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Annex I
Table 1. Reaction buffers compositions.
1x PBS
137 mM Sodium Chloride, 10 mM phosphate, 2.7 mM
Potassium Chloride, pH 7.4
Gliadin immobilization buffer
PBS buffer, 70% ethanol, 10% DMSO
Binding buffer
20 mM Tris-HCl, 2 mM EDTA, 2 M NaCl, 0.2% (v/v)
Tween 20, pH 7.4
His tag binding buffer
20 mM sodium phosphate buffer, 0.5 M NaCl, 5 mM
imidazole, pH 7.4
His tag wash buffer
20 mM sodium phosphate buffer, 0.5 M NaCl, 20 mM
imidazole, pH 7.4
His tag elution buffer
20 µL, 20 mM sodium phosphate buffer, 0.5 M NaCl, 250
mM imidazole, pH 7.4
SDS/DTT elution buffer
1% (v/v) SDS, 50 mM DTT, 50 mM Tris-HCl, 0.5 M
NaCl, 1mM EDTA, 0.05% (v./v) Tween 20, pH 7.4
Table 2. Sequences of the DNA fragments used in constructing VHH DNA.
sequence（5’ to 3’）
Fragment name
T7 promoter, omega (Ω) GATCCCGCGAAATTAATACGACTCACTATAGGGGAAGTAT
enhancer,
Kozak TTTTACAACAATTACCAACAACAACAACAAACAACAACA
consensus sequence
His-tag,
and
ACATTACATTTTACATTCTACAACTACAAGCCACCATG
linker GGGGGAGGCAGCCATCATCATCATCATCACGGCGGAAGC
83
hybridization
region AGGACGGGGGGCGGCGTGGAAA
(LHR)
Table 3. Primers used in the study.
Primer name
sequence（5’ to 3’）
CTGCTCCTCGCGGCCCAGCCGGCCATGGCTSAGKTGCAG
Alp-Sfi-VHH-F1
CTCGTGGAGTC
TTTGCTCTGCGGCCGCAGAGGCCGTGGGGTCTTCGCTGT
Alp-Sfi-SHinge-R
GGTGCG
TTTGCTCTGCGGCCGCAGAGGCCGATTGTGGTTTTGGTGT
Alp-LHinge-R
CTTGGG
Alp-FR3-overlap-F
TCCGTGAAGGGCCGATTC
Alp-FR3-overlap-R
GAATCGGCCCTTCACGGA
New left-F
GATCCCGCGAAATTAATACGACTCACTATAGGG
cnvKpolyA-R
TTTCCACGCCGCCCCCCGTCCT
qPCR VHH 1 Forward
CAAGGCCAAGAACACGGTGTATC
qPCR VHH 1 Reverse
TCGAGATCTTCCTCTCCACGTACTAC
qPCR VHH 2 Forward
CGAATGGCGGCACTATCACA
qPCR VHH 2 Reverse
CTCCAGCTGATAGCGGCTACAA
qPCR VHH 3 Forward
GCTCTAATTCGATGGCCTGGTT
qPCR VHH 3 Reverse
CCACATAGCCCGTGTTACCATTTC
BDA_qPCR (+)
CTACAAGCCACCATGGATAAC
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BDA_qPCR (-)
GCTTGGGTCATCTTTTAGGC
GFPVHHqPCRFw
AACACCATCCTGGGCGATAG
GFPVHHqPCRRv
GTGTTTTTGGCGCGATCAC
Table 4. Amino Acid sequences of the candidate VHHs.
