Click Chemistry-Assisted Bioconjugates for Hapten Immunodiagnostics

Article abstract of DOI:10.1021/acs.bioconjchem.0c00099

Bioorthogonal reactions have revolutionized the way low-molecular-weight compounds are coupled to biomolecules. Organic chemistry, polymer science, and chemical biology are among the disciplines that have benefited the most from this breakthrough. Despite the reliability of the click chemistry concept for the efficient and chemoselective functionalization of biomacromolecules with haptens at preferred positions, the fact that azide-alkyne cycloaddition reactions originate new chemical moieties as part of the linker may have delayed their application in the immunodiagnostic field. Using the mycotoxin ochratoxin A as a model compound, we herein demonstrate for the first time that bioconjugates arising from the ligation between an azido-bearing hapten and an alkyne-modified carrier protein are able to elicit the generation of high-affinity monoclonal antibodies suitable for the development of rapid methods for the immunodetection of small organic molecules.

Full text of DOI:10.1021/acs.bioconjchem.0c00099

Subscriber access provided by Karolinska Institutet, University Library
Article
Click chemistry-assisted bioconjugates for hapten immunodiagnostics
Daniel López-Puertollano, Consuelo Agulló, Josep Vicent
Mercader, Antonio Abad-Somovilla, and Antonio Abad-Fuentes
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.0c00099 • Publication Date (Web): 25 Feb 2020
Downloaded from pubs.acs.org on February 28, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 26
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
Click chemistry-assisted bioconjugates for
hapten immunodiagnostics
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Daniel López-Puertollano,a Consuelo Agulló,a Josep V. Mercader,b Antonio Abad-Somovilla,a
Antonio Abad-Fuentes b,*
a Department of Organic Chemistry, Universitat de València, Doctor Moliner 50, 46100 Burjassot,
València, Spain
b Institute of Agrochemistry and Food Technology (IATA), Spanish National Research Council (CSIC),
Agustí Escardino 7, 46980 Paterna, València, Spain
* Corresponding author email: aabad@iata.csic.es; tel. +34-963900022; fax +34-963636301.
1
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 2 of 26
1
2
3
4
ABSTRACT
5
6
7
8
9
Bioorthogonal reactions have revolutionized the way low molecular weight compounds are
coupled to biomolecules. Organic chemistry, polymer science, and chemical biology are among
the disciplines that are benefited the most from this breakthrough. Despite the reliability of the
click chemistry concept for the efficient and chemoselective functionalization of
biomacromolecules with haptens at preferred positions, the fact that azide–alkyne
cycloaddition reactions originate new chemical moieties as part of the linker may have delayed
their application in the immunodiagnostic field. Using the mycotoxin ochratoxin A as a model
compound, we herein demonstrate for the first time that bioconjugates arising from the ligation
between an azido-bearing hapten and an alkyne-modified carrier protein are able to elicit the
generation of high-affinity monoclonal antibodies suitable for the development of rapid
methods for the immunodetection of small organic molecules.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
ACS Paragon Plus Environment

Page 3 of 26
Bioconjugate Chemistry
1
2
3
4
INTRODUCTION
5
6
7
8
9
Low molecular weight compounds are unable to elicit a secondary, adaptive immune
response by their own. For these so-called haptens to trigger the cascade of events that
ultimately lead to the proliferation of plasma and memory B cells and to the production of high-
affinity IgG antibodies, the compound has to be coupled to an entity carrying epitopes for helper
T-cells.1-8 Those carriers are most commonly proteins of different origin, like hemocyanins,
albumins, globulins, virus-like particles, or toxoids.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The most extended approach for bioconjugation of small compounds is through the
formation of stable amide bonds between carboxylate groups of the hapten and amine groups
of the protein. N-hydroxysuccinimidyl esters prepared by chemistry-mediated carbodiimides are
able to efficiently react, under mild conditions, with solvent-accessible lysine residues, which
are abundant in most carrier proteins.9-10 Very often, total synthetic approaches are required in
order to insert the appropriate functionalized linker at the desired position of the molecular
framework. Nevertheless, chemical targets occasionally possess a carboxylate group that can be
used for direct bioconjugation, like in the case of the mycotoxin ochratoxin A, several
fluoroquinolone antibiotics, and some medicinal drugs like statins. The strategy of using already
available functional groups for coupling, although simple and straightforward, obviates that
those moieties are potent antigenic determinants enabling electrostatic interactions and
hydrogen bonds between the analyte and amino acid residues in the antibody binding pocket.
Accordingly, it looks wise to attach the chemical to the carrier through a different position, so
the native carboxylate group is kept unaltered, thus increasing the chance of obtaining specific
and high-affinity antibodies. The challenge underlying this approach is the need for
protection/deprotection steps in order to introduce a functional group at the selected site of
the analyte skeleton. In this respect, bioorthogonal reactions enable the chemoselective
covalent coupling of two moieties in the presence of other functionalities.11 The prototypical
3
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 4 of 26
1
2
3
4
5
6
7
8
9
example of bioorthogonal reactions in chemical biology is most likely the copper(I)-catalyzed
Huisgen 1,3-dipolar azide–alkyne cycloaddition (CuAAC).12-14 First codiscovered in 2002 by the
groups of Meldal15 and Sharpless,16 this click chemistry reaction allows the ligation of an azide
group and a terminal alkyne group to form a triazole-bridged product under mild, biologically
compatible conditions – aqueous solutions, physiological pH, and ambient temperature.17 These
features, together with the fact that azide and alkyne tags may be easily introduced into small
molecules and proteins via chemical synthesis and common in vitro protein modification
procedures, have revolutionized bioconjugate chemistry, particularly when Cu(I)-chelating
ligands are employed in the coupling reaction.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The main concern arising from using CuAAC reactions for the preparation of bioconjugates
intended for vaccination is the presence of the triazole moiety in the linker region connecting
the hapten with the protein, because this rigid and aromatic moiety may elicit undesirable
antibodies by driving the immune response away from the target compound. Most examples
reporting immunization experiments with bioconjugates from click chemistry-mediated
approaches concern carbohydrate-based vaccines, either for infectious diseases or for cancer
therapeutics. In those studies with rather structurally complex haptens, authors reported that
the triazole group is essentially immunosilent.18-23 However, when it comes to immunoanalysis
of organic chemicals, antibodies with excellent binding properties are strongly desirable because
these sorts of analytes are found at very low levels in complex matrices. Accordingly, even
minimum disturbances in the antibody-analyte interaction coming from artificially introduced
neoepitopes may result in antibodies with insufficient affinity and/or specificity, and therefore
unsuitable for the development of rapid analytical tests. In fact, click chemistry approaches have
not found wide application in the immunochemistry field yet, and the few reported attempts
gave rise to rather disappointing results. Sanders et al. tried to generate antibodies to the
mycotoxin deoxynivalenol by introducing a propargyl moiety at the C-8 position of the
molecule.24 The derivative was coupled to azido-modified bovine serum albumin (BSA), but the
4
ACS Paragon Plus Environment

