Characterization of Four Medically Important Toxins from Centruroides huichol Scorpion Venom and Its Neutralization by a Single Recombinant Antibody Fragment

Abstract

Centruroides huichol scorpion venom is lethal to mammals. Analysis of the venom allowed the characterization of four lethal toxins named Chui2, Chui3, Chui4, and Chui5. scFv 10FG2 recognized well all toxins except Chui5 toxin, therefore a partial neutralization of the venom was observed. Thus, scFv 10FG2 was subjected to three processes of directed evolution and phage display against Chui5 toxin until obtaining scFv HV. Interaction kinetic constants of these scFvs with the toxins were determined by surface plasmon resonance (SPR) as well as thermodynamic parameters of scFv variants bound to Chui5. In silico models allowed to analyze the molecular interactions that favor the increase in affinity. In a rescue trial, scFv HV protected 100% of the mice injected with three lethal doses 50 (LD50) of venom. Moreover, in mix-type neutralization assays, a combination of scFvs HV and 10FG2 protected 100% of mice injected with 5 LD50 of venom with moderate signs of intoxication. The ability of scFv HV to neutralize different toxins is a significant achievement, considering the diversity of the species of Mexican venomous scorpions, so this scFv is a candidate to be part of a recombinant anti-venom against scorpion stings in Mexico.

Full text

Citation: Valencia-Martínez, H.;
Olamendi-Portugal, T.;
Restano-Cassulini, R.;
Serrano-Posada, H.; Zamudio, F.;
Possani, L.D.; Riaño-Umbarila, L.;
Becerril, B. Characterization of
Four Medically Important Toxins
from Centruroides huichol Scorpion
Venom and Its Neutralization by
a Single Recombinant Antibody
Fragment. Toxins 2022,14, 369.
https://doi.org/10.3390/
toxins14060369
Received: 22 April 2022
Accepted: 23 May 2022
Published: 26 May 2022
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4.0/).
toxins
Article
Characterization of Four Medically Important Toxins from
Centruroides huichol Scorpion Venom and Its Neutralization by
a Single Recombinant Antibody Fragment
Hugo Valencia-Martínez 1, Timoteo Olamendi-Portugal 1, Rita Restano-Cassulini 1, Hugo Serrano-Posada 2,
Fernando Zamudio 1, Lourival D. Possani 1, Lidia Riaño-Umbarila 3, * and Baltazar Becerril 1, *
1Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional
Autónoma de México, Apartado Postal 510-3, Cuernavaca 62250, Mexico;
hugo.valencia@ibt.unam.mx (H.V.-M.); timoteo.olamendi@ibt.unam.mx (T.O.-P.);
rita.restano@ibt.unam.mx (R.R.-C.); fernando.zamudio@ibt.unam.mx (F.Z.);
lourival.possani@ibt.unam.mx (L.D.P.)
2
Investigador por México, CONACyT-Laboratorio de Biología Sintética, Estructural y Molecular, Laboratorio
de Agrobiotecnología-Tecnoparque CLQ, Universidad de Colima, Carretera Los Limones-Loma de Juárez,
Colima 28627, Mexico; hserrano0@ucol.mx
3Investigadora por México, CONACyT-Instituto de Biotecnología, Universidad Nacional Autónoma de
México, Apartado Postal 510-3, Cuernavaca 62250, Mexico
*Correspondence: lidia.riano@ibt.unam.mx (L.R.-U.); baltazar.becerril@ibt.unam.mx (B.B.);
Tel.: +52-(777)-329-1669 (B.B.)
Abstract:
Centruroides huichol scorpion venom is lethal to mammals. Analysis of the venom allowed
the characterization of four lethal toxins named Chui2, Chui3, Chui4, and Chui5. scFv 10FG2
recognized well all toxins except Chui5 toxin, therefore a partial neutralization of the venom was
observed. Thus, scFv 10FG2 was subjected to three processes of directed evolution and phage display
against Chui5 toxin until obtaining scFv HV. Interaction kinetic constants of these scFvs with the
toxins were determined by surface plasmon resonance (SPR) as well as thermodynamic parameters
of scFv variants bound to Chui5. In silico models allowed to analyze the molecular interactions that
favor the increase in affinity. In a rescue trial, scFv HV protected 100% of the mice injected with
three lethal doses 50 (LD
50
) of venom. Moreover, in mix-type neutralization assays, a combination
of scFvs HV and 10FG2 protected 100% of mice injected with 5 LD
50
of venom with moderate signs
of intoxication. The ability of scFv HV to neutralize different toxins is a significant achievement,
considering the diversity of the species of Mexican venomous scorpions, so this scFv is a candidate to
be part of a recombinant anti-venom against scorpion stings in Mexico.
Keywords:
affinity maturation; kinetic and thermodynamic parameters; phage display; scFv;
scorpion toxins
Key Contribution:
Four lethal toxins were identified in the venom of C. huichol: Chui2, Chui3, Chui4,
and Chui5. Affinity maturation of scFv 10FG2 led to scFv HV, which neutralized the whole venom.
1. Introduction
In Mexico, during 2020, the number of sting accidents in humans caused by scorpions
was approximately 270,000 [
1
]. The most recent reports indicate that 14 species of scorpion
of the genus Centruroides are possibly responsible for envenoming cases [
2
]. However, apart
from the 14 species described, there are at least seven other species responsible for these
intoxications [
3
]. Such is the case of Centruorides huichol scorpion located in Nayarit, Mexico,
where, in 2020, 14,712 intoxications were reported [
1
]. This scorpion, previously classified
within C. noxius species, was reclassified through a taxonomic analysis that allowed to name
C. huichol as an independent species [
4
]. Due to the great diversity of scorpion species and
Toxins 2022,14, 369. https://doi.org/10.3390/toxins14060369 https://www.mdpi.com/journal/toxins

Toxins 2022,14, 369 2 of 20
the novel strategies to obtain specific antibodies directed against the toxic components, in
the cases of species not yet studied, systematic work is required to identify and characterize
the toxic components present in the venoms. The recovery of sufficient biological material
to implement the generation of antibodies and/or fragments thereof, which are capable of
neutralizing toxic components, is also important.
Various groups have ventured into using single-chain antibody variable fragments
(scFv) as an alternative for generating anti-venoms against venomous animals [
5
–
7
]. This
format has the advantage of being relatively small (27 kDa), which allows it to have better
bioavailability and faster elimination [
8
]. On the other hand, its affinity can be improved
relatively quickly (even to a higher level than natural immunization). Additionally, it is
possible to extend the cross-reactivity through several cycles of directed evolution using
site-directed [
9
] or random [
10
] mutagenesis. The characterization of its kinetic constants
and thermodynamic interaction parameters can be evaluated efficiently through surface
plasmon resonance [11].
From a phage-displayed repertoire of human scFvs and screened against Cn2 toxin
(main toxin of C. noxius venom), scFvs 3F and C1 [
12
] were isolated, which recognize
different epitopes of this toxin [
13
]. From scFv 3F, scFvs 6009F and 9004G were gener-
ated by directed evolution against Cn2 and Css2 (from C. noxius and C. suffusus, respec-
tively). Through a combination of mutations, scFv LR was obtained, which contains V101F
(HCDR3) mutation of scFv 6009F within the context of scFv 9004G, which is an efficient
antibody capable of neutralizing the main toxins (Cn2 and Css2 respectively) and venoms
of C. noxius and C. suffussus [
10
]. On the other hand, from the scFv C1, scFv 10FG2 was gen-
erated, by directed evolution, random and site-directed mutagenesis, and a combination of
mutations [
12
–
16
]. The individual or joint use of these scFvs allowed to neutralize various
scorpion venoms and toxins [
10
,
16
,
17
]. However, there are venoms such as C. limpidus
whose neutralization by scFv 10FG2 is partial due to the presence of the Cl13 toxin, which
is not recognized by 10FG2 [
17
]. Recently, the generation of scFv 11F of murine origin
was published. This scFv recognized and neutralized Cl13 toxin and mixed with 10FG2,
neutralized the whole venom of C. limpidus [18].
Identification of the toxic components for mammals of C. huichol venom, led to identify
Chui5 toxin, whose sequence differs significantly from the one corresponding to toxins
already recognized and neutralized by scFv 10FG2, explaining why it is not capable of
efficiently neutralizing the whole venom. In this work, through the affinity maturation of
10FG2 toward Chui5 toxin by means of the construction of different libraries generated
by random and/or site-directed mutagenesis, scFv HV was obtained. This scFv could
neutralize Chui5 toxin and helped, in conjunction with scFv 10FG2 to optimally rescue
intoxicated mice with the whole venom.
2. Results
C. huichol scorpion (Supplementary Figure S1) was recently identified as a new
species [
4
], and therefore a characterization process was initiated to confirm whether
it is of public health importance, due to its potential toxicity to mammals. The toxicity of
the venom in CD1 mice was evaluated and the LD
50
of the fresh venom was determined
using the “up & down” method [
19
] described in Materials and Methods section. This was
15.4
µ
g/20 g of mouse, which is in the intermediate range of toxicity with respect to the
LD50 of other Mexican scorpion venoms [2].
2.1. Preliminary Neutralization Assays of C. huichol Venom with scFvs 10FG2 and LR
The scFvs generated in our group so far, have shown significant cross-neutralization
capacity against various scorpion toxins. Based on these observations, mix-type assays
were carried out [
16
], where 1 LD
50
of fresh C. huichol venom was mixed with scFvs LR and
10FG2 at a molar ratio of 1:10:10 (venom: scFv LR: scFv 10FG2). To estimate the amount of
scFv to be used, in this experiment it was assumed that the proportion of toxins corresponds
to 10% of the venom (average value with respect to the venoms studied so far [
16
,
18
,
20
]).

