臺灣博碩士論文加值系統

金黃色葡萄球菌（Staphylococcus aureus）是臨床上常見的革蘭氏陽性細菌。近年來許多研究發現在金黃色葡萄球菌表面會出現α-烯醇酶（Eno1）蛋白，然而Eno1原本是一種分布在細胞質中的糖解酶，其功能主要是在糖解作用中負責催化2-磷酸甘油酸（2-phosphoglycerate）轉換成磷酸烯醇式丙酮酸（phosphoenolpyruvate）。更進一步的研究發現金黃色葡萄球菌的Eno1 （SaEno1）中能夠作為多種細胞外基質（extracellular matrix）的受體，進而提升病原菌感染及侵入宿主的能力。以SaEno1作為抗原開發其相對應的單鏈抗體（scFv）具有作為醫療診斷以及治療藥物的潛力與價值。先前我們將重組SaEno1免疫母雞，取其脾臟細胞建構anti-SaEno1 scFv的抗體基因庫，再藉由噬菌體展現技術篩選出了10個anti-SaEno1 scFv單株抗體。本篇論文進一步針對這10個抗體進行表現與純化，但其中只有9個anti-SaEno1 scFv能夠正確表現。以Western以及ELISA確定9個anti-SaEno1 scFv能與重組SaEno1結合，而這9個單株抗體的解離常數（Kd value）分別為SaS1＝8.76 x 10-7；SaS2＝6.16 x 10-7；SaS14＝1.46 x 10-5；SaS15＝1.01 x 10-5；SaS18＝6.93 x 10-6；SaS38＝2.11 x 10-5；SaL2＝2.93 x 10-7；SaL4＝1.07 x 10-4；SaL7＝3.93 x 10-5（M）。後續也針對結合能力較好的SaS1、SaS2、SaL2以epitope mapping的方式得知抗其原結合位在SaEno1序列上的位置，分別為序號302~375、6~146、201~3012的胺基酸區間。另外也透過與Streptococcus pneumoniae、Candida albicans、Homo sapiens、murine的Eno1交叉反應實驗發現SaL2會與SpEno1結合，SaS1、SaS2則不與其他物種的Eno1結合。最後我們也驗證SaS1、SaS2、SaL2是真的能辨識S. aureus臨床菌株的SaEno1，且無論MSSA、MRSA都能辨識。同時，也進行Cell ELISA以及Flow cytometry確定SaS1、SaS2、SaL2是能100 %與S. aureus表面的SaEno1結合且抗體在flow cytometry有90 %的陽性訊號。綜合本篇論文的研究成果，我們認為這三個anti-SaEno1 scFv：SaS1、SaS2、SaL2，在未來具有潛力開發為臨床鑑定用或作為抗生素的替代藥物。

Staphylococcus aureus is well known as a common gram-positive bacterium in clinic. In recent years, many studies have found that α-enolase (Eno1) protein appears on the surface of Staphylococcus aureus. However, Eno1 is used to be a glycolytic enzyme distributed in the cytoplasm, and its function is mainly to catalyze 2- phosphoglycerate into phosphoenolpyruvate during glycolysis. Further studies have found that Staphylococcus aureus Eno1 (SaEno1) can act as a receptor for multiple ECMs, thereby enhancing the pathogen's ability to infect and invade the host. Therefore, we believe that base on SaEno1 to develop its corresponding single-chain antibody (scFv) has its potential and value. Therefore, we immunized recombinant SaEno1 into hens and constructed anti-SaEno1 scFv antibody gene library through the spleen cells. Then selected 10 anti-SaEno1 scFv monoclonal antibodies by phage display technology. This thesis has further performed the expression and purification of these 10 antibodies, but only 9 anti-SaEno1 scFvs were able to expression. 9 anti-SaEno1 scFv has shown the binding ability against recombinant SaEno1 during Western blot and ELISA. The Kd value of these 9 monoclonal antibodies are SaS1＝8.76 x 10-7；SaS2＝6.16 x 10-7；SaS14＝1.46 x 10-5；SaS15＝1.01 x 10-5；SaS18＝6.93 x 10-6；SaS38＝2.11 x 10-5；SaL2＝2.93 x 10-7；SaL4＝1.07 x 10-4；SaL7＝3.93 x 10-5 (M). SaS1, SaS2, and SaL2 had shown better binding ability and the region of their epitope on the SaEno1 was obtained by epitope mapping, which are 302~375, 6~146, and 