will be determined by UV absorption. A comparison with the concentration of the product produced in Target 1 will provide an indication of the relative abundance of propofol-specific antibodies in serum relative to propofol and metabolite-reactive antibodies.

[0173] Objective 3: Confirmation of the reactivity of the final product The reactivity of the elution products from targets 1 and 2 will be assessed using a proof-of-concept assay developed in Phase 3. The products and crude antiserum will be run in the assay against standard curves for propofol, sulfate, and glucuronide metabolites. The working strength of the elution products will be estimated based on the known working strength of the crude antiserum and the product concentrations determined for targets 1 and 2. The products will be evaluated relative to the crude antiserum, and cross-reactivity with the metabolites will be estimated based on the displacements observed relative to propofol itself.

[0174] If metabolite reactivity remains in the product of Target 2, further removal steps, such as with glucuronide metabolites, can be considered, depending on the observed yield.

[0175] Propofol monoclonal antibody Monoclonal antibodies have been produced using hybridoma technology. This involves fusing antibody-producing B cells with myeloma cells, which are cancer cells that proliferate indefinitely. The resulting hybrid cells, called hybridomas, can produce large quantities of the same mAbs targeting specific antigens.

[0176] The general process of creating a hybridoma involves several steps, including immunizing the animal with a target antigen, harvesting B cells from the spleen, fusing the B cells with myeloma cells using chemical or electrical methods, selecting hybridomas that produce the desired antibodies, and finally growing and maintaining the selected hybridomas in culture.

[0177] Four monoclonal antibodies have been produced and sequenced. These antibodies have been named: cAb11051 (also known as CE8); cAb11052 (also known as CE7); cAb11053 (also known as FH10); and cAb11131 (also known as GH5).

[0178] Sequencing was performed using whole transcriptome shotgun sequencing (RNA-Seq).

