Invest Clin 63(1): 57 - 69, 2022 https://doi.org/10.54817/IC.v63n1a05

Corresponding author: Alexis Rodríguez-Acosta. Instituto Anatómico, Ciudad Universitaria de Caracas, Universidad

Central de Venezuela, Caracas 1041. Email: rodriguezacosta1946@yahoo.es

A new approach of immunotherapy against

Crotalus snakes envenoming: ostrich

(Struthio camelus) egg yolk antibodies

(IgY-technology).

Carlos Bello1, Fátima Torrico1, Juan C. Jiménez1,2, Mariana V. Cepeda1,

Miguel A. López1 and Alexis Rodríguez-Acosta1,3

1Biotecfar C.A, Facultad de Farmacia, Universidad Central de Venezuela, Caracas,

República Bolivariana de Venezuela.

2Instituto de Inmunología, Facultad de Medicina, Universidad Central de Venezuela,

Caracas, República Bolivariana de Venezuela.

3Laboratorio de Inmunoquímica y Ultraestructura, Instituto Anatómico, Universidad

Central de Venezuela, Caracas, República Bolivariana de Venezuela.

Key words: antivenom; Crotalus snakes; ostrich egg yolk; IgY; Struthio camelus; venom.

Abstract. Crotalid envenomation is a neglected collective health problem

involving many countries in America, which need secure and inexpensive snake

anti-venom treatments. Here, high antibody titers (IgY) were raised in the Os-

trich (Struthio camelus) egg yolk by immunizing with the venom of Venezuelan

venomous Crotalus snakes. Ostriches were immunized with a pool of venoms

from common rattlesnake (Crotalus durissus cumanensis), Uracoan rattle-

snake (Crotalus vegrandis), Guayana rattlesnake (Crotalus durissus ruruima)

and black rattlesnake (Crotalus pifanorum). The anti-snake venom antibodies

were prepared from egg yolk by the water dilution method, enriched by the ad-

dition of caprylic acid (CA) and precipitation with ammonium sulfate at 30%

(W/V). The purity and molecular mass of the final product was satisfactory,

yielding a single ∼ 175 kDa band in SDS-PAGE gels ran under non-reducing

conditions. In the immunoblot analysis, specific binding of the antivenom was

observed with most venom proteins. The LD50 was 16.5 g/mouse (825 µg/kg

body weight). High titers of IgY against Crot/pool venom were shown by ELISA.

The median effective dose (ED50) was 19.66 mg/2LD50. IgY antibodies neutral-

ized efficiently the Crot/pool venom lethality. As far as we know, this is the first

anti-snake venom produced in ostriches, which could make this technology an

affordable alternative for low-income countries, since it is likely to produce

58 Bello et al.

Investigación Clínica 63(1): 2022

Un nuevo enfoque de inmunoterapia contra el envenenamiento

de serpientes Crotalus: anticuerpos de yema de huevo de

avestruz (Struthio camelus) (tecnología IgY).

Invest Clin 2022; 63 (1): 57 – 69

Palabras clave: antiveneno; avestruz Crotalus; IgY; Struthio camelus; veneno.

Resumen. El envenenamiento por crotálidos es un problema de salud

colectiva desatendido, que involucra a muchos países del continente ameri-

cano, los cuales necesitan tratamientos seguros y económicos. En este tra-

bajo, se obtuvieron títulos altos de anticuerpos (IgY) producidos en yema

de huevo de avestruz (Struthio camelus) mediante la inmunización con

el veneno de serpientes venezolanas del genero Crotalus. Se inmunizaron

avestruces con una colección de veneno de serpientes de cascabel común

(Crotalus durissus cumanensis), cascabel de Uracoa (Crotalus vegrandis),

cascabel de Guayana (Crotalus durissus ruruima) y cascabel negra (Crota-

lus pifanorum). Los anticuerpos anti-veneno de serpiente se prepararon a

partir de yema de huevo por el método de dilución en agua, enriquecidos

mediante la adición de ácido caprílico (CA), seguido de una precipitación

con sulfato de amonio al 30% (P/V). La pureza y masa molecular de los

anticuerpos (IgY) se definieron mediante ensayos de SDS-PAGE nativos y

las masas moleculares se establecieron electroforéticamente, obteniéndose

una única banda de IgY de ∼ 175 kDa. El análisis de inmunotransferencia

mostró la unión específica del antiveneno con la mayoría de las proteínas

del veneno. La DL50 fue de 16,5 µg/ratón (825 µg / kg de peso corporal);