201~3012 amino acids, respectively. In addition, through the cross-reaction assay with Eno1 of Streptococcus pneumoniae, Candida albicans, Homo sapiens, and murine, we found that SaL2 would cross-react to SpEno1, while SaS1 and SaS2 would not bind to Eno1 of other species. Finally, we also verified that SaS1, SaS2, and SaL2 can really identify the SaEno1 of clinical strain of S. aureus, and both MSSA & MRSA can be identified. At the same time, Cell ELISA and Flow cytometry were also performed to confirm that SaS1, SaS2, and SaL2 can bind to SaEno1 on the surface of S. aureus and the antibody response well. Based on the research results of this thesis, we believe that these three anti-SaEno1 scFvs: SaS1, SaS2, and SaL2 have the potential to be developed for clinical diagnosis or as an alternative to antibiotics in the future.

目錄
中文摘要 1
英文摘要 2
壹、背景 3
1.1 金黃色葡萄球菌 3
1.2 烯醇酶蛋白 4
1.3 金黃色葡萄球菌表面α-烯醇酶蛋白的重要性 5
1.4 抗原免疫來亨雞與IgY抗體 6
1.5 單鏈抗體 6
貳、研究目的 8
參、實驗材料 9
3.1化學藥品 9
3.2抗體類 10
3.3其他類 11
3.4培養基 11
3.5緩衝溶液 12
肆、實驗方法 15
4.1 表現anti-SaEno1 scFv蛋白與分析測試 15
4.2 純化anti-SaEno1 scFv蛋白與分析測試 16
4.3 測試anti-SaEno1 scFv與重組SaEno1之結合 18
4.4 分析anti-SaEno1 scFv與重組SaEno1之結合效價 20
4.5 推估anti-SaEno1 scFv的解離常數 20
4.6 分析SaS1, SaS2, SaL2之抗原結合位 22
4.7 測試SaS1, SaS2, SaL2與其它物種的重組Eno1蛋白之交叉反應 24
4.8 測試SaS1, SaS2, SaL2與S. aureus的Eno1之結合 25
4.9 測試anti-SaEno1 scFv（SaS1, SaS2, SaL2）與S. aureus表面Eno1之結合 26
伍、實驗結果 29
5.1 表現anti-SaEno1 scFv蛋白與分析測試 29
5.2純化anti-SaEno1 scFv蛋白與分析測試 30
5.3測試anti-SaEno1 scFv與重組SaEno1之結合 31
5.4 分析anti-SaEno1 scFv與重組SaEno1之結合效價 33
5.5 推估anti-SaEno1 scFv的解離常數 33
5.6 分析SaS1, SaS2, SaL2之抗原結合位 35
5.7 測試SaS1, SaS2, SaL2與其它物種的重組Eno1蛋白之交叉反應 37
5.8 測試anti-SaEno1 scFv（SaS1, SaS2, SaL2）與S. aureus之結合 38
5.9 測試anti-SaEno1 scFv（SaS1, SaS2, SaL2）與S. aureus表面Eno1之結合 39
陸、結論與討論 41
柒、圖表 49
捌、表格 68
玖、附錄 71
拾、參考資料 78

 
圖目錄
Figure 1. Expression of anti-SaEno1 scFvs by SDS-PAGE & Western bolt 49
Figure 2. Purification of anti-SaEno1 scFvs 50
Figure 3. Identification of purified anti-SaEno1 scFvs 51
Figure 4. Binding assay of anti-SaEno1 scFvs by Western 52
Figure 5. Binding assay of anti-SaEno1 scFvs by ELISA 53
Figure 6. Titration assay of anti-SaEno1 scFvs 54
Figure 7. Kd determination of anti-SaEno1 scFvs by competitive ELISA 55
Figure 8. Inhibitory percentage of binding activity of anti-SaEno1 scFvs competitive ELISA 56
Figure 9. Schematic illustration of truncated SaEno1 fragments 57
Figure 10. Epitope mapping on SaEno1 using anti-SaEno1 scFvs by Western 58
Figure 11. Epitope mapping on SaEno1 using anti-SaEno1 scFvs by ELISA 59
Figure 12. Predicted epitope of SaS1 60
Figure 13. Predicted epitope of SaS2 61
Figure 14. Predicted epitope of SaL2 62
Figure 15. Cross reaction of anti-SaEno1 scFvs against 5 species ENO1 by Western 63