[0179] Sequence Report 1. ID: cAb11051 Clone name: CE8.G9.C12.F6.B11 HC quantity: 1 LC quantity: 1 Primary heavy chain protein / DNA sequence HC type: Mouse IgG1 Completeness: Complete Signal peptide: SEQ ID NO: 1 MGWSWIFLFLLSGTAGVHS SEQ ID NO: 2 ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCT VH: SEQ ID NO: 3 EVQLQQSGPELVKPGASMKISCKASGYSFTDYTMNWVNWVKQSHGKNLEWIGHINPYNGGSSYNQKFRGKATLTVDKSSTTAYMELLSLTSEDSAVYYCAVIYYDYDGDIFAYWGQGTLVTVSA SEQ ID NO: 4 GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACACCATGAACTGGGTGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACATATTAATCCTTACAATGGTGGTTCTAGCTACAACCAGAAGTTCAGGGGCAAGGCCACATTAACTGTAGACAAGTCATCCACCACAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCTGTAATCTACTATGATTACGACGGGGATATTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA Constant region: SEQ ID NO: 5 AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 6 GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA TPM: 301891.64 %: 41.3 Primary light chain protein / DNA sequence LC type: mouse kappa Integrity: complete Signal peptide: SEQ ID NO: 7 MSVPTQVLGLLLLWLTGARC SEQ ID NO: 8 ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGT VL: SEQ ID NO: 9 DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNSKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPFTFGSGTKLEIK SEQ ID NO: 10 GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAACTCCTGGTCTATAATTCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTACTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA Constant region: SEQ ID NO: 11 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 12 CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAACCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGA ACAGTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT TPM: 429016.43 %: 58.7 2. ID: cAb11052 Clone name: CE7.E4.G9.F5.E11 HC quantity: 1 LC quantity: 1 Primary heavy chain protein / DNA sequence HC type: Mouse IgG1 Completeness: Complete Signal peptide: SEQ ID NO: 13 MRVLILLCLFTAFPGILS SEQ ID NO: 14 ATGAGAGTGCTGATTCTTTTGGTGCCTGTTCACAGCCTTTCCTGGTATCCTGTCT VH: SEQ ID NO: 15 DVQLQESGPDLVKPSQSLSLTCTVTGYSLTSGFTWHWIRQFPGNKLEWMGYLHYSGDTNYNPSLRSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGGITSALWGQGTLVTVSA SEQ ID NO: 16 GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCCCTCACCTGCACTGTCACTGGCTACTCCCTCACCAGTGGTTTTACCTGGCACTGGATCCGGCAGTTTCCAGGGAACAAACTGGAGTGGATGGGCTACCTACACTACAGTGGTGACACTAACTACAACCCATCTCTCAGAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGAGGCGGGATTACGTCGGCTCTCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA Constant region: SEQ ID NO: 17 AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 18 GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA TPM: 234944.43 %: 35.64 Primary light chain protein / DNA sequence LC type: Mouse kappa Integrity: Complete Signal peptide: SEQ ID NO: 19 MSVPTQVLGLLLLWLTDARC SEQ ID NO: 20 ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGATGCCAGATGT VL: SEQ ID NO: 21 DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYGATNLADGVPSRFSGSGSGTQFSLKINRLQSEDFGIYYCHHLWGIPYTFGGGTKLEIK SEQ ID NO: 22 GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGTATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTAATTTAGCATGGTATCAGCAGAAGCAGGGAAAATCTCCTCAGCTCCTGGTCTATGGTGCTACAAACTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCCCTCAAGATCAACAGGTTGCAGTCTGAAGATTTTGGGATTTACTACTGTCACCATTTATGGGGTATACCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA Constant region: SEQ ID NO: 23 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 24 CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAACCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGA ACAGTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT TPM: 424218.12 %: 64.36 3. ID: cAb11053 Clone name: FH10.G2.G7.H5.D8 HC quantity: 1 LC quantity: 1 Primary heavy chain protein / DNA sequence HC type: Mouse IgG1 Completeness: Complete Signal peptide: SEQ ID NO: 25 MRVLILLWLFTAFPGILS SEQ ID NO: 26 ATGAGAGTGCTGATTCTTTTGGTGGCTGTTCACAGCCTTTCCTGGTATCCTGTCT VH: SEQ ID NO: 27 DVQVQESGPGLVKPSQSLSLTCTVTGYSITSNYAWNWIRQFPGDKLEWMGFITYSGSSTYNPSLKSRISITRDTSKNQFFLQLNSVTSEDTATYYCASVYYDYDAWFAYWGQGTLVTVSA SEQ ID NO: 28 GATGTGCAGGTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTAACTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAGACAAACTGGAGTGGATGGGCTTCATAACCTACAGTGGTAGTTCTACCTACAACCCCTCTCTCAAGAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTTCAATTGAATTCTGTGACTTCTGAGGACACAGCCACTTATTACTGTGCAAGTGTCTATTATGATTACGACGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA Constant region: SEQ ID NO: 29 AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 30 GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA TPM: 319943.2 %: 38.2 Primary light chain protein / DNA sequence LC type: mouse kappa Integrity: complete Signal peptide: SEQ ID NO: 31 MSVPTQVLGLLLLWLTGARC SEQ ID NO: 32 ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTACTGTGGCTTACAGGTGCCAGATGT VL: SEQ ID NO: 33 DIQMTQSPASLSASVGETVTITCRASENIYTYLAWYQLKQGKSPQLLVYNAKTLAGGVPSRFSASGSGTQFSLKINSLQPEDFGSFYCHHHYHTPFTFGSGTRLEIN SEQ ID NO: 34 GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACACTTATTTAGCATGGTATCAGCTGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCGGGAGGTGTGCCATCAAGGTTCAGTGCCAGTGGATCAGGCACACAATTTTCTCTGAAGATCAACAGCCTGCAGCCTGAGGATTTTGGGAGTTTCTACTGTCACCATCATTATCATACTCCTTTCACGTTCGGCTCGGGGACAAGGTTGGAGATAAAC Constant region: SEQ ID NO: 35 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 36 CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAACCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGA ACAGTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT TPM: 517805.2 %: 61.8 4. ID: cAb11131 Clone name: GH5.G4.C8.C4.3.G5.2.H8 HC quantity: 1 LC quantity: 1 Primary heavy chain protein / DNA sequence HC type: Mouse IgG1 Completeness: Complete Signal peptide: SEQ ID NO: 37 MGWSWIFLFLLSGTAGVHS SEQ ID NO: 38 ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCT VH: SEQ ID NO: 39 EVQLQQSGPELVKPGASMKISCKASDYSFTDYTMTWINWVKQSHEKNLEWIGHINPYNGGTSYNQKFRGKATLTVDKSSSAAYMELLSLTSEDSAVYYCAVVYYDYGGDIFAYWGQGTLVTVSA SEQ ID NO: 40 GAGGTCCAACTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGATTACTCATTCACTGACTACACCATGACCTGGATCAACTGGGTGAAGCAGAGCCATGAAAAGAACCTTGAGTGGATTGGACATATTAATCCTTACAATGGTGGTACTAGTTACAACCAGAAGTTCAGGGGCAAGGCCACATTAACTGTAGACAAGTCATCCAGCGCAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCTGTAGTCTACTATGATTACGGCGGGGATATTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA Constant region: SEQ ID NO: 41 AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 42 GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA TPM: 365138.3 %: 47.5 Primary light chain protein / DNA sequence LC type: mouse kappa Integrity: complete Signal peptide: SEQ ID NO: 43 MSVPTQVLGLLLLWLTGARC SEQ ID NO: 44 ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGT VL: SEQ ID NO: 45 DIQMTQSPASLSASVGETVTITCRASENIYNYLAWYQQRQGKSPQLLVYNSKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGNYFCQHHSGTPFTFGSGTKLDLK SEQ ID NO: 46 GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACCGTCACCATCACATGTCGAGCAAGTGAAAATATTTACAATTATTTAGCATGGTATCAACAGAGACAGGGAAAATCTCCTCAACTCCTGGTCTATAATTCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAATTATTTCTGTCAACATCATTCTGGTACTCCATTCACGTTCGGCTCGGGGACAAAGTTGGATTTAAAA Constant region: SEQ ID NO: 47 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 48 CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAACCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGA ACAGTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT TPM: 403810.6 %: 52.5 CDR authentication The complementarity determination region (CDR) was automatically identified using an Excel formula based on the Kabat definition. CDR identification was performed on the primary VH and VL sequences.

