Immunoassay for propofol

Abstract

An antibody for binding to propofol is provided. Also provided is an immunoassay for assaying propofol and a rapid, point-of-care immune-biosensor for propofol.

Description

[0001] IMMUNOASSAY FOR PROPOFOL

[0002] The present invention relates generally to devices, methods, antigens and antibodies for conducting immunoassays and particularly, although not exclusively, to an immuno-biosensor for propofol.

[0003] Immuno-biosensors are a type of biosensor to detect the formation of an immunocomplex using an antibody or antigen as a bioreceptor.

[0004] Propofol (2,6-diisopropylphenol, formula I) is a hypnotic alkylphenol derivative and is a parenteral anaesthetic agent commonly used to produce sedation and general anaesthesia.

[0005] In general anaesthetic practise, propofol can be used for both the induction and maintenance phases, in a process known as total intravenous anaesthesia (TIVA). There is a growing body of evidence of the advantages of TIVA over the more conventional volatile-based anaesthesia, including: reduced short-term side-effects (e.g. nausea and vomiting), reduced cognitive effects, the potential for improved long-term survival rates for cancer patients, and a significantly reduced environmental impact. i One significant obstacle to a greater exploitation of TIVA is the lack of suitable methods for the continuous, real-time monitoring of blood propofol concentration in patients undergoing anaesthesia.

[0006] Similarly, the optimisation of sedation using propofol is limited by the lack of suitable methods for the real-time monitoring of blood propofol concentration in at the point of care of patients receiving sedation.

[0007] The aim of delivering real-time monitoring of blood propofol concentration during general anaesthesia or sedation places several requirements on any potential propofol sensing technique. For instance, any method must be capable of returning results within a sufficiently narrow window of time to provide information that is of practical use to anaesthetists or other healthcare professionals.

[0008] To be of use for patient monitoring in a surgical context, any sensor system needs to be capable of producing stable results over the duration of a surgical procedure, potentially 8 hours or longer. Furthermore, it has been shown that propofol will slowly redistribute between the plasma and blood cell membranes over time, meaning that the time between the collection and measurement of a sample needs to be tightly controlled. As such, any technique for real-time propofol monitoring should be suitable for automation, with minimal sample processing.

[0009] It is likely that propofol will be co-administered with other drugs (e.g. lidocaine, opioid analgesics such as fentanyl or morphone, competitive neuromuscular blocking agents such as atracurium or rocuronium, antibiotics, and anti-inflammatory agents), and as such it is necessary that any propofol assay possess a sufficient degree of specificity. Immunosensors possess high selectivity and sensitivity due to the specific binding between an antibody and corresponding antigen, making them a suitable platform for applications in the medical field.

[0010] To generate antibodies via traditional methods, the antigen is injected into an animal and the resulting antibodies, generated as part of the immune response, are extracted. Propofol is a small molecule: a small molecule is a low molecular weight (< 1000 Da) organic compound.

[0011] Due to their size, small molecules are non-immunogenic, meaning no immune response is raised and so no antibodies generated.

[0012] This problem can be overcome by conjugating the small molecule to a carrier protein, in which case it is termed a hapten (half antigen). Carrier proteins, such as keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA), are conjugated to multiple copies of the small molecule. The presence of the larger protein molecule raises an immune response in the injected animal, with a portion of the generated antibodies specific to the hapten.

[0013] Some of these antibodies are able to bind both the protein-conjugated and unconjugated small molecule, though the majority only bind to the conjugated form and so have to be screened. Once screened some of these antibodies exhibit good affinity for their small molecule target, with reported KD values in the nanomolar range; though this is often only achievable following extensive, stringent in vitro selection from immune libraries and affinity maturation.

[0014] Despite these successes, the failure rate for anti-hapten antibodies in discovery remains high. It is estimated that 50-75% of all anti-hapten antibody development programmes fail to deliver reagents with sufficient affinity or specificity. Unfortunately, the use of carrier molecules often leads to the developed antibodies being specific to the hapten-carrier molecule linker region rather than to the desired hapten. Conjugation of the small molecule to a carrier protein also limits the available epitopes for antibody binding, which result in variation in the performance of the antibody in binding the conjugated target compared to when free in solution, with much lower affinity for the free molecule in solution.

[0015] An aspect of the present invention provides an antibody produced by immunisation of a nonhuman animal with propofol-4-carboxylic acid conjugated to a carrier protein. An aspect of the present invention provides an antibody produced by immunisation of a nonhuman animal with an antigen consisting of propofol-4-carboxylic acid conjugated to a carrier protein.

[0016] A further aspect of the present invention provides an antibody produced by immunisation of a non-human animal with a propofol-4-carboxylic acid conjugated to a carrier protein.

[0017] A further aspect of the present invention provides an antibody produced by immunisation of a non-human animal with a propofol-4-carboxylic acid or an analogue or functional equivalent thereof conjugated to a carrier protein.

[0018] A further aspect of the present invention provides an antibody produced by immunisation of a non-human animal with a propofol-4-carboxylic acid, including salts and esters thereof, conjugated to a carrier protein.

[0019] A further aspect provides an antibody produced by immunisation of a non-human animal with HS357 conjugated to a carrier protein.

[0020] A further aspect provides an antibody which binds specifically to propofol, said antibody produced against an antigen comprising propofol-4-carboxylic acid conjugated to a carrier protein.

[0021] A further aspect provides an antibody which binds specifically to propofol, said antibody produced against an antigen comprising HS357 conjugated to a carrier protein.

[0022] In some aspects and embodiment the carrier is KLH.

[0023] In some aspects and embodiments the carrier is BSA.

[0024] Antibodies formed in accordance with the present invention may be monoclonal or polyclonal. In some aspects and embodiments the antibody is selected to be specific to the hapten.

[0025] The present invention also provides an antibody binding to propofol, which comprises 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, SEQ ID NO: 56.

[0026] The present invention also provides an antibody binding to propofol, which comprises 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, SEQ ID NO: 64.

[0027] The present invention also provides an antibody binding to propofol, which comprises 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 , SEQ ID NO: 72.

[0028] The present invention also provides an antibody binding to propofol, which comprises 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, SEQ ID NO: 80.

[0029] The present invention also provides a monoclonal antibody for binding to propofol, the antibody comprising one or more of SEQ ID NO: 1 to SEQ ID NO: 80.

[0030] A further aspect provides an immunoassay for measuring propofol in a sample, comprising an antibody as defined herein.

[0031] The immunoassay may be provided in a plate-based ELISA format.

[0032] A further aspect provides a rapid, point-of-care immune-biosensor for propofol in blood plasma based on an antibody as defined herein. uence Listings SEQ ID NO: 1 CE8_HSnaa

[0033] Clone CE8 Heavy Chain Signal Peptide Protein Sequence

[0034] MGWSWIFLFLLSGTAGVHS

[0035] SEQ ID NO: 2 CE8_HCSnuc

[0036] Clone CE8 Heavy Chain Signal Peptide DNA Sequence

[0037] ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCT

[0038] SEQ ID NO: 3 CE8_HCVaa

[0039] Clone CE8 Heavy Chain Variable Protein Sequence

[0040] EVQLQQSGPELVKPGASMKISCKASGYSFTDYTMNWVNWVKQSHGKNLEWIGHINPYNGGS

[0041] SYNQKFRGKATLTVDKSSTTAYMELLSLTSEDSAVYYCAVIYYDYDGDIFAYWGQGTLVTVSA

[0042] SEQ ID NO: 4 CE8_HCVnuc

[0043] Clone CE8 Heavy Chain Variable DNA Sequence

[0044] GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATA

[0045] TCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACACCATGAACTGGGTGAACTGGGTGA

[0046] AGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACATATTAATCCTTACAATGGTGGTTC

[0047] TAGCTACAACCAGAAGTTCAGGGGCAAGGCCACATTAACTGTAGACAAGTCATCCACCACA

[0048] GCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCTGTAA

[0049] TCTACTATGATTACGACGGGGATATTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGT CTCTGCA

[0050] SEQ ID NO: 5 CE8_HCCaa

[0051] Clone CE8 Heavy Chain Constant Protein Sequence

[0052] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT

[0053] LSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVL

[0054] TITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWL

[0055] NGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITV

[0056] EWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKS

[0057] LSHSPGK SEQ ID NO: 6 CE8_HCCnuc

[0058] Clone CE8 Heavy Chain Constant DNA Sequence

[0059] GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACT

[0060] CCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCT

[0061] GGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACC

[0062] TCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCA

[0063] CCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGG

[0064] GATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCC

[0065] CCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTA

[0066] GACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTG

[0067] CACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGT

[0068] GAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAAC

[0069] AGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAG

[0070] GCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGT

[0071] CTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATG

[0072] GGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACT

[0073] TCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCT

[0074] GCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCC

[0075] TGGTAAA

[0076] SEQ ID NO: 7 CE8_LCSaa

[0077] Clone CE8 Light Chain Signal Peptide Protein Sequence

[0078] MSVPTQVLGLLLLWLTGARC

[0079] SEQ ID NO: 8 CE8_LCSnuc

[0080] Clone CE8 Light Chain Signal Peptide DNA Sequence

[0081] ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGT

[0082] SEQ ID NO: 9 CE8_LCVaa

[0083] Clone CE8 Light Chain Variable Protein Sequence DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNSKTLAEGVPSRFSG

[0084] SGSGTQFSLKINSLQPEDFGSYYCQHHYGTPFTFGSGTKLEIK

[0085] SEQ ID NO: 10 CE8_LCVnuc

[0086] Clone CE8 Light Chain Variable DNA Sequence

[0087] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCA

[0088] TCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGG

[0089] AAAATCTCCTCAACTCCTGGTCTATAATTCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGT

[0090] TCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAG

[0091] ATTTTGGGAGTTATTACTGTCAACATCATTATGGTACTCCATTCACGTTCGGCTCGGGGACA

[0092] AAGTTGGAAATAAAA

[0093] SEQ ID NO: 11 CE8_LCCaa

[0094] Clone CE8 Light Chain Constant Protein Sequence

[0095] RADAAPTVSIFPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKD

[0096] STYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0097] SEQ ID NO: 12 CE8_LCCnuc

[0098] Clone CE8 Light Chain Constant DNA Sequence

[0099] CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTG

[0100] GAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG

[0101] GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAG

[0102] CAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACG

[0103] ACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGC

[0104] TTCAACAGGAATGAGTGT

[0105] SEQ ID NO: 13 CE7_HCSaa

[0106] Clone CE7 Heavy Chain Signal Peptide Protein Sequence

[0107] MRVLILLCLFTAFPGILS SEQ ID NO: 14 CE7_HCSnuc

[0108] Clone CE7 Heavy Chain Signal Peptide DNA Sequence

[0109] ATGAGAGTGCTGATTCTTTTGTGCCTGTTCACAGCCTTTCCTGGTATCCTGTCT

[0110] SEQ ID NO: 15 CE7_HCVaa

[0111] Clone CE7 Heavy Chain Variable Protein Sequence

[0112] DVQLQESGPDLVKPSQSLSLTCTVTGYSLTSGFTWHWIRQFPGNKLEWMGYLHYSGDTNYNP

[0113] SLRSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGGITSALWGQGTLVTVSA

[0114] SEQ ID NO: 16 CE7_HCVnuc

[0115] Clone CE7 Heavy Chain Variable DNA Sequence

[0116] GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCCCTCA

[0117] CCTGCACTGTCACTGGCTACTCCCTCACCAGTGGTTTTACCTGGCACTGGATCCGGCAGTT

[0118] TCCAGGGAACAAACTGGAGTGGATGGGCTACCTACACTACAGTGGTGACACTAACTACAA

[0119] CCCATCTCTCAGAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTG

[0120] CAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGAGGCGGGATTA

[0121] CGTCGGCTCTCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0122] SEQ ID NO: 17 CE7_HCCaa

[0123] Clone CE7 Heavy Chain Constant Protein Sequence

[0124] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT

[0125] LSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVL

[0126] TITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWL

[0127] NGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITV

[0128] EWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKS LSHSPGK

[0129] SEQ ID NO: 18 CE7_HCCnuc

[0130] Clone CE7 Heavy Chain Constant DNA Sequence

[0131] GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACT

[0132] CCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCT GGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACC

[0133] TCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCA

[0134] CCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGG

[0135] GATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCC

[0136] CCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTA

[0137] GACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTG

[0138] CACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGT

[0139] GAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAAC

[0140] AGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAG

[0141] GCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGT

[0142] CTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATG

[0143] GGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACT

[0144] TCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCT

[0145] GCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCC

[0146] TGGTAAA

[0147] SEQ ID NO: 19 CE7_LCSaa

[0148] Clone CE7 Light Chain Signal Peptide Protein Sequence

[0149] MSVPTQVLGLLLLWLTDARC

[0150] SEQ ID NO: 20 CE7_LCSnuc

[0151] Clone CE7 Light Chain Signal Peptide DNA Sequence

[0152] ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGATGCCAGATGT

[0153] SEQ ID NO: 21 CE7_LCVaa

[0154] Clone CE7 Light Chain Variable Protein Sequence

[0155] DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYGATNLADGVPSRFS

[0156] GSGSGTQFSLKINRLQSEDFGIYYCHHLWGIPYTFGGGTKLEIK

[0157] SEQ ID NO: 22 CE7_LCVnuc io Clone CE7 Light Chain Variable DNA Sequence

[0158] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGTATCTGTGGGAGAAACTGTCACCA