Toxins 2022,14, 369 3 of 20
Control mice treated with 1 LD
50
of venom, showed signs of severe intoxication 15 min after
injection and three died within 2 h. The animals injected with a mixture of venom and the
scFvs 10FG2 and LR showed a 5 h delay in the appearance of signs of intoxication, which
were mild to moderate, and one animal died 12 h post-injection (Table 1). These results
indicate that not all the medically important toxins present in the venom were efficiently
neutralized, so it was necessary to identify the toxic components of this venom.
Table 1. C. huichol venom neutralization assay with scFvs LR and 10FG2.
Venom µg/20 g of Mouse LD50 Molar Ratio Survivors (Alive/Total)
Control Mix-Type Assay
C. huichol 15.4 1 1:10:10 1/4 *** 3/4 **
Signs of intoxication are indicated by ** moderate (tail wagging, abdominal twitching); *** strong (sialorrhea,
difficult breathing, paralysis of hind legs, death). Control group was injected with 1 LD
50
of venom. The molar
ratio shown refers to venom: scFv LR: scFv 10FG2. For this assay, a proportion of 10 % of the toxins within the
venom was assumed [16,18,20].
2.2. Characterization of Toxic Components
In order to know the characteristics of the toxic components of C. huichol venom, dif-
ferent chromatographic procedures were used. Venom extraction allowed obtaining 107 mg
of soluble component, which was separated by molecular exclusion chromatography on
a Sephadex G-50 column (see Materials and Methods details) obtaining 4 fractions
(Figure 1a). To identify the toxic components, the toxicity of the fractions and sub-fractions
that typically contain the toxins isolated in the different chromatographic steps was eval-
uated in mice. It is known that the FII fraction contains the peptides that are toxic
to mammals [
21
–
23
]. This toxic fraction was subsequently processed by ion exchange
chromatography on a carboxymethylcellulose (CMC) column, obtaining 14 sub-fractions
(Figure 1b). Sub-fractions FII.9, FII.10 and FII.12 were toxic to mice at a dose of 30
µ
g
of total protein/20 g mouse. These sub-fractions were subjected to reverse phase HPLC
chromatography on a C18 column. Fraction FII.9 contains three main peaks, while fractions
FII.10 and FII.12 only have one peak (Figure 1c). Each of these peaks were subjected to mass
spectrometry to determine their purity and molecular masses. The FII.9-1 fraction (named
Chui1), showed a molecular mass of 7405.8 Da, typical of toxins that affect voltage-gated
sodium channels; however, mice injected with Chui1 did not show signs of intoxication.
For this reason, Chui1 was not further analyzed and described here. Fractions FII.9-2,
FII.9-3, FII.10-1, and FII.12-1, with masses in the same range, were lethal at a concentration
of 1.7
µ
g/20 g mouse (Table 2). It was also possible to estimate their proportion within
the venom. Toxins present in fractions FII.10-1 (Chui4 with 6.2%) and FII.12-1 (Chui5 with
5.7%), were the most abundant while the least abundant were FII.9-3 (Chui3) and FII.9-2
(Chui2) with abundances of 1.8% and 0.3% respectively.
To know the primary structure of the toxins, the pure peaks were processed for
sequencing using the Edman degradation methodology [
17
]. The amino-terminal was
directly sequenced to know the first 34, 35, 40, and 40 amino acids of the Chui2, Chui3,
Chui4, and Chui5 toxins, respectively. Carboxyl-terminal sequence was determined by
digesting each toxin with Asp-N and V8 proteases. The generated peptides, once purified,
were sequenced. Identification of the cysteine residues (Figure 1d) was carried out by
means of reduction and alkylation reactions [17].
To better understand the action of the venom, the activity of Chui3, Chui4, and Chui5
toxins was tested on human voltage gated sodium ion channels (hNav) of type: hNav1.1,
hNav1.2, hNav1.3, hNav1.4, hNav1.5, hNav1.6, and hNav1.7. The three toxins act on
hNav1.6 as
β
-type toxins, modifying the voltage dependence of the channels activation
process. This means that, during toxins exposure, the hNav1.6 channels open at more
negative membrane potential; meanwhile the maximal current is reduced (Figure 2). The
three toxins were tested at a concentration of 200 nM and under these conditions Chui5
was the most active on the hNav1.6 channels, followed by Chui3 and finally Chui4. Chui5

Toxins 2022,14, 369 4 of 20
toxin also had a weak
β
-effect on hNav1.5 channels, reducing the total current of these
channels. Chui4 was also able to weakly shift the activation curves of the hNav1.2 and
hNav1.4 (Supplementary Figure S2). The values of the fitting parameters and of the current
reduction are summarized in the Supplementary Table S1. Finally, in these conditions, it
can be considered that the action of Chui3, Chui4, and Chui5 is mainly toward hNav1.6
channels. The amount of purified Chui2 was not enough for electro physiological assays.
Toxins 2022, 14, x FOR PEER REVIEW 4 of 21
Figure 1. Characterization of the toxic components of C. huichol venom. A sample corresponding to
107 mg of protein from fresh venom of the C. huichol scorpion was processed to purify the toxic
components. (a) Molecular exclusion chromatogram of the fresh venom. (b) Ion exchange chroma-
togram of the FII fraction showing 14 sub-fractions. (c) Reversed-phase chromatograms of subfrac-
tions FII.9 (Chui1, Chui2 and Chui3), FII.10 (Chui4), and FII.12 (Chui5). (d) Alignment of the se-
quences of Chui2, Chui3, Chui4, and Chui5 toxins showing the direct sequencing (D) of the first 34–
40 amino acids and of the fragments generated by the Asp-N and V8 proteases. Cysteine residues
are indicated in bold.
Table 2. Characteristics of C. huichol toxins.
Sub-Fraction
Name Toxicity
Experimental MW
(Da)
Theoretical MW
(Da)
Relative
Abundance (%)
FII.9-1
Chui1
No
7405.8
ND
5.9
FII.9-2
Chui2
Yes
7633.5
7633.7
0.3
FII.9-3
Chui3/Cn3
Yes
7543.9
7544.7
1.8
FII.10-1
Chui4
Yes
7496.7
7497.6
6.2
FII.12-1
Chui5
Yes
7725.6
7726.8
5.7
Toxicity of all fractions was evaluated at a dose of 1.7 µg/20 g of mice. ND: not determined; because
the amino acid sequence was not obtained. Chui3 toxin has the same sequence as the Cn3 toxin of
C. noxius.
To better understand the action of the venom, the activity of Chui3, Chui4, and Chui5
toxins was tested on human voltage gated sodium ion channels (hNav) of type: hNav1.1,
hNav1.2, hNav1.3, hNav1.4, hNav1.5, hNav1.6, and hNav 1.7. The three toxins act on
Figure 1.
Characterization of the toxic components of C. huichol venom. A sample corresponding
to 107 mg of protein from fresh venom of the C. huichol scorpion was processed to purify the
toxic components. (
a
) Molecular exclusion chromatogram of the fresh venom. (
b
) Ion exchange
chromatogram of the FII fraction showing 14 sub-fractions. (
c
) Reversed-phase chromatograms of
subfractions FII.9 (Chui1, Chui2 and Chui3), FII.10 (Chui4), and FII.12 (Chui5). (
d
) Alignment of the
sequences of Chui2, Chui3, Chui4, and Chui5 toxins showing the direct sequencing (D) of the first
34–40 amino acids and of the fragments generated by the Asp-N and V8 proteases. Cysteine residues
are indicated in bold.
Table 2. Characteristics of C. huichol toxins.
Sub-
Fraction Name Toxicity Experimental MW
(Da)
Theoretical MW
(Da)
Relative
Abundance (%)
FII.9-1 Chui1 No 7405.8 ND 5.9
FII.9-2 Chui2 Yes 7633.5 7633.7 0.3
FII.9-3
Chui3/Cn3
Yes 7543.9 7544.7 1.8
FII.10-1 Chui4 Yes 7496.7 7497.6 6.2
FII.12-1 Chui5 Yes 7725.6 7726.8 5.7
Toxicity of all fractions was evaluated at a dose of 1.7
µ
g/20 g of mice. ND: not determined; because the amino
acid sequence was not obtained. Chui3 toxin has the same sequence as the Cn3 toxin of C. noxius.