Page 5 of 26
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
resulting conjugate provided low titers. Negative results were also obtained by Vasylieva et al.
when addressing the generation of antibodies to tetramethylenedisulfotetramine with an azido-
bearing derivative of this neurotoxin.25 Therefore, the question arising is whether bioconjugates
from alkyne‒azide cycloaddition reactions may be an actually effective approach for the
generation of anti-hapten antibodies with the outstanding binding properties that are required
in immunodiagnostics or, otherwise, the presence of a rigid aromatic ring in the linker
connecting the chemical with the carrier may severely impair that goal. With the aim to shed
some light on this issue, we choose the mycotoxin ochratoxin A (OTA) as a model compound.
This potent toxic fungal metabolite may be encountered in crops infected by Penicillium and
Aspergillus species.26-27 The undesirable presence of OTA in food commodities like cereals,
raisins, coffee, beer, and wine, constitutes a worldwide problem of public health, so its levels
are strictly regulated and controlled.28-30 A particular chemical feature of OTA is that it bears a
carboxylate group. As mentioned above, although this moiety is certainly suitable for coupling
to proteins, its capacity to act as a powerful antigenic determinant makes it advisable to link the
hapten to the carrier through a different site, and we thought that CuAAC chemistry can greatly
assist in facilitating the synthesis of the appropriate derivatives.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
RESULTS AND DISCUSSION
5
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 6 of 26
1
2
3
4
5
6
It is well known that the position of the functionalized spacer arm in the hapten may exert a
decisive effect on the antibody binding properties.31-35 Accordingly, we decided to introduce the
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aliphatic linker with a terminal azido group at two alternative sites of the OTA structure, i.e., the
C-4 position of the benzene ring or the C-3 position of the dihydroisocoumarin ring. The
synthetic routes enabling the preparation of the so-called hapten a (with the linker at the aryl
moiety) and hapten c (with the linker at the CH3 group) are concisely depicted in Scheme 1
(detailed synthesis procedures can be found in the Supporting Information file).
6
ACS Paragon Plus Environment

Page 7 of 26
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
Scheme 1. Synthetic route for the preparation of haptens a and c. Both haptens and all intermediates of
their synthesis containing the dihydroisocumarine moiety are 1:1 mixtures of the epimeric
diastereomers at the stereogenic center of this moiety (marked with an asterisk in the hapten
structures).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Preparation of both haptens followed a similar strategy for the construction of the common
OTA core consisting of the preparation of the conveniently functionalized L-phenylalanine and
dihydroisocoumarin moieties and their coupling through an amide bond. Due to chemical
compatibility with reactions in the designed synthetic route leading to hapten a, we chose a
linker with a terminal azide group for the functionalization of the L-phenylalanine moiety. For
synthetic consistency, the same functional group was selected for the preparation of hapten c,
so the proteins were accordingly modified with alkyne groups for the subsequent cycloaddition
reaction.
Racemic OTα (5) was employed as the starting material for the synthesis of hapten a.
Although this chemical is commercially available, we decided to prepare it by an optimized
synthetic route previously described by our group.36 The rationale behind this decision is that
compound 3, an intermediate in the pathway leading to 5, was the key material for the synthesis
of hapten c.
The first step in the synthesis of hapten a was the condensation of the dihydroisocoumarin
moiety with an iodinated derivative of L-phenylalanine to give 7, which was further subjected to
a Sonogashira reaction to introduce the linker at the C-4 position of the phenyl ring. After
hydrogenation of the acetylenic triple bond of 9 under homogeneous conditions using
Wilkinson’s catalyst, the hydroxyl group was replaced by the azide group, thus completing the
required functionalization of the linker chain in two steps: first, transformation of alcohol 10
into alkyl halide 11, under Appel’s reaction conditions,37 followed by substitution of the bromine
7
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 8 of 26
1
2
3
4
5
6
7
8
9
atom by the highly nucleophilic azide anion. Finally, the desired compound was obtained
following deprotection of the native carboxylic group of OTA with trifluoroacetic acid. With this
strategy, hapten a was obtained in 9 synthetic steps from 1 with a 15% overall yield, or in 5 steps
with 31% yield starting from commercially available racemic OTα.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The synthesis of hapten c started with the construction of the functionalized
dihydroisocoumarin moiety. Metalation of the methyl group of compound 3 with lithium
diisopropylamide (LDA), followed by reaction of the generated benzylic anion with aldehyde 12
and acidic work-up, afforded the dihydroisocoumarin derivative 13, which already incorporated
the azide-terminal linker for further coupling by click chemistry. Basic hydrolysis of the methyl
ester group and condensation reaction between the resulting carboxylic acid 14 and (t-Boc)-
protected L-phenylalanine 15 completed the construction of the hapten skeleton. Finally, as
with hapten a, the acidic deprotection of the native carboxylic group of OTA afforded the desired
functionalized derivative. This synthetic pathway provided hapten c in 6 steps from 1 with a 28%
overall yield.
With the two haptens at hand, we undertook the modification of the proteins with propargyl
groups (Scheme 2). BSA was chosen for the preparation of immunizing bioconjugates, whereas
ovalbumin (OVA) and horseradish peroxidase (HRP) were selected for the preparation of the
8
ACS Paragon Plus Environment