[0159] TCACATGTCGAGCAAGTGAGAATATTTACAGTAATTTAGCATGGTATCAGCAGAAGCAGGG

[0160] AAAATCTCCTCAGCTCCTGGTCTATGGTGCTACAAACTTAGCAGATGGTGTGCCATCAAGG

[0161] TTCAGTGGCAGTGGATCAGGCACACAGTTTTCCCTCAAGATCAACAGGTTGCAGTCTGAAG

[0162] ATTTTGGGATTTACTACTGTCACCATTTATGGGGTATACCGTACACGTTCGGAGGGGGGAC

[0163] CAAGCTGGAAATAAAA

[0164] SEQ ID NO: 23 CE7_LCCaa

[0165] Clone CE7 Light Chain Constant Protein Sequence

[0166] RADAAPTVSIFPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKD

[0167] STYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0168] SEQ ID NO: 24 CE7_LCCnuc

[0169] Clone CE7 Light Chain Constant Protein Sequence

[0170] CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTG

[0171] GAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG

[0172] GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAG

[0173] CAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACG

[0174] ACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGC

[0175] TTCAACAGGAATGAGTGT

[0176] SEQ ID NO: 25 FH10_HCSaa

[0177] Clone FH10 Heavy Chain Signal Peptide Protein Sequence

[0178] MRVLILLWLFTAFPGILS

[0179] SEQ ID NO: 26 FH10_HCSnuc

[0180] Clone FH10 Heavy Chain Signal Peptide DNA Sequence

[0181] ATGAGAGTGCTGATTCTTTTGTGGCTGTTCACAGCCTTTCCTGGTATCCTGTCT

[0182] SEQ ID NO: 27 FH10_HCVaa Clone FH10 Heavy Chain Variable Protein Sequence

[0183] DVQVQESGPGLVKPSQSLSLTCTVTGYSITSNYAWNWIRQFPGDKLEWMGFITYSGSSTYNPS

[0184] LKSRISITRDTSKNQFFLQLNSVTSEDTATYYCASVYYDYDAWFAYWGQGTLVTVSA

[0185] SEQ ID NO: 28 FH10_HCVnuc

[0186] Clone FH10 Heavy Chain Variable DNA Sequence

[0187] GATGTGCAGGTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTC

[0188] ACCTGCACTGTCACTGGCTACTCAATCACCAGTAACTATGCCTGGAACTGGATCCGGCAGT

[0189] TTCCAGGAGACAAACTGGAGTGGATGGGCTTCATAACCTACAGTGGTAGTTCTACCTACAA

[0190] CCCCTCTCTCAAGAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTTC

[0191] AATTGAATTCTGTGACTTCTGAGGACACAGCCACTTATTACTGTGCAAGTGTCTATTATGAT

[0192] TACGACGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0193] SEQ ID NO: 29 FH10_HCCaa

[0194] Clone FH10 Heavy Chain Constant Protein Sequence

[0195] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT

[0196] LSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVL

[0197] TITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWL

[0198] NGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITV

[0199] EWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKS LSHSPGK

[0200] SEQ ID NO: 30 FH10_HCCnuc

[0201] Clone FH10 Heavy Chain Constant DNA Sequence

[0202] GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACT

[0203] CCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCT

[0204] GGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACC

[0205] TCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCA

[0206] CCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGG

[0207] GATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCC

[0208] CCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTA

[0209] I2 GACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTG

[0210] CACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGT

[0211] GAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAAC

[0212] AGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAG

[0213] GCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGT

[0214] CTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATG

[0215] GGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACT

[0216] TCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCT

[0217] GCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCC TGGTAAA

[0218] SEQ ID NO: 31 FH10_LCSaa

[0219] Clone FH10 Light Chain Signal Peptide Protein Sequence

[0220] MSVPTQVLGLLLLWLTGARC

[0221] SEQ ID NO: 32 FH10_LCSnuc

[0222] Clone FH10 Light Chain Signal Peptide DNA Sequence

[0223] ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTACTGTGGCTTACAGGTGCCAGATGT

[0224] SEQ ID NO: 33 FH10_LCVaa

[0225] Clone FH10 Light Chain Variable Protein Sequence

[0226] DIQMTQSPASLSASVGETVTITCRASENIYTYLAWYQLKQGKSPQLLVYNAKTLAGGVPSRFSA

[0227] SGSGTQFSLKINSLQPEDFGSFYCHHHYHTPFTFGSGTRLEIN

[0228] SEQ ID NO: 34 FH10_LCVnuc

[0229] Clone FH10 Light Chain Variable DNA Sequence

[0230] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCA

[0231] TCACATGTCGAGCAAGTGAGAATATTTACACTTATTTAGCATGGTATCAGCTGAAACAGGGA

[0232] AAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCGGGAGGTGTGCCATCAAGGT

[0233] I 3 TCAGTGCCAGTGGATCAGGCACACAATTTTCTCTGAAGATCAACAGCCTGCAGCCTGAGGA

[0234] TTTTGGGAGTTTCTACTGTCACCATCATTATCATACTCCTTTCACGTTCGGCTCGGGGACAA

[0235] GGTTGGAGATAAAC

[0236] SEQ ID NO: 35 FH10_LCCaa

[0237] Clone FH10 Light Chain Constant Protein Sequence

[0238] RADAAPTVSIFPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKD

[0239] STYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0240] SEQ ID NO: 36 FH10_LCCnuc

[0241] Clone FH10 Light Chain Constant DNA Sequence

[0242] CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTG

[0243] GAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG

[0244] GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAG

[0245] CAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACG

[0246] ACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGC

[0247] TTCAACAGGAATGAGTGT

[0248] SEQ ID NO: 37 GH5_HCSaa

[0249] Clone GH5 Heavy Chain Signal Peptide Protein Sequence

[0250] MGWSWIFLFLLSGTAGVHS

[0251] SEQ ID NO: 38 GH5_HCSnuc

[0252] Clone GH5 Heavy Chain Signal Peptide DNA Sequence

[0253] ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCT

[0254] SEQ ID NO: 39 GH5_HCVaa

[0255] Clone GH5 Heavy Chain Variable Protein Sequence

[0256] EVQLQQSGPELVKPGASMKISCKASDYSFTDYTMTWINWVKQSHEKNLEWIGHINPYNGGTSY

[0257] NQKFRGKATLTVDKSSSAAYMELLSLTSEDSAVYYCAVVYYDYGGDIFAYWGQGTLVTVSA SEQ ID NO: 40 GH5_HCVnuc

[0258] Clone GH5 Heavy Chain Variable DNA Sequence

[0259] GAGGTCCAACTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATA

[0260] TCCTGCAAGGCTTCTGATTACTCATTCACTGACTACACCATGACCTGGATCAACTGGGTGA

[0261] AGCAGAGCCATGAAAAGAACCTTGAGTGGATTGGACATATTAATCCTTACAATGGTGGTAC

[0262] TAGTTACAACCAGAAGTTCAGGGGCAAGGCCACATTAACTGTAGACAAGTCATCCAGCGCA

[0263] GCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCTGTAG

[0264] TCTACTATGATTACGGCGGGGATATTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGT CTCTGCA

[0265] SEQ ID NO: 41 GH5_HCCaa

[0266] Clone GH5 Heavy Chain Constant Protein Sequence

[0267] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT

[0268] LSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVL

[0269] TITLTPKVTCVWDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWL

[0270] NGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITV

[0271] EWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKS LSHSPGK

[0272] SEQ ID NO: 42 GH5_HCCnuc

[0273] Clone GH5 Heavy Chain Constant DNA Sequence

[0274] GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACT

[0275] CCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCT

[0276] GGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACC

[0277] TCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCA

[0278] CCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGG

[0279] GATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCC

[0280] CCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTA

[0281] GACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTG

[0282] CACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGT

[0283] GAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAAC

[0284] I 5 AGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAG

[0285] GCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGT

[0286] CTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATG

[0287] GGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACT

[0288] TCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCT

[0289] GCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCC

[0290] TGGTAAA

[0291] SEQ ID NO: 43 GH5_LCSaa

[0292] Clone GH5 Light Chain Signal Peptide Protein Sequence

[0293] MSVPTQVLGLLLLWLTGARC

[0294] SEQ ID NO: 44 GH5_LCSnuc

[0295] Clone GH5 Light Chain Signal Peptide DNA Sequence

[0296] ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGT

[0297] SEQ ID NO: 45 GH5_LCVaa

[0298] Clone GH5 Light Chain Variable Protein Sequence

[0299] DIQMTQSPASLSASVGETVTITCRASENIYNYLAWYQQRQGKSPQLLVYNSKTLAEGVPSRFS

[0300] GSGSGTQFSLKINSLQPEDFGNYFCQHHSGTPFTFGSGTKLDLK

[0301] SEQ ID NO: 46 GH5_LCVnuc

[0302] Clone GH5 Light Chain Variable DNA Sequence

[0303] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACCGTCACCA

[0304] TCACATGTCGAGCAAGTGAAAATATTTACAATTATTTAGCATGGTATCAACAGAGACAGGGA

[0305] AAATCTCCTCAACTCCTGGTCTATAATTCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTT

[0306] CAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGA

[0307] TTTTGGGAATTATTTCTGTCAACATCATTCTGGTACTCCATTCACGTTCGGCTCGGGGACAA

[0308] AGTTGGATTTAAAA SEQ ID NO: 47 GH5_LCCaa

[0309] Clone GH5 Light Chain Constant Protein Sequence

[0310] RADAAPTVSIFPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKD

[0311] STYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0312] SEQ ID NO: 48 GH5_LCCnuc

[0313] Clone GH5 Light Chain Constant DNA Sequence

[0314] CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTG

[0315] GAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG

[0316] GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAG

[0317] CAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACG

[0318] ACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGC

[0319] TTCAACAGGAATGAGTGT

[0320] SEQ ID NO: 49 CE8_VHaa

[0321] Clone CE8 Primary VH Sequence

[0322] EVQLQQSGPELVKPGASMKISCKASGYSFTDYTMNWVNWVKQSHGKNLEWIGHINPYNGGS

[0323] SYNQKFRGKATLTVDKSSTTAYMELLSLTSEDSAVYYCAVIYYDYDGDIFAYWGQGTLVTVSA

[0324] SEQ ID NO: 50 CDR-H1aa

[0325] Clone CE8 CDR-H1 Sequence

[0326] DYTMN

[0327] SEQ ID NO: 51 CDR-H2aa

[0328] Clone CE8 CDR-H2 Sequence

[0329] WIGHINPYNGGSSYNQKFRG

[0330] SEQ ID NO: 52 CDR-H3aa

[0331] Clone CE8 CDR-H3 Sequence

[0332] IYYDYDGDIFAY

[0333] I 7 SEQ ID NO: 53 CE8_VLaa

[0334] Clone CE8 Primary VL Sequence

[0335] DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNSKTLAEGVPSRFSG

[0336] SGSGTQFSLKINSLQPEDFGSYYCQHHYGTPFTFGSGTKLEIK

[0337] SEQ ID NO: 54 CDR-L1aa

[0338] Clone CE8 CDR-L1 Sequence

[0339] RASENIYSYLA

[0340] SEQ ID NO: 55 CDR-L2aa

[0341] Clone CE8 CDR-L2 Sequence

[0342] NSKTLAE

[0343] SEQ ID NO: 56 CDR-L3aa

[0344] Clone CE8 CDR-L2 Sequence

[0345] QHHYGTPFT

[0346] SEQ ID NO: 57 CE7_VHaa

[0347] Clone CE7 Primary VH Sequence

[0348] DVQLQESGPDLVKPSQSLSLTCTVTGYSLTSGFTWHWIRQFPGNKLEWMGYLHYSGDTNYNP

[0349] SLRSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGGITSALWGQGTLVTVSA

