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A NEW IMMUNOCHEMISTRY PROCESS THAT TRANSFORM A NON-
IMMUNOGENIC CROTAMINE-LIKE ANTIGEN FROM RATTLESNAKE
(Crotalus durissus cumanensis) VENOM, IN IMMUNOGENIC TO
PRODUCE ANTI-CROTAMINE-LIKE ANTIBODIES
UN NUEVO PROCESO INMUNOQUÍMICO QUE CONVIERTE CROTAMINA DEL VENENO
DE SERPIENTE DE CASCABEL (Crotalus durissus cumanensis), UN ANTÍGENO
NO-INMUNOGÉNICO, EN INMUNOGÉNICO PARA PRODUCIR ANTICUERPOS ANTI-CROTAMINA
María Magdalena Pulido-Mendez
1
, María Eugenia Acosta
2
and Alexis Rodríguez-Acosta
2*
1
Laboratory of Immunology, Experimental Medicine Institute, Universidad Central de Venezuela, Caracas, Bolivarian Republic of
Venezuela.
2
Immunochemistry and Ultrastructural Laboratory, Anatomical Institute, Universidad Central de Venezuela, Caracas,
Bolivarian Republic of Venezuela. *Correspondence to: Laboratorio de Inmunoquímica y Ultraestructura, Instituto Anatómico de la
Universidad Central de Venezuela, Caracas 1041, Venezuela. E-mail address: rodriguezacosta1946@yahoo.es
ABSTRACT
The making of antibodies in animals can be demanding due
to that several antigens, mostly of low molecular masses,
provoke imperceptible immune response or are even totally non-
immunogenic. The transformation of non-immunogenic molecules
into eective antigens represent an important immunological
tasks. The crotamine from the rattlesnake Crotalus durissus
cumanensis snake venom was puried by a Mono S HR 10/10
chromatography column and used to immunise C57/B mice, after
to be polymerised with glutaraldehyde. The murine polyclonal
antibodies directed against native crotamine-like (NCL) treated
with glutaraldehyde and their product crotamine-like polymer
(CLP) were generated by immunisation injecting CLP via lymph
node cells. These antibodies were capable of detecting CLP
in an enzyme-linked immunosorbent assay. The SDS-PAGE
of NCL and CLP showed bands of molecular masses ~ 3 kDa
and ~18 kDa, respectively. These results oer evidence that the
polyclonal antibodies recognise specic putative original and
post-polymerisation epitopes on the CLP molecule, which were
maintained following the process of polymerisation. The results
are discussed in relation to the preservation of a functional post-
polymerisation epitopes on CLP.
Key words: Crotalus durissus cumanensis; crotamine-like;
glutaraldehyde; polyclonal antibody;
polymerisation; venom
RESUMEN
La producción de anticuerpos en animales puede ser una actividad
ardua, debido a que muchos antígenos, principalmente los de baja
masa molecular, provocan una respuesta inmune imperceptible o
aún son totalmente no-inmunogénicos. La transformación de una
molécula no inmunogénica, en un antígeno efectivo representa un
importante reto inmunológico. La crotamina obtenida del veneno
de la serpiente de cascabel (Crotalus durissus cumanensis) fue
puricada a través de una columna de cromatografía Mono S
HR 10/10 (Biorad, EUA) y usada para inmunizar ratones de la
cepa C57/B, luego de ser polimerizada con glutaraldehido. Los
anticuerpos policlonales dirigidos contra la crotamina nativa
tratada con glutaraldehido, y su producto el polímero obtenido
de la crotamina (CLP) se lograron mediante inmunización vía
ganglios linfáticos con polímeros de CLP. Esos anticuerpos
policlonales fueron capaces de detectar el CLP, en un ensayo de
ELISA. Los perles de migración (SDS-PAGE) de la crotamina
nativa y la CLP mostraron bandas de masa molecular ~ 3 kDa y
~18 kDa, respectivamente. Estos resultados ofrecen evidencia de
que los anticuerpos policlonales reconocen epítopes especícos
originales y posteriores a la polimerización en la molécula de CLP,
que se mantuvieron luego del proceso de polimerización. Los
efectos se discuten en relación con la preservación de epítopes
funcionales post-polimerización en CLP.