Fragment name
Amino Acid sequence
AEVQLVESGGGLVQAGGSLRLSCAINDRTFSNYSMGWFR
QAPGKEREFVAAITHNGSTNFPDSVKGRFTISVDKAKNTV
VHH1
YLQMNSLKPEDTAVYYCAVDHSFITVVRGEEDLEVWGQ
GTLVTVSSAHHSEDPT
AEVQLVESGGGSVQAGGSLRLSCSASGPEWRHYHMGWF
RQPPGKEREFVAAISWSGGTTMYADSVKGRFTISRDNVK
VHH2
NTVYLQMNSLKPEDTAVYYCAAGDTVVALLDYRAYWG
QGTQVTVSSEPKTPKPQS
AEVQLVESGGDLVQPGGSLNLSCVADATIFGSNSMAWFR
QYPGKQRDLLATVARNGNTGYVDSVKGRFTISRDDGQNI
VHH3
VYLQMNSLKPEDTALYTCNLKRYRMGFILDGDYWGQGT
QVTVSSEPKTPKPQS
Table 5. Abbreviations
VHH
Camelid heavy-chain antibody VH
cDNA
Complementary DNA
85
PCR
Polymerase chain reaction
cD-iPCR
cDNA display mediated immuno-polymerase chain reaction
mRNA
Messenger RNA
ELISA
Enzyme-linked immunosorbent assay
CDR
complementarity determining region
cnvK
3-cyanovinylcarbazole nucleoside
qPCR
quantitative polymerase chain reaction
PAGE
Polyacrylamide gel electrophoresis
SDS
sodium dodecyl sulfate
TBE
Tris/Borate/EDTA
MES
2-(N-morpholino)ethanesulfonic acid
PBS
Phosphate-buffered saline
UV
Ultraviolet
FITC
Fluorescein isothiocyanate
SA beads
Streptavidin Magnetic Beads
Ni-NTA
nickel-charged affinity resin
DTT
Dithiothreitol
NGS
Next-generation Sequencing
BSA
Bovine serum albumin
HPR
horseradish peroxidase enzyme
Ct
Cycle threshold
DMSO
Dimethyl sulfoxide
GFP
Green Fluorescent Protein
86
BDA
B Domain protein A
IgG
Immunoglobulin G
Table 6. ELISA absorbance data (for Figure 2.11A)
VHH
Gliadin
concentration
VHH1
+ Gliadin
+ Gliadin
VHH2
VHH3
µg/mL
+ Gliadin
Anti-BSA
+ BSA + AntiBSA VHH
VHH
0.049
0.044
0.048
0.036
0.031
0.15
0.087
0.090
0.037
0.033
0.258
0.31
0.124
0.116
0.041
0.033
0.391
0.62
0.194
0.151
0.043
0.033
0.625
1.25
0.298
0.227
0.051
0.038
0.955
2.5
0.458
0.354
0.084
0.051
1.581
0.535
0.484
0.120
0.054
2.024
10
0.733
0.684
0.218
0.072
2.148
Table 7. Standard deviation data for Figure 3.5
Spiked
Concentration VHH1
VHH2
(ng/mL)
Rice
Buffer
Rice
Buffer
0.175
0.116
0.032
0.045
0.05
0.202
0.067
0.112
1.259
0.5
0.087
0.282
0.342
0.195
0.627
0.050
0.813
0.227
87
Table 8. Primers used in the NGS analysis.
Primer name
sequence（5’ to 3’）
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNN
prRd1N4VHH-F
NATGGCTGAGGTGCAGCTCGTG
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNN
prRd2N4VHH-R
NNTGATGATGATGGCTACCACCTCCCG
CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCT
prNexteraXTIndex1(N701)
CGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCT
prNexteraXTIndex1(N702)
CGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCT
prNexteraXTIndex1(N703)
CGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCT
prNexteraXTIndex1(N704)
CGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCT
prNexteraXTIndex1(N705)
CGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCT
prNexteraXTIndex1(N706)
CGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCT
prNexteraXTIndex1(N707)
CGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATCCTCTCTGGTCT
prNexteraXTIndex1(N708)
CGTGGGCTCGG
88
CAAGCAGAAGACGGCATACGAGATAGCGTAGCGTCT
prNexteraXTIndex1(N709)
CGTGGGCTCGG
AATGATACGGCGACCACCGAGATCTACACTAGATCGC
prNexteraXTIndex2(N501)
TCGTCGGCAGCGTC
Materials and Reagents
Note: All reagents should be of molecular biology grade to avoid contamination of ribonuclease.
1. Polystyrene microtiter plate (MICROLON, 96 Well Single-Break Strip Plate, PS), Greiner
Bio-One 705071.
2. PCR tubes (End point PCR tubes; TreffLab Laboratory Consumables, catalog number:
96.09852.9.01, qPCR strip tubes; SSIBio, catalog number: 3248-00).
3. DNA oligos can be obtained from custom DNA synthesis service. Eurofins Genomics (Otaku, Tokyo, Japan), Store at -20 °C.
4. PrimeSTAR HS DNA polymerase (Takara, catalog number: R010A). Store at -20 °C.
5. FavorPrep PCR Clean-Up Mini Kit (Favorgen). Store at room temperature.
6. RiboMAXTM Large-Scale RNA Production System T7 (Promega, catalog number: P1300).
Store at -20 °C.