[0350] SEQ ID NO: 58 CDR-H1aa

[0351] Clone CE7 CDR-H1

[0352] SGFTWH

[0353] SEQ ID NO: 59 CDR-H2aa

[0354] Clone CE7 CDR-H2

[0355] YLHYSGDTNYNPSLRS

[0356] I 8 SEQ ID NO: 60 CDR-H3aa

[0357] Clone CE7 CDR-H3

[0358] GGITSAL

[0359] SEQ ID NO: 61 CE7_VLaa

[0360] Clone CE7 Primary VL Sequence

[0361] DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYGATNLADGVPSRFS

[0362] GSGSGTQFSLKINRLQSEDFGIYYCHHLWGIPYTFGGGTKLEIK

[0363] SEQ ID NO: 62 CDR-L1aa

[0364] Clone CE7 CDR-L1

[0365] RASENIYSNLA

[0366] SEQ ID NO: 63 CDR-L2aa

[0367] Clone CE7 CDR-L2

[0368] GATNLAD

[0369] SEQ ID NO: 64 CDR-L3aa

[0370] Clone CE7 CDR-L3

[0371] HHLWGIPYT

[0372] SEQ ID NO: 65 FH10_VHaa

[0373] Clone FH10 Primary VH Sequence

[0374] DVQVQESGPGLVKPSQSLSLTCTVTGYSITSNYAWNWIRQFPGDKLEWMGFITYSGSSTYNPS

[0375] LKSRISITRDTSKNQFFLQLNSVTSEDTATYYCASVYYDYDAWFAYWGQGTLVTVSA

[0376] SEQ ID NO: 66 CDR-H1aa

[0377] Clone FH10 CDR-H1

[0378] SNYAWN SEQ ID NO: 67 CDR-H2aa

[0379] Clone FH10 CDR-H2

[0380] FITYSGSSTYNPSLKS

[0381] SEQ ID NO: 68 CDR-H3aa

[0382] Clone FH10 CDR-H3

[0383] VYYDYDAWFAY

[0384] SEQ ID NO: 69 FH10_VLaa

[0385] Clone FH10 Primary VL Sequence

[0386] DIQMTQSPASLSASVGETVTITCRASENIYTYLAWYQLKQGKSPQLLVYNAKTLAGGVPSRFSA

[0387] SGSGTQFSLKINSLQPEDFGSFYCHHHYHTPFTFGSGTRLEIN

[0388] SEQ ID NO: 70 CDR-L1aa

[0389] Clone FH10 CDR-L1

[0390] RASENIYTYLA

[0391] SEQ ID NO: 71 CDR-L2aa

[0392] Clone FH10 CDR-L2

[0393] NAKTLAG

[0394] SEQ ID NO: 72 CDR-L3aa

[0395] Clone FH10 CDR-L3

[0396] HHHYHTPFT

[0397] SEQ ID NO: 73 GH5_VHaa

[0398] Clone GH5 Primary VH Sequence

[0399] EVQLQQSGPELVKPGASMKISCKASDYSFTDYTMTWINWVKQSHEKNLEWIGHINPYNGGTSY

[0400] NQKFRGKATLTVDKSSSAAYMELLSLTSEDSAVYYCAVVYYDYGGDIFAYWGQGTLVTVSA

[0401] SEQ ID NO: 74 CDR-H1aa Clone GH5 CDR-H1aa

[0402] DYTMT

[0403] SEQ ID NO: 75 CDR-H2aa

[0404] Clone GH5 CDR-H2

[0405] WIGHINPYNGGTSYNQKFRG

[0406] SEQ ID NO: 76 CDR-H3aa

[0407] Clone GH5 CDR-H3

[0408] VYYDYGGDIFAY

[0409] SEQ ID NO: 77 GH5_VLaa

[0410] Clone GH5 Primary VL Sequence

[0411] DIQMTQSPASLSASVGETVTITCRASENIYNYLAWYQQRQGKSPQLLVYNSKTLAEGVPSRFS

[0412] GSGSGTQFSLKINSLQPEDFGNYFCQHHSGTPFTFGSGTKLDLK

[0413] SEQ ID NO: 78 CDR-L1aa

[0414] Clone GH5 CDR-L1

[0415] RASENIYNYLA

[0416] SEQ ID NO: 79 CDR-L2aa

[0417] Clone GH5 CDR-L2

[0418] NSKTLAE

[0419] SEQ ID NO: 80 CDR-L3aa

[0420] Clone GH5 CDR-L3

[0421] QHHSGTPFT

[0422] Different aspects and embodiments of the invention may be used separately or together.

[0423] 2I Further particular and preferred aspects of the present invention are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with the features of the independent claims as appropriate, and in combination other than those explicitly set out in the claims. Each aspect can be carried out independently of the other aspects or in combination with one or more of the other aspects.

[0424] There is no intent to limit to the particular forms disclosed. On the contrary, all modifications, equivalents, and alternatives falling within the scope of the appended claims should be included.

[0425] Unless otherwise defined, all terms (including technical and scientific terms) used herein are to be interpreted as is customary in the art. It will be further understood that terms in common usage should also be interpreted as is customary in the relevant art and not in an idealised or overly formal sense unless expressly so defined herein.

[0426] PHASE 1 - DEVELOPMENT OF A SPECIFIC MONOCLONAL ANTIBODY FOR PROPOFOL

[0427] The size of Propofol puts severe limitations on the success of raising a good quality antibody.

[0428] Propofol will be conjugated to both KLH and BSA carrier proteins for immunisation and screening.

[0429] Objective

[0430] To couple propofol to carrier proteins (KLH and BSA) for use as immunogen and screening material for any subsequently raised antibodies.

[0431] Immunise 4 mice with KLH-conjugated Propofol and then select antibodies that are specific for Propofol using the BSA conjugated material during the screening phase. Once candidates are screened, expand selected clonal cell lines, confirm stable antibody expression, and upon successful confirmation of clones undertake antibody production to a scale of 10mg.

[0432] Report - preparation of Propofol conjugates & mouse immunisation

[0433] Materials

[0434] Propofol - purchased from Sigma Aldrich P1643-1 ML lot LRAC4221 Pharmaceutical secondary standard reference material.

[0435] KLH Imject KLH #77653 Thermo Fisher lot 1902219 cBSA Imject cBSA - cBSA #77150 Thermo Fisher lot VH312389

[0436] Formaldehyde solution Sigma Aldrich F8776 lot MKPC0535

[0437] MES Sigma Aldrich M8250 lot SLBQ4750V

[0438] Absolute Ethanol Fisher E / 0650DF / 17 lot 2189134

[0439] NaCI Fisher S / 3160 / 63 lot 2033832

[0440] Sodium Hydroxide Acros 308215000 lot A0384089

[0441] HCI SevernBio 20-555-50 lot 21331

[0442] DPBS GIBCO 14190136 lot 2234628

[0443] PD10 columns GE17-0851-01 lot 17179054

[0444] Methods

[0445] Mannich Condensation reaction from Hermanson Bioconjugate techniques (second edition) and initially the method listed on pages 776 to 779 was attempted as given. The Third Edition does not list this method in detail, possibly because it is not reliable. It was noted that the recommended buffer 0.1 M MES / 0.15M NaCI does not have significant buffering capacity at the required pH 4.50.

[0446] Initial methodology BSA and KLH dissolved to 10mgml in MES buffer.

[0447] Propofol dissolved to 10mgml in ethanol

[0448] Reaction mixtures added as follows to glass vials the cBSA and KLH were supplied in: cBSA - 1000ul at 10mgml

[0449] +250ul ETOH

[0450] +1 OOOul Propofol in ETOH at 10mgml

[0451] +250ul 37% formaldehyde

[0452] KLH - 2 X 200ul at 10mgml (separate vials)

[0453] +50ul ETOH per vial

[0454] +200ul propofol in ETOH at 10mgml

[0455] +50ul formaldehyde

[0456] Components gently but thoroughly mixed together with swirling, placed at 37°C overnight. Mixtures were noted to be a little milky at start of reaction, and by following morning had aggregated and precipitated.

[0457] A separation of conjugated protein from the reaction mixture was attempted using PD10 columns, but no protein could be recovered. Materials discarded and fresh cBSA and KLH purchased.

[0458] Investigation of precipitation

[0459] 1 ) Buffer concentration - Further conjugates were prepared using conventional Fraction V BSA, varying the MES / NaCI buffer concentration, amount of ethanol added and the relative concentration of formaldehyde. All variations showed precipitation, but the standard method as already tried was the least bad.

[0460] 2) Buffer type - Buffers prepared using Acetate, Citrate and Citrate-phosphate formulations to 0.1 M around pH4.50. A variety of concentrations of BSA, formaldehyde and ETOH were evaluated. All were found to have precipitated after incubation at 37°C, but less so that MES buffer system. 3) Buffer type - variations evaluated again using the 3 previous buffers with variations on the volumes and concentrations required, using plastic reaction vessels in place of glass. Some variations found to precipitate more slowly than others.

[0461] 4)Variations of buffer type and volumes / concentrations / temperature using cBSA and KLH based on previous work. Citrate-phosphate buffer showed the least precipitation under various conditions.

[0462] 5)Final run:

[0463] 0.1 M Citrate-Phosphate buffer:

[0464] 39.45ml 0.1 M citric acid (21g / l) + 44.1 ml 0.2M Na2HPO4 (32g / l) + 0.165M NaCI. pH4.48 cBSA and KLH dissolved in CP buffer for 30min on roller. Reaction performed in polypropylene tubes.

[0465] KLH tubes 200ul / tube at 10mgml + 50ul formaldehyde + 25ul of ethanol containing propofol at 80mgml. Mixed. cBSA tubes 200ul / tube at 10mgml + 200ul CP buffer + 50ul formaldehyde + 75ul ethanol containing propofol at 80mgml.

[0466] Reaction performed at 37°C overnight. All materials found to be substantially precipitated and would not pass through PD10 columns.

[0467] Protein-propofol material collected by centrifugation at 17,000g for 10min. Pellets resuspended in DPBS and stored frozen.

[0468] Pellets thawed, resuspended and aggregates broken up using a sonication bath. Given 2 additional washed with DPBS to remove any remaining formaldehyde and un-coupled propofol. Finally aggregates re-suspended using water bath and caps left off the tubes to allow any remaining formaldehyde to escape by evaporation.

[0469] Final estimated recovery of proteins: KLH 2.8mg at 1.68mgml combining 2 coupling reactions. cBSA 3.2mg at 1.86mgml combining 2 coupling reactions.

[0470] Materials sent for use as immunogen. It was not possible to generate soluble conjugates using the methodology. However, aggregated proteins will also work for this purpose.

[0471] Screening of test bleeds after immunisation

[0472] Plate plan

[0473] +ve minus -ve OD values Findings

[0474] A weak clear positive response was seen in mouse 4, suggesting the propofol was successfully coupled, but it was not possible to raise a strong antibody response. This is a small, measurable response but this is probably 100 times lower than we would expect it to be in order to take the project further forward.

[0475] There were no issues with the preparation of the antigen or immunisation process and all mice were immunised correctly showing no significant signs of illness.

[0476] Other mice from a similar cohort (M-NAC-25 mice) showed a strong response as expected, therefore we do not believe there were any issues with the animals themselves or the immunisation process.

[0477] Regarding the assay, these same reagents were used for analysis of other test sera and again these demonstrated a strong response indicating there were no issues with the assay reagents used.

[0478] It is concluded that the small propofol molecule is either not very immunogenic; or that the antibodies raised can only weakly bind the small target.

[0479] Discussion

[0480] The method used for conjugate synthesis:

[0481]

[0482] This route did not produce a sensitive or specific antibody, possibly because of steric hinderance.

[0483] No linker was used. Consider a 6-carbon linker between propofol and the protein conjugate to give a better antigenic response. Consider using a propofol analogue e.g.:

[0484] 2-(3-ethyl-4-hydroxy-5-isopropyl-phenyl)-3,3,3-trifluoro-2-hydroxypropionamide; HS357.5

[0485]

[0486] Compound structures and design. Propofol, HS245, and HS357. HS245 includes a hydroxyamide moiety, whereas HS357 incorporates a six-carbon linker between the phenyl and hydroxyamide moieties.

[0487] The use of propofol-BSA for screening may be problematic since there are two pocket binding sites for propofol in serum albumin. Will this be beneficial and produce two sets of antibodies specific for free propofol and bound propofol? Propofol-BSA cannot really be used in assay development for the same reason; it will non- specifically bind propofol in the sample and give a bias in measurement of low concentrations of propofol.

[0488] Proposal: propofol analogue e.g., 2-(3-ethyl-4-hydroxy-5-isopropyl-phenyl)-3,3,3-trifluoro-2- hydroxypropionamide.

[0489] PHASE 2 - CONJUGATION OF A PROPOFOL DERIVATIVE AND GENERATION OF A MONOCLONAL MOUSE ANTI-PROPOFOL ANTIBODY

[0490] A program of work was initiated to produce a monoclonal antibody against a biomarker of interest; propofol (CAS:2078-54-8). For conjugation purposes an analogue with a suitable functional handle was proposed, e.g. propofol-4-carboxylic acid to afford “propofol” conjugated to carrier protein.

[0491] Propofol

[0492]

[0493] Propofol-4-carboxylic acid

[0494] Stage 1 . Preparation of Protein - Propofol-4-carboxylic acid Conjugates

[0495] At 10 mg protein start scales, Propofol-4-carboxylic acid 2 (TRC, P829760-1G) will be conjugated to KLH (Sigma, H7017) and BSA (Sigma, A7030) using EDC / NHS coupling chemistry, to generate the propofol immunogen and screening conjugate, respectively. After conjugation, the products will be purified by gel filtration (e.g. Sephadex G25) eluting with PBS pH 6.7, to remove unreacted hapten and coupling reagents. Protein concentrations will be ascertained by UV-vis spectrophotometry. A qualitative assessment of hapten incorporation will be made by TNBS assay and UV-vis spectrophotometry. The products will be normalised at >1 mg / ml and terminally filtered to 0.2 pm.

[0496] Stage 2. Generation of anti-propofol monoclonal antibody

[0497] An appropriate quantity of immunogen and screening conjugate is provided from Stage 1 , and an immunisation program is carried out to generate monoclonal antibody against propofol.