Page 9 of 26
Bioconjugate Chemistry
1
2
3
4
5
6
bioconjugates employed in competitive assays – OVA for the antigen-coated ELISA format and
HRP for the antibody-coated ELISA format.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Preparation of bioconjugates. Proteins were first modified with propargyl groups by reaction
with compound 17, and then coupled to haptens by CuAAC reaction.
Compound 17 was chosen for the alkynylation of the proteins because it was successfully
used by other research groups for the modification of cowpea mosaic virus, poly(lysine) chains,
hemocyanin, and albumins.38-42 The molar ratio of 17 to BSA in the initial reaction mixture was
established at 25 in order to modify a sufficient number of lysine residues as to later achieve a
click chemistry-assisted bioconjugate with a rather high hapten loading for immunization. The
alkyne-modified BSA was purified by size-exclusion chromatography, and the number of
introduced linkers was determined by MALDI-TOF‒MS. As shown in Figure 1, the increase in
molecular weight with respect to the intact protein (2935 Da) corresponds to an average
incorporation of 17.2 alkyne chains into each molecule of BSA.
The CuAAC conditions for the ligation of haptens a and c to BSA–alkyne where essentially
based on procedures developed by Finn’s group.43-44 The coupling reaction was carried out with
6 equivalents of hapten per alkyne residue in 100 mM sodium phosphate, pH 7.4, under
nitrogen. After 20 h at room temperature, the bioconjugates were concentrated and purified by
size-exclusion chromatography. Hapten-to-protein loadings of 14.9 and 15.1 were found for
bioconjugates BSA–a and BSA–c, respectively (Scheme 2), as determined by MALDI-TOF‒MS
(Figure 1). Concerning OVA and HRP, wherein lower cargos are preferred, initial alkyne-to-
protein molar ratios of 12 and 10, respectively, were employed. Under these conditions, 7.6
alkyne residues were introduced into OVA and 0.8 into HRP (Figure S1). After CuAAC reaction
under similar conditions to those used with BSA, OVA was modified with an average number of
9
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 10 of 26
1
2
3
4
4.5 and 5.0 residues of hapten a and hapten c, respectively, whereas 0.7 residues of both
5
6
haptens were introduced into HRP (Scheme 2).
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. MALDI-TOF mass spectra of BSA (singly charged ions) in their unmodified form (blue), modified
with propargyl groups (green), and coupled to haptens a (orange) and c (light blue).
Evaluation of the immune response was first assessed by immunizing two rabbits with each
bioconjugate, BSA–a and BSA–c. As judged by antibody-coated ELISA using HRP–a and HRP–c
enzyme tracers, excellent immune responses, with titres higher than 3×104, were observed.
Concerning antiserum capability to recognize OTA in solution, high affinity was observed by
competitive ELISA. IC50 values of 9.4 nM and 12.9 nM were found with antisera a#I and a#II,
respectively, and of 2.1 nM and 0.5 nM with antisera c#I and c#II, respectively (Figure 2).
These results clearly prove the suitability of click chemistry-assisted bioconjugates with
triazole-bearing linkers for the generation of polyclonal antibodies with excellent binding
properties to a small organic compound like OTA. It is also worthy to note that slightly higher
affinity to OTA was found with antisera from hapten c, that is, the derivative holding the spacer
arm at a distal position from the carboxylic acid moiety of OTA. Regarding specificity, no
noticeable recognition of ochratoxin B or ochratoxin C, the two main OTA metabolites, was
found – the highest cross-reactivity value was 3.3% for ochratoxin B with antisera c#II. This result
10
ACS Paragon Plus Environment