Se mostraron títulos altos de IgY contra el veneno de Crot / pool median-

te ELISA. La dosis mediana efectiva (DE50) fue de 19,66 mg/2 LD50. Los

anticuerpos IgY neutralizaron eficazmente la letalidad del veneno de Crot

/ pool. Hasta donde sabemos, se trata del primer antídoto de serpiente

producido en avestruces, lo que podría abaratar la producción de este tra-

tamiento en países del tercer mundo. Ya que es probable que se obtengan

alrededor de 2-4 g de IgY por huevo de avestruz. Por lo tanto, se podrían

purificar casi 400 g de IgY de un solo avestruz durante un año. Asimismo,

debido a las enormes diferencias en el costo de inversión en el manteni-

about 2-4 g of IgY per ostrich egg. Hence, almost 400 g of IgY can be purified

from only one ostrich during a year. In addition, there are enormous differences

in the cost of investment in the maintenance of horses, from the points of view

of infrastructure, feeding and veterinary care, in which the cost can reach USD

100 per animal per day, compared to a maintenance cost of USD 146 per month

per producing bird. These results are encouraging and could easily be extrapo-

lated to the manufacturing of other antivenoms and antitoxins as well, as they

could be applied to the manufacturing of potential diagnostic tools.

Crotalus snakes envenoming treated with ostrich (Struthio camelus) IgY 59

Vol. 63(1): 57 - 69, 2022

INTRODUCTION

Venomous snakebite is a worldwide

problem, especially in tropical and subtropi-

cal geographical regions. There are more

than five million snakebite accidents annual-

ly worldwide; the members of the Viperidae

family 1 produce the most common snake-

bites that take place in the American conti-

nent (~ 98%).

Traditional antivenom production

is based on purified antibodies extracted

from hyperimmunized horses’ plasma 2. In

recent years, several authors report that

bird’s (mainly chicken) antibodies produced

against snake venoms, presented venom

effective neutralization activities 2,3. How-

ever, despite their potential considering

their body size and egg-laying advantages,

ostriches (Struthio camelus) have not been

previously tested for snake anti-venom pro-

duction. Clinical assays will be need to as-

sess their security as an antidote to human

victims of ophitoxemia and further experi-

mentation addressing IgY-based antivenoms

safety and effectiveness is required.

MATERIALS AND METHODS

Reagents

Polyvalent anti-ophidic serum (PAOS)

(Biotecfar C.A, Universidad Central de Vene-

zuela, Pharmacy Faculty, Caracas, República

Bolivariana de Venezuela). Sodium chloride,

sodium citrate, sodium azide, tris, hydro-

chloric acid, sodium hydroxide, Coomassie

blue, acrylamide, bis-acrylamide, ammoni-

um per-sulphate, glacial acetic acid, temed,

glycerol, sodium bicarbonate, caprylic acid

(Sigma-Aldrich, Missouri, USA). Saran wrap

(S.C. Johnson & Son, Inc, USA). Complete

Freund’s and incomplete Freund’s adjuvants

(GIBCO, USA). Antibody goat anti-avian (in-

cluded anti-ostrich) (Laboratories ABCAM,

USA). Peroxidase substrate (TMB)(Vector

Lab, USA). 30 kDa cassette filtration unit

(Vivaflow 50 R, Sartorius, Germany). Fil-

ter (Sartorious Laboratories, Germany).

Microtitration plates (Corning® ELISA mi-

croplates, USA). Automatic ELISA reader

(Bio-Tek Laboratories, USA). Mini-Protean

II system (Bio-Rad Laboratories, USA).

Trans Blot SD system (Bio-Rad Laboratories,

USA). Molecular mass standards for SDS-

PAGE were from Bio-Rad Laboratories Ltd

(California, USA).

Ostriches

Two female healthy adult ostriches

(Struthio camelus) were obtained from a lo-

cal ostrich farm (Villa de Cura town, Aragua

state, República Bolivariana de Venezuela).

They were housed under standard environ-

ment (humidity, lighting and temperature)

and fed ad libitum with standard ostrich diet

and potable water (Fig. 1).

Venom

A pool of venoms from common rat-

tlesnake (Crotalus durissus cumanensis)

(0.15 mg/mL), Uracoan rattlesnake (Crota-

lus vegrandis) (0.03 mg/mL), Guayana rat-

miento de los caballos desde el punto de vista de infraestructura, alimenta-

ción y atención veterinaria, en los que el costo puede llegar a los 100 USD

por día, frente a los 146 USD por mes de mantenimiento de la producción

de aves. Estos resultados abren un campo terapéutico, para la fabricación

de otros antivenenos contra un amplio espectro de toxinas y también como

probables herramientas de diagnóstico.

Received: 03-08-2021 Accepted: 04-11-2021

60 Bello et al.

Investigación Clínica 63(1): 2022

tlesnake (Crotalus durissus ruruima) (0.02

mg/mL) and black rattlesnake (Crotalus pi-

fanorum) (0.012mg/mL) were obtained by

milking the snakes and then crystallized un-

der vacuum in a desiccator containing CaCl2

as a desiccant and maintained at 4°C until

use (Fig. 1).

Ethical statement

Skilled staff prepared all the experi-

mental methods relating to the use of live

animals. These methods were permitted by

the Institute of Anatomy Ethical Committee

of the Universidad Central de Venezuela un-

der assurance number (Protocol N° 190619)

and followed the norms obtained from the

Guidelines for the Care and Use of Labora-

tory Animals, published by the USA National

Institute of Health 4.