[0498] Monoclonal antibody development programme is outlined below:

[0499] Phase 1 : Immunisation and test bleed analysis - 4 * mice. Phase 2: Fusion of 2 * mice spleen and screening.

[0500] Phase 3: Limiting dilution cloning, per round, per cell line.

[0501] Phase 4: Final production per cell line, up to 5mg approx. Hybridomas - Pilot scale up and purification.

[0502] Stage 3. Evaluate polyclonal antisera produced using the immunogen produced in Stage 2 and carry out proof of concept development in an immunoassay format.

[0503] DEVELOPMENT OF PROPOFOL ASSAY

[0504] 1. Introduction

[0505] The aim of this phase was to evaluate the polyclonal antisera produced using the immunogen produced and carry out proof of concept development in an immunoassay format.

[0506] This involved the development of a plate-based ELISA format to demonstrate specificity, sensitivity, and precision with the goal of ultimately pursuing monoclonal antiserum development.

[0507] The polyclonal antisera has been raised to be sensitive to Propofol, also known as Diprivan, a commonly used anaesthetic involved in the starting and maintenance of general anaesthesia as well as other medical applications.

[0508] Development involved the optimisation of a competitive format ELISA using the screenogen (same hapten as immunogen coupled to an alternative carrier protein) as a coating as well as the testing for the interference of several commonly used anaesthetics and a preliminary investigation of precision.

[0509] 2. Summary of the Program of Work

[0510] The program of work was split into three objectives: Objective 1 - Initial Optimisation

[0511] Objective 2 - Characterisation of antibody reactivity

[0512] Objective 3 - Matrix tolerance and preliminary assay precision.

[0513] Objective 1 :

[0514] The polyclonal antisera was tested before assay development was undertaken using the Propofol-BSA conjugate showing comparable results for both hosts. These were then titrated at FBL at a range of concentrations (Propofol-BSA: 20- 0.156pg / ml, Antisera: 1 / 250 - 1 / 512000) using an Anti-Rabbit IgG-HRP detection conjugate.

[0515] This format was then tested for displacement with Propofol-4-Carboxylic acid (the hapten used to generate the immunogen) to get a general idea of the assay sensitivity in advance of receipt of Propofol from Merck.

[0516] Once Propofol arrived this was used for all subsequent experiments (DV305 / 197 onwards) as standard material.

[0517] It was found that standards in the 1 g / ml to 15.625 ng / ml range produced a dose response however high background was observed. This high background remained when tested with no Propofol-BSA coating indicating non-specific binding was occurring (DV305 / 195, 197, 199). Different coating and antisera concentrations were then tested with standards and without coating to see if this could reduce background (DV337 / 01 ).

[0518] Several different solutions were tested as blocking buffers with a 30minute blocking step added. Of these, the lowest background was found using a 0.1% Milk Powder solution (DV337 / 07). This blocking step was effective in removing the non-specific binding that had been seen.

[0519] The secondary antibody was then titrated at 2 different coating concentrations (20pg / ml and 10pg / ml) looking at concentrations from 1 / 1000 to 1 / 100,000 (DV337 / 10, 12). The ideal conditions from this were then used for all further studies. Objective 2:

[0520] The assay conditions which were optimised in Objective 1 were used to test serial dilutions of each of the interferents identified. These were tested in duplicate with a control on each plate. All interferents were made up at the same concentration as the control series to calculate relative displacement (DV337 / 17.24). This was only necessary for two of the interferents, Propofol-O-Glucuronide and Propofol-O-Sulphate. All other potential interferents tested produced no antibody displacement over the assessed concentration range.

[0521] Objective 3:

[0522] Different dilutions of serum were tested on propofol curves using the final assay format and compared to look at displacement, signal, dynamic range and non-specific binding. Minimal difference was found between the control curve and curves in 1 / 5, 1 / 15 and 1 / 20 serum, 1 / 10 serum did not align with these curves but as 1 / 5 and 1 / 15 did not show the same trend this was considered anomalous (DV337 / 15). As such it was considered that the prototype assay is able to tolerate up to 20% serum.

[0523] QC samples were made up at 30pg / ml, 2pg / ml and 0.2pg / ml in pooled human serum to be diluted 1 in 20 in assay buffer. These were stored in single use aliquots at <-20°C until required for testing.

[0524] These samples were tested on the finalised format on 3 different days in triplicate with standard curves on each plate. The concentration values for each of the samples were calculated from these curves using 4PL fit and compared for intra- and inter-assay precision (DV337 / 32,34,37).

[0525] Table 1 : Summary of experiments performed during testing

[0526] 3. Materials and Methods

[0527] 3.1 Reagents:

[0528] Table 2: Reagent Specifications

[0529] 3.2 Method Reagent Descriptions:

[0530] ■ Anti-Propofol Antibody: Anti-Propofol serum (Harvest Bleed, Rabbit 1 ) sourced the antibody generation program (lot number: 22:05 / 1551-3).

[0531] ■ Anti-Rabbit HRP: Anti-rabbit HRP sourced from Merck, (catalogue code: lot number: 3920753).

[0532] ■ Analytical Standard: Propofol sourced from TCI (Catalogue code: D0617, lot number: MTBK7900V)

[0533] ■ Propofol- BSA solution: Propofol-BSA conjugate prepared by FBL and provided by SOM / APS (lot number: SOM1 / 2), stock concentration 1.7mg / ml.

[0534] ■ Buffer 21 : See appendix for formulation.

[0535] ■ Buffer 111 : See appendix for formulation.

[0536] ■ Buffer 58: See appendix for formulation.

[0537] Key Equipment:

[0538] ■ Pipettes: RAININ- P20 (5-20ul, PIP085), P200 (20-200ul, PIP088), P1000 (200-1000UI, PIP090); EPPENDORF- P5000 (1000-5000ul, PIP091 )

[0539] ■ Microtubes: VWR- Microcentrifuge tubes, 2.0 ml (catalogue code: 211-2606) I Microcentrifuge tubes, 0.5ml (catalogue code: 525-1157)

[0540] ■ Plates: VWR - multi-well flat bottom plates (96 well) (catalogue code: 734- 2327)

[0541] ■ Balances: Sartorius R160D (<100g, BAL 1 ), Sartorius LP3200D (100-2000g, BAL 2), Sartorius ISI 10 (>2000g, BAL3)

[0542] ■ Plate Incubators: Grant Bio PHMP (PINCU2)

[0543] ■ Plate Washer: Biotek ELx50 (WASH2)

[0544] ■ Plate Reader: Thermofisher Multiskan FC (READ1)

[0545] Preparation of Working Solutions:

[0546] Propofol-BSA preparation: Optimal assay concentration was found to be 20pg / ml for Propofol-BSA coating.

[0547] This was prepared in Buffer 21 from the stock concentration on the day of use.

[0548] Anti-Propofol Antibody preparation:

[0549] Optimal assay concentration was found to be a 1 / 500 dilution.

[0550] This was prepared in Buffer 111 on the day of use.

[0551] Anti-Rabbit HRP Antibody preparation:

[0552] Optimal assay concentration was found to be a 1 / 5000 dilution.

[0553] This was prepared in Buffer 111 from a single use aliquot on the day of use.

[0554] Propofol Standard preparation:

[0555] Standard series started from a concentration of 4pg / ml.

[0556] Initial stock made up on day of use by weighing out an amount of Propofol (2- 3mg) and diluting in Methanol to 1 mg / ml.

[0557] A 1 / 20 dilution of pooled human serum in Buffer 111 was made up.

[0558] 1 / 20 serum matrix was used to dilute Propofol to 4pg / ml.

[0559] A serial dilution of 1 in 4 was performed to produce 7 standards in the range 4pg / ml to 0.98ng / ml.

[0560] 1 / 20 serum used for blank.

[0561] Blocking Buffer preparation:

[0562] 0.1% Milk Powder w / w buffer made up in Buffer 58.

[0563] Assay Procedure

[0564] 1. 100 L Propofol-BSA added to each well.

[0565] 2. Incubated at 37°C for 1 hour.

[0566] 3. Washed with Buffer 58 using Assay 3 protocol.

[0567] 4. 150 L Blocking buffer (0.1% Milk Powder) added to each well. 5. Incubated at 37°C for 10 minutes.

[0568] 6. Washed with Buffer 58 using Assay 3 protocol.

[0569] 7. 50 pL Propofol standards and samples added at required concentrations

[0570] 8. 50 pL Anti-Propofol antibody added to each well.

[0571] 9. Incubated at 37°C for 1 hour, 600rpm.

[0572] 10. Washed with Buffer 58 using Assay 3 protocol.

[0573] 11. 100 pL Anti-Rabbit HRP added to each well.

[0574] 12. Incubated at 37°C for 1 hour.

[0575] 13. Washed with Buffer 58 using Assay 3 protocol.

[0576] 14. 100 pL Sureblue reserve added to each well.

[0577] 15. Incubated for 10 minutes at room temperature.

[0578] 16. 100 pL Hydrochloric Acid added to each well to quench.

[0579] 17. Plate read at 450nm and results saved to project folder.

[0580] Results

[0581] Objective 1 - Initial Optimisation

[0582] Initial Coat-Antisera titration - Figure 1

[0583] In the initial titration of the coating and antisera, coating concentrations from 20-0.156pg / ml were tested with antisera concentrations from 1 / 125 to 1 / 512,000. Testing had shown that this combination could be used to produce a signal but had not been tested with in-house reagents. An Anti-Rabbit IgG HRP was used at a 1 / 1000 dilution. Both host's antisera were used for this test and showed minimal difference between them. No propofol was used in this titration which looked at the maximum signal for each condition. Titre was similar between the two host animals but marginally higher with Rabbit 1 which was used for subsequent investigations. The best 3 coating conditions and best 2 antisera concentrations were then tested using standards to look at displacement.

[0584] Using a standard curve starting at 2pg / ml and a 1 in 4 dilution series, a dose response was observed with high background. This was tested using 1 / 125 antisera and a coating concentration of 20pg / ml, the highest which was tested.

[0585] Background Titration - Figures 2 and 3

[0586] In order to reduce the background observed different coatings and antisera were tested again with a standard curve and a row without coating. While a small reduction in background was seen with decreased concentrations, the lowest background observed was 0.65 Abs with a coat of 5pg / ml and antisera concentration of 1 / 2000.

[0587] A blocking step was then introduced with 6 different blocking solutions tested. Of these, a 0.1% Milk Powder blocking solution provided the best reduction in background. This was tested with an antisera concentration of 1 / 500 and coating concentration of 10pg / ml. Pierce ‘Clear Milk’ buffer (Fisher Scientific, 13494209) was diluted 1 in 10 per manufacturer's instructions while Pierce Blocking buffer (Fisher Scientific, 37572) and ‘Superblock’ (ThermoScientific, 37515). Milk Powder, BSA and Pluronic F-127 solutions were made at 0.1% in Buffer 58.

[0588] For this assay (DV337 / ) and all further, the bought-in Anti-Rabbit HRP (Merck, 12-348) was used.

[0589] Coat-Secondary Antibody Titration - Figure 4

[0590] Coating concentrations of 2-20pg / ml and secondary antibody concentrations of 1 / 1000 to 1 / 100,000 were tested to optimise the assay range and maximum signal. For these 1 / 500 antisera was used which was decided to be the final antisera concentration. The relative displacement of each combination was compared and the final conditions of 1 / 500 primary antibody, 1 / 5000 secondary antibody and a coat of 20pg / ml Propofol-BSA were established.

[0591] Objective 2 - Characterisation of Antibody reactivity

[0592] Figures 5 to 7

[0593] In order to assess the cross reactivity of the antisera, standard curves in the same range as the Propofol control were made up of 16 common compounds including several anaesthetics and over the counter pain medications. These were made up as a stock in methanol before dilution to a 100|jg / ml intermediate also in methanol. This was then diluted to 4pg / ml in assay buffer.

[0594] Of these, several were Home Office controlled drugs which were handled in accordance with relevant procedures. Ketamine, Midazolam, Morphine and Fentanyl are controlled drugs, and classified by the Home Office as schedule 2, 3, 2 and 2 respectively. The use of controlled drugs required in this project is covered by COSHH assessment FBL006, FBL007 and FBL064 while SOP091 details instructions for the management of Home Office scheduled controlled drugs in line with Home Office regulatory requirements. These were all tested in a single plate excluding Thiopental and Etomidate which was delayed due to shipping issues. The Etomidate / Thiopental results were negative.

[0595] Of the 14 interferents tested there were two compounds which also interacted with the antisera. These were Propofol-O-glucuronide and Propofol-O-Sulphate, metabolites of Propofol which have a similar molecular structure to Propofol. Propofol-O-Sulphate showed a greater displacement than Propofol-O-Glucuronide due to greater structural similarity. These were compared at a 50% level for relative displacement. Propofol-O-Sulphate was calculated to have a relative displacement of 23% at a 50% level.

[0596] Displacement for Propofol-O-Glucuronide was not calculable at a 50% level so was calculated for 30% displacement. At 30% this showed 2% displacement. T able 3 - see the drawings.