Figure 16. Cross reaction of anti-SaEno1 scFvs against 5 species ENO1 by ELISA 64
Figure 17. Binding assay of anti-SaEno1 scFvs against S. aureus 65
Figure 18. Cell ELISA of anti-SaEno1 scFvs against S. aureus 66
Figure 19. Anti-SaEno1 scFvs against S. aureus by Flow cytometry 67


表目錄
Table 1. Prediction of dissociation constant (Kd value) 68
Table 2. Identity percentage of CaEno1, SpEno1, SaEno1, hEno1, mEno1 amino acid sequence comparison 69
Table 3. Summarization of the experiment 70

1.Masalha, M., et al., Analysis of transcription of the Staphylococcus aureus aerobic class Ib and anaerobic class III ribonucleotide reductase genes in response to oxygen. J Bacteriol, 2001. 183(24): p. 7260-72.
2.Kluytmans, J., A. van Belkum, and H. Verbrugh, Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev, 1997. 10(3): p. 505-20.
3.Tong, S.Y., et al., Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev, 2015. 28(3): p. 603-61.
4.Matthews, K.R., et al., Identification and Differentiation of Coagulase-Negative Staphylococcus aureus by Polymerase Chain Reaction. J Food Prot, 1997. 60(6): p. 686-688.
5.Munir, M.T., et al., Experimental Parameters Influence the Observed Antimicrobial Response of Oak Wood (Quercus petraea). Antibiotics (Basel), 2020. 9(9).
6.Curran, J.P. and F.L. Al-Salihi, Neonatal staphylococcal scalded skin syndrome: massive outbreak due to an unusual phage type. Pediatrics, 1980. 66(2): p. 285-90.
7.Bath-Hextall, F.J., et al., Interventions to reduce Staphylococcus aureus in the management of atopic eczema: an updated Cochrane review. Br J Dermatol, 2011. 164(1): p. 228.
8.Wakabayashi, Y., et al., Staphylococcal food poisoning caused by Staphylococcus argenteus harboring staphylococcal enterotoxin genes. Int J Food Microbiol, 2018. 265: p. 23-29.
9.Latha, T., et al., MRSA: the leading pathogen of orthopedic infection in a tertiary care hospital, South India. Afr Health Sci, 2019. 19(1): p. 1393-1401.
10.Murray, R.J., Staphylococcus aureus infective endocarditis: diagnosis and management guidelines. Intern Med J, 2005. 35 Suppl 2: p. S25-44.
11.Silversides, J.A., E. Lappin, and A.J. Ferguson, Staphylococcal toxic shock syndrome: mechanisms and management. Curr Infect Dis Rep, 2010. 12(5): p. 392-400.
12.Olson, M.E. and A.R. Horswill, Staphylococcus aureus osteomyelitis: bad to the bone. Cell Host Microbe, 2013. 13(6): p. 629-31.
13.Rasmussen, R.V., et al., Future challenges and treatment of Staphylococcus aureus bacteremia with emphasis on MRSA. Future Microbiol, 2011. 6(1): p. 43-56.
14.Mitchell, D.H. and B.P. Howden, Diagnosis and management of Staphylococcus aureus bacteraemia. Intern Med J, 2005. 35 Suppl 2: p. S17-24.