Immunization procedure

Two female ostriches were injected in-

tramuscularly intro the bird’s anterior thigh

muscles on day 0 with the Crot/pool of the

Venezuelan rattlesnake venoms (1µg/kg

body weight) emulsified with an equivalent

volume of complete Freund’s adjuvant. The

primary booster dose was administered two

weeks later in an incomplete Freund’s ad-

juvant. Successively, the second and third

boosters emulsified with an equivalent vol-

ume of incomplete Freund’s adjuvant were

administered at 45-day intervals in order to

sustain high antibody titers. Blood was col-

lected through the wing vein, arranged to

obtain the serum. Sera were separated by

centrifugation (1500 G for 15 min). Pre-

immune serum and egg yolk replicas col-

lected from ostriches were used as negative

controls. They were stored in a freezer at

−20°C, until used.

Purification of antibodies from egg yolk

Isolation of IgY from egg yolk was per-

formed with a modified method 5. Briefly,

once the egg shell was opened, the yolk was

softly detached from the white of the egg,

washing it with abundant distilled water, un-

til all the white egg disappeared. Then, the

yolk was punctured with a syringe and all

its contents were extracted and mixed com-

pletely with a five-fold dilution with PBS pH

7.4 and slow addition of caprylic acid (CA),

then the pH was adjusted to 5.0 with 10 M

Fig. 1. (A) Ostriches at bird’s farm, Villa de Cura, Aragua state, Venezuela. (B) Crotalus durissus cumanensis.

Crotalus snakes envenoming treated with ostrich (Struthio camelus) IgY 61

Vol. 63(1): 57 - 69, 2022

hydrochloric acid. Concisely, the CA was

slowly added dropwise at an approximate

rate of 0.6 mL/min, until a final concentra-

tion of 6% (v/v) was obtained.

After the first step of the process, the

sample was filtered through a 15 µm and

then 0.45 µm filter.

The preparation was kept to room tem-

perature, and afterward ammonium sulfate

at 30% (W/V) concentration was added,

keeping it under constant stirring for one

hour at room temperature and then filtered

through a 2-µm filter. In order to eliminate

the ammonium sulfate, a tangential filtra-

tion process was applied using a cassette

of 30 kDa. The sample was resuspended in

saline solution and kept under stirring for

one hour- Subsequently, filtered through a

2-µm filter to remove remaining solids and

a diafiltration process was started until

eight diafiltration volumes were completed.

When the ammonium sulfate was removed,

the washed sediment was dissolved in 200

mL saline pH 6.3.

Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) of IgY

Purified IgY under non-reduced and re-

duced conditions were electrophoresed with

a MINIPROTEAN II (BioRad, USA) cham-

ber. SDS-PAGE was performed using 12%

gels. Wide range molecular weight markers

(Bio-Rad) were run in parallel and gels were

stained with Comassie blue (National Diag-

nostic, USA).

Determination of antibody titers by an

indirect ELISA

The immunoglobulins titers in serum

and egg yolk of immunized ostriches were

tested by enzyme-linked immunosorbent

assay (ELISA). Titer was considered as the

concentration of an antibody, as determined

by finding the highest dilution at which it

was still able to cause recognition of the

antigen. The uncomplicated way to do this

is as follows: selecting the highest respond-

ing serum and another that shows low re-

sponse. Do readings for serial dilutions as we

have done (Fig. 3). Looking for one dilution

were both sera are in the steepest part of

the curve, are clearly different (generally a

1:1000 – 1:4000 dilutions will do it) and the

OD450nm of the most responding serum is

nearly 1.0. Then it is possible to define the

titer as the OD450nm for each serum at the

dilution obtained before.

All ELISA incubations were carried out

at 24-26°C. Briefly, aliquots (100 µL) of the

Crot/pool venom (1 µg/mL PBS) were pi-

petted into the wells of the micro-titration

plates in overlay phosphate buffer saline

(PBS) pH 7.4 that were protected with saran

wrap and stored overnight at 4°C. The wells

content was aspirated, and washed three

times with washing buffer (PBS, pH 7.4 con-

taining 0.05% Tween-20), the wells were then

overflowed with blocking buffer (skimmed

milk 2% in PBS-T) and left for one hour. After

the aspiration of blocking buffer, the ostrich

sera or purified IgY immunoglobulins sam-

ples were diluted properly in blocking buffer

and 100 µL added to the wells before incu-

bating at 37°C for one hour. Later, the plates

were washed three times with washing buffer

and incubated with goat anti-avian (includ-

ed anti-ostrich) IgY peroxidase (1:10.000)

at 37°C for one hour. The contents of the

wells were subsequently aspirated, the wells

washed three times with PBS-0.05% Tween

20 (PBS-TW), and 100 µL of peroxidase sub-

strate (TMB)(Vector Lab, USA) was added

to each well. The plates were maintained in

the dark, at room temperature for 20 min

for the progress of dye. The reaction was

stopped with 50 µL of sulfuric acid 1 M. The

absorbance of solutions at X = 450 nm was

determined after the addition of substrate

by an automatic ELISA reader.