[0597] Table 3: Table showing relative displacement of interferents (DV337 / 24)

[0598] <Etomidate / Thiopental results>

[0599] Objective 3- Matrix tolerance and preliminary assay precision

[0600] Figure 11 : Serum curves were run at 1 / 5, 1 / 10,1 / 15 and 1 / 20 serum and compared to a control curve in buffer (DV337 / 15). Minimal difference was seen between the curves in serum and the control curves aside from the 1 / 10 serum dilution which was considered as anomalous. Based on these findings, it was considered that the assay is capable of tolerating up to 20% serum. However, it should be noted that a shift in the curve compared to buffer only is seen. For this reason, to ensure accuracy, it is considered best practice for calibration curves to be prepared in diluted matrix. Based upon the assay working range and the target working range for the assay, 5% serum is considered optimal, and this was used for subsequent studies.

[0601] A 1 / 20 dilution was decided to be sufficient to minimise any matrix effects, so controls were made up at 20x concentrations aimed at 1500, 200 and 20ng / ml on the standard curve as a high, mid-range and low QC respectively. These were prepared in single use aliquots and stored frozen. On the day of assay these were diluted 1 in 20 using assay buffer and run as samples.

[0602] To test these the samples were tested in triplicate on 3 different days in independent runs with the relative values calculated from the associated standard curve. This data was used to compare the inter and intra-assay variation - Figure 12.

[0603] Tables 4-6 - see the drawings.

[0604] Table 4: Summary of Optical Density values for testing of QC samples (DV337 / 32,34,37) Table 5: Summary of signal normalised to mean maximum binding (0 std) (DV337 / 32, 34, 37)

[0605] Table 6: Back calculated values for undiluted QC samples based on 4PL fit of standard curve for the respective day of testing (DV337 / 32,34,37).

[0606] Conclusions

[0607] The aim of this phase of the project was to develop an ELISA based assay to assess the developed Anti-Propofol antisera in human serum.

[0608] Following on antibody development work, we continued development and successfully produced a prototype assay in plate ELISA format suitable for the analysis of clinical samples.

[0609] The assay developed demonstrated an effective range of between 0.02-80pg / ml with a serum dilution of 1 / 20 applied which meets the target range of 0.1-20pg / ml. The assay appears to be tolerant of up to 20% serum, but calibrators should be prepared in diluted matrix pool. If maximising sensitivity is required, then using a 1 / 5 sample dilution gives an effective assay range of 0.005-20pg / mL.

[0610] With regards to the co-administered substances and common interferents tested, no crossreactivity or interference was observed with any of them. It is only with the structurally similar metabolites that any cross-reactivity is seen. For the propofol-glucuronide this was about 2% cross-reactivity, so a fifty-fold higher concentration of the glucuronide metabolite would be required to produce the same response in the assay as a given concentration of propofol. For propofol-sulphate, the cross-reactivity was 23% so a four to five fold excess of the sulphate metabolite would produce the same assay response as a given concentration of propofol. Whilst there is a relatively low level of cross-reactivity with the metabolites, particularly given that the glucuronide is the primary metabolite, the antiserum is not 100% specific for propofol only. Whilst it had been hoped that the design of the immunogen would give something that was reactive with propofol only, the low level of cross-reactivity, particularly for the glucuronide metabolite, does give options. It should be remembered that this is crude polyclonal antiserum and so contains antibodies with a range of different specificities. For a lateral flow type test to be manufactured, a monoclonal antibody would be the preferred embodiment. In which case, this data gives guidance for the clone screening approach to be used. The same immunogen can be used for the immunisation, but for screening hits and clones a tiered approach would need to be taken. The initial screen would be for binding to the screening conjugate, after which subsequent testing would be done to exclude those clones which displace with the metabolites. In this way the clones for investigation can be selected on the basis of minimal cross-reactivity.

[0611] For further assay development using the polyclonal antiserum, affinity purification is carried out - see Phase 4. The screening conjugate would be immobilised on a column and antibodies allowed to bind. The column would then be washed with a solution containing the metabolite to remove those antibodies which are cross-reactive before eluting with a pH change to claim the propofol specific portion.

[0612] Preliminary assay precision was considered acceptable (<20%) at this stage in the assay development. Both intra- and inter-assay imprecision peak at high propofol concentrations. The highest level of imprecision seen for calibrators was around 16%. Whilst there is a degree of signal variation between plates, normalisation to the maximum binding signal (Ong / mL standard) limits this. Such practice would be considered standard for competitive assays of this type. Imprecision for back calculated concentration of QC samples using 4PL curve fit peaked at 12%, and the maximum deviation seen from expected values was 14%.

[0613] Based on the findings of this Phase, it is concluded that proof of concept has been demonstrated.

[0614] PHASE 4 - ANTIGEN AFFINITY PURIFICATION OF POLYCLONAL ANTISERA Following completion of a program to raise polyclonal antisera against propofol and proof of concept assay development using said antisera, a program of work was undertaken to carry out affinity purification of the antisera.

[0615] During the proof of concept work, the antisera had shown a degree of reactivity to the sulphate and, to a lesser extent, glucuronide metabolites of propofol. The aim of this phase of the project is to attempt to deplete the polyclonal antiserum of the unwanted metabolite reactivity whilst maintaining reactivity with the parent compound.

[0616] Objective 1 Generation and testing of affinity column

[0617] Propofol-BSA hapten conjugate will be produced using the method established in project S0M1. The resulting conjugate will then be immobilised on Cytiva HiTrap NHS-activated HP 1 mL column. Such columns can be readily scaled if required in the future for process scale up. 1 mL of crude antiserum will be passed down the column and allowed to bind. Captured antibodies will be eluted by acidification. This will demonstrate utility of the column and provide a baseline for the proportion of the antiserum which is propofol specific. The concentration of eluted antibodies will be determined by UV absorbance.

[0618] Objective 2 Depletion of metabolite reactivity

[0619] An additional 1 mL aliquot of the antiserum will be passed down the column generated in objective 1. After allowing the propofol specific antibodies to bind, the column will be washed with a solution containing the sulphate metabolite of propofol. Antibodies reactive with metabolite will be displaced and collected in the flow through. The remaining metabolite non- reactive antibodies will be eluted by acidification.

[0620] Concentration of eluate will be determined by UV absorbance. Comparison with the concentration of the product generated in objective 1 will give an indication of the relative abundance of propofol only specific antibodies in the serum compared to propofol and metabolite reactive. Objective 3 Confirmation of final product reactivity

[0621] The eluate products from objectives 1 and 2 will be assessed for reactivity using the proof of concept assay developed under Phase 3. The products and crude antiserum will be run in the assay against standard curves of propofol, and the sulphate and glucuronide metabolites. Working strengths for the eluate products will be estimated based on the known working strength of the crude antiserum and the product concentrations determined under objectives 1 and 2. The products will be assessed relative to the crude antiserum and cross reactivity with the metabolites estimated based on the displacement seen relative to propofol itself.

[0622] If metabolite reactivity remains in the product of objective 2 then further depletion steps could be considered, e.g. with glucuronide metabolite, depending on the yields seen.

[0623] Propofol Monoclonal Antibodies

[0624] Monoclonal antibodies have been generated using a hybridoma technology approach. It involves fusing B cells, which produce antibodies, with myeloma cells, which are cancerous cells that grow indefinitely. The resulting hybrid cells, known as hybridomas, can produce a large quantity of identical mAbs that target a specific antigen.

[0625] The general process of creating hybridomas involves several steps, including immunization of an animal with the antigen of interest, harvesting B cells from the spleen, fusing the B cells with myeloma cells using a chemical or electrical method, selecting the hybridomas that produce the desired antibody, and finally, growing and maintaining the selected hybridomas in culture.

[0626] Four monoclonal antibodies have been produced and sequenced. These antibodies have been designated: cAb11051 (aka CE8); cAb11052 (aka CE7); cAb11053 (aka FH10); and cAb11131 (aka GH5).

[0627] Sequencing was performed by whole transcriptome shotgun sequencing (RNA-Seq). Sequence Reports

[0628] 1. ID: cAb11051

[0629] Clone name: CE8.G9.C12.F6.B11

[0630] No of HCs: 1

[0631] No of LCs: 1

[0632] Primary Heavy Chain Protein / DNA Sequences

[0633] HC type: Mouse lgG1

[0634] Integrity: Complete

[0635] Signal Peptide:

[0636] SEQ ID NO: 1

[0637] MGWSWIFLFLLSGTAGVHS

[0638] SEQ ID NO: 2

[0639] ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCT

[0640] VH:

[0641] SEQ ID NO: 3

[0642] EVQLQQSGPELVKPGASMKISCKASGYSFTDYTMNWVNWVKQSHGKNLEWIGHINPYNGGS

[0643] SYNQKFRGKATLTVDKSSTTAYMELLSLTSEDSAVYYCAVIYYDYDGDIFAYWGQGTLVTVSA

[0644] SEQ ID NO: 4

[0645] GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATA

[0646] TCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACACCATGAACTGGGTGAACTGGGTGA

[0647] AGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACATATTAATCCTTACAATGGTGGTTC

[0648] TAGCTACAACCAGAAGTTCAGGGGCAAGGCCACATTAACTGTAGACAAGTCATCCACCACA

[0649] GCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCTGTAA

[0650] TCTACTATGATTACGACGGGGATATTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGT CTCTGCA

[0651] Constant:

[0652] SEQ ID NO: 5

[0653] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT

[0654] LSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVL

[0655] TITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWL

[0656] NGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITV

[0657] EWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKS LSHSPGK

[0658] SEQ ID NO: 6 GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACT

[0659] CCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCT

[0660] GGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACC

[0661] TCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCA

[0662] CCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGG

[0663] GATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCC

[0664] CCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTA

[0665] GACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTG

[0666] CACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGT

[0667] GAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAAC

[0668] AGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAG

[0669] GCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGT

[0670] CTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATG

[0671] GGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACT

[0672] TCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCT

[0673] GCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCC TGGTAAA

[0674] TPM: 301891.64

[0675] %: 41.3

[0676] Primary light chain protein / DNA sequence

[0677] LG Type: Mouse kappa

[0678] Integrity: Complete

[0679] Signal Peptide:

[0680] SEQ ID NO: 7

[0681] MSVPTQVLGLLLLWLTGARC

[0682] SEQ ID NO: 8

[0683] ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGT

[0684] VL:

[0685] SEQ ID NO: 9

[0686] DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNSKTLAEGVPSRFSG

[0687] SGSGTQFSLKINSLQPEDFGSYYCQHHYGTPFTFGSGTKLEIK

[0688] SEQ ID NO: 10

[0689] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCA

[0690] TCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGG

[0691] AAAATCTCCTCAACTCCTGGTCTATAATTCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGT

[0692] TCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAG

[0693] ATTTTGGGAGTTATTACTGTCAACATCATTATGGTACTCCATTCACGTTCGGCTCGGGGACA

[0694] AAGTTGGAAATAAAA

[0695] Constant:

[0696] SEQ ID NO: 11

[0697] RADAAPTVSIFPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKD

[0698] STYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 12

[0699] CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTG

[0700] GAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG

[0701] GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAG

[0702] CAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACG

[0703] ACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGC

[0704] TTCAACAGGAATGAGTGT

[0705] TPM: 429016.43

[0706] %: 58.7

[0707] 2. ID: cAb11052

[0708] Clone name: CE7.E4.G9.F5.E11

[0709] No of HCs: 1

[0710] No of LCs: 1

[0711] Primary Heavy Chain Protein / DNA Sequence

[0712] HC type: Mouse lgG1

[0713] Integrity: Complete

[0714] Signal Peptide:

[0715] SEQ ID NO: 13

[0716] MRVLILLCLFTAFPGILS

[0717] SEQ ID NO: 14

[0718] ATGAGAGTGCTGATTCTTTTGTGCCTGTTCACAGCCTTTCCTGGTATCCTGTCT

[0719] VH:

[0720] SEQ ID NO: 15

[0721] DVQLQESGPDLVKPSQSLSLTCTVTGYSLTSGFTWHWIRQFPGNKLEWMGYLHYSGDTNYNP

[0722] SLRSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGGITSALWGQGTLVTVSA

[0723] SEQ ID NO: 16

[0724] GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCCCTCA

[0725] CCTGCACTGTCACTGGCTACTCCCTCACCAGTGGTTTTACCTGGCACTGGATCCGGCAGTT

[0726] TCCAGGGAACAAACTGGAGTGGATGGGCTACCTACACTACAGTGGTGACACTAACTACAA

[0727] CCCATCTCTCAGAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTG

[0728] CAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGAGGCGGGATTA

[0729] CGTCGGCTCTCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0730] Constant:

[0731] SEQ ID NO: 17 AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT

[0732] LSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVL

[0733] TITLTPKVTCVWDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWL

[0734] NGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITV

[0735] EWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKS

[0736] LSHSPGK

[0737] SEQ ID NO: 18

[0738] GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACT

[0739] CCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCT

[0740] GGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACC

[0741] TCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCA

[0742] CCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGG

[0743] GATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCC

[0744] CCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTA

[0745] GACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTG

[0746] CACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGT

[0747] GAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAAC

[0748] AGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAG

[0749] GCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGT

[0750] CTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATG

[0751] GGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACT

[0752] TCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCT

[0753] GCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCC

[0754] TGGTAAA

[0755] TPM: 234944.43

[0756] %: 35.64

[0757] Primary light chain protein / DNA sequence

[0758] LC Type: Mouse kappa

[0759] Integrity: Complete

[0760] Signal Peptide:

[0761] SEQ ID NO: 19

[0762] MSVPTQVLGLLLLWLTDARC

[0763] SEQ ID NO: 20

[0764] ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGATGCCAGATGT

[0765] VL:

[0766] SEQ ID NO: 21

[0767] DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYGATNLADGVPSRFS

[0768] GSGSGTQFSLKINRLQSEDFGIYYCHHLWGIPYTFGGGTKLEIK

[0769] SEQ ID NO: 22

[0770] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGTATCTGTGGGAGAAACTGTCACCA

[0771] TCACATGTCGAGCAAGTGAGAATATTTACAGTAATTTAGCATGGTATCAGCAGAAGCAGGG

[0772] AAAATCTCCTCAGCTCCTGGTCTATGGTGCTACAAACTTAGCAGATGGTGTGCCATCAAGG

[0773] TTCAGTGGCAGTGGATCAGGCACACAGTTTTCCCTCAAGATCAACAGGTTGCAGTCTGAAG ATTTTGGGATTTACTACTGTCACCATTTATGGGGTATACCGTACACGTTCGGAGGGGGGAC

[0774] CAAGCTGGAAATAAAA

[0775] Constant:

[0776] SEQ ID NO: 23

[0777] RADAAPTVSIFPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKD

[0778] STYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0779] SEQ ID NO: 24

[0780] CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTG

[0781] GAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG

[0782] GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAG CAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACG ACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGC

[0783] TTCAACAGGAATGAGTGT

[0784] TPM: 424218.12

[0785] %: 64.36

[0786] 3. ID: cAb11053

[0787] Clone name: FH10.G2.G7.H5.D8

[0788] No of HCs: 1

[0789] No of LCs: 1

[0790] Primary Heavy Chain Protein / DNA Sequence

[0791] HC type: Mouse lgG1

[0792] Integrity: Complete

[0793] Signal Peptide:

[0794] SEQ ID NO: 25

[0795] MRVLILLWLFTAFPGILS

[0796] SEQ ID NO: 26

[0797] ATGAGAGTGCTGATTCTTTTGTGGCTGTTCACAGCCTTTCCTGGTATCCTGTCT

[0798] VH:

[0799] SEQ ID NO: 27

[0800] DVQVQESGPGLVKPSQSLSLTCTVTGYSITSNYAWNWIRQFPGDKLEWMGFITYSGSSTYNPS

[0801] LKSRISITRDTSKNQFFLQLNSVTSEDTATYYCASVYYDYDAWFAYWGQGTLVTVSA

[0802] SEQ ID NO: 28

[0803] GATGTGCAGGTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTC

[0804] ACCTGCACTGTCACTGGCTACTCAATCACCAGTAACTATGCCTGGAACTGGATCCGGCAGT

[0805] 5 I TTCCAGGAGACAAACTGGAGTGGATGGGCTTCATAACCTACAGTGGTAGTTCTACCTACAA

[0806] CCCCTCTCTCAAGAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTTC

[0807] AATTGAATTCTGTGACTTCTGAGGACACAGCCACTTATTACTGTGCAAGTGTCTATTATGAT

[0808] TACGACGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0809] Constant:

[0810] SEQ ID NO: 29

[0811] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT

[0812] LSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVL

[0813] TITLTPKVTCVWDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWL

[0814] NGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITV

[0815] EWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKS

[0816] LSHSPGK

[0817] SEQ ID NO: 30

[0818] GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACT

[0819] CCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCT

[0820] GGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACC

[0821] TCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCA

[0822] CCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGG

[0823] GATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCC

[0824] CCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTA

[0825] GACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTG

[0826] CACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGT

[0827] GAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAAC

[0828] AGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAG

[0829] GCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGT

[0830] CTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATG

[0831] GGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACT

[0832] TCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCT

[0833] GCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCC TGGTAAA

[0834] TPM: 319943.2

[0835] %: 38.2

[0836] Primary light chain protein / DNA sequence

[0837] LC Type: Mouse kappa

[0838] Integrity: Complete

[0839] Signal Peptide:

[0840] SEQ ID NO: 31

[0841] MSVPTQVLGLLLLWLTGARC

[0842] SEQ ID NO: 32

[0843] ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTACTGTGGCTTACAGGTGCCAGATGT

[0844] VL:

[0845] SEQ ID NO: 33 DIQMTQSPASLSASVGETVTITCRASENIYTYLAWYQLKQGKSPQLLVYNAKTLAGGVPSRFSA

[0846] SGSGTQFSLKINSLQPEDFGSFYCHHHYHTPFTFGSGTRLEIN

[0847] SEQ ID NO: 34

[0848] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCA

[0849] TCACATGTCGAGCAAGTGAGAATATTTACACTTATTTAGCATGGTATCAGCTGAAACAGGGA

[0850] AAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCGGGAGGTGTGCCATCAAGGT

[0851] TCAGTGCCAGTGGATCAGGCACACAATTTTCTCTGAAGATCAACAGCCTGCAGCCTGAGGA

[0852] TTTTGGGAGTTTCTACTGTCACCATCATTATCATACTCCTTTCACGTTCGGCTCGGGGACAA GGTTGGAGATAAAC

[0853] Constant:

[0854] SEQ ID NO: 35

[0855] RADAAPTVSIFPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKD

[0856] STYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0857] SEQ ID NO: 36

[0858] CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTG

[0859] GAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG

[0860] GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAG

[0861] CAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACG

[0862] ACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGC TTCAACAGGAATGAGTGT

[0863] TPM: 517805.2

[0864] %: 61.8

[0865] 4. ID: cAb11131

[0866] Clone name: GH5.G4.C8.C4.3.G5.2.H8

[0867] No of HCs: 1

[0868] No of LCs: 1

[0869] Primary Heavy Chain Protein / DNA Sequence

[0870] HC type: Mouse lgG1

[0871] Integrity: Complete

[0872] Signal Peptide:

[0873] SEQ ID NO: 37

[0874] MGWSWIFLFLLSGTAGVHS

[0875] SEQ ID NO: 38

[0876] ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCT

[0877] VH: SEQ ID NO: 39

[0878] EVQLQQSGPELVKPGASMKISCKASDYSFTDYTMTWINWVKQSHEKNLEWIGHINPYNGGTSY NQKFRGKATLTVDKSSSAAYMELLSLTSEDSAVYYCAWYYDYGGDIFAYWGQGTLVTVSA

[0879] SEQ ID NO: 40

[0880] GAGGTCCAACTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATA

[0881] TCCTGCAAGGCTTCTGATTACTCATTCACTGACTACACCATGACCTGGATCAACTGGGTGA

[0882] AGCAGAGCCATGAAAAGAACCTTGAGTGGATTGGACATATTAATCCTTACAATGGTGGTAC TAGTTACAACCAGAAGTTCAGGGGCAAGGCCACATTAACTGTAGACAAGTCATCCAGCGCA GCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCTGTAG

[0883] TCTACTATGATTACGGCGGGGATATTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGT CTCTGCA

[0884] Constant:

[0885] SEQ ID NO: 41

[0886] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT LSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVL

[0887] TITLTPKVTCVWDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWL

[0888] NGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITV EWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKS LSHSPGK

[0889] SEQ ID NO: 42

[0890] GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACT

[0891] CCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCT

[0892] GGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACC TCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCA CCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGG

[0893] GATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCC

[0894] CCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTA

[0895] GACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTG

[0896] CACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGT

[0897] GAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAAC

[0898] AGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAG

[0899] GCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGT CTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATG GGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACT

[0900] TCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCT GCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCC TGGTAAA

[0901] TPM: 365138.3

[0902] %: 47.5

[0903] Primary light chain protein / DNA sequence

[0904] LC Type: Mouse kappa

[0905] Integrity: Complete

[0906] Signal Peptide:

[0907] SEQ ID NO: 43 MSVPTQVLGLLLLWLTGARC

[0908] SEQ ID NO: 44

[0909] ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGT

[0910] VL:

[0911] SEQ ID NO: 45

[0912] DIQMTQSPASLSASVGETVTITCRASENIYNYLAWYQQRQGKSPQLLVYNSKTLAEGVPSRFS

[0913] GSGSGTQFSLKINSLQPEDFGNYFCQHHSGTPFTFGSGTKLDLK

[0914] SEQ ID NO: 46

[0915] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACCGTCACCA

[0916] TCACATGTCGAGCAAGTGAAAATATTTACAATTATTTAGCATGGTATCAACAGAGACAGGGA

[0917] AAATCTCCTCAACTCCTGGTCTATAATTCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTT

[0918] CAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGA

[0919] TTTTGGGAATTATTTCTGTCAACATCATTCTGGTACTCCATTCACGTTCGGCTCGGGGACAA

[0920] AGTTGGATTTAAAA

[0921] Constant:

[0922] SEQ ID NO: 47

[0923] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKD

[0924] STYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0925] SEQ ID NO: 48

[0926] CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTG

[0927] GAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG

[0928] GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAG

[0929] CAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACG

[0930] ACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGC

[0931] TTCAACAGGAATGAGTGT

[0932] TPM: 403810.6

[0933] %: 52.5

[0934] OPR IDENTIFICATION

[0935] The complementarity determining regions (CDRs) have been automatically identified using Excel formulas working to the Kabat definition for CDRs. CDR identification is performed for the primary VH and VL sequences. cAb11051

[0936] Primary VH Sequence SEQ ID NO: 49

[0937] EVQLQQSGPELVKPGASMKISCKASGYSFTDYTMNWVNWVKQSHGKNLEWIGHINPYNGGS

[0938] SYNQKFRGKATLTVDKSSTTAYMELLSLTSEDSAVYYCAVIYYDYDGDIFAYWGQGTLVTVSA

[0939] CDR-H1

[0940] SEQ ID NO: 50

[0941] DYTMN

[0942] CDR-H2

[0943] SEQ ID NO: 51

[0944] WIGHINPYNGGSSYNQKFRG

[0945] CDR-H3

[0946] SEQ ID NO: 52

[0947] IYYDYDGDIFAY

[0948] Primary VL Sequence

[0949] SEQ ID NO: 53

[0950] DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNSKTLAEGVPSRFSG

[0951] SGSGTQFSLKINSLQPEDFGSYYCQHHYGTPFTFGSGTKLEIK

[0952] CDR-L1

[0953] SEQ ID NO: 54

[0954] RASENIYSYLA

[0955] CDR-L2

[0956] SEQ ID NO: 55

[0957] NSKTLAE CDR-L3

[0958] SEQ ID NO: 56

[0959] QHHYGTPFT cAb11052

[0960] Primary VH Sequence

[0961] SEQ ID NO: 57

[0962] DVQLQESGPDLVKPSQSLSLTCTVTGYSLTSGFTWHWIRQFPGNKLEWMGYLHYSGDTNYNP

[0963] SLRSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGGITSALWGQGTLVTVSA

[0964] CDR-H1

[0965] SEQ ID NO: 58

[0966] SGFTWH

[0967] CDR-H2

[0968] SEQ ID NO: 59

[0969] YLHYSGDTNYNPSLRS

[0970] CDR-H3

[0971] SEQ ID NO: 60

[0972] GGITSAL

[0973] Primary VL Sequence

[0974] SEQ ID NO: 61

[0975] DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYGATNLADGVPSRFS

[0976] GSGSGTQFSLKINRLQSEDFGIYYCHHLWGIPYTFGGGTKLEIK CDR-L1

[0977] SEQ ID NO: 62

[0978] RASENIYSNLA

[0979] CDR-L2

[0980] SEQ ID NO: 63

[0981] GATNLAD

[0982] CDR-L3

[0983] SEQ ID NO: 64

[0984] HHLWGIPYT cAb11053

[0985] Primary VH Sequence

[0986] SEQ ID NO: 65

[0987] DVQVQESGPGLVKPSQSLSLTCTVTGYSITSNYAWNWIRQFPGDKLEWMGFITYSGSSTYNPS

[0988] LKSRISITRDTSKNQFFLQLNSVTSEDTATYYCASVYYDYDAWFAYWGQGTLVTVSA

[0989] CDR-H1

[0990] SEQ ID NO: 66

[0991] SNYAWN

[0992] CDR-H2

[0993] SEQ ID NO: 67

[0994] FITYSGSSTYNPSLKS CDR-H3

[0995] SEQ ID NO: 68

[0996] VYYDYDAWFAY

[0997] Primary VL Sequence

[0998] SEQ ID NO: 69

[0999] DIQMTQSPASLSASVGETVTITCRASENIYTYLAWYQLKQGKSPQLLVYNAKTLAGGVPSRFSA

[1000] SGSGTQFSLKINSLQPEDFGSFYCHHHYHTPFTFGSGTRLEIN

[1001] CDR-L1

[1002] SEQ ID NO: 70

[1003] RASENIYTYLA

[1004] CDR-L2

[1005] SEQ ID NO: 71

[1006] NAKTLAG

[1007] CDR-L3

[1008] SEQ ID NO: 72

[1009] HHHYHTPFT cAb11131

[1010] Primary VH Sequence

[1011] SEQ ID NO: 73 EVQLQQSGPELVKPGASMKISCKASDYSFTDYTMTWINWVKQSHEKNLEWIGHINPYNGGTSY

[1012] NQKFRGKATLTVDKSSSAAYMELLSLTSEDSAVYYCAWYYDYGGDIFAYWGQGTLVTVSA

[1013] CDR-H1

[1014] SEQ ID NO: 74

[1015] DYTMT

[1016] CDR-H2

[1017] SEQ ID NO: 75

[1018] WIGHINPYNGGTSYNQKFRG

[1019] CDR-H3

[1020] SEQ ID NO: 76

[1021] VYYDYGGDIFAY

[1022] Primary VL Sequence

[1023] SEQ ID NO: 77

[1024] DIQMTQSPASLSASVGETVTITCRASENIYNYLAWYQQRQGKSPQLLVYNSKTLAEGVPSRFS

[1025] GSGSGTQFSLKINSLQPEDFGNYFCQHHSGTPFTFGSGTKLDLK

[1026] CDR-L1

[1027] SEQ ID NO: 78

[1028] RASENIYNYLA

[1029] CDR-L2

[1030] SEQ ID NO: 79

[1031] NSKTLAE CDR-L3

[1032] SEQ ID NO: 80

[1033] QHHSGTPFT

[1034] Affinity Purification

[1035] Affinity Purification report: the displacement work in ELISA for the four propofol mAbs.