15.Hussain, Z., et al., Evaluation of screening and commercial methods for detection of methicillin resistance in coagulase-negative staphylococci. J Clin Microbiol, 1998. 36(1): p. 273-4.
16.Ng, C.Y., et al., Risks for Staphylococcus aureus colonization in patients with psoriasis: a systematic review and meta-analysis. Br J Dermatol, 2017. 177(4): p. 967-977.
17.McDonald, L.C., et al., The status of antimicrobial resistance in Taiwan among Gram-positive pathogens: the Taiwan Surveillance of Antimicrobial Resistance (TSAR) programme, 2000. Int J Antimicrob Agents, 2004. 23(4): p. 362-70.
18.Jean, S.S., et al., Susceptibility of clinical isolates of meticillin-resistant Staphylococcus aureus and phenotypic non-extended-spectrum beta-lactamase-producing Klebsiella pneumoniae to ceftaroline in Taiwan: Results from Antimicrobial Testing Leadership and Surveillance (ATLAS) in 2012-2018 and Surveillance of Multicentre Antimicrobial Resistance in Taiwan (SMART) in 2018-2019. Int J Antimicrob Agents, 2020. 56(1): p. 106016.
19.McGuinness, W.A., N. Malachowa, and F.R. DeLeo, Vancomycin Resistance in Staphylococcus aureus. Yale J Biol Med, 2017. 90(2): p. 269-281.
20.Wold, F. and C.E. Ballou, Studies on the enzyme enolase. I. Equilibrium studies. J Biol Chem, 1957. 227(1): p. 301-12.
21.Wold, F. and C.E. Ballou, Studies on the enzyme enolase. II. Kinetic studies. J Biol Chem, 1957. 227(1): p. 313-28.
22.Clegg, N., et al., Molecular characterization of prostatic small-cell neuroendocrine carcinoma. Prostate, 2003. 55(1): p. 55-64.
23.Peshavaria, M. and I.N. Day, Molecular structure of the human muscle-specific enolase gene (ENO3). Biochem J, 1991. 275 ( Pt 2): p. 427-33.
24.Pancholi, V., Multifunctional alpha-enolase: its role in diseases. Cell Mol Life Sci, 2001. 58(7): p. 902-20.
25.Petrak, J., et al., Deja vu in proteomics. A hit parade of repeatedly identified differentially expressed proteins. Proteomics, 2008. 8(9): p. 1744-9.
26.Thu Nguyen, T.T., et al., alpha-Enolase as a novel vaccine candidate against Streptococcus dysgalactiae infection in cobia (Rachycentron canadum L.). Fish Shellfish Immunol, 2020. 98: p. 899-907.
27.Krucinska, J., et al., Structural and Functional Studies of Bacterial Enolase, a Potential Target against Gram-Negative Pathogens. Biochemistry, 2019. 58(9): p. 1188-1197.
28.Satala, D., et al., Structural Insights into the Interactions of Candidal Enolase with Human Vitronectin, Fibronectin and Plasminogen. Int J Mol Sci, 2020. 21(21).
29.Toledo, A., et al., The enolase of Borrelia burgdorferi is a plasminogen receptor released in outer membrane vesicles. Infect Immun, 2012. 80(1): p. 359-68.
30.Floden, A.M., J.A. Watt, and C.A. Brissette, Borrelia burgdorferi enolase is a surface-exposed plasminogen binding protein. PLoS One, 2011. 6(11): p. e27502.
31.Placzkiewicz, J., et al., Lactobacillus crispatus and its enolase and glutamine synthetase influence interactions between Neisseria gonorrhoeae and human epithelial cells. J Microbiol, 2020. 58(5): p. 405-414.
32.Kishimoto, N., et al., Alpha-enolase in viral target cells suppresses the human immunodeficiency virus type 1 integration. Retrovirology, 2020. 17(1): p. 31.
33.Song, Z., et al., alpha-Enolase, an adhesion-related factor of Mycoplasma bovis. PLoS One, 2012. 7(6): p. e38836.