Immunoblot analysis

Recognition of IgY antibodies was per-

formed by western blot according to the

modified method 6 (n=3). Briefly, to deter-

mine the specificity of the immunoglobulins

against Crot/pool snake venoms, the anti-

62 Bello et al.

Investigación Clínica 63(1): 2022

bodies were tested with a total of six venoms

Crotalus durissus cumanensis, Crotalus veg-

randis, Crotalus durissus ruruima, and Cro-

talus pifanorum venoms.

These venoms were electrophoresed on

a 10% SDS gel using a Mini-Protean II system

at 150V (Bio-Rad PowerPac Basic) for one

hour. Then, were transferred onto a 0.2 µm

nitrocellulose membrane (Millipore) using

a Trans Blot SD system at 100 mA for one

hour. After blocking the membrane for one

hour at room temperature with 5% skimmed

dry milk in PBS buffer pH 7.4 containing

0.05% (w/v) Tween 20, the nitrocellulose

membrane was incubated under stirring for

one hour with anti-Crot/pool ostrich IgY an-

tivenom diluted to 1:1000 in PBS-Tween 20.

After the rinses, the secondary antibody goat

anti-avian IgY (coupled to horseradish perox-

idase) diluted 1:20000 in PBS-Tween 20 was

complemented. Finally, blots electrophoret-

ic bands identified by Crot/pool ostrich IgY

antivenom were colorimetric visualized us-

ing the peroxidase substrate (TMB), and the

image was analyzed.

Lethality Dose (LD50)

Five groups of five NIH female mice

(Mus musculus) for Crot/pool venom were

maintained in plastic boxes (Tecniplast, Ita-

ly) and observed throughout the quarantine

period and experiments. The endpoint of le-

thality of the mice was established after 48

hr. The venom was suspended in 0.85% saline

at the maximum test dose per mouse. Serial

dilutions of 2-fold using saline solution were

prepared to obtain four extra concentra-

tions. All solutions throughout the experi-

ment were kept at 0°C and warmed to 37°C

before being injected into mice. The lethal

toxicity was determined by injecting 0.2 mL

of venom (containing dosages ranging be-

tween 38.0 to 11.6 µg/mouse) into the peri-

toneum of 18–20 g female NIH mice. The in-

jections were dispensed using a 1-mL syringe

fitted with a 25-gauge, 0.5-inch needle. Sa-

line as normal controls were used. The lethal

dose fifty (LD50) was calculated following the

Spearman-Kärber (n = 3 ± SD) method 7.

The estimated LD50 was then used for testing

median effective dose fifty (ED50/2 LD50).

Antivenom neutralization test: median

effective dose fifty (ED50/2 LD50) assays of

yielded antibodies (anti-Crot/pool ostrich

IgY antivenom neutralizing lethal toxic

activity of Crot/pool venom)

The median effective dose value (ED50)

of the anti-Crot/pool ostrich IgY antivenom

from ostrich egg yolk was measured for analy-

sis of quantitative and categorical data, with

the aid of the Prism 8 program (Graphpad,

USA). Five groups of five female NIH mice

(18–20 g) were confronted with a combina-

tion of serial dilutions of a certain amount of

anti-Crot/pool venoms (1.488, 1.395, 1.321,

1.145, 0.843), containing constant concen-

tration of Crot/pool venom (33 µg). 2LD50:

(LD50=16.5 µg/mouse x 2 = 33 µg venom/20

g mouse). The Ostrich IgY anti-Crot/pool

venom/ Crot/pool venom combinations were

pre-incubated for 30 min at 37°C, then was in-

travenously injected into mice for each dose.

Negative control mice were injected with two

LD50 of venom alone. The neutralizing poten-

cy reproduces the ratio of mL of anti-Crot/

pool venom /mg of Crot/pool venom or mg of

antivenom/mg of venom. The control group

was also injected with venom pre-incubated

with normal ostrich serum.

RESULTS

Immune response

Two female ostriches were immunized

as described in Materials and Methods. De-

tectable specific IgY anti-venom respons-

es were not observed in sera until 15 days

from the initial dose, by the time of the first

booster-dose. This secondary response was

sustained thereafter by successive booster

injections given at 45-days pauses.

IgY purification

At a pH of 5-6 most of the immunoglob-

ulins were recovered and the caprylic acid

Crotalus snakes envenoming treated with ostrich (Struthio camelus) IgY 63

Vol. 63(1): 57 - 69, 2022

precipitation leads to a maximum recovery

of IgY, with minimum contaminating pro-

teins. After ammonium sulfate purification,

the obtained sample under reduction con-

ditions gave two mains electrophoretic IgY

bands of ∼65 and ∼20 kDa. Otherwise, under

non-reducing conditions a IgY single band of

∼175 kDa was observed (Fig. 2). The average

recovery of IgY from a single egg yolk was ~

3800 mg (N=5).