Page 11 of 26
Bioconjugate Chemistry
1
2
3
4
points out the influence of the chlorine atom and mainly the carboxylic group of OTA in the
5
6
antibody‒antigen interaction.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Recognition of OTA by polyclonal antibodies from rabbits immunized with bioconjugate BSA–a
(blue lines) and BSA–c (black lines). Each line represents the immune response of a different animal.
Experimental points by competitive ELISA are the average of three replicates on different days.
At this point of our study, we decided to gain further insights into the participation of the
triazole group in the binding event. To this purpose, two haptens previously synthesized by our
group bearing carboxylated aliphatic linkers – without the triazole group – at the same positions
of the OTA framework than haptens a and c (Scheme S1) were coupled to HRP.36 When these
two bioconjugates, so-called HRP–b and HRP–d, were tested by immunoassay, they were
perfectly recognized by the antisera from hapten a and antisera from hapten c, respectively,
although concentrations slightly higher than those required with the homologous bioconjugates
HRP–a and HRP–c were needed in order to attain the same signal. Concerning recognition of
OTA, the use of bioconjugates HRP–b and HRP–d in competitive assays caused a 2‒4 times
decrease of the IC50 values, thus resulting in better OTA detectability, probably because of the
hapten heterology effect. Altogether, these data evidence that, although there is certainly an
influence of the triazole moiety in antibody binding, its contribution is of small relevance and it
does not handicap the generation of antibodies suitable for immunodiagnostics.
11
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 12 of 26
1
2
3
4
5
6
7
8
9
This conclusion was further confirmed by immunizing mice with bioconjugate BSA–c in order
to produce monoclonal antibodies (mAbs) by hybridoma technology. From two independent cell
fusion experiments, 12 hybridoma cell lines producing antibodies with IC50 values to OTA lower
than 10 nM were identified. A subset of 5 hybridomas were cloned and stabilized, and the
antibodies they produce were comprehensively characterized by both the antigen-coated and
the antibody-coated competitive ELISA. Remarkably, mAbs displaying subnanomolar affinity to
OTA in both ELISA formats were identified (Table 1). It is also worth mentioning that the affinity
to OTA of this group of mAbs was similar to that of the antibodies derived from hapten d which,
as already mentioned, was coupled to BSA through the same position that hapten c but through
NHS ester chemistry instead of click chemistry. In addition, whereas the preparation of hapten
d required 10 steps with an overall yield of 18.8%, the synthesis of hapten c herein reported
took 6 steps with an overall yield of 28%. Therefore, bioconjugation through CuAAC chemistry
arises as an advantageous approach, even though further studies with other analytes are
required to ascertain the scope of this finding.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Characterization of monoclonal antibodies for
binding to OTA by competitive ELISAa.
Antigen-coated ELISA
Antibody-coated ELISA
mAb [mAb] [OVA–c] IC50
[mAb] [HRP–c] IC50
c#12
c#29
c#116
c#117
c#134
30
30
30
100
30
10
10
10
10
100
0.39
0.69
0.58
0.48
0.93
30
30
30
100
300
10
10
10
10
10
0.31
0.57
0.85
0.29
1.13
a Data are the average of three independent determinations.
Concentrations of antibodies and bioconjugates are in ng/mL, whereas
IC50 values are in nanomolar units.
The IC50 values exhibited by the group of antibodies generated from the bioconjugate BSA–c,
in particular by mAbs c#12 and c#117, make them ideal reagents for the development of rapid
immunoanalytical methods for on-site OTA detection. As a proof-of-concept of this assessment,
two immunoassays, an antibody-coated competitive ELISA and a chromatographic immunostrip,
were tentatively developed (Table 2). With analytical standards in buffer, the ELISA has a LOQ,
12
ACS Paragon Plus Environment

Page 13 of 26
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
defined as the analyte concentration causing a 20% signal inhibition, of 0.04 ng/mL, a value well
below common regulated levels of OTA in foodstuff. As in regular commercial kits, one microtiter
plate would allow the simultaneous determination of 42 samples and a 6-point standard curve,
both in duplicated wells, in 70 min. Regarding immunostrips, signal intensities of the control line
(upper stripe) and of the test line (lower stripe) in the membrane visually equals at an OTA
concentration of 1‒3 ng/mL, while a common desktop scanner for reading signal intensities
enable to quantify OTA at levels higher than 0.04 ng/mL by using the ratio between the signal of
both lines.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Analytical parameters of the developed
immunoanalytical methods for OTAa.
Antibody-coated ELISA
Immunostrip
c#117
(100 ng/mL)
HRP–c
c#117
(1 µg/mL)
BSA–c
Antibody
Bioconjugate
(10 ng/mL)
(0.5 µg/cm)
Maximum signal
IC50 (ng/mL)
Slope
LOD (ng/mL)b
LOQ (ng/mL)c
1.21
0.12
22.60
0.18
−1.82
0.02
−1.03
0.02
0.04
0.04
Assay time
70 min
15 min
a
Data are the mean of three independent experiments for the direct
ELISA, and of a representative experiment for the immunostrip. b The LOD
was calculated from the signal at 10% inhibition. c The LOQ was calculated
from the signal at 20% inhibition.
CONCLUSIONS
13
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 14 of 26
1
2
3
4
5
6
7
8
9
This study demonstrates for the first time, using OTA as a model compound, that CuAAC
chemistry is a convenient approach for the preparation of protein–hapten bioconjugates
intended for the generation of suitable antibodies for immunodiagnostics of small organic
chemicals. Our results point out that the participation of the triazole moiety in antibody binding
is not as critical as it could be thought, so its presence in the spacer arm does not preclude the
production of antibodies with apparent affinity constants for the analyte in the subnanomolar
range. In our opinion, this finding opens the door to obtaining highly valuable biotechnological
immunoreagents for other relevant compounds, like antibiotics, biotoxins, and drugs, through
unexplored chemical approaches involving innovative positions for linker attachment and
hapten functionalization. Further studies should certainly contribute to clarify the
generalizability of the herein reported strategy.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL PROCEDURES
Preparation of click chemistry-assisted bioconjugates.
Modification of proteins with propargyl groups. Compound 17 was dissolved in N,N’-
dimethylformamide (DMF) and drop wise added to a solution of protein in phosphate buffer
(PB, pH 7.4). The conjugation reaction was carried out with a DMF maximum content of 10%
(v/v). After stirring overnight at room temperature, the protein conjugates were purified by size-
exclusion chromatography using PB as eluent at a flow rate of 5 mL/min.
Coupling of haptens to alkyne-modified proteins by CuAAC reaction. Solutions of haptens a
and c in dimethylsulphoxide were drop wise added to alkyne-modified protein solutions in PB,
pH 7.4. After adding a premixed solution of THPTA/Cu(I) complex (5:1) in Milli-Q H2O, the
mixture was purged by repeated vacuum‒nitrogen cycles. Finally, a solution of sodium
ascorbate in Milli-Q H2O was added and the final reaction volume was adjusted with PB, pH 7.4,
followed by vacuum‒nitrogen cycles. The solutions were incubated 20 h at room temperature,
14
ACS Paragon Plus Environment