34.Lopez-Lopez, M.J., et al., Biochemical and Biophysical Characterization of the Enolase from Helicobacter pylori. Biomed Res Int, 2018. 2018: p. 9538193.
35.Silva, R.C., et al., Extracellular enolase of Candida albicans is involved in colonization of mammalian intestinal epithelium. Front Cell Infect Microbiol, 2014. 4: p. 66.
36.Chandran, V. and B.F. Luisi, Recognition of enolase in the Escherichia coli RNA degradosome. J Mol Biol, 2006. 358(1): p. 8-15.
37.Yan, H., et al., Both Enolase and the DEAD-Box RNA Helicase CrhB Can Form Complexes with RNase E in Anabaena sp. Strain PCC 7120. Appl Environ Microbiol, 2020. 86(13).
38.Wu, Y., et al., Octameric structure of Staphylococcus aureus enolase in complex with phosphoenolpyruvate. Acta Crystallogr D Biol Crystallogr, 2015. 71(Pt 12): p. 2457-70.
39.Lopes, J.D., M. dos Reis, and R.R. Brentani, Presence of laminin receptors in Staphylococcus aureus. Science, 1985. 229(4710): p. 275-7.
40.Molkanen, T., et al., Enhanced activation of bound plasminogen on Staphylococcus aureus by staphylokinase. FEBS Lett, 2002. 517(1-3): p. 72-8.
41.Boyle, M.D. and R. Lottenberg, Plasminogen activation by invasive human pathogens. Thromb Haemost, 1997. 77(1): p. 1-10.
42.Antikainen, J., et al., Enolases from Gram-positive bacterial pathogens and commensal lactobacilli share functional similarity in virulence-associated traits. FEMS Immunol Med Microbiol, 2007. 51(3): p. 526-34.
43.Glowalla, E., et al., Proteomics-based identification of anchorless cell wall proteins as vaccine candidates against Staphylococcus aureus. Infect Immun, 2009. 77(7): p. 2719-29.
44.Artini, M., et al., A new anti-infective strategy to reduce adhesion-mediated virulence in Staphylococcus aureus affecting surface proteins. Int J Immunopathol Pharmacol, 2011. 24(3): p. 661-72.
45.Hajighahramani, N., et al., Immunoinformatics analysis and in silico designing of a novel multi-epitope peptide vaccine against Staphylococcus aureus. Infect Genet Evol, 2017. 48: p. 83-94.
46.Xu, Y., et al., Application of chicken egg yolk immunoglobulins in the control of terrestrial and aquatic animal diseases: a review. Biotechnol Adv, 2011. 29(6): p. 860-8.
47.Zhang, W.W., The use of gene-specific IgY antibodies for drug target discovery. Drug Discov Today, 2003. 8(8): p. 364-71.
48.Schade, R., et al., [Avian egg yolk antibodies. The egg laying capacity of hens following immunisation with antigens of different kind and origin and the efficiency of egg yolk antibodies in comparison to mammalian antibodies]. ALTEX, 1994. 11(2): p. 75-84.
49.Larsson, A., D. Carlander, and M. Wilhelmsson, Antibody response in laying hens with small amounts of antigen. Food and Agricultural Immunology, 1998. 10(1): p. 29-36.
50.Gassmann, M., et al., Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. FASEB J, 1990. 4(8): p. 2528-32.
51.Huston, J.S., et al., Protein engineering of single-chain Fv analogs and fusion proteins. Methods Enzymol, 1991. 203: p. 46-88.
52.Bird, R.E., et al., Single-chain antigen-binding proteins. Science, 1988. 242(4877): p. 423-6.
53.Takkinen, K., et al., An active single-chain antibody containing a cellulase linker domain is secreted by Escherichia coli. Protein Eng, 1991. 4(7): p. 837-41.
54.Argos, P., An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol, 1990. 211(4): p. 943-58.
55.Whitlow, M., et al., An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability. Protein Eng, 1993. 6(8): p. 989-95.
56.Smith, G.P., Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science, 1985. 228(4705): p. 1315-7.