Immune-specific recognition of crotalic

venom pool by IgY

The anti- Crot/pool venom-specific ac-

tivity of IgY in partially purified preparations

was assayed by a home-designed indirect

ELISA as described in Materials and Meth-

ods. The purification procedure resulted in

venom-specific IgY recognition of Crot/pool

venoms (Fig. 3). Microtiter plates with pre-

immune serum showed no binding.

Fig. 2. IgY tested under native and reduction conditions. (1) Molecular mass markers. (2) IgY (native condi-

tions); (3) Under reducing conditions HC: Heavy chain; DHC: Detritus heavy chain; LC: Light chain;

VH: Variable fraction of heavy chain; VL: Portion variable light chain; CL: Constant fraction of light

chain.

Fig. 3. ELISA immuno-specific recognition of IgY. The anti- Crot/pool venom-specific activity of IgY showed

that final product (rhombus) and final product dialyzed (square) resulted in the venom-specific IgY

recognition of Crot/pool venoms. The pre-immune serum (triangle) was negative.

64 Bello et al.

Investigación Clínica 63(1): 2022

Specificity of Crot/pool antivenom (IgY)

to Crotalus venoms via Western blot

The identity of the Crot/pool venom pro-

teins was confirmed with the Crot/pool antive-

nom (IgY) by Western-blot, identifying most of

the fraction venoms used in the present work

(Fig. 4). As a reference, the polyvalent antive-

nom of equine origin produced by BIOTECFAR

C.A (Caracas, Venezuela) was used.

Antivenom effective dose (ED50)

In order to estimate the ED50 of the final

Crot/pool antivenom (IgY), different amounts

of this antivenom were preincubated with 2LD50

(33 µg) of a Crot/venom pool, as described in

Materials and Methods. An ED50 of 19.66 mg

was calculated for the Crot/pool antivenom, as

the minimum amount of purified IgY prepara-

tion able to protect 50% of the mice popula-

tion. There were no survivals in the control

group (Table 1, Fig. 5).

DISCUSSION

Snakebite accidents, categorized as

a neglected tropical disease by the World

Health Organization (WHO) is responsible

for nearly 50.000 deaths annually, mostly in

Third World countries 8.

In the current work specific anti-Crota-

lus snake venoms antibodies were obtained

by an immunization schedule in female os-

triches (Struthio camelus). The eggs from

bird species have demonstrated being a de-

sirable basis for the production of antibod-

ies, without invasive methods, which present

a predominant class of IgY immunoglobulin

3, 9,10. This IgY has been used in diagnosis,

research, and immunotherapy 11-13. Further-

more, bird’s immunoglobulins production

poses several benefits over mammalian an-

tibodies with respect to the antigenic speci-

ficity and low manufacturing expenses 3,12. It

was possible to obtain about 2-4 g of IgY per

ostrich egg. Hence, almost 400 g of IgY can

be purified from only one ostrich per year.

For that reason, ostrich eggs could repre-

sent an exceptional source of immunoglobu-

lins for antivenom production.

A great amount of cross-reactive and

neutralizing antibodies (IgY) were produced

in the egg yolk of ostriches (Struthio cam-

elus) by immunizing with the venom of four

Crotalus (Crot/pool) using a simple and

Fig. 4. Western blot. (A) Crot/pool venom+ PAOS (Biotecfar C.A, Venezuela); (B) Crot/pool venom+ immu-

nized ostrich IgY; (C) Negative control (Non-immunized ostrich IgY+PBS).

Crotalus snakes envenoming treated with ostrich (Struthio camelus) IgY 65

Vol. 63(1): 57 - 69, 2022

Table 1

Determination of the survive percentage of mice after the injection of 2LD50 of Crot/pool venom with different

concentrations of ostrich IgY. Evaluation of the neutralizing capacity of anti-crotalic IgY antibodies.

LD50

16.5

(µg/mouse)

Concentration

(mg/mL)

Vol.

(mL)

Amount of

proteins

(mg)

Vol.

(mL)

Venom

Amount of

injected

venom (µg)

Vol.

(mL)

Saline

solution

Final Vol.

(mL)

Inoculated

animals Dead Alive Accumulated

Deaths

Accumulated

Alive

% survival

18.6

1.655 30.78 0.330

0.015 2 5 0 5 0 13 100.00

1.335 24.83

0.165

0 1.5 5 1 4 1 8 88.89

1.125 20.93 0.375 1.5 5 2 3 3 4 57.14

0.750 13.95 0.750 1.5 5 4 1 7 1 12.50

0.375 6.98 1.125 1.5 5 5 0 12 0 0.00

0.190 3.53 1.310 1.5 5 5 0

LD50: Lethal dose fifty; Vol: Volume.