Page 15 of 26
Bioconjugate Chemistry
1
2
3
4
5
6
and the reaction was then stopped using Amicon Ultra 4 10K centrifugal filters. The protein
conjugates were purified by size-exclusion chromatography.
7
8
9
MALDI-TOF mass spectrometry analysis of modified proteins. 100 μL of native protein, alkyne-
modified protein or protein–hapten bioconjugate (0.5‒1 mg/mL) was dialyzed against Milli-Q
water and then lyophilized. The samples were dissolved in Milli-Q H2O to a final concentration
of 1 μg/μL. Then, 1 μL of every sample solution was spotted onto the MALDI plate. After the
droplets were air dried at room temperature, 1 μL of matrix [10 mg/mL sinapinic acid in 0.1%
trifluoroacetic acid‒CH3CN/H2O (7:3 v/v)] was added and allowed to air-dry at room
temperature. Samples were analysed in a 5800 MALDI TOF/TOF (ABSciex) in positive linear mode
(1500 shots every position) in a mass range of 15000‒100000 m/z. Previously, the plate was
calibrated with 1 μL of the TOF/TOF calibration mixture (ABSciex), in 13 positions. Every sample
was calibrated by ‘close external calibration’ method with a BSA, OVA, or HRP spectrum acquired
in a close position.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Antibody generation
Female New Zealand white rabbits were immunized at 21-day intervals by subcutaneous
injection with 0.3 mg of BSA–hapten conjugate in 1 mL of a 1:1 emulsion between sterile 100
mM phosphate buffer, pH 7.4, and Freund’s adjuvant (complete for the first dose and
incomplete for subsequent boosts). Ten days after the fourth injection, rabbits were
exsanguinated by intracardiac puncture. Blood samples were allowed to coagulate overnight at
4 °C, and sera were separated by centrifugation (3000×g, 20 min). Finally, antibodies were
precipitated with one volume of saturated ammonium sulphate solution. Salting out was
performed twice, and precipitates were stored at 4 °C.
Two-month old female Balb/c mice were immunized by intraperitoneal injection of 0.1 mg of
BSA‒hapten conjugate in 200 µL of a water-in-oil emulsion that was prepared using Freund’s
adjuvants as described above. Three doses were applied at 21-day intervals, and a fourth
15
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 16 of 26
1
2
3
4
5
6
7
8
9
injection in sterile PBS with the same amount of bioconjugate was given at least 21 days after
the third injection and 4 days before the animal was sacrificed. Hybridomas were prepared by
fusion of mouse myeloma cells with splenocytes from two equally-immunized mice following
standard procedures.45 Antibody-producing cells were identified by a double screening
procedure. Twelve days after cell fusion, hybridoma culture supernatants were first screened by
differential antigen-coated competitive ELISA on microtiter plates coated with 0.1 µg/mL (100
µL per well) of the homologous OVA–hapten bioconjugate. Fifty microliter of each supernatant
was added to two adjacent wells of an ELISA plate, one containing 50 µL of PBS (blank) and the
other one containing 50 µL of 200 nM OTA in PBS. The ratio between the signals of both wells
was used as the criterion for selecting the antibodies with the highest affinity. Fresh culture
medium was added to those culture plate wells performing best, and next day they were re-
evaluated by checkerboard competitive ELISA. This second screening assay was carried out with
two coating concentrations of the homologous OVA–hapten conjugate (0.01 and 0.1 µg/mL),
four supernatant dilutions (1/10, 1/50, 1/250, and 1/1250), and three OTA levels (0, 10, and 100
nM).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Competitive ELISA
Antibody-coated direct format. 96-well plates were coated with 100 μL per well of capture
antibody [goat anti-mouse for monoclonal antibodies (GAM) and goat anti-rabbit for polyclonal
antibodies (GAR)] diluted in 50 mM carbonate‒bicarbonate buffer, pH 9.6, by overnight
incubation at 4 °C. Next day, plates were washed four times with washing solution (150 mM
NaCl containing 0.05% (v/v) Tween 20), anti-OTA antibodies in PBST were added (100 µL per
well), and plates were incubated for 1 h at room temperature. After washing as described
before, the competitive step was carried out by mixing 50 µL per well of OTA solution in PBS and
50 µL per well of HRP‒hapten tracer in PBST. After 1 h incubation at room temperature and
washing, signal was generated by adding 100 μL per well of enzyme substrate solution [2 mg/mL
16
ACS Paragon Plus Environment