7. Wheat gliadin (RT) (Sigma, catalog number: G3375)
8. Dynabeads MyOne carboxylic acid beads (Invitrogen, catalog number: 65012).
9. RNA Clean-Up Kit (Favorgen, catalog number: FAPCK001), store at 4 °C.
10. Nuclease treated Rabbit Reticulocyte Lysate System, (Promega, catalog number: L4960).
Amino acid mixtures for translation are included. Store at -80 °C.
11. RNasin® Ribonuclease Inhibitor (Promega; catalog number: N2111). Store at -20 °C.
89
12. Dynabeads MyOne streptavidin C1 (VERITAS; catalog number: DB65002). Store at 4 °C.
13. ReverTra Ace® (Toyobo, catalog number: TRT-101). 5× RT Buffer and 2.5 mM each dNTP
mix are included. Store at -20 °C.
14. His Mag Sepharose Ni (GE Healthcare, catalog number: 2896390)
15. RNase T1 (1,000 U/L) (Thermo Fisher Scientific, catalog number: EN0541)
16. RNase H (Takara, catalog number: 2150A, 10 U) and 10× NE buffer 2 (NEB)
17. DL-Dithiothreitol ≥ 99.0% (RT) (Sigma, catalog number: 4381)
18. Quick-Load 100 bp DNA Ladder, (Biolabs: catalog number: N0467), NEB
19. SYBR Gold Nucleic Acid Gel Strain (Invitrogen, catalog number: S11494)
20. THUNDERBIRD SYBR qPCR Mix (Toyobo, catalog number: QPS-201)
21. 10x PBS (Phosphate-buffered saline -) (Wako, catalog number: 163-25265)
22. Skim milk (Wako, catalog number: 190-12865)
23. NaCl (Wako, catalog number: 191-01665)
24. Tris-HCl (Wako, catalog number: 208-14691)
25. KCl (Wako, catalog number: 163-03545)
26. MgCl2 (Wako, catalog number: 136-03995)
27. EDTA (Invitrogen, catalog number: 15575-038)
28. Urea (Wako, catalog number: 217-01215)
29. Bromophenol Blue (BPB) (Wako, catalog number: 021-02911)
30. Xylene Cyanol (XC) (Wako, catalog number: 244-00461)
31. UltraPure DNase/RNase-Free Distilled Water (UPDW)
32. Ammonium persulfate (Wako, catalog number: 012-20503)
33. Acrylamide (Nacalai tesque, catalog number: 00807-05)
90
34. Tetramethylethylenediamine (TEMED) (Wako, catalog number: 205-06313)
35. Tween20 (Sigma-Aldrich, catalog number: P9416)
36. Sodium phosphate Dibasic (Wako, catalog number: 042-29445)
37. Imidazole (Wako, catalog number: 095-05392)
38. 40% (w/v) Acrylamide-Bis (Nacalai tesque, catalog number: 00857-55)
39. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 320331)
40. Tris-borate-EDTA (TBE) Buffer (10X) (Invitrogen, catalog number: B52)
41. Tris-EDTA (TE) buffer (Invitrogen, catalog number: AM9849)
42. N-hydroxysuccinimide (NHS) (Tokyo chemical industry, catalog number: B0249)
43. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (Tokyo chemical
industry, catalog number: D1601)
Equipment
1. Thermal cycler (Biometra, Model: TRIO48)
2. Biomolecular imager (GE healthcare, Model: Typhoon FLA9500)
3. NanoDrop Spectrophotometer (Thermo Scientific, Model: 1000 V3.3)
4. NanoDrop Spectrophotometer (Thermo Scientific, Model: 3300 V2.7, FITC)
5. Heat block (ANATECH, Model: cool stat 5200)
6. UV Crosslinker (UVP, Model: CL-1000)
7. Vortex mixer (IWAKI, Model: TM 2000)
8. Magnetic separator (Invitrogen, Model: 12320D)
9. Thermo block rotator (NISSIN, Model: SNP 24B)
10. StepOne Real-Time PCR System, (Thermo Fisher Scientific, Model: 4376600)
11. High speed refrigerated microcentrifuge (KITMAN, Model: Tomy Tech MX-301)
91
12. Gel electrophoresis apparatus (PAGE/SDS-PAGE) (ATTA, Model: AE 6510)
13. Pipettes (Gilson Pipetman, Model P2- P1000)
Software
1. Quantity One 1-D Analysis Software (Bio-Rad, Version 4.6.6)
2. Primer
Express
(Thermo
Fisher
92
Scientific,
Version
3.01)
...
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