Palabras clave: Crotalus durissus cumanensis; crotamina-
similar; glutaraldehido; anticuerpo policlonal;
polimerización; veneno
Recibido: 20/03/2020 Aceptado: 28/09/2020
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A new immunochemistry process that transform a non-immunogenic crotamine-like antigen from rattlesnake / Pulido, M. y col.
INTRODUCTION
The study of antivenomic methodology about the immunoreac-
tivity of Crotalus antivenoms against subspecies of rattlesnakes
showed that many Crotalus antivenoms lack of recognising and
binding competent antibodies against crotamine [5]. Based on the
classical knowledge that less than 10 kilodalton (kDa) molecular
mass proteins are not good immunogenic molecules, it is though
that crotamine (∼4.8 kDa in average) must be an insignicant
immunogenic in horses (Equus ferus caballus)
The transformation of non-immunogenic molecules into
eective antigens remains important immunological tasks. The
proposal of transforming derived-glutaraldehyde precipitated
crotamine into eective antigens are both basic and applied
provocative deance. Here, it is supplied information on
glutaraldehyde molecule, mainly its mode action on proteins.
Glutaraldehyde is a di-aldehyde composed of ve highly reactive
carbons, which has been isolated as oil and stored as an aqueous
solution. Its storage forms a carbohydrate, pyrans (oxines) and
polymers mixtures [4]. From the chemical point of view, it is a
very reactive product, which polymerises in water and acidic
aqueous solutions are stable. In alkaline medium, the reactivity
is higher, in this environment, at room temperature, reacts rapidly
with amino terminals of proteins and produce insoluble cross-
linked aggregates. In correspondence with the conditions here
described, yield covalently linked soluble protein oligomers and
polymers were obtained. Glutaraldehyde is well known for its
ability to react with proteins and this process is valid to a wide
range of proteins, and by slight variation in the reaction conditions,
soluble polymers in the molecular mass range from 1x10
4
x 100
4
were produced.
Crotamine is a basic∼ 42-amino-acid polypeptide with an
isoelectric point of 10.3 and a ∼ 4.8 kDa molecular mass,
originally isolated from the venom of the South American
rattlesnake Crotalus durissus terricus [11]. This toxin is regarded
as a potential cell-inltrating vehicle competent of gathering into
replicating cells [19]. Here, it has been puried a ∼3 kDa molecule
with crotamine properties from Crotalus durissus cumanensis, in
order to be used in all the experiments.
Currently, glutaraldehyde has been used for producing
insoluble protein aggregates [2, 16], and insoluble products of
some enzymes, e.g. carboxypeptidase [28], trypsin [13], papain
[17, 26] and catalase [31], maintaining its enzymatic activity.
This project intended to report the eect of molecular size on
immunogenicity of proteins, which have been investigating
procedures planned to produce high-molecular mass protein
aggregates, via intermolecular cross-bridges. To this purpose, it
was studied the reaction of glutaraldehyde with the crotamine-
like non-immunogenic protein, in order to make a crotamine-
like polymer (CLP), using a special lymph node via, to get an
immunological response.
MATERIALS AND METHODS
Reagents
Glutaraldehyde (25%), bovine serum albumin (crystalline),
acrylamide, methylene-bis-acrylamide and NNN’N’-
tetramethylene-diamine, ammonium persulphate, trichloroacetic
acid, sodium dodecyl sulphate and Coomassie Brilliant Blue
R-250, Complete Freund’s adjuvant (CFA) and incomplete
Freund’s adjuvant (IFA) (GIBCO, USA), Trypan blue solution,
Dimethyl sulfoxide (DMSO), Goat anti-mouse IgG conjugated
with alkaline phosphatase and alkaline phosphatase substrate
(pNPP) were all purchased from Sigma/Aldrich, USA. All stock
reagents were stored as recommended by the manufacturer.