Page 17 of 26
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
o-phenylendiamine in 25 mM citrate and 62 mM phosphate buffer, pH 5.4, containing 0.012%
(v/v) H2O2] and incubation during 10 min at room temperature. Finally, the enzymatic reaction
was stopped with 100 μL per well of 1 M H2SO4, and the absorbance was immediately read at
492 nm using the absorbance at 650 nm as a reference value.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Antigen-coated indirect format. Microplates were coated with 100 μL per well of OVA–
hapten bioconjugate solution in 50 mM carbonate‒bicarbonate buffer, pH 9.6. After overnight
incubation at room temperature, plates were washed four times with washing solution. The
competitive step was carried out during 1 h at room temperature with 50 µL per well of OTA
solution in PBS plus 50 µL per well of antibody dilution in PBST. After washing, 100 µL per well
of HRP-labelled secondary antibody in PBST was added, and the plates were incubated at room
temperature for 1 h. Finally, microwells were washed as before and the signal was obtained as
described for direct assays.
Competitive LFIA protocol
Preparation of AuNPGAM@mAb. Commercial 40-nm gold nanoparticles coated with GAM (OD
= 10) were diluted ten times in 10mM HEPES buffer (pH 7.4). Monoclonal antibody was added
to the desired concentration from a stock solution in 10 mM HEPES buffer (pH 8.0) and the
mixture was incubated for 1 h at room temperature. After addition of Tween20 to obtain a final
concentration of 0.05% (v/v), nanoparticles were stored at 4 °C.
Preparation of immunostrips. Solutions of GAM (control line) and BSA‒hapten bioconjugate
(test line) in 100 mM phosphate buffer containing 150 mM NaCl, pH 7.4, were dispensed on a
nitrocellulose membrane (300x25 mm) at a flow rate of 0.5 µL/cm using a BioDot ZX1010
machine. BSA‒hapten bioconjugate and GAM solution were dispensed at 10 mm and 15 mm
from the base of the membrane, respectively. Membranes were dried at room temperature for
1 h and were placed 15 mm from the base of the backing card (300x78 mm). Nitrocellulose
17
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 18 of 26
1
2
3
4
5
6
sample pad (300x17 mm) and adsorbent pad (300x43 mm) were assembled overlapping 2 mm
and 3 mm, respectively, with the membrane.
7
8
9
Assay protocol. Dipstick lateral flow immunoassays were carried out in 300-µL wells by mixing
12 µL of the suspension of antibody-coated gold nanoparticles and 88 µL of OTA solution in 100
mM TRIS buffer containing 30 mM NaCl, pH 8.0. The mixtures were incubated 5 min at room
temperature and then the immunostrips were plunged into the well. After incubating the
immunostrips in vertical position for 10 min, sample pads were removed, the membranes were
dried for 3 min under a cold airflow, and signals were registered with a desktop scanner at a
resolution of 1200 dpi.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACKNOWLEDGMENTS
This work was supported by the Spanish Ministerio de Economía y Competitividad (AGL2015-
64488 and RTI2018-096121), Conselleria d’Educació, Investigació, Cultura i Esport de la
Generalitat Valenciana (AICO/2018/111), and cofinanced by European Regional Development
Funds. D.L.-P. was supported by a FPU predoctoral grant (FPU14/05266) from the Spanish
Ministerio de Educación, Cultura y Deporte. The proteomic analysis was performed at the
Proteomics Section of SCSIE of the University of Valencia which belongs to ProteoRed, PRB2-3,
and was supported by grant PT17/0019, of the PE I+D+i 2013‒2016, and funded by ISCIII and
ERDF. The technical assistance by Paula Peña Murgui is greatly appreciated. Animal
manipulation was carried out at the Animal Production Section, also belonging to the SCSIE of
the University of Valencia.
Samples of the immunoreagents herein reported are available upon request from the
corresponding authors.
ASSOCIATED CONTENT
18
ACS Paragon Plus Environment