[0180] cAb11051 Primary VH sequence SEQ ID NO: 49 EVQLQQSGPELVKPGASMKISCKASGYSFTDYTMNWVNWVKQSHGKNLEWIGHINPYNGGSSYNQKFRGKATLTVDKSSTTAYMELLSLTSEDSAVYYCAVIYYDYDGDIFAYWGQGTLVTVSA CDR-H1 SEQ ID NO: 50 DYTMN CDR-H2 SEQ ID NO: 51 WIGHINPYNGGSSYNQKFRG CDR-H3 SEQ ID NO: 52 IYYDYDGDIFAY Primary VL sequence SEQ ID NO: 53 DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNSKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPFTFGSGTKLEIK CDR-L1 SEQ ID NO: 54 RASENIYSYLA CDR-L2 SEQ ID NO: 55 NSKTLAE CDR-L3 SEQ ID NO: 56 QHHYGTPFT cAb11052 Primary VH sequence SEQ ID NO: 57 DVQLQESGPDLVKPSQSLSLTCTVTGYSLTSGFTWHWIRQFPGNKLEWMGYLHYSGDTNYNPSLRSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGGITSALWGQGTLVTVSA CDR-H1 SEQ ID NO: 58 SGFTWH CDR-H2 SEQ ID NO: 59 YLHYSGDTNYNPSLRS CDR-H3 SEQ ID NO: 60 GGITSAL Primary VL sequence SEQ ID NO: 61 DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYGATNLADGVPSRFSGSGSGTQFSLKINRLQSEDFGIYYCHHLWGIPYTFGGGTKLEIK CDR-L1 SEQ ID NO: 62 RASENIYSNLA CDR-L2 SEQ ID NO: 63 GATNLAD CDR-L3 SEQ ID NO: 64 HHLWGIPYT cAb11053 Primary VH sequence SEQ ID NO: 65 DVQVQESGPGLVKPSQSLSLTCTVTGYSITSNYAWNWIRQFPGDKLEWMGFITYSGSSTYNPSLKSRISITRDTSKNQFFLQLNSVTSEDTATYYCASVYYDYDAWFAYWGQGTLVTVSA CDR-H1 SEQ ID NO: 66 SNYAWN CDR-H2 SEQ ID NO: 67 FITYSGSSTYNPSLKS CDR-H3 SEQ ID NO: 68 VYYDYDAWFAY Primary VL sequence SEQ ID NO: 69 DIQMTQSPASLSASVGETVTITCRASENIYTYLAWYQLKQGKSPQLLVYNAKTLAGGVPSRFSASGSGTQFSLKINSLQPEDFGSFYCHHHYHTPFTFGSGTRLEIN CDR-L1 SEQ ID NO: 70 RASENIYTYLA CDR-L2 SEQ ID NO: 71 NAKTLAG CDR-L3 SEQ ID NO: 72 HHHYHTPFT cAb11131 Primary VH sequence SEQ ID NO: 73 EVQLQQSGPELVKPGASMKISCKASDYSFTDYTMTWINWVKQSHEKNLEWIGHINPYNGGTSYNQKFRGKATLTVDKSSSAAYMELLSLTSEDSAVYYCAVVYYDYGGDIFAYWGQGTLVTVSA CDR-H1 SEQ ID NO: 74 DYTMT CDR-H2 SEQ ID NO: 75 WIGHINPYNGGTSYNQKFRG CDR-H3 SEQ ID NO: 76 VYYDYGGDIFAY Primary VL sequence SEQ ID NO: 77 DIQMTQSPASSLSASVGETVTITCRASENIYNYLAWYQQRQGKSPQLLVYNSKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGNYFCQHHSGTPFTFGSGTKLDLK CDR-L1 SEQ ID NO: 78 RASENIYNYLA CDR-L2 SEQ ID NO: 79 NSKTLAE CDR-L3 SEQ ID NO: 80 QHHSGTPFT Affinity purification Affinity purification report: Displacement of four propofol mAbs in ELISA.

[0181] introduction Affinity purification was performed on the antiserum used to develop the proof-of-concept ELISA. This involved affinity purification of the antiserum and evaluation of the purified product in the proof-of-concept assay to confirm the presence of specific antibodies and the removal of non-specific antibodies. The polyclonal antiserum had been cultured to be sensitive to propofol (also known as diprivan), a commonly used anesthetic agent involved in the initiation and maintenance of general anesthesia and other medical applications.