[1036] Introduction

[1037] Carry out an affinity purification of the antisera used to develop a proof-of-concept ELISA. This involved the affinity purification of the antisera as well as the evaluation of the products of the purification on the proof-of-concept assay to confirm the presence of specific antibody and the depletion of non-specific antibody. The polyclonal antisera has been raised to be sensitive to Propofol, also known as Diprivan, a commonly used anaesthetic involved in the starting and maintenance of general anaesthesia as well as other medical applications.

[1038] Summary of the Program of Work

[1039] Objective 1 : Generation and testing of affinity column

[1040] Objective 2: Depletion of metabolite reactivity

[1041] Objective 3: Confirmation of final product reactivity

[1042] Objective 1-

[1043] Multiple affinity columns were made using two different techniques to try and find what would be the best mechanism of action. Five columns were made as part of this objective, 3 of them using a Propofol-BSA conjugate, and the other 2 by using a direct-coupling mechanism. The first method involved using the Propofol-BSA conjugates generated and later of DV333 / 59 - the conjugates were immobilised on Cytiva HiTrap NHS-activated HP 1 ml columns. Initially, two different columns were made using the conjugates generated as part of SOM1 ; however, due to the poor yield of anti-propofol antibodies after the AKTA run, two alternatives were investigated. The first one was to make a 3rdcolumn, using a fresh Propofol-BSA conjugate, and the other one was to try a completely different method of binding the propofol to the column.

[1044] The direct-coupling column was made by reacting the NHS-activated HP column with adipic dihydrazine, activating the Propofol 4-COOH by using EDC and NHS, and conjugating this directly to propofol via the added NHS.

[1045] All the columns were used to determine which mechanism is the best at depleting the antipropofol antibodies as part of Objective 2.

[1046] Objective 2-

[1047] The columns generated in Objective 1 were further used to run anti-propofol antibody rich serum down them in the attempt to deplete it. The columns were tested in turn, by using 1 ml of buffered serum. Both the flowthrough and the antibody elutions were given to the Assay team for testing to determine which method would be the best to continue with. After some of the testing in Objective 3, it was determined that the direct-coupling columns were better than the BSA ones, and it was decided that, for the final experiment, the 2 direct-coupling columns would be used in series. Furthermore, the serum would be recirculated on them to allow for the highest binding probability.

[1048] The method described above would also be used for the testing of antibody specificity to propofol over propofol sulfate.

[1049] Objective 3- The products generated from Objective 1 were tested using the prototype assay format developed herein without the target analyte to look at the relative concentration of antibody specific to the Propofol-BSA conjugate present. This was tested at a range of dilutions to compare the absorbance across the range. The flowthrough from the affinity purification was also tested to investigate the amount of antibody which had been removed and it's concentration relative to the product eluted. The products of the affinity purification were evaluated compared to the crude antiserum looking at the maximum absorbance.

[1050] The final products of the affinity purification were tested against the propofol metabolites to look at cross-reactivity. Testing using the same method showed a 23% relative displacement for

[1051] Propofol-Sulphate at a 50% level and a 2% relative displacement for Propofol-Glucuronide at a 30% level. These metabolites were compared to the displacement of Propofol at the same concentration to work out relative displacement. T able 1 : Summary of experiments performed during testing.

[1052] 1. Materials and Methods 1.1 Reagents:

[1053] Table 2: Reagent Specifications

[1054] Method - Chemistry Reagent Descriptions:

[1055] ■ Anti-Propofol Antibody: Anti-Propofol serum (Harvest Bleed, Rabbit 1) sourced from

[1056] APS from S0M1 antibody generation program (lot number: 22:05 / 1551-3).

[1057] ■ Propofol-BSA Conjugates: Prepared by FBL and provided by SOM (lot numbers: SOM 1 / 2 and DV333 / 59-1 ).

[1058] ■ Buffer 3: See appendix for formulation.

[1059] ■ Buffer 33: See appendix for formulation.

[1060] ■ Buffer 97: See appendix for formulation. ■ 0.1 M Glycine Buffer, pH 2.5: 7.5 g Glycine added to 11 of RO / DI water, pH adjusted with 1 M HCI. Filtered to 0.22 pm.

[1061] ■ Glycine Buffer. pH 2.5, with 5% Ethanol: 3.75 g Glycine added to 500 ml of RO / DI Water, pH adjusted with 1 M HCI, then ethanol added to make up 5% of the final solution. Filtered to 0.22 pm.

[1062] Protocol for Propofol-BSA Conjugated Column Packing:

[1063] 1. 6 ml of ice-cold 1mM HCI added to the column.

[1064] 2. Immediately after, 2 ml of the BSA-Propofol Conjugate added to the column. Column left to incubate at 20°C for 30 minutes.

[1065] 3. Column washed with 6 ml of 0.5M Ethanolamine + 0.5M NaCI (Buffer A), followed by 6 ml of 0.1 M Sodium Acetate + 0.5M NaCI (Buffer B), followed by 6 ml of Buffer A. Column was then left to stand at 20°C for 30 minutes.

[1066] 4. Column washed with 6 ml of Buffer B, followed by 6 ml of Buffer A and 6 ml of Buffer B.

[1067] 5. 2 ml of Buffer 3 added to the column, and column stored at 2-8°C overnight.

[1068] Protocol for Direct Coupling Column Packing:

[1069] 1. Column activated by reacting it with 20 ml of 0.1 mg / ml Adipic Dihydrazine solution in Buffer 3 as follows: 5 ml of the dihydrazine solution injected onto the column, and column left to stand at 20°C for 10 minutes. Procedure repeated 3 more times, until the 20 ml have been run through.

[1070] 2. Column washed with Buffer 3 at 3 ml / min for 5 minutes.

[1071] 3. Activated Propofol (0.1 mg / ml solution) added to the column as follows: 5 ml washed through column dropwise - column then left at 20°C for 10 minutes. Procedure repeated 2 more times, until 15 ml of Activated Propofol were run through the column.

[1072] 4. Column washed as follows: a. 10 ml of Buffer 3, 1 ml / min b. 10 ml of Glycine, pH 2.5 Buffer, 1 ml / min c. 10 ml of Buffer 3, 1 ml / min d. 10 ml of Glycine, pH 2.5 Buffer, 1 ml / min e. 10 ml of Buffer 3, 1 ml / min 5. Column stored at 2-8°C.

[1073] Protocol for Capture of Anti-Propofol Antibodies:

[1074] 1. A 96-well plate was prepared, by adding 20 l of 1 M Tris Buffer to the wells where the antibody would be eluted into.

[1075] 2. 1 ml of serum was buffered with Di-Potassium Hydrogen Phosphate and Potassium DiHydrogen Phosphate (by doing a 1 :10 dilution into Buffer 3, resulting in a total volume of 10 ml).

[1076] 3. The 10 ml diluted serum was run on the column and recirculated for 1 hour by using an external peristaltic pump.

[1077] 4. The AKTA unit was set up to collect fractions of the flowthrough.

[1078] 5. After the baseline equilibrated, the pumps on the AKTA unit were switched into a Glycine, pH 2.5 buffer, which would allow the antibody that was bound to the column to be eluted into fractions.

[1079] 6. Both types of fractions collected and desalted into Buffer 33 by using Zeba 2 ml Spin Columns, then handed over to the Assay Team for testing - the antibody fraction was also filtered through a 0.22 pm spin filter.

[1080] * For one of the runs, the same serum was rerun onto the column after the antibody elution, to ensure that the column’s capacity is not the limiting factor when it comes to the amount of antibody that is captured.

[1081] Protocol for Capture of Specific Anti-Propofol Antibodies:

[1082] 1. A 96-well plate was prepared, by adding 20 pl of 1 M Tris Buffer to the wells where the antibody would be eluted into

[1083] 2. 10 ml of the diluted buffered serum (1 :10 dilution in Buffer 3) was run on the column and recirculated for one hour by using an external peristaltic pump.

[1084] 3. The AKTA unit’s pump was then switched to a 200 ng / ml Propofol Sulfate in Buffer 3 dilution to strip down any non-specific antibodies that were bound to the Propofol on the column. This elution was collected by fractionation.

[1085] 4. After the non-specific antibodies are eluted, the AKTA pump switches to a Glycine, pH 2.5 buffer, to elute the antibody that is still bound to the column. 5. Both types of fractions were desalted into Buffer 33 by using Zeba 5 ml Spin Columns, then handed over to the Assay Team for testing after being filtered through a 0.22 pm spin filter.

[1086] Method - Assay

[1087] Reagent Descriptions:

[1088] ■ Anti-Propofol Antibody: Anti-Propofol serum (Harvest Bleed, Rabbit 1 ) sourced from APS from SOM1 antibody generation program (lot number: 22:05 / 1551-3).

[1089] ■ Anti-Rabbit HRP: Anti-rabbit HRP sourced from Merck, (catalogue code: lot number: 3920753).

[1090] ■ Analytical Standard: Propofol sourced from TCI (Catalogue code: D0617, lot number: MTBK7900V)

[1091] ■ Propofol- BSA solution: Propofol-BSA conjugate prepared by FBL and provided by SOM / APS (lot number: SOM1 / 2), stock concentration 1.7mg / ml.

[1092] ■ Coating Buffer

[1093] ■ Assay Buffer

[1094] ■ Wash Buffer

[1095] Preparation of Working Solutions:

[1096] Propofol-BSA preparation:

[1097] Optimal assay concentration was found to be 20ug / ml for Propofol-BSA coating.

[1098] This was prepared in Coating Buffer from the stock concentration on the day of use.

[1099] Anti-Propofol Antibody preparation:

[1100] Optimal assay concentration was found to be a 1 / 500 dilution.

[1101] This was prepared in Assay Buffer on the day of use.

[1102] Anti-Rabbit HRP Antibody preparation: Optimal assay concentration was found to be a 1 / 5000 dilution.

[1103] This was prepared in Assay Buffer from a single use aliquot on the day of use.

[1104] Propofol Standard preparation:

[1105] Standard series started from a concentration of 4ug / ml.

[1106] Initial stock made up on day of use by weighing out an amount of Propofol (2-3mg) and diluting in Methanol.

[1107] Assay Buffer was used to dilute Propofol to 4ug / ml.

[1108] A serial dilution of 1 in 4 was performed to produce 7 standards in the range 4ug / ml to 0.98ng / ml.

[1109] Assay Buffer used for blank.

[1110] Blocking Buffer preparation:

[1111] 0.1% Milk Powder w / w buffer made up in Buffer 58.

[1112] Assay Procedure

[1113] 1 . 10Oul Propofol-BSA added to each well.

[1114] 2. Incubated at 37°C for 1 hour, no shaking.

[1115] 3. Washed with Assay 3 protocol.*

[1116] 4. 150ul Blocking buffer (0.1% Milk Powder) added to each well.

[1117] 5. Incubated at RT for 30 minutes.

[1118] 6. Washed with Assay 3 protocol.*

[1119] 7. 50ul Propofol standards and samples added at required concentrations (Not required for Objective 2)

[1120] 8. 50ul Anti-Propofol antibody added to each well. (100ul used for assays without standards).

[1121] 9. Incubated at 37°C for 1 hour, 600rpm.

[1122] 10. Washed with Assay 3 protocol.*

[1123] 11. 1 OOul Anti-Rabbit HRP added to each well.

[1124] 12. Incubated at 37°C for 1 hour, no shaking.

[1125] 13. Washed with Assay 3 protocol.* 14. 10Oul Sureblue reserve added to each well.

[1126] 15. Incubated for 10 minutes at room temperature.

[1127] 16. 10Oul Hydrochloric Acid added to each well to quench.