57.McCafferty, J., et al., Phage antibodies: filamentous phage displaying antibody variable domains. Nature, 1990. 348(6301): p. 552-4.
58.Ho, M., S. Nagata, and I. Pastan, Isolation of anti-CD22 Fv with high affinity by Fv display on human cells. Proc Natl Acad Sci U S A, 2006. 103(25): p. 9637-42.
59.Feldhaus, M.J. and R.W. Siegel, Yeast display of antibody fragments: a discovery and characterization platform. J Immunol Methods, 2004. 290(1-2): p. 69-80.
60.Galeffi, P., et al., Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems. J Transl Med, 2006. 4: p. 39.
61.Choo, A.B., et al., Soluble expression of a functional recombinant cytolytic immunotoxin in insect cells. Protein Expr Purif, 2002. 24(3): p. 338-47.
62.Vaks, L. and I. Benhar, Production of stabilized scFv antibody fragments in the E. coli bacterial cytoplasm. Methods Mol Biol, 2014. 1060: p. 171-84.
63.Cariccio, V.L., et al., Phage display revisited: Epitope mapping of a monoclonal antibody directed against Neisseria meningitidis adhesin A using the PROFILER technology. MAbs, 2016. 8(4): p. 741-50.
64.Marks, J.D., et al., By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol, 1991. 222(3): p. 581-97.
65.Griffiths, A.D. and A.R. Duncan, Strategies for selection of antibodies by phage display. Curr Opin Biotechnol, 1998. 9(1): p. 102-8.
66.Lake, D.F., et al., Molecular cloning, expression and mutagenesis of an anti-insulin single chain Fv (scFv). Mol Immunol, 1994. 31(11): p. 845-56.
67.Sharon, J., Structural correlates of high antibody affinity: three engineered amino acid substitutions can increase the affinity of an anti-p-azophenylarsonate antibody 200-fold. Proc Natl Acad Sci U S A, 1990. 87(12): p. 4814-7.
68.Dai, K., H. Zhu, and C. Ruan, Generation and characterization of recombinant single chain Fv antibody that recognizes platelet glycoprotein Ibalpha. Thromb Res, 2003. 109(2-3): p. 137-44.
69.Ravn, P., et al., Multivalent scFv display of phagemid repertoires for the selection of carbohydrate-specific antibodies and its application to the Thomsen-Friedenreich antigen. J Mol Biol, 2004. 343(4): p. 985-96.
70.Sakai, K., et al., Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 1. A new strategy for generation of anti-carbohydrate antibodies. Biochemistry, 2007. 46(1): p. 253-62.
71.Shadidi, M. and M. Sioud, An anti-leukemic single chain Fv antibody selected from a synthetic human phage antibody library. Biochem Biophys Res Commun, 2001. 280(2): p. 548-52.
72.Reiche, N., et al., Generation and characterization of human monoclonal scFv antibodies against Helicobacter pylori antigens. Infect Immun, 2002. 70(8): p. 4158-64.
73.Li, T.W., et al., Development of single-chain variable fragments (scFv) against influenza virus targeting hemagglutinin subunit 2 (HA2). Arch Virol, 2016. 161(1): p. 19-31.
74.Ahmad, Z.A., et al., scFv antibody: principles and clinical application. Clin Dev Immunol, 2012. 2012: p. 980250.
75.Kontermann, R.E., M.G. Wing, and G. Winter, Complement recruitment using bispecific diabodies. Nat Biotechnol, 1997. 15(7): p. 629-31.
76.Emanuel, P.A., et al., Recombinant antibodies: a new reagent for biological agent detection. Biosens Bioelectron, 2000. 14(10-11): p. 751-9.
77.Kerschbaumer, R.J., et al., Single-chain Fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay. Anal Biochem, 1997. 249(2): p. 219-27.
78.Oelschlaeger, P., et al., Fluorophor-linked immunosorbent assay: a time- and cost-saving method for the characterization of antibody fragments using a fusion protein of a single-chain antibody fragment and enhanced green fluorescent protein. Anal Biochem, 2002. 309(1): p. 27-34.