[0182] Work Plan Summary Objective 1: Generation and testing of affinity columns Objective 2: Reactive removal of metabolites Objective 3: Confirmation of the reactivity of the final product Target 1 - Multiple affinity columns were fabricated using two different techniques in an attempt to determine the optimal mechanism of action. Five columns were fabricated as part of this goal, three using propofol-BSA conjugates and two using a direct coupling mechanism.

[0183] The first approach involved using a propofol-BSA conjugate later produced in DV333 / 59 – the conjugate was immobilized on a Cytiva HiTrap NHS-activated HP 1 ml column. Initially, two different columns were fabricated using the conjugate produced as part of SOM1; however, due to low yields of anti-propofol antibodies after AKTA runs, two alternatives were investigated. The first was to fabricate a third column using fresh propofol-BSA conjugate, and the second was to try a completely different method of binding propofol to the column.

[0184] The direct coupling column was prepared by reacting an NHS-activated HP column with adipic acid dihydrazide, activating propofol 4-COOH with EDC and NHS, and then directly coupling it with propofol via the addition of NHS.

[0185] All columns were used to determine which mechanism was most effective at removing anti-propofol antibodies, as part of Target 2.

[0186] Target 2 - The columns produced in Target 1 were further used to flush serum rich in anti-propofol antibodies through them in an attempt to remove them. Each column was tested sequentially using 1 ml of buffered serum. Both the eluent and antibody elution buffer were given to the assay team for testing to determine which method was best suited for continued use. After some testing in Target 3, it was determined that the direct-coupled columns were better than the BSA columns, therefore it was decided to use two direct-coupled columns in tandem in the final experiment. Furthermore, the serum was recycled on these columns to obtain the highest binding probability.

[0187] The above method will also be used to test the specificity of antibodies to propofol relative to propofol sulfate.

[0188] Target 3 - The product generated from Target 1, without the target analyte, was tested using the prototype assay developed in this paper to examine the relative concentration of antibodies specific to the propofol-BSA conjugate present. Tests were performed at a range of dilutions to compare absorbance across the entire range. The affinity-purified elution was also tested to investigate the amount of antibody removed and its concentration relative to the elution product. The affinity-purified product was compared to crude antiserum to examine the maximum absorbance.

[0189] The affinity-purified final products of propofol metabolites were tested to examine cross-reactivity. Tests using the same method showed a relative substitution of 23% for propofol-sulfate at 50% level and 2% for propofol-glucuronide at 30% level. The substitutions of these metabolites were compared to those of propofol at the same concentration to calculate the relative substitutions.

[0190]

[0191] 1. Materials and Methods 1.1 Reagents: Table 2: Reagent Specifications

[0192] Methods – Chemistry Reagent Description: Antipropofol antibody: Antipropofol serum (harvested blood, rabbit 1) was derived from APS’s SOM1 antibody production program (batch number: 22:05 / 1551-3).

[0193] Propofol-BSA conjugates: prepared by FBL and supplied by SOM (lot numbers: SOM1 / 2 and DV333 / 59-1).

[0194] Buffer 3: See Appendix for formulation.

[0195] Buffer 33: See appendix for formulation.

[0196] Buffer 97: See appendix for formulation.

[0197] 0.1 M glycine buffer, pH 2.5: Add 7.5 g glycine to 1 L RO / DI water and adjust the pH with 1 M HCl. Filter to 0.22 µm.

[0198] Glycine buffer, pH 2.5, containing 5% ethanol: Add 3.75 g glycine to 500 mL RO / DI water, adjust the pH with 1M HCl, and then add ethanol to make it 5% of the final solution. Filter to 0.22 µm.

[0199] Propofol-BSA coupling column packing scheme: 1. Add 6 ml of ice-cold 1 mM HCl to the column.

[0200] 2. Next, 2 ml of BSA-propofol conjugate was added to the column. The column was incubated at 20°C for 30 minutes.

[0201] 3. Wash the column with 6 ml of 0.5M ethanolamine + 0.5M NaCl (buffer A), then wash the column with 6 ml of 0.1M sodium acetate + 0.5M NaCl (buffer B), and then wash the column with 6 ml of buffer A. Incubate the column at 20°C for 30 minutes.

[0202] 4. Wash the column with 6 ml of buffer B, then with 6 ml of buffer A and 6 ml of buffer B.

[0203] 6. Add 2 ml of buffer 3 to the column and store the column overnight at 2-8°C.

[0204] Direct coupling column loading scheme: 1. Activate the column by reacting it with 20 ml of 0.1 mg / ml adipic acid dihydrazide solution (in buffer 3) as follows: Inject 5 ml of dihydrazide solution into the column and incubate at 20°C for 10 minutes. Repeat this step 3 times until all 20 ml has passed through.