[1128] 17. Plate read at 450nm and results saved to project folder. * Assay 3 protocol = Aspirate, 3x wash with 400ul wash buffer

[1129] Results

[1130] Objective 1 : Generation and testing of affinity column

[1131] Five affinity columns were generated as part of this objective: 3 Propofol-BSA conjugate columns, and 2 Direct Coupling columns. After feedback from the assay team, it was found that the Direct Coupling columns were better at pulling more antibody from the serum, and as such it was decided that, for the final run, the two of them will be linked in series and the serum recirculated - this would ensure that the column is capturing as much of the antibody as possible.

[1132] Objective 2: Depletion of metabolite reactivity

[1133] UVs of the fractionated flowthroughs and of the eluted conjugate were collected, as follows: Concentrations calculated using an extinction coefficient of 1.4.

[1134] Even though the UVs suggest relatively high concentrations of protein being eluted from the column, these do not correlate to the results obtained from Objective 3. One possible explanation for this would be that the columns were also capturing antibodies or other proteins that were not targeting propofol or propofol sulfate specifically. All the elution products, together with the flowthroughs (where applicable), were handed over to the Assay team for testing.

[1135] Objective 3: Confirmation of final product reactivity

[1136] The products of the first and second antibody purifications (DV333 / 52 & DV333 / 65) were tested in a serial dilution starting at a concentration of 1 / 62.5, 4x higher than the normal assay concentration (1 / 500). Of the 3 products of the first purification, eluant 3 showed the highest maximum signal which was approximately 27% of the maximum for the control curve generated using the crude serum.

[1137] All three of the flowthroughs showed a high concentration of propofol specific antibody ranging from 86% to 76% signal relative to the control curve. From this it was determined that the column had not been fully effective in extracting the specific antibody but of the three methods used, direct coupling (Eluant 3) was most effective (Figure 13 & 14).

[1138] For the third purification (DV333 / 80), because of the dilution of the antisera on the column, the purification products were run at a 1 / 50 dilution while the control was run at 1 / 250, a 5x dilution (Figure 15).

[1139] Despite this, there was minimal response from the eluant of the BSA coupled column and while there was some signal from the antibody of the direct coupled column (approximately 20% relative absorbance), this remained much lower than the antibody in the flowthrough.

[1140] For the fourth and fifth purifications (DV333 / 110 &DV333 / 114), the first eluant showed approximately 25% relative absorbance while the second was approximately 6%. The 7I flowthrough was at an 12.5x dilution with no changes to the dilution used for the assay resulting in approximately 56% relative absorbance when compared to the control at the same dilution (Figure 16).

[1141] To confirm that the final product (DV333 / 120) was specific to Propofol it was compared to the metabolites it was found to cross react with in S0M3. This was tested by running serial dilutions of the interferents (Propofol-O-Sulphate and Propofol-O-Glucuronide) at the same concentrations as the standard, the antibody was used at a 1 / 125 dilution (normal dilution 1 / 500) assuming a signal of approximately 25% of the control.

[1142] The eluted antibody showed no reactivity to Propofol-Glucuronide across the concentration range, but the Propofol-Sulphate showed approximately 43% relative displacement at a 50% level. This was retested for confirmation (DV337 / 89) and showed no improvement.

[1143] Conclusions

[1144] The aim of this project was to produce affinity purified antibody which was less reactive to propofol metabolites when compared using the ELISA assay developed and described herein. Several methods were used to produce an affinity column, the products of which were tested on the assay, producing a low yield relative to the control. The final column design used a direct coupled column and recirculated the serum to maximise the column yield. When the final product was compared to Propofol metabolites it was found to no longer have cross-reactivity with Propofol-Glucuronide but remained cross-reactive with Propofol-Sulphate.

[1145] We looked at displacement in ELISA for the four propofol mAbs. We did an initial checkerboard with CE7 to judge where to pitch things and then based on those results have run curves for all four. These results are using 1 ug / mL propofol-BSA coat (100uL per well) with 5ug / mL mAb (50uL mAb + 50uL std). We are getting displacement with all four mAbs (Figure 18).

[1146] Immunoassay development Optimisation work is comparing different polystreptavidin, different nitrocellulose membranes (to optimise speed), and used spiked serum at different concentrations to show that, as expected, for a competitive immunoassay the signal intensity of the test line increases as the concentration of propofol decreases.

[1147] Experimental procedure for comparing Polystreptavidin from BBI and fleet

[1148] The experiment was conducted using two different polystreptavidins, one from BBI and the other provided by fleet. Both were processed identically: 0.5mg / ml of polystreptavidin was plotted, followed by 0.025mg / ml of biotin propofol on a nitrocellulose membrane. GH5 conjugate was sprayed on the conjugate pad. The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad were all assembled into the device as described below:

[1149] A 60cm backing card used to allow adhesion of the following;

[1150] • The NC is placed with the bottom of the NC at 20mm up the backing card

[1151] • 22mm absorbent pad flushed with the top of the backing card to allow 7mm overlap with the NC

[1152] • The 17mm conjugate pad (sprayed with gold) placed at the bottom of the card with 2mm overlap with the NC

[1153] • The 16mm FR1 pad flushed with the bottom of the backing card overlapping with conjugate pad by 4mm

[1154] • Strips were cut at 5mm length and assembled into one-well Kanani devices

[1155] • Sample addition: 30 I of sample + 30 I of buffer after 30 seconds

[1156] • Buffer addition: 30 l of buffers 5mins after the sample addition to allow the plasma to run up the strip

[1157] • Read time: 10 minutes to 15 minutes

[1158] 1 mg / ml solution of free propofol in 100% ethanol was prepared, and propofol standards were subsequently prepared in PBST at concentrations of 1000,100,10,0 ng / ul. Added 80ul of propofol standards to the sample port and allow the sample to run up the strip.

[1159] Result - Figure 19 Polystreptavidin from fleet works best compared to BBI.

[1160] Experimental procedure for comparing different formats of NC

[1161] The experiment was conducted by using different formats of nitrocellulose membranes (CNPC10, CNPC15, CNPF5, CNPF10, 70CNPH, 200CNPH and CN140. All seven of the NC’s were processed identically: 0.5mg / ml of polystreptavidin was plotted, followed by 0.025mg / ml of biotin propofol on a nitrocellulose membrane. GH5 conjugate was sprayed on the conjugate pad. The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad were all assembled into the device.

[1162] Result - Figure 20

[1163] 1 mg / ml solution of free propofol in 100% ethanol was prepared, and propofol standards were subsequently prepared in PBST at concentrations of 1000,100,10,0 ng / ul. Added 80ul of propofol standards to the sample port and allow the sample to run up the strip.

[1164] Experimental procedure for spiked plasma sample as a test medium in different formats of NC.

[1165] The experiment was conducted by using different formats of nitrocellulose membranes (CNPC10, CNPC15, CNPF5, CNPF10, 70CNPH, 200CNPH and CN140. All seven of the NC’s were processed identically: 0.5mg / ml of polystreptavidin was plotted, followed by 0.025mg / ml of biotin propofol on a nitrocellulose membrane. GH5 conjugate was sprayed on the conjugate pad. The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad were all assembled into the device.

[1166] 1 mg / ml solution of free propofol in 100% ethanol was prepared, and propofol standards were subsequently prepared into a human antibody sera sample at concentrations of 1000,100,10,0 ng / ul and then applied 40 L of the spiked sample to the device and followed by 40 L PBST running buffer as a chase buffer to the sera sample. Result - Figure 21

[1167] The signal intensity in the test line increases as the concentration of propofol in the human antibody sera sample decreases from 1000ng / ul to Ong / ul.

[1168] Lateral Flow

[1169] Methodology

[1170] • 0.5mg / ml of Poly streptavidin plotted onto the nitrocellulose membranes (NC95 & NC140) and on top of the Poly streptavidin 0.01 mg / ml & 0.02mg / ml of biotin-propofol were plotted by using Isoflow dispenser.

[1171] • As a positive control 0.7mg / ml of Goat anti-mouse IgG plotted onto the the nitrocellulose membranes (NC95).

[1172] • By using Biodot each of the plotted nitrocellulose membrane were chopped off into 54 pieces.

[1173] • 1 ul of conjugated antibodies (40ug / ml of 4 Abs) and 39ul of PBST were added into the wells.

[1174] • Placed the nitrocellulose membrane strips to each of the wells.

[1175] Result - Figure 22

[1176] FH10 works best among the 4 Monoclonal Antibodies for this test.

[1177] Methodology

[1178] • 0.5mg / ml of Poly streptavidin plotted onto the nitrocellulose membranes (NC140).

[1179] • By using Biodot the plotted nitrocellulose membrane were chopped off into 54 pieces.

[1180] • 1 ul of conjugated antibodies (40ug / ml of 4 Abs) and 39ul of PBST were added into the wells.

[1181] • Placed the nitrocellulose membrane strips to each of the wells.

[1182] Result - Figure 23 • There were no bands because nitrocellulose membrane is plotted only with polystreptavidin.

[1183] Methodology

[1184] • 0.5mg / ml of Poly streptavidin plotted onto the nitrocellulose membranes (NC140) and on top of the Poly streptavidin 0.02mg / ml of biotin-propofol were plotted by using Isoflow dispenser.

[1185] • By using Biodot each of the plotted nitrocellulose membrane were chopped off into 54 pieces.

[1186] • 1ul of FH10 conjugate with 4 different concentrations (10-40ug / ml) and 39ul of PBST were added into the wells.

[1187] • Placed the nitrocellulose membrane strips to each of the wells.

[1188] Result - Figure 24

[1189] • Intensity of bands in the test line is higher when the concentration increases.

[1190] • 40ug / ml has more intensity compared to 10ug / ml.

[1191] Methodology

[1192] • 0.5mg / ml of Poly streptavidin plotted onto the nitrocellulose membranes (NC140) and on top of the Poly streptavidin 0.02mg / ml of biotin-propofol were plotted by using Isoflow dispenser.

[1193] • By using Biodot each of the plotted nitrocellulose membrane were chopped off into 54 pieces.

[1194] • 1 ul of 5EZ conjugate (40ug / ml) and 39u I of PBST were added into the wells.

[1195] • Placed the nitrocellulose membrane strips to each of the wells.

[1196] Result - Figure 25

[1197] 5EZ conjugate which is already were there in Creonate and taken as a negative control and there were no bands in the test line. Methodology

[1198] • 0.5mg / ml of Poly streptavidin plotted on the nitrocellulose membrane (NC140) and on above the Poly streptavidin 0.02mg / ml of biotin-propofol were plotted by using Isoflow dispenser.

[1199] • By using Biodot the plotted nitrocellulose membrane were chopped off into 54 pieces.

[1200] • Prepared 1 mg / ml of free propofol in 100% ethanol and then prepared propofol standards in PBST at different concentrations (10,000, 1000, 100, 10, 1 ,0ng / ul).

[1201] • 1ul of conjugated antibodies (40ug / ml of 4 Abs) and 39ul of propofol standards (6 concentrations) were added into the wells.

[1202] • Placed the nitrocellulose membrane strips to each of the wells.

[1203] Result - Figure 26

[1204] • We can see the bands in the test line with higher intensity when the concentration of propofol reduces from 10,000ng / ul to Ong / ul in CE7, CE8 and GH5 antibodies but not in the FH10.

[1205] • Again FH10 repeated one more time but the result was same.

[1206] • Again repeated with 10ug / ml of FH10 conjugate and two concentrations of propofol (10,000ng / ul & 0 ng / ul). And the result were same that of 40ug / ul FH10 conjugate.

[1207] Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiments shown and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.

Claims

CLAIMS1. An antibody produced by immunisation of a non-human animal with a propofol-4- carboxylic acid or a functional equivalent thereof conjugated to a carrier protein.

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

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

4. An antibody which binds specifically to propofol, said antibody produced against an antigen comprising HS357 conjugated to a carrier protein.

5. An antibody which binds to propofol, said antibody produced against an antigen comprising an analogue of propofol conjugated to a carrier protein.

6. An antibody as claimed in any preceding claim, in which the carrier is KLH.

7. An antibody as claimed in any of claims 1 to 5, in which the carrier is BSA.

8. An antibody as claimed in any preceding claim and being a monoclonal antibody.

9. An antibody as claimed in any of claims 1 to 7 and being a polyclonal antibody.

10. An antibody as claimed in any preceding claim, in which a 6-carbon linker is provided between the antigen and the carrier protein.

11. An antibody as claimed in any preceding claim and selected to be specific to the hapten.

12. An antibody binding to propofol, which comprises 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, SEQ ID NO: 56.

13. An antibody binding to propofol, which comprises 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, SEQ ID NO: 64.

14. An antibody binding to propofol, which comprises 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 , SEQ ID NO: 72.

15. An antibody binding to propofol, which comprises 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, SEQ ID NO: 80.

16. A monoclonal antibody for binding to propofol, the 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 claimed in any preceding claim.

18. An immunoassay as claimed in claim 17 and configured in a plate-based ELISA format.

19. A rapid, point-of-care immune-biosensor for propofol based on an antibody as claimed in any of claims 1 to 16.

20. An immune-biosensor as claimed in claim 19, configured to measure propofol in a blood or blood plasma sample.

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