Page 19 of 26
Bioconjugate Chemistry
1
2
3
4
Supporting information
5
6
7
8
9
General reagents and equipment, detailed synthesis procedures, NMR spectra, and mass
spectrometry data. This material is available free of charge via the Internet at
http://pubs.acs.org.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
REFERENCES
(1)
antigen processing in phagosomes. Curr. Opin. Immunol. 21, 98—104.
(2) Burgdorf, S., Kurts, C. (2008) Endocytosis mechanisms and the cell biology of antigen
presentation. Curr. Opin. Immunol. 20, 89—95.
(3) Yoshida, T., Mei, H., Doerner, T., Hiepe, F., Radbruch, A., Fillatreau, S., Hoyer, B. F.
(2010) Memory B and memory plasma cells. Immunol. Rev. 237, 117—139.
(4) Bachmann, M. F., Jennings, G. T. (2010) Vaccine delivery: a matter of size, geometry,
kinetics and molecular patterns. Nat. Rev. Immunol. 10, 787—96.
(5) Costantino, P., Rappuoli, R., Berti, F. (2011) The design of semi-synthetic and synthetic
glycoconjugate vaccines. Expert Opin. Drug Dis. 6, 1045—1066.
(6) Yin, Z., Comellas-Aragonés, M., Chowdhury, S., Bentley, P., Kaczanowska, K.,
Ramachandra, L., Simmons, D., Harding, C. V. (2009) MHC molecules and microbial
Benmohamed, L., Gildersleeve, J. C., Finn, M. G., Huang, X. (2013) Boosting immunity to small
tumor-associated carbohydrates with bacteriophage qbeta capsids. ACS Chem. Biol. 8, 1253—
1262.
(7)
Ceballos-Alcantarilla, E., Abad-Somovilla, A., Agulló, C., Abad-Fuentes, A., Mercader, J.
V. (2017) Protein-free hapten-carbon nanotube constructs induce the secondary immune
response. Bioconjug. Chem. 28, 1630—1638.
(8)
Parra, J., Abad-Somovilla, A., Mercader, J. V., Taton, T. A., Abad-Fuentes, A. (2013)
Carbon nanotube-protein carriers enhance size-dependent self-adjuvant antibody response to
haptens. J. Control Release 170, 242—251.
(9)
Pickens, C. J., Johnson, S. N., Pressnall, M. M., Leon, M. A., Berkland, C. J. (2018)
Practical considerations, challenges, and limitations of bioconjugation via azide-alkyne
cycloaddition. Bioconjug. Chem. 29, 686—701.
(10)
click chemistry for amine bioconjugation. Bioconjug. Chem. 28, 1422—1433.
(11) Thirumurugan, P., Matosiuk, D., Jozwiak, K. (2013) Click chemistry for drug
Elahipanah, S., O'Brien, P. J., Rogozhnikov, D., Yousaf, M. N. (2017) General dialdehyde
development and diverse chemical-biology applications. Chem. Rev. 113, 4905—4979.
19
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 20 of 26
1
2
3
4
(12)
Dirks, A. J., Cornelissen, J. J. L. M., Nolte, R. J. M. (2009) Monitoring protein-polymer
conjugation by a fluorogenic Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition. Bioconjug.
Chem. 20, 1129—1138.
5
6
7
8
9
(13)
selectivity in the labeling of biological molecules. Biochemistry 48, 6571—6584.
(14) Lallana, E., Riguera, R., Fernández-Megía, E. (2011) Reliable and efficient procedures
Best, M. D. (2009) Click chemistry and bioorthogonal reactions: unprecedented
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
for the conjugation of biomolecules through Huisgen azide-alkyne cycloadditions. Angew.
Chem. Int. Ed. Engl. 50, 8794—8804.
(15)
Tornoe, C. W., Christensen, C., Meldal, M. (2002) Peptidotriazoles on solid phase:
[1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal
alkynes to azides. J. Org. Chem. 67, 3057—3064.
(16)
Rostovtsev, V. V., Green, L. G., Fokin, V. V., Sharpless, K. B. (2002) A stepwise Huisgen
cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal
alkynes. Angew. Chem. Int. Ed. Engl. 41, 2596—2599.
(17)
semisynthetic enzymes to new click enzymes. Org. Biomol. Chem. 10, 9309—9318.
(18) Adamo, R., Hu, Q.-Y., Torosantucci, A., Crotti, S., Brogioni, G., Allan, M., Chiani, P.,
Palomo, J. M. (2012) Click reactions in protein chemistry: from the preparation of
Bromuro, C., Quinn, D., Tontini, M., et. al. (2014) Deciphering the structure–immunogenicity
relationship of anti-Candida glycoconjugate vaccines. Chem. Sci. 5, 4302—4311.
(19)
Lipinski, T., Luu, T., Kitov, P. I., Szpacenko, A., Bundle, D. R. (2011) A structurally
diversified linker enhances the immune response to a small carbohydrate hapten. Glycoconj. J.
28, 149—164.
(20)
Yin, Z., Nguyen, H. G., Chowdhury, S., Bentley, P., Bruckman, M. A., Miermont, A.,
Gildersleeve, J. C., Wang, Q., Huang, X. (2012) Tobacco mosaic virus as a new carrier for tumor
associated carbohydrate antigens. Bioconjug. Chem. 23, 1694—1703.
(21)
Hu, Q.-Y., Allan, M., Adamo, R., Quinn, D., Zhai, H., Wu, G., Clark, K., Zhou, J., Ortiz, S.,
Wang, B., et. al. (2013) Synthesis of a well-defined glycoconjugate vaccine by a tyrosine-
selective conjugation strategy. Chem. Sci. 4, 3827–3832.
(22)
Cai, H., Shukla, S., Wang, C., Masarapu, H., Steinmetz, N. F. (2019) Heterologous prime-
boost enhances the antitumor immune response elicited by plant-virus-based cancer vaccine.
J. Am. Chem. So.c 141, 6509—6518.
(23)
Nilo, A., Morelli, L., Passalacqua, I., Brogioni, B., Allan, M., Carboni, F., Pezzicoli, A.,
Zerbini, F., Maione, D., Fabbrini, M., et al. (2015) Anti-group B Streptococcus glycan-conjugate
vaccines using pilus protein GBS80 as carrier and antigen: comparing lysine and yyrosine-
directed conjugation. ACS Chem. Biol. 10, 1737—1746.
20
ACS Paragon Plus Environment