[0205] 2. Wash the column with buffer 3 at 3 ml / min for 5 minutes.

[0206] 3. Add activated propofol (0.1 mg / ml solution) to the column as follows: wash the column dropwise with 5 ml – then incubate the column at 20°C for 10 minutes. Repeat this step twice more until 15 ml of activated propofol has passed through the column.

[0207] 4. Wash the column as follows: a. 10 ml buffer solution, 3, 1 ml / min b. 10 ml glycine, pH 2.5 buffer, 1 ml / min c. 10 ml buffer solution, 3, 1 ml / min d. 10 ml glycine, pH 2.5 buffer, 1 ml / min e. 10 ml buffer 3, 1 ml / min 5. Store the columns at 2-8°C.

[0208] Antipropofol antibody capture regimen: 1. Prepare a 96-well plate and add 20 µl of 1M Tris buffer to the wells where the antibody will be eluted.

[0209] 2. 1 ml of serum was buffered with dipotassium hydrogen phosphate and potassium dihydrogen phosphate (by diluting 1:10 in buffer 3 to produce a total volume of 10 ml).

[0210] 3. Run 10 ml of diluted serum on the column and recirculate it for 1 hour using an external peristaltic pump.

[0211] 4. The AKTA device is configured to collect the effluent fraction.

[0212] 5. After baseline equilibration, switch the pump on the AKTA device to glycine, pH 2.5 buffer, which will elute the antibody bound to the column into the fraction.

[0213] 6. Collect both types of fractions and desalt them into buffer 33 using a Zeba 2 ml rotary column before handing them over to the assay team for testing – the antibody fraction was also filtered through a 0.22 µm rotary filter.

[0214] * For one run, the same serum is rerun onto the column after antibody elution to ensure that column capacity is not a limiting factor for the amount of antibody captured.

[0215] Capture-specific anti-propofol antibody regimen: 1. Prepare a 96-well plate and add 20 µl of 1M Tris buffer to the wells where the antibody will be eluted. 2. Run 10 ml of diluted buffered serum (1:10 diluted in buffer 3) on the column and recirculate it for one hour using an external peristaltic pump.

[0216] 3. The pump of the AKTA apparatus was then switched to 200 ng / ml propofol sulfate (diluted in buffer 3) to strip any nonspecific antibodies bound to propofol on the column. The eluent was collected by fractionation.

[0217] 4. After elution of nonspecific antibodies, switch the AKTA pump to glycine, pH 2.5 buffer to elute antibodies still bound to the column.

[0218] 5. Both types of fractions were desalted into buffer 33 using a Zeba 5 ml rotary column, then filtered through a 0.22 µm rotary filter before being delivered to the assay team for testing.

[0219] Methods - Determination Reagent Description: Antipropofol antibody: Antipropofol serum (harvested blood, rabbit 1) was derived from APS’s SOM1 antibody production program (batch number: 22:05 / 1551-3).

[0220] Anti-rabbit HRP: Anti-rabbit HRP is sourced from Merck (catalog code: batch number: 3920753).

[0221] Analytical standard: Propofol is derived from TCl (catalog code: D0617, batch number: MTBK7900V). Propofol-BSA solution: The propofol-BSA conjugate was prepared by FBL and supplied by SOM / APS (lot number: SOM1 / 2), with a storage concentration of 1.7 mg / ml.

[0222] Coating buffer Assay buffer Washing buffer Preparation of working solution: Preparation of propofol-BSA: The optimal assay concentration for propofol-BSA coating was found to be 20 µg / ml.

[0223] Prepare the coating buffer at the storage concentration on the day of use.

[0224] Preparation of anti-propofol antibody: The optimal concentration for assay was found to be a 1 / 500 dilution.

[0225] Prepare in the assay buffer on the day of use.

[0226] Preparation of anti-rabbit HRP antibody: The optimal concentration for assay was found to be a 1 / 5000 dilution.

[0227] Prepare an aliquot of the assay buffer on the day of use.

[0228] Preparation of propofol standards: The standard series starts at a concentration of 4 µg / ml.

[0229] On the day of use, prepare an initial stock solution by weighing a certain amount of propofol (2-3 mg) and diluting it with methanol.

[0230] Dilute propofol to 4 µg / ml using assay buffer.

[0231] Perform 1 / 4 serial dilutions to produce seven standards ranging from 4 µg / ml to 0.98 ng / ml.

[0232] The assay buffer is used as a blank.

[0233] Preparation of blocking buffer: 0.1% milk powder w / w buffer solution was prepared in Buffer 58.

[0234] Measurement Procedure 1. Add 100 μL of propofol-BSA to each well.

[0235] 2. Incubate at 37°C for 1 hour without shaking.

[0236] 3. Wash using the measurement procedure 3. 4. Add 150 μL of blocking buffer (0.1% milk powder) to each well.