Snake and venom collection
Pool from six female and male adults of the rattlesnake
(Crotalus durissus cumanensis) (CDC) captured in Santa
Teresa del Tuy Town (Miranda State, Venezuela) and housed
at the Tropical Medicine Institute of the Universidad Central de
Venezuela (Caracas, Venezuela) were used in the experiments.
Venom was extracted by allowing the snake to bite into a Para-
lm® extended over a disposable plastic cup. The venom sample
was centrifuged (Beckman Avanti 30, USA) at 5000 G for 10
minutes (min), and ltered through 0.45 micrometres (μm) lter.
The venom was frozen at -90 °C (Thermo Scientic™ Revco™
UxF, USA). and then lyophilised.
Mice
C57/B inbred strains of mice (Mus musculus) between 18
and 22 grams (g), bred under specic healthy conditions were
obtained from the Animal House of the Instituto Venezolano de
Investigaciones Cienticas (IVIC)(Venezuela).
Purication of crotamine-like
Isolation and purication of (CDC) crotamine-like was carried
out by one chromatographic step. Crude venom (30 milligrams
(mg) by protein estimation) was diluted to 1.0 millilitres (mL of
50 millimol (mM) Tris–HCl buer, pH 8.2, and then applied on a
Mono S HR 10/10 (GE Healthcare Biosciences Ltd, USA) column
equilibrated with same buer. Attached proteins were eluted with
a 0–1 M NaCl linear gradient in equal buer over 60 minutes (min)
at a ow rate of 1.5 mililiters (mL)/min. Proteins were monitored
at 280 nm. The chromatogram displayed 8 fractions, which were
identied agreeing to their elution (FIG. 1). Clear spastic hind-limb
paralysis action was apparent in fraction 5, which was dialysed
against water at 4
0
C, lyophilised and stored at -20°C (Frigidaire
FGVU21F8QF Vertical Freezer, USA) until used. This fraction
was chosen for the experimental assays.
Protein concentration
The CLP concentration was spectrophotometrically (Beckman,
USA) calculated by assuming that 1 unit of absorbance/
centimetres (cm) of path length at 280 nm corresponds to 1
milligram (mg) protein/mL [35].
Concentration of the crotamine-like under polymerisation
Equal volumes of Cdc crotamine-like (2 mL) at 5 mg/mL in
phosphate saline buer ,(PBS) pH 7.4, were treated with con-
stant proportions (by weight) of glutaraldehyde at 2.5, 6.25 and
12.5% (2 mL); as a negative control PBS was added to the cro-
tamine-like without glutaraldehyde. Reaction procedures were
as follow: products were diluted to 5mg/mL; the crotamine-like
and the glutaraldehyde solutions were mixed on a vortex mixer
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Revista Cientíca, FVC-LUZ / Vol. XXX, N° 4, 173 - 174, 2020
(SI™ Vortex-Genie™ 2T, Scientic Industries, USA). Mixing was
sustained for 1 min and the solutions were incubated (Shel Lab
General Purpose Incubator, Global Lab, CA, USA) at 5
0
C for 24
hours (h). All samples turned yellow after 10 min of addition of
glutaraldehyde. Then, the mixtures were dialysed during 24 h at
6
0
C, against several changes of 0.01 M PBS, pH 7.4, until yellow
colour disappeared; the yields were checked and the molecular
mass of crotamine-like and CLP solutions were determined using
a 12.5% SDS-PAGE as described below.
Polyacrylamide gel electrophoresis (SDS-PAGE)
Twenty micrograms (ug) of crotamine-like and CLP were run on
a 12.5% SDS-PAGE under non-reduced conditions. The gel was
stained with Comassie brilliant Blue R-250 for 1 h.