Page 21 of 26
Bioconjugate Chemistry
1
2
3
4
(24)
Sanders, M., Guo, Y., Lyer, A., García, Y. R., Galvita, A., Heyerick, A., Deforce, D.,
Risseeuw, M. D., Van Calenbergh, S., Bracke, M., et. al. (2014) An immunogen synthesis
strategy for the development of specific anti-deoxynivalenol monoclonal antibodies. Food
Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 31, 1751—1759.
5
6
7
8
9
(25)
Vasylieva, N., Barnych, B., Rand, A., Inceoglu, B., Gee, S. J., Hammock, B. D. (2017)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
sensitive immunoassay for detection and quantification of the neurotoxin,
tetramethylenedisulfotetramine. Anal. Chem. 89, 5612—5619.
(26)
available data. Toxins (Basel) 7, 4253—4282.
(27) Tao, Y., Xie, S., Xu, F., Liu, A., Wang, Y., Chen, D., Pan, Y., Huang, L., Peng, D., Wang, X.,
Heussner, A. H., Bingle, L. E. (2015) Comparative ochratoxin toxicity: a review of the
et al. (2018) Ochratoxin A: toxicity, oxidative stress and metabolism. Food Chem. Toxicol. 112,
320—331.
(28)
Bellver Soto, J., Fernández-Franzón, M., Ruiz, M. J., Juan-García, A. (2014) Presence of
ochratoxin A (OTA) mycotoxin in alcoholic drinks from southern European countries: wine and
beer. J. Agric. Food Chem. 62, 7643—7651.
(29)
Amézqueta, S., Schorr-Galindo, S., Murillo-Arbizu, M., González-Peñas, E., López de
Cerain, A., Guiraud, J. P. (2012) OTA-producing fungi in foodstuffs: a review. Food Control 26,
259—268.
(30)
occurrence in beer produced in several European countries. Food Control 22, 2059—2064.
(31) Quiñones-Reyes, G., Agulló, C., Mercader, J. V., Abad-Somovilla, A., Abad-Fuentes, A.
Bertuzzi, T., Rastelli, S., Mulazzi, A., Donadini, G., Pietri, A. (2011) Mycotoxin
(2019) Synthetic haptens and monoclonal antibodies to the cyanotoxin anatoxin-a. Angew.
Chem. Int. Ed. Engl. 58, 9134—9139.
(32)
Mercader, J. V., Agulló, C., Abad-Somovilla, A., Abad-Fuentes, A. (2011) Synthesis of
site-heterologous haptens for high-affinity anti-pyraclostrobin antibody generation. Org.
Biomol. Chem. 9, 1443—1453.
(33)
Parra, J., Mercader, J. V., Agulló, C., Abad-Somovilla, A., Abad-Fuentes, A. (2012)
Generation of anti-azoxystrobin monoclonal antibodies from regioisomeric haptens
functionalized at selected sites and development of indirect competitive immunoassays. Anal.
Chim. Acta 715, 105—112.
(34)
Esteve-Turrillas, F. A., Agulló, C., Mercader, J. V., Abad-Somovilla, A., Abad-Fuentes, A.
(2018) Rationally designed haptens for highly sensitive monoclonal antibody-based
immunoanalysis of fenhexamid. Analyst 143, 4057—4066.
21
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 22 of 26
1
2
3
4
5
(35)
Ceballos-Alcantarilla, E., López-Puertollano, D., Agulló, C., Abad-Fuentes, A., Abad-
Somovilla, A., Mercader, J. V. (2018) Combined heterologies for monoclonal antibody-based
6
7
immunoanalysis of fluxapyroxad. Analyst 143, 5718—5727.
8
9
(36)
López-Puertollano, D., Mercader, J. V., Agulló, C., Abad-Somovilla, A., Abad-Fuentes, A.
(2018) Novel haptens and monoclonal antibodies with subnanomolar affinity for a classical
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
analytical target, ochratoxin A. Sci. Rep. 8, 9761.
(37)
van Kalkeren, H. A., van Delft, F. L., Rutjes, F. P. J. T. (2012) Catalytic Appel reactions.
Pure Appl. Chem. 85, 817—828.
(38)
Wang, Q., Chan, T. R., Hilgraf, R., Fokin, V. V., Sharpless, K. B., Finn, M. G. (2003)
Bioconjugation by copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc.
125, 3192—3193.
(39)
Aime, S., Dastru, W., Gobetto, R., Viale, A. (2005) para-Hydrogenation of unsaturated
moieties on poly(lysine) derived substrates for the development of novel hyperpolarized MRI
contrast agents. Org. Biomol. Chem. 3, 3948—3954.
(40)
Kaltgrad, E., Gupta, S., Punna, S., Huang, C. Y., Chang, A., Wong, C. H., Finn, M. G., Blixt,
O. (2007) Anti-carbohydrate antibodies elicited by polyvalent display on a viral scaffold.
ChemBioChem 8, 1455—1462.
(41)
Shi, W., Dolai, S., Averick, S., Fernando, S. S., Saltos, J. A., L'Amoreaux, W., Banerjee, P.,
Raja, K. (2009) A general methodology toward drug/dye incorporated living copolymer-protein
hybrids: (NIRF dye-glucose) copolymer-avidin/BSA conjugates as prototypes. Bioconj. Chem.
20, 1595—1601.
(42)
and immunogenicity of a glycopolymer conjugate. Bioconjug. Chem. 22, 274—281.
(43) Hong, V., Presolski, S. I., Ma, C., Finn, M. G. (2209) Analysis and optimization of copper-
Lipinski, T., Kitov, P. I., Szpacenko, A., Paszkiewicz, E., Bundle, D. R. (2011) Synthesis
catalyzed azide-alkyne cycloaddition for bioconjugation. Angew. Chem. Int. Ed. Engl. 48,
9879—9883.
(44)
Presolski, S. I., Hong, V. P., Finn, M. G. (2011) Copper-catalyzed azide-alkyne click
chemistry for bioconjugation. Curr. Proto.c Chem. Biol. 3, 153—162.
(45)
Mercader, J. V., Suárez-Pantaleón, C., Agulló, C., Abad-Somovilla, A., Abad-Fuentes, A.
(2008) Production and characterization of monoclonal antibodies specific to the strobilurin
pesticide pyraclostrobin. J. Agric. Food Chem. 56, 7682—7690.
22
ACS Paragon Plus Environment

Page 23 of 26
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Synthetic route for the preparation of haptens a and c. Both haptens and all intermediates of
their synthesis containing the dihydroisocumarine moiety are 1:1 mixtures of the epimeric diastereomers at
the stereogenic center of this moiety (marked with an asterisk in the hapten structures).
266x261mm (300 x 300 DPI)
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 24 of 26
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Preparation of bioconjugates. Proteins were first modified with propargyl groups and then
coupled to haptens by CuAAC reaction
241x158mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 25 of 26
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. MALDI-TOF mass spectra of BSA (singly charged ions) in their unmodified form (blue), modified
with propargyl groups (green), and coupled to haptens a (orange) and c (light blue).
132x97mm (144 x 144 DPI)
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 26 of 26
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Recognition of OTA by polyclonal antibodies from rabbits immunized with bioconjugate BSA–a
(blue lines) and BSA–c (black lines). Each line represents the immune response of a different animal.
Experimental points by competitive ELISA are the average of three replicates on different days.
152x122mm (600 x 600 DPI)
ACS Paragon Plus Environment