[0237] 5. Incubate at room temperature for 30 minutes.

[0238] 6. Wash using the measurement procedure 3. 7. Add 50 μL of propofol standard and sample at the required concentration (not required for target 2). 8. Add 50 μL of anti-propofol antibody to each well. (Use 100 μL for assays without standards).

[0239] 9. Incubate at 37°C for 1 hour at 600 rpm.

[0240] 10. Wash using the Measurement 3 procedure. * 11. Add 100 μL of anti-rabbit HRP to each well.

[0241] 12. Incubate at 37°C for 1 hour without shaking.

[0242] 13. Wash using the determination procedure 3. * 14. Add 100 μL of Sureblue stock solution to each well.

[0243] 15. Incubate at room temperature for 10 minutes.

[0244] 16. Add 100 μL of hydrochloric acid to each well to terminate the reaction.

[0245] 17. Read the plate at 450nm and save the results to the project folder.

[0246] *Assay Procedure 3 = Aspirate, wash 3 times with 400 μL washing buffer result Objective 1: Generation and testing of affinity columns As part of this goal, five affinity columns were developed: three propofol-BSA-coupled columns and two directly coupled columns. Following feedback from the assay team, it was found that the directly coupled columns were better at pulling more antibodies from the serum, so it was decided that in the final run, the two would be tandemly coupled and the serum would be recycled – this would ensure that the columns capture as many antibodies as possible.

[0247] Objective 2: Reactive removal of metabolites

[0248] Although UV analysis indicated a relatively high concentration of proteins eluted from the column, these were not correlated with the results obtained for Target 3. One possible explanation is that the column also captured antibodies or other proteins that do not specifically target propofol or propofol sulfate. All elution products, along with the eluent (if applicable), were submitted to the assay team for testing.

[0249] Objective 3: Confirmation of the reactivity of the final product The products from the first and second antibody purifications (DV333 / 52 and DV333 / 65) were tested with serial dilutions starting at 1 / 62.5 concentration, which is 4-fold higher than the normal assay concentration (1 / 500). Among the three products from the first purification, elution 3 showed the highest maximum signal, approximately 27% of the maximum value of the control curve generated using crude serum.

[0250] All three elutions showed high concentrations of propofol-specific antibodies, with signal strength ranging from 86% to 76% relative to the control. This indicates that the column was not entirely effective in extracting specific antibodies, but direct coupling (elution buffer 3) was the most effective of the three methods used. Figure 13 and 14 ).

[0251] For the third purification (DV333 / 80), due to the dilution of the antiserum on the column, the purified product was run at a 1 / 50 dilution, while the control was run at a 1 / 250 dilution, i.e., a 5-fold dilution. Figure 15 ).

[0252] Nevertheless, the elution buffer of BSA-conjugated columns showed minimal reaction, although antibodies directly conjugated to columns exhibited some signal (approximately 20% relative absorbance), which was still far lower than that of antibodies in the effluent.

[0253] For the fourth and fifth purifications (DV333 / 110 and DV333 / 114), the first eluent showed a relative absorbance of approximately 25%, while the second showed approximately 6%. The eluent was diluted 12.5-fold, and the dilution used for the determination remained unchanged; compared to the control at the same dilution, the relative absorbance was approximately 56%. Figure 16 ).

[0254] To confirm the specificity of the final product (DV333 / 120) for propofol, it was compared with metabolites found to be cross-reactive in SOM3. This was tested by running serial dilutions of the interfering substances (propofol-O-sulfate and propofol-O-glucuronide) at the same concentrations as the standards, with the antibody diluted 1 / 125 (normal dilution 1 / 500), assuming a signal of approximately 25% of the control.

[0255] The eluted antibody was unresponsive to propofol-glucuronide across the entire concentration range, but propofol-sulfate showed a relative substitution of approximately 43% at the 50% level. This was retested for confirmation (DV337 / 89), with no improvement observed.

[0256] in conclusion The aim of this project was to produce affinity-purified antibodies with low reactivity to propofol metabolites for comparison using the ELISA assay developed and described herein. Several methods were used to produce affinity columns whose products, tested in the assay, produced low yields relative to controls. The final column design employed direct column coupling and serum recycling to maximize column yield. When the final product was compared with propofol metabolites, it was found that it no longer cross-reacted with propofol-glucuronide but still cross-reacted with propofol-sulfate.

[0257] We examined the displacement of four propofol mAbs in ELISA. We performed an initial checkerboard analysis using CE7 to determine localization, and then ran curves for all four based on these results. These results were obtained using 1 µg / mL propofol-BSA coating (100 μL per well) and 5 µg / mL mAb (50 μL mAb + 50 μL standard). We obtained displacement in all four mAbs ( Figure 18 ).

[0258] Immunoassay Development The optimization work compared different polystreptavidins, different nitrocellulose membranes (to optimize speed), and used different concentrations of spiked serum to show that, as expected, for competitive immunoassays, the signal intensity of the test line increased as the propofol concentration decreased.