66 Bello et al.

Investigación Clínica 63(1): 2022

inexpensive method. Knowledge of venom

variations permits the selection of suitable

specimens for production of more effective

antivenoms and biological substances.

The anti- Crot/pool venom immuno-

globulins were carried out from egg yolk by

the McLaren RD et al. 5 method and given a

single pure electrophoretic IgY band of 174

kDa (native conditions) and 65/20 kDa (un-

der reduced conditions) in the SDS-PAGE.

Neutralization experiments demon-

strated a high neutralization capacity of the

anti- Crot/pool preparation, as a preincu-

bated mixture of both purified antivenom

(19.66 mg) with two LD50 dose of Crot/pool

venom (33 µg) protected 50% of the mice.

It has been showed since the early nine-

ties (1990) that chicken egg yolk and their

IgY immunoglobulins were able to neutral-

ize scorpion and rattlesnake venoms, con-

firmed by in vivo experiments performed in

rodents 13. Comparing hens with ostriches,

the ostrich is one of the most primitive liv-

ing avian suggesting that the diverse fea-

tures of the bird Ig genes appeared very

early during the divergence of the avian spe-

cies and are thus common by most, if not

all, bird’s species 14.

Antibodies are competent of explicitly

recognizing a wide diversity of antigens with

different affinities. Affinity, simultaneously

with avidity is closely related to sensitivity,

which is an experimentally measurable value

in terms of antibody titer. Affinity is the con-

centration of the antigen that is needed to

occupy the binding sites, of half of the anti-

body molecules present in an antibody solu-

tion, while avidity is the universal summation

of the affinities of various antibodies (biva-

lent, multivalent and of different isotypes)

fixed to all places to all available epitopes,

considering conformational and valence as-

pects of the antibody 15. Authors 3 have devel-

oped successful anti-coral snake venom IgY

antibodies, which were carried out in chick-

en egg yolks and their neutralizing action

was similarly presented in mice by in vivo

counteraction experiments. These antive-

nom immunoglobulins neutralized the toxic

and lethal consequences of venom and, ac-

cordingly, could function to treat coral snake

envenomed victims. Similarly, authors 16 have

also produced hen antibodies against Scolo-

pendra gigantea toxins with high neutraliza-

tion titers and antivenom for the treatment

of scolopendrism 17.

Fig.5. Curve of Log doses of mg of protein with the percentage of survival animals. An ED50 of 19.66 mg Crot/

pool antivenom (IgY) was calculated from the represented data as the mass of IgY that was able to

protect at least 50% of the mice population against a 2LD50 (33 µg) challenge of Crot/pool venom. In

the control group there were no survivals. (%): percentage. (Log): logarithm.

Crotalus snakes envenoming treated with ostrich (Struthio camelus) IgY 67

Vol. 63(1): 57 - 69, 2022

The present experimental work refers

to the production of anti-(Crot/pool) anti-

venom in ostrich egg yolk and its ability in

deactivating the lethal consequences of the

above mentioned Crotalus venoms. Immu-

nization of ostriches with Crot/pool venom

provoked a characteristic primary humoral

response of low antibody titer in the ostrich

sera and the egg yolks at 15 days, followed

for a higher secondary response. The immu-

noglobulins titres amplified after the second

booster and the intensities were sustained

by continuing boosters. Ostrich eggs kept at

5°C during a year exhibited no substantial

reduction in the antibody titers.

The caprylic acid 18, 19 method showed

that under controlled pH conditions, at

a constricted pH range of 5.3–6.3 and low

ionic strength of acidified water simplified

the separation of IgY, after the yolk lipids

aggregation, producing a clear IgY enriched

supernatant, which was confirmed by SDS-

PAGE analysis. This technique has been used

for experimental purposes 18, but actually

is used in the production of horse antiven-

oms 19, with novel fractionation approaches

for antivenom production with the purpose

of achieving antivenoms of higher purity,

which would stimulate less allergic reactions

in snake bitten patients treated with this

product.

In the current work, it has been es-

tablished by an immunoblot and/or ELISA

assays that the anti-(Crot/pool) antivenom

IgY recognized and responded to Crot/pool

venom proteins. Immunoblot analysis re-

vealed not only the specific binding of the

antivenom but also dose-dependent blocking

of antivenom by venom proteins.

The outcomes of inhibition studies

show a specific neutralizing capacity of ven-

om activity in the experimental anti-(Crot/

pool) IgY antivenom. This ending activity

was regularly dose dependent, presenting

ample inhibition at a concentration of 2LD50

(33µg) of Crot/pool venom, being indicative

of specific binding of the IgY antibodies to

venom proteins to which they were devel-

oped and their capacity to block the lethal

effects of Crot/pool venoms. These types of

immuno-neutralization help reverse toxicity

and define the kinetics of toxins and anti-

bodies.