Toxins 2022,14, 369 5 of 20
Toxins 2022, 14, x FOR PEER REVIEW 5 of 21
hNav1.6 as β-type toxins, modifying the voltage dependence of the channels activation
process. This means that, during toxins exposure, the hNav1.6 channels open at more neg-
ative membrane potential; meanwhile the maximal current is reduced (Figure 2). The
three toxins were tested at a concentration of 200 nM and under these conditions Chui5
was the most active on the hNav1.6 channels, followed by Chui3 and finally Chui4. Chui5
toxin also had a weak β-effect on hNav1.5 channels, reducing the total current of these
channels. Chui4 was also able to weakly shift the activation curves of the hNav1.2 and
hNav1.4 (Supplementary Figure S2). The values of the fitting parameters and of the cur-
rent reduction are summarized in the Supplementary Table S1. Finally, in these condi-
tions, it can be considered that the action of Chui3, Chui4, and Chui5 is mainly toward
hNav1.6 channels. The amount of purified Chui2 was not enough for electro physiological
assays.
Figure 2. Effect of the C. huichol toxins on sodium channels. The figure shows the voltage depend-
ence of activation (circles) and inactivation (squares) process of the hNav 1.6 channel under control
condition (closed symbols) and for toxins at 200 nM (open symbols). Data were fitted by means of
Boltzmann equation (grey line). For hNav 1.6 with Chui3, Chui4, and Chui5 toxins, data were fitted
with double Boltzmann equations, where one of them has the same parameter of the corresponding
control condition. The activation and inactivation fitting parameters, as well as the fractional resid-
ual current values are summarized in Supplementary Table S1.
2.3. Evaluation of the Recognition of scFvs LR and 10FG2 toward C. huichol Toxins
A mixture of scFvs LR and 10FG2 generated a delay in the time of the onset of signs
of envenoming in mice treated with these scFvs, as well as a higher survival rate of the
mice with respect to the control (Table 1). Due to these observations, the individual capac-
ity of each scFv to interact with the different toxins of C. huichol was evaluated. Toxins
were covalently immobilized on CM5 chips reaching bindings between 90 and 417 reso-
nance units (RU). Molecular interactions with scFvs were determined with the
BIAcoreX100. Figure 3 shows the sensorgrams of the interaction of the scFvs with the tox-
ins. The evaluation of the scFvs was performed using a concentration of 100 nM for the
scFv 10FG2 and 25 nM for the scFv LR. The recognition level of the scFv 10FG2 against
Chui2, Chui3, and Chui4 toxins is relatively high while the interaction with Chui5 is much
lower. In this last case, despite showing a good level of association it dissociates quickly
(Figure 3).
In the analyses of scFv LR at 25 nM, it is observed that it also binds to three toxins to
a good level (Chui2, Chui3, and Chui4); however, it dissociates much faster than scFv
10FG2. It is important to note for an antibody fragment to be neutralizing, it must show a
minimal dissociation. Moreover, LR does not interact with Chui5 while scFv 10FG2 does.
These results showed that scFv 10FG2 contributes to a higher extent in the neutralization
of the toxins and for this reason, the kinetic constants of interaction were determined, as
well as the evaluation of the neutralizing capacity was carried out. The determination of
the affinity was achieved by evaluating the interaction of scFv 10FG2 by SPR at different
concentrations (between 10 and 225 nM) with the four toxins at a temperature of 25 °C
(Table 3). The KD of scFv 10FG2 for Chui2 and Chui3 toxins are in the nM range and for
Chui4 in the sub-nM range. From the koff, it is possible to calculate the residence time (TR)
Figure 2.
Effect of the C. huichol toxins on sodium channels. The figure shows the voltage dependence
of activation (circles) and inactivation (squares) process of the hNav 1.6 channel under control
condition (closed symbols) and for toxins at 200 nM (open symbols). Data were fitted by means of
Boltzmann equation (grey line). For hNav 1.6 with Chui3, Chui4, and Chui5 toxins, data were fitted
with double Boltzmann equations, where one of them has the same parameter of the corresponding
control condition. The activation and inactivation fitting parameters, as well as the fractional residual
current values are summarized in Supplementary Table S1.
2.3. Evaluation of the Recognition of scFvs LR and 10FG2 toward C. huichol Toxins
A mixture of scFvs LR and 10FG2 generated a delay in the time of the onset of signs of
envenoming in mice treated with these scFvs, as well as a higher survival rate of the mice
with respect to the control (Table 1). Due to these observations, the individual capacity
of each scFv to interact with the different toxins of C. huichol was evaluated. Toxins were
covalently immobilized on CM5 chips reaching bindings between 90 and 417 resonance
units (RU). Molecular interactions with scFvs were determined with the BIAcoreX100.
Figure 3shows the sensorgrams of the interaction of the scFvs with the toxins. The
evaluation of the scFvs was performed using a concentration of 100 nM for the scFv 10FG2
and 25 nM for the scFv LR. The recognition level of the scFv 10FG2 against Chui2, Chui3,
and Chui4 toxins is relatively high while the interaction with Chui5 is much lower. In this
last case, despite showing a good level of association it dissociates quickly (Figure 3).
Toxins 2022, 14, x FOR PEER REVIEW 6 of 21
or the interaction time between the scFv and the toxins. The results showed that they are
higher than previously determined for the scFv 10FG2 with other toxins, which were only
16 min [16]. The level of interaction of 10FG2 with Chui5 toxin is low, since it showed a
KD value of 12.2 nM and a TR of less than 5 min.
Subsequently, neutralization assays of the Chui3, Chui4, and Chui5 toxins were per-
formed with scFv 10FG2. Chui2 toxin was not evaluated because its proportion in the
venom was very low (0.3%), thus we assumed that it would have a limited role in enven-
oming. Furthermore, its yield was insufficient to implement toxicity assays. For the other
toxins, 1 LD100 was assayed with scFv 10FG2 in a 1:10 molar ratio (toxin: scFv). While con-
trols showed 100% mortality with all toxins, scFv 10FG2 was able to completely neutralize
the toxic effect of Chui3 and Chui4 since no signs of envenoming were observed and all
individuals survived (Table 4). In view of the KD and TR of the scFv toward these two
toxins and the Chui2 toxin (Table 3), it is very likely that the latter is also neutralized by
10FG2. In contrast, scFv 10FG2 with Chui5 toxin only managed to neutralize 33.3% of the
mice, with severe signs of intoxication being observed (Table 4). Taking into account that
Chui5 corresponds to 5.7% of the venom, the incomplete neutralization of C. huichol
venom by scFv 10FG2 is explained.
Figure 3. Sensorgrams of the interaction of the indicated scFvs with the toxins of C. huichol. The
toxins were bound to a CM5 Chip and the interaction of the scFvs 10FG2 and HV at a concentration
of 100 nM and of LR at 25 nM was evaluated. Sensorgrams were obtained at the standard tempera-
ture of 25 °C.
Table 3. Kinetic parameters of the interaction of scFvs 10FG2 and HV with C. huichol toxins.
scFv
Toxin
k
on
(×10
5
M
−1
s
−1
)
k
off
(×10
−4
s
−1
)
T
R
(min)
K
D
(nM)
10FG2
Chui2
2.8
2.8
60.4
1.1
Chui3
1.8
3.1
53.1
1.8
Chui4
2.1
0.7
241.5
0.3
Chui5
2.6
34.1
4.9
12.2
HV
Chui2
1.6
4.9
34.2
3.1
Chui3
0.9
3.4
48.8
3.8
Chui4
1.8
1.6
104.8
0.9
Chui5
1.6
10.8
15.5
6.9
Figure 3.
Sensorgrams of the interaction of the indicated scFvs with the toxins of C. huichol. The
toxins were bound to a CM5 Chip and the interaction of the scFvs 10FG2 and HV at a concentration of
100 nM and of LR at 25 nM was evaluated. Sensorgrams were obtained at the standard temperature
of 25 ◦C.
In the analyses of scFv LR at 25 nM, it is observed that it also binds to three toxins
to a good level (Chui2, Chui3, and Chui4); however, it dissociates much faster than scFv
10FG2. It is important to note for an antibody fragment to be neutralizing, it must show

Toxins 2022,14, 369 6 of 20
a minimal dissociation. Moreover, LR does not interact with Chui5 while scFv 10FG2 does.
These results showed that scFv 10FG2 contributes to a higher extent in the neutralization
of the toxins and for this reason, the kinetic constants of interaction were determined, as
well as the evaluation of the neutralizing capacity was carried out. The determination of
the affinity was achieved by evaluating the interaction of scFv 10FG2 by SPR at different
concentrations (between 10 and 225 nM) with the four toxins at a temperature of 25
◦
C
(Table 3). The K
D
of scFv 10FG2 for Chui2 and Chui3 toxins are in the nM range and for
Chui4 in the sub-nM range. From the k
off
, it is possible to calculate the residence time (T
R
)
or the interaction time between the scFv and the toxins. The results showed that they are
higher than previously determined for the scFv 10FG2 with other toxins, which were only
16 min [
16
]. The level of interaction of 10FG2 with Chui5 toxin is low, since it showed a K
D
value of 12.2 nM and a TRof less than 5 min.
Table 3. Kinetic parameters of the interaction of scFvs 10FG2 and HV with C. huichol toxins.
scFv Toxin kon (×105M−1s−1)koff (×10−4s−1) TR(min) KD(nM)
10FG2
Chui2 2.8 2.8 60.4 1.1
Chui3 1.8 3.1 53.1 1.8
Chui4 2.1 0.7 241.5 0.3
Chui5 2.6 34.1 4.9 12.2
HV
Chui2 1.6 4.9 34.2 3.1
Chui3 0.9 3.4 48.8 3.8
Chui4 1.8 1.6 104.8 0.9
Chui5 1.6 10.8 15.5 6.9
Subsequently, neutralization assays of the Chui3, Chui4, and Chui5 toxins were per-
formed with scFv 10FG2. Chui2 toxin was not evaluated because its proportion in the
venom was very low (0.3%), thus we assumed that it would have a limited role in en-
venoming. Furthermore, its yield was insufficient to implement toxicity assays. For the
other toxins, 1 LD
100
was assayed with scFv 10FG2 in a 1:10 molar ratio (toxin: scFv).
While controls showed 100% mortality with all toxins, scFv 10FG2 was able to completely
neutralize the toxic effect of Chui3 and Chui4 since no signs of envenoming were observed
and all individuals survived (Table 4). In view of the K
D
and T
R
of the scFv toward these
two toxins and the Chui2 toxin (Table 3), it is very likely that the latter is also neutralized
by 10FG2. In contrast, scFv 10FG2 with Chui5 toxin only managed to neutralize 33.3% of
the mice, with severe signs of intoxication being observed (Table 4). Taking into account
that Chui5 corresponds to 5.7% of the venom, the incomplete neutralization of C. huichol
venom by scFv 10FG2 is explained.
Table 4. Neutralization of C. huichol toxins with scFv 10FG2.
Toxin 1 LD100 (µg/20 g of Mouse) Molar Ratio Survivors (Alive/Total)
Control Mix-Type Assay
Chui3 2 1:10 0/6 *** 6/6
Chui4 2.5 1:10 0/6 *** 6/6
Chui5 2 1:10 0/6 *** 2/6 ***
Control and experimental groups were injected with 1 LD
100
of the different toxins. Signs of intoxication are
indicated by *** strong (sialorrhea, difficult breathing, paralysis of hind legs, death).
2.4. Affinity Maturation of scFv 10FG2 against Chui5 Toxin
scFv 10FG2 turned out to be the best candidate to be matured since it recognizes and
partially neutralized Chui5 toxin. Based on these characteristics, it was decided to improve
its interaction properties until it became a neutralizing scFv. For the first maturation process,
a library constructed by site-directed mutagenesis at positions Y53 and S57 of HCDR2, Y59
of HFR3 and D102, as well as L105 of HCDR3, with a size of 3
×
10
7
transformants was used.
These positions were chosen from the analysis of the interaction of the scFv 10FG2 with