Testing CLP on mice
In order to know if the polymer conserved the crotamine
neurotoxic activity (posterior limb paralysis), three mice were
intravenously injected with CLP and observed for this posterior
limb paralysis. Three mice were also injected with the native
crotamine-like as positive control.
Immunisation
Five male C57/B mice were immunised with CLP by injections
at day (d) 0, in the lower hock of right limb (50 ug / 50 microliters
(µg)/50 microliters (µL) of a 1:1 emulsion of Freund’s complete
adjuvant and the CLP dissolved in PBS, pH 7.4.
One injections using incomplete Freund’s adjuvant were
given after 4 d in the same place. Afterward, with 4 d’ intervals
the immunisation was carried out for three more times with CLP
dissolved in PBS in the same location. Developed immunisation
process, mice blood was obtained by cardiac puncture, collected,
centrifuged and serum samples frozen at -20°C; at this point all
surviving mice were ethically euthanised by neck dislocation and
used to feed snakes.
ELISA test for specic antibodies
The ELISA [25] was implemented using antibodies against
both CLP and native crotamine-like, which were used as antigens
capturing and detecting antibody, respectively. The optimal activity
of these antibodies was determined comparing with a negative-
and positive-control serum samples in each plate.
Polystyrene microplate 96 wells (Nunc, USA) were sensitised
overnight at room temperature with both native crotamine-like
and CLP (10 μg/well in PBS, pH 7.4). For each step, 100 μL/
well was added unless mentioned otherwise. A normal mice
serum was used as negative control and immunised mice sera
as experimental sample (1:200). Remaining binding sites were
blocked by incubation with 1% Bovine serum albumin (BSA) in
PBS for 1 h. The total volume of the well was kept constant (100/
µL). The plates were exhaustively washed with PBS/Tween (2
min/wash), and unbound sites were blocked with 200 μL/well of
0.5% BSA diluted in PBS, pH 7.4 and 0.05% Tween 20 diluted in
milli Q water. After 2 h incubation at 37°C, the plates were emptied
by suction. Diluted anti-mouse serum (1:200) was added and the
plates were incubated for 1 h at 37°C. After thorough washing as
described above, horse-radish (HR) peroxidase-conjugated goat
anti-mouse (10 μg/mL of PBS/Tween) was added. The plates
were incubated for 1 h at 37°C and washed with PBS/Tween.
The TMB (3,3’,5,5’-tetramethyl-benzidine) was added and the
plates were incubated for 1 h in the dark at room temperature.
The enzyme reaction was blocked with 50 μL/well of 8 N H
2
SO
4
.
The absorbance at 450nm wavelength (A
450
) of the plates was
read using a microplate ELISA reader (Bio-Rad, USA).
RESULTS AND DISCUSSION
Several procient reagents for cross-linking proteins have
been described [9], but in common, they have been utilised
to generate intramolecular quite than intermolecular bridges.
The information about the use of glutaraldehyde in biomedical
research is vast. Several authors have described the basis for
insoluble protein aggregates production [2,15,16]. Others had
demonstrated that insoluble derivatives of numerous enzymes
such as carboxypeptidase [28,29], trypsin [13] and catalase [31]
could preserve signicant enzymatic activity. Dierent authors
[1, 26, 37] showed that it was possible to conjugate proteins
and enzymes and coupling proteins to diverse matrices as well
as evident cases, where soluble protein derivatives have been
described [12,14]. The great anity could be related to a signicant
success of congurational entropy after the polymerisation, which
would be the case, if the folding and assembly processes are
coupled.
In the present work, the crotamine-like from CDC venom was
chromatographic isolated generating 8 peaks, where only the
fraction 5 produced the classical spastic inferior limbs paralysis
activity in mice (FIG. 1).