Until now, traditional treatment of

snakebite accidents is based on the use of

antivenoms from horse’s origin. Neverthe-

less, equine serum in theory could activate

complement cascade and initiate acute hy-

persensitivity reactions in patients formerly

sensitized to horse serum proteins 20. The

immunoglobulin IgY has the capacity of

eliminating this possible side effect since it

does not react as the mammalian IgG per-

forms and it does not activate the mamma-

lian complement factors 9. For that reason,

the horse serum mammalian IgG activating

mammalian complement factors 20 does not

permit physicians to give out much larger

doses of horse antivenom.

Regarding recent developments of

chicken antivenoms, Latin American authors

17 have characterized IgY antivenoms capable

of neutralizing the lethal activity of B. alter-

natus snake venom, at a preclinical level. An

antivenom, as an alternative to the conven-

tional antivenom production with egg yolk

antibodies (IgY-technology) was proposed.

Similarly, to the results presented in the cur-

rent work, antivenom efficacy assays were

carried out by them and after successive im-

munizations, levels of specific IgY reached a

maximum that was maintained throughout

the observation period; IgY antivenoms ob-

tained after several immunizations neutral-

ized 35.65 µg of B. alternatus venom per mg

of antivenom. Other authors 21 proposed that

birds were excellent hosts for the produc-

tion of neutralization antibodies at low cost.

These antibodies could be applied in the de-

velopment of diagnostic kits or as an alterna-

tive for snakebite envenomation handling in

the immediate future.

In conclusion, the specificity and spe-

cific activity of the antibody were scrutinized

by western blotting and confirmed the pres-

ence of highly specific antibodies to Crot/

68 Bello et al.

Investigación Clínica 63(1): 2022

pool venoms in the treated ostrich egg yolk.

Ostrich’s antivenom can represent an excel-

lent alternative for producing high amounts

of antivenoms at very low costs 22. Thus, they

could be a very good option to treat these

accidents in countries with low economic

resources where the ophitoxemia is a collec-

tive health problem. Therefore, the cleanli-

ness, efficiency, and simplicity of producing

antivenoms in ostriches, and the inability

of these antibodies (IgY) to bind to the hu-

man complement formulates an interesting

alternative to other antivenoms produced in

mammals. These findings point out that os-

trich egg antibody can be helpful as a thera-

peutic instrument to treat snakebites in hu-

mans, cattle and domestic animals.

In addition, these results open a thera-

peutic field, for the manufacturing of other

antivenoms against the broad spectrum of

toxins and also as a probable diagnostic

tool.

ACKNOWLEDGEMENTS

The authors are grateful to Lic. Rosa

Gutierrez Sánchez and her husband Juan C.

Da Silva, owners of the ostrich’s farm “Inver-

siones en Avestruces Rosa Gutierrez F.P” for

their generous help, managing the animal’s

upkeep. We would like to thank the helpful

commentaries from two anonymous refer-

ees, which allowed us to improve the manu-

script.

Declaration of conflict of interest

The authors pronounce that they have no

known competing financial interests or per-

sonal associations that could give the idea to

influence the work described in this work.

Funding

This work was funded by Biotecfar C.A

(Grant Ostrich #1), Facultad de Farmacia,

Universidad Central de Venezuela, Caracas,

República Bolivariana de Venezuela.

Authors’ ORCID numbers

• Carlos Bello (CB)

0000-0002- 5689-9284

• Fática Torrico (FT):

0000-0003-3388-8299

• Mariana Cepeda (MC):

0000-00025396-8483

• Miguel A. López (MAL):

0000-0002-6162-711X

• Alexis Rodriguez-Acosta (ARA):

0000-0003-1234-7522

• Juan Carlos Jimenez (JCJ):

0000-0002-1554-4292

Authors' contribution

MAL, FT and ARA envisioned and

planned research; CB, JCJ, MVC and ARA

carried out experiments; CB, FT, JCJ and

ARA analysed data; CB, FT, JCJ and ARA elu-

cidated results of experiments; CB and ARA

drafted manuscript; MAL, CB, FT, JCJ and

ARA edited and revised manuscript; MAL,

CB, FT, JCJ, MVC and ARA accepted final

version of manuscript.

REFERENCES

1. Rengifo C, Rodríguez-Acosta A. Serpien-

tes, Veneno y Tratamiento Médico en Ve-

nezuela. Caracas: Universidad Central de

Venezuela, 2019; 1-272.

2. Aguilar I, Sánchez EE, Girón ME, Estrella

A, Guerrero B. Rodríguez-Acosta A. Co-

ral snake antivenom produced in chickens

(Gallus domesticus). Rev Inst Med Trop Sao

Paulo 2014; 56: 61-66.

3. Almeida CM, Kanashiro MM, Rangel-Filho

FB, Mata MF, Kipnis TL, da Silva WD. De-

velopment of snake antivenom antibodies

in chickens and their purification from

yolk. Vet Rec 1998; 143: 579–584.

4. NIH, Principles of laboratory animal care.

National Institute of Health of United Sta-

tes of America, Pub. 85-23, Maryland; 1985;

1–112.