[0259] Comparison of experimental procedures for BBI and fleet polystreptavidin The experiment used two different polystreptavidins, one from BBI and the other from Fleet. Both were treated in the same manner: 0.5 mg / ml of polystreptavidin was spotted onto the nitrocellulose membrane, followed by 0.025 mg / ml of biotinylated propofol. GH5 conjugate was sprayed onto the coupling pad. The sample pad, coupling pad, nitrocellulose membrane, and absorbent pad were assembled into the apparatus as described below: Use a 60cm backing clip to allow the following components to adhere; The NC is placed on the backplate, with its bottom 20 mm from the top of the backplate. The 22mm absorbent pad is flush with the top of the backing card, allowing for a 7mm overlap with the NC. A 17mm coupling pad (gold-plated) is placed on the bottom of the card, overlapping the NC by 2mm. The 16mm FR1 pad is flush with the bottom of the backing card and overlaps the coupling pad by 4mm. The strips were cut to 5mm lengths and assembled into a single-hole Kanani device. Sample addition: 30µl sample + 30µl buffer after 30 seconds Buffer addition: Add 30 µl of buffer 5 minutes after sample addition to allow plasma to rise along the band. Reading time: 10 to 15 minutes A 1 mg / mL free propofol solution was prepared in 100% ethanol, and then propofol standards were prepared in PBST at concentrations of 1000, 100, 10, and 0 ng / μL. 80 μL of propofol standards were added to the sample port, allowing the sample to rise along the band.

[0260] result- Figure 19 Polystreptavidin from Fleet showed the best efficacy compared to BBI.

[0261] Experimental procedures for comparing different forms of NC Experiments were conducted using different types of nitrocellulose membranes (CNPC10, CNPC15, CNPF5, CNPF10, 70CNPH, 200CNPH, and CN140). All seven NCs were treated identically: 0.5 mg / ml of polystreptavidin was spotted onto the nitrocellulose membrane, followed by 0.025 mg / ml of biotinylated propofol. GH5 conjugate was sprayed onto the coupling pad. The sample pad, coupling pad, nitrocellulose membrane, and absorbent pad were all assembled into the apparatus.

[0262] result- Figure 20 A 1 mg / mL free propofol solution was prepared in 100% ethanol, and then propofol standards were prepared in PBST at concentrations of 1000, 100, 10, and 0 ng / μL. 80 μL of propofol standards were added to the sample port, allowing the sample to rise along the band.

[0263] Experimental procedures using spiked plasma samples as different forms of NC test media.

[0264] Experiments were conducted using different types of nitrocellulose membranes (CNPC10, CNPC15, CNPF5, CNPF10, 70CNPH, 200CNPH, and CN140). All seven NCs were treated identically: 0.5 mg / ml of polystreptavidin was dotted onto the membrane, followed by dotting 0.025 mg / ml of biotin-propofol onto the nitrocellulose membrane. GH5 conjugate was sprayed onto the coupling pad. The sample pad, coupling pad, nitrocellulose membrane, and absorbent pad were all assembled into the apparatus.

[0265] A 1 mg / mL free propofol solution was prepared in 100% ethanol. Propofol standards were then prepared in human antibody serum samples at concentrations of 1000, 100, 10, and 0 ng / μL. 40 µL of spiked sample was then added to the device, followed by 40 µL of PBST running buffer as a catch-up buffer for the serum sample.

[0266] result- Figure 21 When the concentration of propofol in human antibody serum samples decreased from 1000 ng / μL to 0 ng / μL, the signal intensity of the test line increased.

[0267] Lateral flow report method Polystreptavidin at a concentration of 0.5 mg / ml was spotted onto nitrocellulose membranes (NC95 and NC140). Above the polystreptavidin, biotin-propofol at concentrations of 0.01 mg / ml and 0.02 mg / ml was spotted using an Isoflow dispenser.

[0268] As a positive control, 0.7 mg / ml of goat anti-mouse IgG was spotted onto a nitrocellulose membrane (NC95).

[0269] Using Biodot, each dotted nitrocellulose membrane was cut into 54 pieces.

[0270] 1 μL of conjugated antibody (40 µg / ml of 4 types of Ab) and 39 μL of PBST were added to the well.

[0271] Place the nitrocellulose membrane strip into each well.

[0272] result- Figure 22 In this test, FH10 performed best among the four monoclonal antibodies.

[0273] method 0.5 mg / ml of polystreptavidin was spot-coated onto a nitrocellulose membrane (NC140).

[0274] The dotted nitrocellulose membrane was cut into 54 pieces using Biodot.

[0275] 1 μL of conjugated antibody (40 µg / ml of 4 types of Ab) and 39 μL of PBST were added to the well.

[0276] Place the nitrocellulose membrane strip into each well.