FIGURE.1. CATION EXCHANGE CROMATOGRAPHY OF
RATTLESNAKE (Crotalus durissus cumanensis) VENOM.
Crotamine (arrow). Absorbance at 280 nm. Elution with % 0.5 M
NaCl linear gradient
To maintain natural protein conformation, guaranteeing
the solubility of the secondary polymers by preservation of
original hydrophilic amino acid residues, in the current work
was required to produce cross-linked polymers with minimum
amino acid substitution. With this objective, low glutaraldehyde
concentrations were utilised, jointly with high protein
concentrations, in order to make easy intermolecular reactivity.
It was used crotamine-like protein concentration at 1.97 ± 0.02
mg/mL. In the past, many experimental studies had used high
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A new immunochemistry process that transform a non-immunogenic crotamine-like antigen from rattlesnake / Pulido, M. y col.
concentrations of glutaraldehyde, which produced an extensive
formation of derivatives, but without extensive crosslinking [2]. It
has been proposed by Lee et al. [22] that crotamine is able to
delivery molecules into mammalian cells without needing special
receptors. Several crotamine isoforms have been described
[10,21,25,27,30,32,34,36]. Crotamine hold three disulphide
bridges [5] and the main crotamine folding is similar to many
proposed as anti-bacterial defence peptides, which have been
described to belong to defensins families (α-defensin, β-defensin
and insect defensin) [7]. Although these molecules show a great
structural diversity and a broad spectrum of activity, they have in
common the ability to induce the permeation of liquids (by osmosis
or diusion) through the bacterial cytoplasmic membranes [6,8],
as crotamine does in other cell membranes [19].
Immunoglobulins have the faculty to identify structures
exclusively present on oligomeric assemblies [18,20]. The
expression oligomers can, on the other hand, be applied to
grouping ranging from a dimer to a much higher assembly [33].
Here, it was used 12% of polyacrylamide gel electrophoresis
(SDS-PAGE) under non-reduced conditions of both native
crotamine-like and CLP showed a molecular mass bands of ~3
and ~ 18 kDa, respectively, to show both proteins (FIG. 2).
FIGURE. 2. SDS-PAGE ELECTROPHORESIS OF NATIVE AND
CROTAMINE POLYMER. A SDS-PAGE (12.5 %) under non-
reduced conditions of both native crotamine-like and crotamine
polymer. (Lane 1): Molecular mass markers; (Lane 2): native
crotamine-like ~3 kDa; (Lane 3) polymer ~ 18 kDa
Due to this intrinsic heterogeneity, and because it did not have
a previous structural polymer crotamine information, the directed
design of oligomer-specic antibodies was frequently randomly
determined. They had a random probability distribution or pattern
that may be analysed statistically, but may not be predicted
specically events. These limitations obstruct development in
the eld. Nevertheless, here, it is showed that the multivalent
architecture of polymeric CLP, having multiple independent binding
sites, can be used as a discriminating binder for an antibody,
which is appropriate for the exposure of multiple epitopes, on
the oligomeric and polymeric assemblies. This was successfully
used for the immune system (through lymph node immunisation)
that could recognise and build up an immune response of specic
antibodies against CLP. As can be seen in TABLE 1, immunised
mice sera reacted only with CLP, being capable of recognising the
antigen in the ELISA assay. An immunisation was considered to
be successful when an ELISA positive test with mouse anti-CLP
mouse serum diluted at least 1:200 was obtained.
TA B L E I
ELISA REACTIVITY OF NATIVE CDC CROTAMINE-LIKE AND
CLP WITH IMMUNISED MICE POLYCLONAL ANTIBODIES.