Toxins 2022,14, 369 7 of 20
Cl13 toxin from an in silico model (see Materials and Methods). Three rounds of screening
against Chui5 toxin were performed, and from the third round, 180 random colonies were
evaluated by ELISA assays. From this process, variant 9a was selected, which was the
one that showed the highest signal in an ELISA assay against the toxin (Supplementary
Figure S3). The sequence of this variant beared the L105Q change in HCDR3, identifying
position 105 as important for the interaction with the toxin. When determining the kinetic
parameters of the interaction, a decrease in the value of the dissociation rate (k
off
) was
observed, resulting in an improvement in both the affinity constant (K
D
; 8.2 nM) and the T
R
(14 min; Figure 4a; Table 5). With this improvement, it was decided to determine the LD
50
of Chui5 toxin and the neutralizing capacity of the 9a variant. Again, the “up & down”
method was used, as in ref [
24
], and it was determined that the LD
50
of Chui5 toxin was
1.1
µ
g/20 g of CD1 mice. For the neutralization assay, 2 LD
50
of the toxin were used with
a molar ratio of scFv 1:10. scFv 9a increased the extent of protection to 60% of the mice and
with an average delay time of the onset of signs around 99 min, while scFv 10FG2 protected
16.7% of mice and the onset of signs at around 18 min after injection (Table 6).
Toxins 2022, 14, x FOR PEER REVIEW 8 of 21
Figure 4. Affinity maturation cycles of scFv 10FG2 against Chui5 toxin. (a) Sensorgrams showing
the interaction of scFv 10FG2 and the three variants obtained through corresponding three matura-
tion processes at a concentration of 100 nM against Chui5 toxin. The adjacent table describes the
mutations acquired by each of the variants, as well as the positions in which they are found. (b)
Scorpion toxins aligned according to the level of recognition they show against scFv 10FG2. The
analysis was performed evaluating the amino acid changes that the toxins present in the different
reported interaction sites (asterisks) with the scFv 10FG2. In this way, positions 8 and 9 in the toxins
(as well as amino acids K65 and L105 in the scFv) whose changes affect the interaction (indicated by
the plus sign) were identified.
Table 5. Determination of the kinetic parameters of Chui5-scFvs interaction.
scFv
k
on
(×10
5
M
−1
s
−1
)
k
off
(×10
−4
s
−1
)
T
R
(min)
K
D
(nM)
Yield (mg/L)
10FG2
2.6
34.1
4.9
12.2
3.1
9a
1.5
11.9
14.0
8.2
2.3
B15a
1.3
9.7
17.1
7.3
1.5
HV
1.6
10.8
15.5
6.9
1.7
Table 6. Neutralization assays of the matured scFvs against Chui5 toxin.
scFv LD50
μg/20 g of
Mouse
Molar Ratio
Appearance of
Signs (min)
Survivors (Alive/Total)
Control
Mix-Type Assay
-
2
2.2
-
8
0/6 ***
-
10FG2
2
2.2
1:10
18
0/6 ***
1/6 ***
9a
2
2.2
1:10
99
0/4 ***
3/5 **
B15a
2
2.2
1:10
125
0/6 ***
2/6 **
HV
2
2.2
1:10
138
0/4 ***
3/4 *
HV
1
1.1
1:20
NS
3/6 ***
6/6
Control groups with a 1:10 molar ratio were injected with 2 LD50 of toxin. Control group with molar
ratio 1:20 was injected with 1 LD50 of toxin. The molar ratio represents toxin: scFv. NS: no signs of
Figure 4.
Affinity maturation cycles of scFv 10FG2 against Chui5 toxin. (
a
) Sensorgrams showing the
interaction of scFv 10FG2 and the three variants obtained through corresponding three maturation
processes at a concentration of 100 nM against Chui5 toxin. The adjacent table describes the mutations
acquired by each of the variants, as well as the positions in which they are found. (
b
) Scorpion
toxins aligned according to the level of recognition they show against scFv 10FG2. The analysis
was performed evaluating the amino acid changes that the toxins present in the different reported
interaction sites (asterisks) with the scFv 10FG2. In this way, positions 8 and 9 in the toxins (as well as
amino acids K65 and L105 in the scFv) whose changes affect the interaction (indicated by the plus
sign) were identified.
For the following maturation process, clone 9a was used as a template from which
a library was generated by random mutagenesis [
14
] with a size of 2.8
×
10
7
transformants.
After the third round of screening, 180 random clones were evaluated and the B15a variant
was selected which maintained L105Q change and incorporated K65Q mutation in HFR3.
The protein was expressed and evaluated for determining its kinetics interaction with Chui5.

Toxins 2022,14, 369 8 of 20
The parameter that changed was the dissociation rate whose value is lower resulting in
a better interaction with a K
D
= 7.3 nM (Figure 4a, Table 5). This result represents a slight
increase in T
R
(17 min). Its ability to neutralize 2 LD
50
of Chui5 in mice using a 1:10 molar
ratio (toxin: scFv) was also evaluated. The result showed that B15a variant, despite further
delaying the onset of signs (around 125 min), only managed to neutralize 33.3% of the
mice (Table 6).
Table 5. Determination of the kinetic parameters of Chui5-scFvs interaction.
scFv kon (×105M−1s−1)koff (×10−4s−1) TR(min) KD(nM) Yield (mg/L)
10FG2 2.6 34.1 4.9 12.2 3.1
9a 1.5 11.9 14.0 8.2 2.3
B15a 1.3 9.7 17.1 7.3 1.5
HV 1.6 10.8 15.5 6.9 1.7
Table 6. Neutralization assays of the matured scFvs against Chui5 toxin.
scFv LD50 µg/20 g
of Mouse
Molar
Ratio
Appearance of
Signs (min)
Survivors (Alive/Total)
Control Mix-Type Assay
- 2 2.2 - 8 0/6 *** -
10FG2 2 2.2 1:10 18 0/6 *** 1/6 ***
9a 2 2.2 1:10 99 0/4 *** 3/5 **
B15a 2 2.2 1:10 125 0/6 *** 2/6 **
HV 2 2.2 1:10 138 0/4 *** 3/4 *
HV 1 1.1 1:20 NS 3/6 *** 6/6
Control groups with a 1:10 molar ratio were injected with 2 LD
50
of toxin. Control group with molar ratio 1:20
was injected with 1 LD
50
of toxin. The molar ratio represents toxin: scFv. NS: no signs of envenoming. Signs of
intoxication are indicated by * minimal (ruffled hair, itching); ** moderate (tail wagging, abdominal twitching);
*** strong (sialorrhea, difficult breathing, paralysis of hind legs, death).
From an analysis of cross-reactivity level that 10FG2 showed with other toxins from
scorpions of the genus Centruroides (Figure 4b), it was determined that positions 8 and 9 in
the toxins are important for the interaction with amino acids K65 and L105 of the scFv
10FG2 [
14
–
16
] which are different in Chiu5 toxin. For this reason, it was decided to mu-
tagenize positions 65 and 105 to saturation, including random mutagenesis using variant
9a as template. Of the three rounds of screening carried out, the second and third ones
were done under more stringent conditions incubating the phage-antibodies bound to
toxin absorbed in the immunotube in the presence of 0.5 M and 1 M guanidinium chloride
solution in PBS, respectively, for 30 min at 37
◦
C (See Materials and Methods). Likewise,
from the third round, 528 clones were evaluated, of which the best 18 clones were subjected
to sequencing. The sequences were aligned, mainly observing variants with changes at
positions 65 and 105. In addition, other mutations (of lower recurrence) were detected at
other positions, product of random mutagenesis (Supplementary Figure S4a). The variant
called HV was the one that showed the highest signal in the ELISA assay and did not
present amber-type stop codons. This scFv, relative to B15a, kept K65Q change and acquired
three changes: F29Y in HCDR1, L105G in HCDR3, and G154E in LFR1. HV variant was
sub-cloned into the pSyn1 vector and expressed. With the purified protein, the kinetic
parameters of interaction with the toxin were determined. At 25
◦
C, HV variant shows
minimal k
on
enhancement over B15a variant; however, k
off
shows a slight improvement
resulting in a K
D
of 6.9 nM and a T
R
of 15.5 min (Figure 4a, Table 5). Neutralization of
2 LD
50
of Chui5 with HV at a 1:10 molar ratio (toxin: scFv) protected 75% of mice and
showed a delay in the onset of symptoms around 138 min (Table 6). This result is sig-
nificantly better than that obtained with variants 9a and B15a, turning scFv HV into
a candidate for further characterization. To achieve a better neutralization of Chui5 toxin,
the amount of scFv HV was increased, 1 LD
50
was assayed in a molar ratio of 1:20 reaching
100% protection of mice and the absence of envenoming signs (Table 6).