[0277] result- Figure 23 There are no stripes because the nitrocellulose membrane was only dotted with polystreptavidin.

[0278] method 0.5 mg / ml of polystreptavidin was dotted onto a nitrocellulose membrane (NC140), and above the polystreptavidin, 0.02 mg / ml of biotin-propofol was dotted using an Isoflow dispenser.

[0279] Using Biodot, each dotted nitrocellulose membrane was cut into 54 pieces.

[0280] 1 μL of FH10 conjugate was added to the wells at four different concentrations (10–40 µg / ml) and 39 μL of PBST.

[0281] Place the nitrocellulose membrane strip into each well.

[0282] result- Figure 24 As the concentration increases, the strength of the test strip becomes higher.

[0283] 40µg / ml is stronger than 10µg / ml.

[0284] method 0.5 mg / ml of polystreptavidin was dotted onto a nitrocellulose membrane (NC140), and above the polystreptavidin, 0.02 mg / ml of biotin-propofol was dotted using an Isoflow dispenser.

[0285] Using Biodot, each dotted nitrocellulose membrane was cut into 54 pieces.

[0286] 1 μL of 5EZ conjugate (40 µg / ml) and 39 μL of PBST were added to the well.

[0287] Place the nitrocellulose membrane strip into each well.

[0288] result- Figure 25 The 5EZ conjugate was already present in Creonate as a negative control; no band was observed in the test line.

[0289] method 0.5 mg / ml of polystreptavidin was dotted onto a nitrocellulose membrane (NC140), and above the polystreptavidin, 0.02 mg / ml of biotin-propofol was dotted using an Isoflow dispenser.

[0290] The dotted nitrocellulose membrane was cut into 54 pieces using Biodot.

[0291] Free propofol at a concentration of 1 mg / mL was prepared in 100% ethanol, and then propofol standards were prepared in PBST at different concentrations (10,000, 1,000, 100, 10, 1, 0 ng / μL).

[0292] 1 μL of conjugated antibodies (4 Abs at 40 µg / ml) and 39 μL of propofol standards (6 concentrations) were added to the wells.

[0293] Place the nitrocellulose membrane strip into each well.

[0294] result- Figure 26 When the propofol concentration was reduced from 10,000 ng / μL to 0 ng / μL, we could see stronger bands in the test lines of CE7, CE8, and GH5 antibodies, but not in FH10.

[0295] Repeat FH10 once more, but the result is the same.

[0296] The experiment was repeated with 10 µg / mL FH10 conjugate and two concentrations of propofol (10,000 ng / μL and 0 ng / μL). The results were the same as with 40 µg / μL FH10 conjugate.

[0297] Although illustrative embodiments of the invention have been disclosed in detail herein with reference to the accompanying drawings, it should be understood that the invention is not limited to the precise embodiments shown, and those skilled in the art can make various changes and modifications to it without departing from the scope of the invention as defined by the appended claims and their equivalents.

Claims

1. An antibody produced by immunizing a non-human animal with propofol-4-carboxylic acid or a functional equivalent thereof conjugated to a carrier protein.

2. An antibody produced by immunizing a non-human animal with HS357 or a functional equivalent thereof conjugated to a carrier protein.

3. An antibody that specifically binds to propofol, said antibody being generated against an antigen comprising propofol-4-carboxylic acid conjugated to a carrier protein.

4. An antibody that specifically binds to propofol, said antibody being generated against an antigen comprising HS357 conjugated to a carrier protein.

5. An antibody that binds to propofol, said antibody being generated against an antigen comprising a propofol analog conjugated to a carrier protein.

6. The antibody as claimed in any of the preceding claims, wherein the carrier is KLH.

7. The antibody according to any one of claims 1 to 5, wherein the carrier is BSA.

8. The antibody as claimed in any of the preceding claims, wherein the antibody is a monoclonal antibody.

9. The antibody according to any one of claims 1 to 7, wherein the antibody is a polyclonal antibody.

10. The antibody as claimed in any of the preceding claims, wherein a 6-carbon linker is provided between the antigen and the carrier protein.

11. The antibody as claimed in any of the preceding claims, wherein the antibody is selected to be specific for the hapten.

12. An antibody that binds to propofol, comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO:

56.

13. An antibody that binds to propofol, comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO:

64.

14. An antibody that binds to propofol, comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO:

72.

15. An antibody that binds to propofol, comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 79 and SEQ ID NO:

80.

16. A monoclonal antibody for binding propofol, said antibody comprising one or more of SEQ ID NO: 1 to SEQ ID NO:

80.

17. An immunoassay for measuring propofol in a sample, comprising an antibody as described in any of the preceding claims.

18. The immunoassay of claim 17, configured as a plate ELISA.

19. An immunobiosensor for rapid, immediate care of propofol, based on an antibody as described in any one of claims 1 to 16.

20. The immunobiosensor of claim 19, configured to measure propofol in a blood or plasma sample.

Images