THE CONCENTRATIONS OF THE SAMPLES NATIVE AND
POLYMER CLP WERE CALCULATED TO 5µg/ML
Normal Mice serum (1:200) 0.055
Controls
BSA 0.040
No primary Ab 0.060
No Secondary Ab 0.048
Medium Control 0.038
Immunised
Mice serum (1:200) NCL 0.065
Mice serum (1:200) CLP 0.328
NCL: native crotamine-like. CLP: crotamine-like polymers
The polymers running above 18 kDa (crotamine-like monomers
were ∼4 kDa) showed that separation on the basis of size
was achieved and fractions containing oligomers of particular
molecular mass distribution were obtained, the progression
throughout the fractions of large polymers from monomers was
revealed (FIG. 2).
It is thought that the development of a technique that allows
transforming a non-immunogenic molecule into an immunogenic
one was performed by conjunction of both methods: the making
of the glutaraldehyde polymers, but mainly because was used
an innovative immunological technique via lymph node instead
of spleen immunisation. As it is known, dendritic cells (Decs)
play a crucial role in antigen presentation to CD4+ T cells, which
open developed immune responses. Therefore, dening positive
modulators of dendritic cell activation, to improve immune
responses for low molecular masses antigens might be useful;
Decs go through a route of dierentiation identied as maturing.
This advance considerably increases their ability for antigen
handling and presentation [3]. The broad-spectrum facets of Decs
progress are well empathised and comprise the redistribution
of MHC-II molecules from the intracellular sections to the cell
membrane, increased co-stimulatory molecules expression, such
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Revista Cientíca, FVC-LUZ / Vol. XXX, N° 4, 173 - 174, 2020
as several cytokines (TNF-α and IL-12) [23,24].
It is believed that the immunisation pathway through the lymph
node, which necessarily involved dendritic cells, makes the
low molecular mass antigen well recognised and processed,
as stated above. Thus, the both events, the production of a
crotamine polymer and the intervention of dendritic cells, made
the procedure successful.
This methodology would be of great importance in the eld
of immunology, not only for crotamine, but for other antigens of
very low molecular masses. Probably, this original protocol of
lymph node immunisation would permit a better immunological
recognition and higher production of IgG, representing an
alternative to the traditional protocol of splenic production, which
generates a larger quantity of IgM.
The production of polyclonal antibodies anti- CLP is not only
an original nding, as far as it is known, this is the rst time that
has been developed, allowing us to have a very useful tool in
experimental processes, which could be carried out to track
crotamine with labelled antibodies. This also opens an interesting
eld for the production of monoclonal antibodies, since it was
demonstrated that there was an immunisation process and
necessarily had lymphocytes recognising the antigen, which
can be fused with myeloma cells, in order to obtain hybridomas
producing monoclonal antibodies.
CONCLUSIONS
An initial aim of this project was to produce a large polymer
of CLP through polymerisation, which was capable of inducing
a humoral immune response by a protocol of lymph node
immunisation, when introduced into C57/B inbred strains of mice.
Successfully producing polyclonal antibodies immunologically
tailored to crotamine, which has no precedent. Subsequent
to constructing a large polymer through protein crosslinking,
the ultimate goals of this endeavour was to produce polyclonal
antibodies specic to CLP secreted by B-lymphocytes. In
the future, the laboratory is planning to produce monoclonal
antibodies using this immunisation protocol as a probe, in various
tissues of vertebrates, and why not in invertebrates too.
ACKNOWLEDGEMENTS
Funding for this project was provided by Grant from the
Science and Technology Fund (FONACIT) programs grant #
PEI201400352 (Universidad Central de Venezuela).
ETHICAL STATEMENT
Skilful workers did all the experimental proceedings regarding
the use of live animals. The applicable regulations as well as
institutional guidelines, according to protocols approved by by the
Institute of Anatomy Ethical Committee of the Universidad Central
de Venezuela, on 18 January 2019, under assurance number
(#18-01-19). The research was carried out in accordance with the
U.K. Animals (Scientic Procedures) Act, 1986 and associated
guidelines, EU Directive 2010/63/EU for animal experiments.
COMPETING INTERESTS
The authors declare that they have no competing interests.
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