Toxins 2022,14, 369 9 of 20
2.5. Determination of the Kinetic Constants of scFv HV against C. huichol Toxins
Due to the neutralizing properties of the scFv HV against Chui5 toxin, it was decided
to evaluate the binding levels with Chui2, Chui3, and Chui4 toxins (Figure 3). It was
observed that the changes present in this antibody fragment modified its interaction toward
these toxins. When comparing the kinetic constants with those of scFv 10FG2, it is observed
that the K
D
are maintained in the same order (Table 3). For the case of Chui2 toxin,
scFv 10FG2 showed a T
R
of 60.4 min whereas for scFv HV it was 34.2 min. Chui3 toxin
showed a small change since the T
R
was 53.1 min with scFv 10FG2, but for scFv HV it was
48.8 min. The most affected kinetic constants were with Chui4, since T
R
values of 241.5 and
104.8 min were obtained with 10FG2 and HV, respectively. Neutralization of Chui5 toxin
with scFv HV was 100%, with a T
R
of 15 min. Therefore, it could be assumed that scFv
HV also neutralized Chui2, Chui3, and Chui4 toxins because scFv HV residence times
with these toxins are higher. This cross-neutralization has also been observed with other
members of this family [16].
2.6. Neutralization of C. huichol Venom with scFv HV
Considering the cross-reactivity of scFv HV toward Chui2, Chui3, Chui4, and Chui5
toxins and their K
D
and T
R
values against them, a fresh venom neutralization assay was
performed with this variant both in mix-type (venom: scFv), as well as rescue assays
(see Materials and Methods). With regard to mix-type assay, the ability of scFv HV to
neutralize 1 LD
50
of venom in a 1:10 molar ratio (venom: scFv) was evaluated, as well as
in combination with scFv 10FG2 in a molar ratio 1:10:10 (venom: scFv 10FG2: scFv HV).
A 100% protection was achieved in both modalities, with the difference being the presence
of mild signs of intoxication in those mice that only received scFv HV and the complete
absence of symptoms in those who received the mixture of the two scFvs. Because of these
results, in the following assay the amount of scFv HV was maintained while the number of
LD
50
was increased. In this way, when neutralizing 2 LD
50
, the molar ratio was 1:5 (venom:
scFv HV) where the neutralization of 66.7% of the mice was reached with the appearance
of mild signs of intoxication in those that survived. Subsequently, 3 LD
50
with a 1:3.3 molar
ratio (venom: scFv) were assayed, reaching a neutralization percentage of 33.3% of the
mice, which presented moderate signs of intoxication. These results showed that molar
ratios of at least 1:10 (venom: scFv) are required to achieve neutralization with scFv HV.
Based on these results and the ones corresponding to the neutralization of the scFv 10FG2
with the other toxins, the capacity of neutralizing 5 LD
50
of venom using the combination
of scFvs 10FG2 and HV in a 1:5:5 molar ratio, 100% of the mice were protected, although
with the presence of moderate signs of intoxication (Table 7).
Table 7. Neutralization of C. huichol venom with scFv HV.
Assay Group LD50 Molar Ratio Survivors (Alive/Total)
Control 2 - 0/6 ***
HV
1 1:10 6/6 *
Mix-type 2 1:5 4/6 *
3 1:3.3 2/6 **
10FG2 + HV 1 1:10:10 6/6
5 1:5:5 6/6 **
Rescue Control 3 - 0/6 ***
HV 3 1:10 6/6 **
Molar ratio represents venom: scFv HV or venom: scFv 10FG2: scFv HV. The molar ratio for this experiment is
given considering that the proportion of the toxins within the venom is 14%. Signs of intoxication are indicated by
* minimal (ruffled hair, itching); ** moderate (tail wagging, abdominal twitching); *** strong (sialorrhea, difficult
breathing, paralysis of hind legs, death).
On the other hand, scFv HV was evaluated in a rescue assay, which more closely
mimics the treatment of a scorpion sting envenomation. The experimental group of mice

Toxins 2022,14, 369 10 of 20
was injected with 3 LD
50
of fresh venom and 10 min post-injection the scFv HV was applied
at a 1:10 molar ratio. One hundred % of the envenomed mice were rescued. Severe signs of
intoxication disappeared around 2 h after the administration of the scFv HV, and the total
disappearance of the signs occurred around 48 h later (Table 7).
2.7. Thermodynamic and Structural Analysis of the Improvement of scFv HV
scFv HV was able to neutralize Chui5 toxin, so the binding characteristics of scFvs
were analyzed through thermodynamic parameters of interaction and by in silico models
of the Chui5-10FG2 and Chui5-HV complexes.
2.7.1. Thermodynamic Analysis
The values of the interaction kinetic constants of the scFvs HV and B15a at 25
◦
C with
Chui5 toxin are very similar (Table 5); however, the differences in terms of neutralizing
capacity were clear, with HV variant being the best (Table 6). To understand this property,
the kinetic constants of interaction were determined at different temperatures and, with
the K
D
from these results, it is possible to calculate thermodynamic parameters through
the Van’t Hoff expression [
11
,
25
,
26
] (See Materials and methods). For each interaction of
Chui5 with scFvs, a Van’t Hoff plot was created (Supplementary Figure S5). The slope of
the straight line in the Van’t Hoff plot determines the stability of the scFv binding with
Chui5 toxin. The absolute value of the slope of scFvs 10FG2 and B15a is very similar
(1316 and 1299 respectively) but the one for scFv HV was the lowest (814.6) suggesting
that Chui5-HV complex establishes a more stable interaction than the Chui5-B15a complex
when the temperature varies. When calculating the thermodynamic parameters of the
binding, we observed in the three complexes that
∆
G is negative, indicating that these
bindings are spontaneous (Table 8). The negative variation of
∆
H indicates the formation
of weak interactions such as hydrogen bonds, salt bridges, etc., [
26
]. Both scFv 10FG2
and B15a showed equal
∆
H values (
−
2.6 kcal
·
mol
−1
) that are more negative than those
calculated for scFv HV (
−
1.6 kcal
·
mol
−1
). These parameters suggest that scFv HV could be
missing interactions with Chui5 toxin. The positive variation of the
∆
S indicates the degree
of freedom of movement between the scFv and the toxin [
26
]. Chui5-HV interaction is the
one with the highest
∆
S (31.9 cal
·
mol
−1·
K
−1
) compared to Chui5-10FG2 and Chui5-B15a
interactions (27.3 and 28.6 cal
·
mol
−1·
K
−1
respectively), suggesting a greater freedom of
movement in the complex Chui5-HV (Table 8).
Table 8. Thermodynamic parameters of Chui5-scFv complexes.
scFv ∆G (kcal·mol−1)∆H (kcal·mol−1)∆S (cal·mol−1·K−1)
10FG2 −10.8 −2.6 27.3
B15a −11.1 −2.6 28.6
HV −11.1 −1.6 31.9
2.7.2. In Silico Models of Chui5 Toxin with scFvs 10FG2 and HV
To understand how the molecular interactions of scFv HV with Chui5 toxin were
improved, two in silico models of the binary Chui5-10FG2 and Chui5-HV complexes were
generated by in silico mutagenesis and modeling using the crystallographic structure of
scFv RU1-Cn2-LR scFv ternary complex (PDB entry 4V1D) [
13
]. The analysis focused on
the mutated positions of scFv HV. Hence, the cation-
π
interaction of 3.3 Å between F29
of HCDR1 and R72 in the scFv 10FG2 is conservatively changed to the interaction with
Y29 in scFv HV. Nevertheless, Y29 residue also forms a hydrogen bond of 2.8 Å with the
main chain of T78 (Figure 5a). In contrast, while G154 of LFR1 has no interactions in 10FG2
variant, the change to E154 in HV generates a hydrogen bond of 3.1 Å with the side chain of
Q219 (Figure 5b). These two new hydrogen bonds may contribute to the intra-chain stability
of scFv HV. Regarding the changes in the toxin-scFv interaction, while K65 of HFR3 forms
a strong salt bridge of 3.0 Å with D62 in scFv 10FG2, the change to Q65 in scFv HV generates