Crotalus snakes envenoming treated with ostrich (Struthio camelus) IgY 69

Vol. 63(1): 57 - 69, 2022

5. McLaren RD, Prosser CG, Grieve RC, Bo-

rissenko M. The use of caprylic acid for

the extraction of the immunoglobulin frac-

tion from egg yolk of chickens immunised

with ovine alpha-lactalbumin. J Immunol

Methods 1994; 177:175-184.

6. Towbin H, Stachelin T, Gordon J. Electro-

phoretic transfer of proteins from polyac-

rylamide gels to nitrocellulose sheets: pro-

cedure and some applications. Proc. Natl

Acad Sci USA 1979; 76: 4350–4354.

7. Spearman- Kärber R. Alternative methods

of analysis for quantal responses. In: Finney

D, editor. Statistical method in biological

assay. London, Charles Griffin; 1978; 1– 78.

8. Bagcchi S. Experts call for snakebite to be

re-established as a neglected tropical disea-

se. BMJ. 2015; 351: h5313.

9. Larsson A, Balow RM, Lindahl TL, Fors-

berg PO. Chicken antibodies: taking advan-

tage of evolution. A Rev Poultry Sci 1993;

72: 1807-1812.

10. Adachi K, Handharyani E, Sari DK, Taka-

ma K, Fukuda K, Endo I, Yamamoto R,

Sawa M, Tanaka M, Konishi I, Tsukamoto

Y. Development of neutralization antibo-

dies against highly pathogenic H5N1 avian

influenza virus using ostrich (Struthio ca-

melus) yolk. Mol Med Rep 2008;1:203–209.

11. Larsson A, Mellstedt H. Chicken antibo-

dies: a tool to avoid interference by human

anti-mouse antibodies in ELISA after in

vivo treatment with murine monoclonal

antibodies. Hybridoma 1992; 11: 33–39.

12. Schade R, Calzado EG, Sarmiento R,

Chacana PA, Porankiewicz-Asplund J,

Terzolo HR. Chicken egg yolk antibodies

(IgY-technology): a review of progress in

production and use in research and human

and veterinary medicine. Altern Lab Anim

2005;33:129-154.

13. Thallay BS, Carroll SB. Rattle snake and

scorpion antivenoms from the egg yolks of

immunized hens. Biotechniques (NY) 1990;

8: 934–938.

14. Huang T, Zhang M, Wei Z, Wang P, Sun Y,

Hu X, Ren L, Meng Q, Zhang R, Guo Y,

Hammarstrom L, Li N, Zhao Y. Analysis of

immunoglobulin transcripts in the ostrich

(Struthio camelus), a primitive avian spe-

cies. PLoS ONE 2012; 7: e34346.

15. Morimoto J, Sarkar M, Kenrick S, Koda-

dek T. Dextran as a generally applicable

multivalent scaffold for improving immu-

noglobulin-binding affinities of peptide and

peptidomimetic ligands. Bioconjug Chem

2014; 25:1479‐1491.

16. Parrilla P, Navarrete LF, Girón M.E, Agui-

lar I, Rodríguez-Acosta A. Use of chicken

egg yolk-derived immunoglobulin against

Scolopendra venom as an alternative to

treat scolopendrism. Rev Cient FCV-LUZ

2008; 18: 385-392.

17. Leiva CL, Cangelosi A, Mariconda V, Fa-

race M, Geoghegan P, Brero L, Fernán-

dez-Miyakawa M, Chacana P. IgY-based

antivenom against Bothrops alternatus:

Production and neutralization efficacy. To-

xicon 2019; 163: 84-92.

18. Dos Santos MC, D’imperio-Lima MR, Fur-

tado GC, Colletto GM, Kipnis TL, Dias da

Silva W. Purification of F(ab’)2 anti-snake

venom by caprylic acid: a fast method for

obtaining IgG fragments with large neutra-

lization activity, purity and yield. Toxicon

1989; 27: 297-303.

19. Rojas G, Jimenez JM, Gutierrez JM. Ca-

prylic acid fractionation of hyper immune

horse plasma: description of a simple pro-

cedure for antivenom production. Toxicon

1994; 32: 351-363.

20. Sutherland SK. Serum reactions. An analy-

sis of commercial antivenoms and the pos-

sible role of anticomplementary activity in

de-novo reactions to antivenoms and anti-

toxins. Med J Aust. 1977; 1: 613–615.

21. Lee CH, Liu CI, Leu SJ, Lee YC, Chiang

JR, Chiang LC, Mao YC, Tsai BY, Hung

CS, Chen CC, Yang YY. Chicken antibodies

against venom proteins of Trimeresurus ste-

jnegeri in Taiwan. J Venom Anim Toxins Incl

Trop Dis 2020; 26: e20200056.

22. Carbajo E. Produccion de avestruces: Una

industria ya establecida. Rev Cien Vet 2007;

23: 3-5.