79.Cho, S.H., et al., Development of novel detection system for sweet potato leaf curl virus using recombinant scFv. Sci Rep, 2020. 10(1): p. 8039.
80.Lichty, J.J., et al., Comparison of affinity tags for protein purification. Protein Expr Purif, 2005. 41(1): p. 98-105.
81.Zhao, Q., et al., One-step expression and purification of single-chain variable antibody fragment using an improved hexahistidine tag phagemid vector. Protein Expr Purif, 2009. 68(2): p. 190-5.
82.Kimple, M.E., A.L. Brill, and R.L. Pasker, Overview of affinity tags for protein purification. Curr Protoc Protein Sci, 2013. 73: p. 9 9 1-9 9 23.
83.Forsstrom, B., et al., Dissecting antibodies with regards to linear and conformational epitopes. PLoS One, 2015. 10(3): p. e0121673.
84.Friguet, B., et al., Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods, 1985. 77(2): p. 305-19.
85.Benedict, C.A., A.J. MacKrell, and W.F. Anderson, Determination of the binding affinity of an anti-CD34 single-chain antibody using a novel, flow cytometry based assay. J Immunol Methods, 1997. 201(2): p. 223-31.
86.Sato, R., et al., Preparation and characterization of anti-tissue factor single-chain variable fragment antibody for cancer diagnosis. Cancer Sci, 2014. 105(12): p. 1631-7.
87.Tadokoro, T., et al., Biophysical characterization and single-chain Fv construction of a neutralizing antibody to measles virus. FEBS J, 2020. 287(1): p. 145-159.
88.Schodin, B.A. and D.M. Kranz, Binding affinity and inhibitory properties of a single-chain anti-T cell receptor antibody. J Biol Chem, 1993. 268(34): p. 25722-7.
89.MacKenzie, C.R., et al., Analysis by surface plasmon resonance of the influence of valence on the ligand binding affinity and kinetics of an anti-carbohydrate antibody. J Biol Chem, 1996. 271(3): p. 1527-33.
90.Sykes, K.F., J.B. Legutki, and P. Stafford, Immunosignaturing: a critical review. Trends Biotechnol, 2013. 31(1): p. 45-51.
91.Avilan, L., et al., Enolase: a key player in the metabolism and a probable virulence factor of trypanosomatid parasites-perspectives for its use as a therapeutic target. Enzyme Res, 2011. 2011: p. 932549.
92.Lagier, J.C., et al., Current and past strategies for bacterial culture in clinical microbiology. Clin Microbiol Rev, 2015. 28(1): p. 208-36.
93.Bouzid, D., et al., Rapid diagnostic tests for infectious diseases in the emergency department. Clin Microbiol Infect, 2021. 27(2): p. 182-191.
94.Challa, S., et al., Diagnosis of filamentous fungi on tissue sections by immunohistochemistry using anti-aspergillus antibody. Med Mycol, 2015. 53(5): p. 470-6.
95.Kozel, T.R. and B. Wickes, Fungal diagnostics. Cold Spring Harb Perspect Med, 2014. 4(4): p. a019299.
96.Foulston, L., et al., The extracellular matrix of Staphylococcus aureus biofilms comprises cytoplasmic proteins that associate with the cell surface in response to decreasing pH. mBio, 2014. 5(5): p. e01667-14.
97.Song, Y., et al., Multifunctional Bismuth Oxychloride/Mesoporous Silica Composites for Photocatalysis, Antibacterial Test, and Simultaneous Stripping Analysis of Heavy Metals. ACS Omega, 2018. 3(1): p. 973-981.
98.Cronin, U.P., et al., Protein A-Mediated Binding of Staphylococcus spp. to Antibodies in Flow Cytometric Assays and Reduction of This Binding by Using Fc Receptor Blocking Reagent. Appl Environ Microbiol, 2020. 86(17).
99.Carneiro, C.R., et al., Identification of enolase as a laminin-binding protein on the surface of Staphylococcus aureus. Microbes Infect, 2004. 6(6): p. 604-8.