Toxins 2022,14, 369 11 of 20
a weak hydrogen bond of 3.8 Å (Figure 5c,d). Moreover, although the side chain of L105
in scFv 10FG2 seems to favor fitting the HCDR3 region with the toxin, this adjustment
shows a steric hindrance between the bulky residues Y8/Y59 of Chui5-10FG2 complex,
respectively (Figure 5e). Accordingly, this observation suggests that the low affinity of
scFv 10FG2 toward Chui5 (Table 5) could be related to this weak surface complementarity
between the scFv and the toxin. Remarkably, L105G change in HV removed the steric
hindrance between Y8/Y59 of Chui5-HV complex (Figure 5f), generating a flexible zone
that favored a proper fitting of the HCDR3 region with the toxin.
Toxins 2022, 14, x FOR PEER REVIEW 12 of 21
Figure 5. Structural comparison of the in silico models of binary Chui5-10FG2 and Chui5-HV com-
plexes. (a) F29 of scFv 10FG2 forms a cation-π interaction with R72 of the same scFv, which is con-
served in HV containing residue Y29. In addition, the HV Y29 forms a new hydrogen bond with the
main chain of T78. (b) The E154 mutation in HV presumably allowed the formation of a new hydro-
gen bond with Q219, which could not be formed with G154 of 10FG2. (c,d) K65 of 10FG2 forms a
salt bridge with D62. The salt bridge is lost by exchanging it for Q65 in HV, but a weak hydrogen
bond with D62 is gained. (e) L105 in 10FG2 promotes a conformational change of Y8 in Chui5, show-
ing a steric hindrance with Y59 of 10FG2. (f) By mutating position 105 to a small amino acid such as
glycine, Y8 is now in a looser condition and without the steric hindrance with Y59. The red and
orange colors represent the variable domains of the heavy and light chains of the scFv 10FG2, re-
spectively; the green and blue colors represent the variable domains of the heavy and light chains
of the scFv HV, respectively; the purple color corresponds to the Chui5 toxin. Distances are indi-
cated in angstroms (Å).
Figure 5.
Structural comparison of the in silico models of binary Chui5-10FG2 and Chui5-HV
complexes. (
a
) F29 of scFv 10FG2 forms a cation-
π
interaction with R72 of the same scFv, which
is conserved in HV containing residue Y29. In addition, the HV Y29 forms a new hydrogen bond
with the main chain of T78. (
b
) The E154 mutation in HV presumably allowed the formation of
a new hydrogen bond with Q219, which could not be formed with G154 of 10FG2. (
c
,
d
) K65 of 10FG2
forms a salt bridge with D62. The salt bridge is lost by exchanging it for Q65 in HV, but a weak
hydrogen bond with D62 is gained. (
e
) L105 in 10FG2 promotes a conformational change of Y8 in
Chui5, showing a steric hindrance with Y59 of 10FG2. (
f
) By mutating position 105 to a small amino
acid such as glycine, Y8 is now in a looser condition and without the steric hindrance with Y59. The
red and orange colors represent the variable domains of the heavy and light chains of the scFv 10FG2,
respectively; the green and blue colors represent the variable domains of the heavy and light chains
of the scFv HV, respectively; the purple color corresponds to the Chui5 toxin. Distances are indicated
in angstroms (Å).

Toxins 2022,14, 369 12 of 20
3. Discussion
In recent years, progress has been made in the study of the diversity of Mexican
scorpion species of medical importance [
2
,
3
]. One of them recently described is the scorpion
C. huichol [
4
]. The present work showed that the venom of this species is lethal to mammals.
The toxicity is due to the presence of four lethal toxins in this venom: Chui2, Chui3, Chui4,
and Chui5. It is noteworthy that Chui3 sequence is the same as the one of Cn3 toxin from
the venom of C. noxius [
27
]. Of the three more abundant toxins, it was confirmed that they
showed activity on hNav1.6 human sodium channel. In the case of Chui5 toxin, it also
modified the activity of hNav1.5 channel. The presence of four components with a total
abundance of 14% is significant since, typically, the abundances of the toxins correspond to
less than 10% in the venom of Mexican scorpions [16,18,20].
C. huichol venom was partially neutralized by scFv 10FG2, which is capable of neu-
tralizing various venoms and toxins from scorpions of the genus Centruroides [
16
]. This
partial neutralization is due to the presence of Chui5 toxin with an abundance of 5.7% and
whose primary structure varies considerably from those already neutralized by this scFv.
In this work, Chui5 toxin was used to perform three
in vitro
affinity maturation processes
of scFv 10FG2. In the first process, variant 9a with L105Q mutation was obtained. The
change in this position, compared to the others obtained, was the most important in terms
of improvement of K
D
and T
R
. This variant delayed the onset of intoxication signs, as
well as their intensity due to a considerable increase in T
R
; however, the toxic effect of
Chui5 was not neutralized. Position 105 of the parental scFv C1 was previously changed in
processes of directed evolution against toxin Cll1 from C. limpidus venom. In that work,
the scFv 202F (with various mutations including M105L) was isolated, whose kinetic and
neutralizing characteristics were improved when compared to its parent (scFv C1) [
14
]. In
addition, position 105 has been shown to be an important point of hydrogen bond-type
interactions between several amino acids of various toxins (positions 5, 6, 8, 33, 55, and 56)
and scFv 10FG2 [
16
]. Variant 9a was used as template for the second maturation process,
obtaining variant B15a that maintains L105Q mutation and incorporates K65Q mutation.
This variant showed an improved K
D
value compared to 9a and a higher T
R
, delaying the
onset of signs; however, its neutralizing capacity was lower. Previously, a model of the
interaction between toxin Cn2 and scFv RJI-2 was generated which showed that position
K65 of the scFv forms a hydrogen bond with N9 of the toxin. This observation prompted
to identify RJI-2 as a target for mutagenesis by applying a rational design strategy. In
this way, ER-5 variant was generated with K65R mutation, which neutralizes toxins Cn2,
Cll1, and Css2, as well as the venom of C. suffussus [
15
]. Initially in this work, L105Q and
K65Q changes were shown to be relevant for the affinity maturation toward Chui5 toxin.
However, the results of the neutralization and the interaction kinetic constants indicated
that the selected changes are not necessarily the optimal ones to obtain a neutralizing scFv.
With this rational, positions 65 and 105 of scFv 9a were again mutagenized to saturation
and random mutagenesis were performed at the same time. From this process 18 clones
were isolated and sequenced, letting to analyze the frequency of occurrence of the amino
acids at positions 65 and 105 (Supplementary Figure S4b). It was observed that 50% of
the sequences showed a glutamine at position 65 and 50% a glycine at position 105. Only
4 clones of the 18 evaluated carried changes K65Q and L105G, which are also present in
the best variant, scFv HV. This scFv showed the highest recognition signal in ELISA assays
(data not shown) and the absence of amber stop codons (E. coli TG1 replaces amber stop
codon by Q). This variant with an improved K
D
despite a decreased T
R
value with respect
to scFv B15a, was able to neutralize Chui5 toxin. The evaluation of scFv HV with the other
toxins of the C. huichol venom showed that the mutations present decreased affinity and T
R
(Table 3). However, these K
D
and T
R
values are similar to those previously reported for
neutralizing members of this family of scFvs [16,17].
Due to the affinity properties, the cross-reactivity and the ability of the scFv HV to
neutralize Chui5 toxin, its neutralization capacity against C. huichol venom was evaluated.
In mix-type assays, the neutralization of 1 LD
50
with scFv HV and with the combination of

Toxins 2022,14, 369 13 of 20
scFvs 10FG2 and HV was evaluated using a 1:10:10 molar ratio (venom: scFv(s)). In both
groups, the neutralization was 100%. The number of LD
50
was also increased, decreasing
the venom: scFv molar ratio and it was observed that the neutralization decreased pro-
portionally to the increase in LD
50
until a partial neutralization of 33% was obtained with
3 LD
50
. These results lead to consider the use scFv 10FG2 as a neutralizing complement
since it neutralizes other toxins except Chui5 (Table 7). At molar ratios 1:10:10, neutraliza-
tion occurred without signs of intoxication; thus, it was tested at lower molar ratios. The
assay was made with 5 LD
50
of venom mixed with scFvs 10FG2 and HV at molar ratio 1:5:5
(venom: scFv 10FG2: scFv HV). A 100% neutralization was reached but with moderate
signs of envenoming as expected due to this extreme challenge. Rescue assays were also
carried out since they are closer to what happens during scorpion sting accidents. Only
scFv HV was used in the rescue assays. Mice were envenomed with 3 LD
50
and 10 min later
injected with the scFv; all mice survived the challenge. The disappearance of the strong
signs of intoxication (sialorrhea, respiratory stress) was evident at around 2 h post injection,
although discomfort in the area of the application of the venom disappeared around 48 h
later. If we consider the mix-type neutralization assay of 5 LD
50
with scFvs 10FG2 and HV,
the presence of 10FG2 in rescue assays can complement the neutralization of the venom
and turn it even more efficient.
When analyzing the mutations of scFv HV, it was found that both the mutations
in the FRs and those of the CDRs of the heavy chain variable domain contribute to the
improvement of the neutralizing properties. Mutations F29Y and G154E seem to contribute
to the stabilization of scFv HV at higher temperatures, which allowed the maintenance
of binding to the toxin observed in the Van’t Hoff plot. It is likely that it is the result of
the formation of new hydrogen bonds between Y29 and E154 with other residues of the
scFv. Possibly, these mutations were favored by the use of guanidinium chloride in the
screening rounds by eliminating unstable variants [
28
] and/or weak interactions. The
differences observed in the values of the slopes in the Van’t Hoff plot correlate with the
better neutralization efficiency of scFv HV compared to that of scFv B15a that do not
possess these changes. Increased stability has also been observed in scFv LR [
10
] and
LER [
29
] scFvs with mutations in the FR region for example. Structural analysis of changes
K65Q and L105G in the interaction area, suggests the conformation of a more flexible
contact area, without steric hindrance for the accommodation of bulky amino acids such
as Y8 of Chui5 toxin and Y59 of scFv HV. This rearrangement of the interaction forces, in
terms of thermodynamic parameters, would be favoring binding. The negative
∆
G of all
interacting complexes indicates that the binding would be spontaneous and would form
a stable complex. The
∆
H of the Chui5-HV complex is slightly less negative than Chui5-
B15a and Chui5-10FG2 complexes, suggesting that original interactions of L105 were
possibly lost [
16
], correlating with the decrease of the interaction of scFv HV with Chui2,
Chui3, and Chui4 (Figure 3and Table 3). This loss of interactions is reflected in the increase
in
∆
S in the Chui5-HV complex, which suggests a greater degree of movement of amino
acid residues in the interaction zone of the toxin and scFv [30].
Cross-reactivity with toxins from the venom of different species is a highly desirable
phenomenon in those scFvs that would be selected to form part of an anti-venom. In
the case of scFv HV, it was observed that it recognized a recently identified C. tecomanus
toxin too (unpublished data). We have successfully expanded the cross-neutralization of
scFv 10FG2, so it would be very useful to have the structure of the Chui5-HV complex
to optimize this interaction and make the neutralization of these and other toxins even
more efficient.
4. Conclusions
The characterization of the venom of C. huichol, the processes of directed evolution,
the characterization of the scFv HV in terms of cross-reactivity, neutralizing capacity of
the venom of C. huichol, and the analysis of kinetic and thermodynamic interactions, have

Toxins 2022,14, 369 14 of 20
allowed to propose this scFv to be part of a novel recombinant anti-venom against the sting
of Mexican scorpions.
5. Materials and Methods
5.1. Venom and Reagents
C. huichol scorpions were purchased from the company Octolab (Xalapa, Veracruz,
Mexico). The venom was obtained by electro stimulation as reported [
21
]. The milked
venom was dissolved in tetra-distilled water and centrifuged at 25,000
×
grpm at 8
◦
C
for 20 min. Soluble protein was quantified by spectrophotometry at
λ
= 280 nm. It was
assumed that 1 AU = 1 mg/mL. All reagents used were of analytical grade.
5.2. Enzymes
Taq polymerase, T4 DNA ligase, and restriction enzymes Not1 and Sfi1 were purchased
from Thermo Fisher (Waltham, MA, USA). V8 and AspN enzymes were purchased from
Roche (Basel, Switzerland).
5.3. Purification, Analysis by Mass Spectrometry, and Sequencing of Toxins
Purification, analysis of pure toxins by mass spectrometry, and Edman degradation
sequencing were performed as reported [
17
]. With the sequence, the theoretical molar
extinction coefficient was obtained: 23,420 M
−1·
cm
−1
for Chui2, 21,930 M
−1·
cm
−1
for
Chui3, and Chui4 and 24,910 M
−1·
cm
−1
for Chui5. These data were used to calculate
the concentration of the toxins with the formula A =
ε
bc where A is the absorbance,
ε
is
the molar extinction coefficient (M
−1·
cm
−1
), b is the width of the cell (cm), and c is the
concentration (M).
5.4. Alkylation and Enzymatic Digestion of Toxins
Alkylation and digestion of the toxins were carried out as reported [
17
] with some
modifications. To digest Chui2 toxin, 0.5
µ
g of the AspN protease was used (specific cut at
the amino side of the Asp residue) and to digest Chui3, Chui4, and Chui5 toxins, 2
µ
g of
the V8 protease were used (specific cut at the carboxyl side of the Asp and Glu residues).
5.5. Electro Physiology Assays
The experimental strategies used for the culture of HEK cells expressing hNav 1.1–hNav 1.6
sodium channels and CHO cells expressing hNav 1.7 sodium channels, as well as the
conditions for the electrophysiological characterization of Chui toxins were previously
described [
17
,
24
]. Data are the mean of 3–7 cells
±
standard error and a Paired Sample
t Assay was applied to calculate the significance at 0.05 level of the difference between
control and toxin conditions.
5.6. scFv Expression
The DNA segments containing the genes that encode the selected scFvs, were sub-
cloned into the pSyn1 (AmpR) vector as reported [
12
]. Briefly, electro competent Escherichia
coli TG1 [F’traD36 proAB lacI
q
Z
∆
M15] bacteria were electroporated with the recombinant
pSyn1 constructs and grown in SOC medium for 1 h to be subsequently cultured in YT2X
solid medium with 200
µ
g/mL ampicillin and 1% glucose. A pre-inoculum was generated
from an individual colony in YT2X/Amp/Glu medium, incubated overnight (ON) and
subsequently inoculated at a 1:100 dilution in YT2x/Amp medium. The culture was incu-
bated up to an optical density (at
λ
= 600 nm) of 1.1 to be induced with 1 mM IPTG for 6 h
at 37
◦
C. The bacterial culture was centrifuged, the supernatant discarded, and the pellet
was resuspended with PPB buffer and 5 mM MgSO
4
. Both supernatants were recovered,
mixed, and dialyzed two times against PBS 1X with agitation at 4
◦
C. The dialysate was
passed through a Ni
2+
-NTA column previously equilibrated with 20 mM imidazole. The
column with the attached scFvs was washed with 35 mM imidazole and subsequently
eluted with 350 mM imidazole. Finally, the eluted samples were subjected to size exclusion

Toxins 2022,14, 369 15 of 20
chromatography using a Superdex TM 75 10/300 G L column (GE Healthcare) recovering
the scFvs in PBS 1X. The molecular weight and molar extinction coefficient of each scFv
was calculated theoretically using its sequence. Protein concentration was calculated by
UV spectrophotometry
λ
= 280 nm with their molar extinction coefficient which were
49,765 M−1·cm−1for three scFvs 10FG2, 9a and B15a and 51,255 M−1·cm−1for HV.
5.7. Directed Evolution and Screening
The affinity maturation process included the generation of phage-displayed antibody
libraries subjected to site-directed and/or random mutagenesis. For all the generated
repertoires: the amplification products obtained were purified by means of a preparative
0.8% agarose gel with 1X TAE buffer. Analytical gels were made with 1% agarose in 1X
TBE buffer. The PCR conditions were the same as earlier reported [
12
]. Bacterial cultures
from the libraries were cryo-preserved with 20% glycerol and stored at
−
35
◦
C until use.
Three rounds of screening were carried out as reported [
12
] with some modifications that
are described below. Bacterial colonies were randomly selected from the 3rd round of
screening and evaluated by ELISA of soluble scFvs. Those clones with a higher signal than
their respective control were prepared for sequencing with the Dir [
12
] primer at Unidad
de Síntesis y Secuenciación de DNA of the IBt-UNAM.
5.7.1. First Library with Site-Directed Mutagenesis
A mutagenic library of the scFv 10FG2 with saturation mutations at positions 53, 57,
59, 102, and 105 was constructed. In separate PCR reactions using 10FG2 as template and
two primer pairs Dir [
12
]-CDR2REVC1 (5
0
-C TGC ATA KYW TTT SNN ACC TCC ACC
MTM TGA TAT AAC-30) and CDR3DIRC1 (50-GCC CGC NNS TGC CTA NNS TGC AGC
GAC-30)-cMyc[12], two mega-primers were generated as reported [12] containing saturat-
ing mutations at positions 53, 57, and 59 as well as 102 and 105 respectively. To obtain the
complete gene, PCR products were used as mega-primers in a second PCR step with the
same template (10FG2). The complete gene was digested with the enzymes Not1 and Sfi1
and ligated with T4 ligase into the phagemid vector pSyn2 previously digested with the
same restriction enzymes. The ligation product was transformed into electro-competent
E. coli TG1 cells and resuspended in SOC medium. After 1 hour of incubation at 37
◦
C,
an aliquot was taken for the determination of the repertoire titer; the rest was resus-
pended in 50 mL of YT2x/amp/glu medium and grown ON at 150 rpm at 30
◦
C for
cryo-preservation. The amount of Chui5 used for biopanning was 4, 1, and 0.2
µ
g, and the
PBS/PBS-Tween20 washes were 10/10, 10/10, and 10/20 for the first, second, and third
rounds, respectively.
5.7.2. Library Construction with Random Mutagenesis
Clone 9a was used to generate the library with random mutagenesis by error-prone
PCR as reported [
14
]. The amount of Chui5 used for biopanning was 4, 2, and 2
µ
g, and
the PBS/PBS-Tween20 washes were 10/10, 12/15, and 15/15 for the first, second, and third
rounds, respectively. The phage-antibodies from the 3rd round had an additional wash
with 100 mM NaCl.
5.7.3. Construction of the Second Library with Site Directed Mutagenesis
In separate PCR reactions, with template 9a and two primer pairs Dir [
12
]-Q65Xrev
(5
0
-GAA TCG GCC SNN CAC GGA GT-3
0
) and Q105XDir (5
0
-GC CTA NNS TGC AGC
GAC TGG-3
0
)-cMyc [
12
], two mega-primers were generated as reported [
12
] containing
saturation mutations at positions 65 and 105 respectively. To obtain the complete gene, the
204 bp and 512 bp PCR products were used as mega-primers in a second PCR with the
same template (9a). Furthermore, this second PCR was done under the same conditions to
generate a library with random mutagenesis [
14
]. For the library construction, the complete
gene was processed as mentioned before. The amounts of Chui5 toxin used for screening
were 2, 1.5, and 1
µ
g for the first, second, and third rounds, respectively. PBS/PBS-Tween20

Toxins 2022,14, 369 16 of 20
washes were 13/13 for the three rounds. For the 2nd and 3rd round, the phage-antibodies
bound to the immunotube and prior to washing with PBS/PBS-Tween20, were incubated
with a 0.5 M and 1 M guanidinium chloride solution in PBS, respectively, for 30 min at 37
◦
C.
5.8. Surface Plasmon Resonance Measurements
The binding kinetic constants of the scFvs 10FG2, 9a, B15a, and HV with the Chui2,
Chui3, Chui4, and Chui5 toxins were determined using the BIAcoreX100 (GE, Uppsala,
Sweden) biosensor system. Total of 500 ng of the different toxins were dissolved in 200
µ
L of
MES buffer [10 mM of 2-(N-morpholino)ethanesulfonic acid pH6]. Twenty microliter of the
mixture with each toxin was attached to cell 2 of a CM5 chip using the amine coupling kit
with a flow rate of 5
µ
L/min. The binding RU were 90, 417, 367, and 170 for Chui2, Chui3,
Chui4, and Chui5 toxins, respectively. Cell 1 (no toxin bound) was used as a reference.
One hundred microliter of serial dilutions (10–225 nM) of each scFv were injected on
each of the chips with a flow rate of 30
µ
L/min. The equipment was set at 25
◦
C. The
dissociation time was 800 s. To regenerate the chips, 10 or 20 mM HCl was used as required.
The sensorgrams used for the determination of the kinetic constants were corrected by
subtracting the signal from the reference cell and injecting buffer/blank in the cases where
it was necessary. The constants were determined with the BIA-evaluation version 3.1
program using the Langmuir mathematical model (1:1); the maximum Chi
2
value allowed
for all assays was 3. All determinations were performed in duplicate.
5.9. Toxin Neutralization Assays
All
in vivo
neutralization assays were approved by the Bioethics Committee of the
Instituto de Biotecnología of the Universidad Nacional Autónoma de México IBt-UNAM
(Project number: 413). The LD
50
of the Chui5 toxin were determined as reported [
24
].
Neutralization assays of scFv 10FG2 were carried out with Chui3, Chui4, and Chui5 toxins.
For the control groups, 6 female CD1 mice of 18–20 g were used and they were injected
with a lethal amount of each toxin (Table 4). For the experimental group, mixtures of the
toxins were made with the scFv 10FG2 at a molar ratio of 1:10 (toxin: scFv). For Chui5
toxin neutralization assays with scFvs 9a, B15a, and HV, the control and experimental
groups ranged from 4 to 6 mice. Control groups were injected with 2 LD
50
(Table 6).
For the experimental groups, scFvs were mixed with Chui5 toxin in a 1:10 molar ratio.
Additionally, a pool was made with a mixture of 1 LD
50
of Chui5 with the scFv HV at
a 1:20 molar ratio. All mixtures were incubated for 30 min at room temperature (~25
◦
C)
and injected intraperitoneally in a volume of 100
µ
L. Surviving animals were observed
for 48 h. In order to avoid unnecessary suffering, the animals that showed irreversible
envenoming effects, were sacrificed according to the bioethical protocols approved by
the IBt-UNAM.
5.10. Neutralization Assays with Whole Fresh Venom of C. huichol
Neutralization assays were performed with freshly collected scorpion venom to ensure
the highest toxicity. The soluble part of the venom was quantified by spectrometry at
λ= 280 nm and its LD50 was determined as reported [2].
5.10.1. Classic Protection (Mix-Type Assays)
Female CD1 mice of 18–20 g weight were used for all assays. For the neutralization
assay with scFvs 10FG2 and LR, a control group of 4 mice was used, which were admin-
istered with 1 LD
50
of venom. The experimental group consisted of 4 mice injected with
1 LD
50
and a mixture of scFvs 10FG2 and LR with a 1:10:10 molar ratio (venom: scFv 10FG2:
scFv LR) assuming that the toxins represent 10% of the venom. For the neutralization assay
with 10FG2/HV and HV alone, a control group of 6 mice injected with 2 LD
50
was used.
For experimental groups of the scFvs 10FG2/HV mix-type assays, 1 LD
50
and 5 LD
50
of
venom were used with molar ratios of 1:10:10 and 1:5:5, respectively. For scFv HV alone
groups 1 LD
50
, 2 LD
50
, and 3 LD
50
of venom were used keeping the amount of scFv fixed

Toxins 2022,14, 369 17 of 20
to obtain different molar ratios of scFv (Table 7) assuming that the toxins represent 14%
of the venom. All mixtures were incubated for 30 min at room temperature (~25
◦
C) and
injected intraperitoneal with a volume of 100 µL.
5.10.2. Rescue Assays
For the rescue assays, 12 CD1 female mice weighing 18–20 g were injected with
3 LD
50
of fresh venom. About 10 min post-injection, 6 of such mice were administered with
scFv HV in a 1:10 molar ratio assuming that the toxins represent 14% of the venom. Both
injections were done intraperitoneal with a volume of 100 µL.
5.11. Calculation of Thermodynamic Parameters
The kinetic constants of scFvs 10FG2, B15a, and HV with Chui5 toxin were generated
as mentioned before. The determinations were made at temperatures of 15, 20, 25, 30, and
35 ◦C. The Van’t Hoff equation in terms of KDis represented mathematically as
∆G=RTlnKD=∆H−T∆S (1)
where
∆
G is the Gibbs free energy, R is the universal ideal gas constant (1.987 cal
·
K
−1·
mol
−1
),
T is the temperature in Kelvin, K
D
is the affinity constant,
∆
H is the enthalpy, and
∆
S the
entropy. So, the equation becomes
lnKD=
∆H
R
1
T−∆S
R(2)
This equation is a representation of the equation of a straight line of the form
y=mx +b (3)
To calculate the values of the thermodynamic parameters, a Van’t Hoff plot was made,
which is obtained by plotting the inverse of the temperature (in Kelvin) on the X-axis
against the natural logarithm of the K
D
on the Y-axis. The values of the slope, obtained with
the linear regression of 1/T vs lnK
D
, allow to calculate the
∆
H and the y-intercept allows
to calculate the
∆
S;
∆
G at 25
◦
C is calculated using Equation (1). Linear regression was
performed with the GraphPad Prims 6 program for Windows version 6.01; the multiple
correlation coefficient R
2
= 0.95 was the minimum value allowed to assume that the graph
is a straight line [30].
5.12. In Silico Mutagenesis and Modeling
The models of the binary complexes were made by in silico mutagenesis based on
the crystallographic structure of the ternary complex scFv RU1-Cn2-LR scFv (PDB entry
4V1D [
13
]) eliminating the LR structure. We used this complex because the antibody
fragments mentioned in this work come from the parental scFv C1, from which the scFv
RU1 was derived. scFv RU1 gave rise to scFv 10FG2, and from this scFv HV was obtained.
The identity between scFvs RU1 and HV is 96% (Supplementary Figure S4c). The models
generated were scFvs 10FG2 and HV with Chui5 toxin. The Coot [
31
] standard rotamer
library was used to generate the models, which were subsequently subjected to energy
minimization using the YASARA [
32
] software. Schemes were built with Pymol version
2.2.2 software and edited with Adobe Photoshop version 22.5.0.
5.13. Access Numbers
The sequences of each of the toxins were deposited in the UniProt knowledgebase
portal under the following access numbers: C0HM15 for Chui2, C0HM16 for Chui3,
C0HM17 for Chui4, and C0HM18 for Chui5.

Toxins 2022,14, 369 18 of 20
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/toxins14060369/s1, Supplementary Figure S1. Representative
specimens of Centruroides huichol scorpion. Supplementary Figure S2. Effect of the C. huichol toxins
on sodium channels. Supplementary Figure S3. Evaluation by ELISA of the colonies isolated from
the different mutagenic libraries. Supplementary Figure S4. Analysis of sequenced clones from the
3rd round of affinity maturation process 2b screening. Supplementary Figure S5. Van’t Hoff plot of
Chui5-scFvs complexes.
Author Contributions:
Conceptualization, L.R.-U. and B.B.; methodology, H.V.-M., T.O.-P., R.R.-C.,
H.S.-P. and F.Z.; software, H.S.-P.; validation, H.V.-M., T.O.-P., R.R.-C. and F.Z.; formal analysis,
H.V.-M., R.R.-C., H.S.-P. and B.B.; investigation, H.V.-M., T.O.-P., R.R.-C., H.S.-P. and F.Z.; resources,
L.D.P., L.R.-U. and B.B.; writing—original draft preparation, H.V.-M. and R.R.-C.; writing—review
and editing, H.V.-M., L.R.-U. and B.B.; visualization, H.V.-M. and R.R.-C.; supervision, L.D.P., L.R.-U.
and B.B.; project administration, H.V.-M., L.R.-U. and B.B.; funding acquisition, B.B. All authors have
read and agreed to the published version of the manuscript.
Funding:
This research was funded by a grant PRONAII Venoms and Antivenoms [grant number
303045] from Consejo Nacional de Ciencia y Tecnología (CONACyT).
Institutional Review Board Statement:
The animal study protocol was approved by the Bioethics
Committee of the Instituto de Biotecnología of the Universidad Nacional Autónoma de México
IBt-UNAM (Project number: 413) (Approval date: 24 September 2021).
Informed Consent Statement: Not applicable.
Data Availability Statement:
All data are included within the article and supplementary materials. Addi-
tional data supporting this study are available from the corresponding author upon reasonable request.
Acknowledgments:
Hugo Valencia-Martínez is a PhD student from the Programa de Doctorado
en Ciencias Bioquímicas at the Universidad Nacional Autónoma de México (UNAM) and received
a fellowship No. 667203 from the Consejo Nacional de Ciencia y Tecnología (CONACyT). The
technical assistance of Cipriano Balderas, Elizabeth Mata, Sergio Gonzales, Leopoldo Güereca, Mario
Trejo, Martin Patiño and Juan Manuel Hurtado are greatly acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
scFv Single chain fragment variable
SPR Surface plasmon resonance
LDLethal dose
HCDR Heavy chain Complementary-determining regions
HFR Heavy chain Framework region
LFR Light chain Framework region
CMC Carboxymethylcellulose
nM nanomolar
KDAffinity constant
TRResidence time
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