Production of Rabbit Anti-Excretory/Secretory Product of Fasciola

gigantica Lombok Isolate Antibody

Made Sriasih

, Sulaiman Ngongu Depamede

, Djoko Kisworo

, Galuh Tresnani

Faculty of Animal Science, Mataram University, Jalan Majapahit No.62 Mataram

Faculty of Mathematics and Science, Mataram University, Jalan Majapahit No. 62 Mataram

Keywords: Excretory/Secretory Product, Fasciola gigantica Lombok Isolate, Polyclonal Antibody, ELISA.

Abstract: The major limiting factor in immunodiagnostic development for Fasciolosis detection is the absence of

specific, monoclonal or polyclonal, antibodies against Fasciola antigens specifically Fasciola gigantica,

which is the main species responsible for Fasciolosis in ruminants in Indonesia. The purpose of this study is

to produce polyclonal antibody against excretory/secretory (ES) product of F. gigantica Lombok isolate using

rabbit. Four months old rabbits were immunized subcutaneously with 0.5ml (300μg/ml) of the ES product

emulsified with 0.5ml Freund’s complete adjuvant and then Freund’s incomplete adjuvant. Serum antibodies

were harvested after two-times booster at 1-month interval and determined using enzyme linked-

immunosorbent assay (ELISA) and Western blotting. Immunization results evaluated by ELISA show that

rabbits could be used as a bioreactor to produce ES antibodies. The anti-ES antibody response could be

detected 4 weeks post-immunization, followed by increased humoral response of rabbit after first booster.

The optical density (OD

450nm

) value in the ELISA increased from 0.4 before immunization to 1.184 and 1.392

depending on the type of blocking agent used. The Western blotting results confirm that the ES protein bands

were only recognized by rabbit serum samples post-immunization and thus confirmed the ELISA test result.

1 INTRODUCTION

One of the strategies in early diagnosis of Fasciolosis

caused by Fasciola sp. is the development of

detection methods that can indicate the presence of

disease before it develops into chronic through

immunologic approaches based on antibody detection

or antigen detection. However, the major limiting

factor in the development of immunodiagnostic

methods to detect Fasciolosis in livestock is the

absence of specific, monoclonal or polyclonal,

antibodies against Fasciola sp. antigens. Thus, the

antibody production will be able to overcome the

major inhibiting factor.

Fasciola sp. (liver fluke) that resides inside the

infected host release a significant amount of

excretory/secretory (ES) products (Spithill et al.,

1999), and they play an important role in the

avoidance of parasites from the host immune system

(Morphew et al., 2007). A one-dimension

electrophoresis gel analysis shows that ES products

are composed of proteins of different molecular

weight. Several studies have reported that geographic

variation and different species of fluke will affect the

components of the ES products produced. Sriasih et

al. (2013) stated that one-dimension gel

electrophoresis of the ES products isolated from

Fasciola gigantica that infest Bali cattle in Lombok

island showed protein bands with molecular weight

between 7-25 kDa. The results of the study indicated

that there is a difference in the components of the ES

proteins of F. gigantica Lombok isolate with the

results of previous studies reported by Meshgi et al.

(2008) and Estuningsih et al. (2004).

Studies show that ES products have potential as

an immunoprophylactic agent against Fasciolosis

(Acosta et al., 2008; Jayaraj et al., 2009). Moreover,

ES products are immunogenic so that are potentially

used as vaccine candidates (Ortiz et al., 2000;

Sethadavit et al., 2009), and are used for the diagnosis

of Fasciola-specific antibodies in livestock (Sriasih et

al., 2005; El Ridi et al., 2007; Kooshan et al., 2010).

This indicates that the ES products from the Fasciola

sp. may be potentially used as an antigen to produce

antibodies.

578

Sriasih, M., Depamede, S., Kisworo, D. and Tresnani, G.

Production of Rabbit Anti-Excretory/Secretory Product of Fasciola gigantica Lombok Isolate Antibody.

DOI: 10.5220/0007547405780582

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 578-582

ISBN: 978-989-758-348-3

Antibody may be produced by inducing

laboratory animals, such as rats, guinea pigs and

rabbits, with an immunogenic antigen. Immune

response induced by antigen after exposure can then

be measured and determined using serological

testing. Serologic tests developed today can be

divided into two categories; primary binding and

secondary binding tests. The primary binding test is a

test that directly measures the antigenic bonding to

antibodies including fluorescence antibody technique

(FAT), radioimmunoassay (RIA) and Enzyme Linked

Immunoassay (ELISA). The secondary binding test is

a test that measures the results of antigen-antibody

interaction in-vitro which includes agar gel

precipitation est (AGPT), serum agglutination test

and complement fixation test (FAT). However, the

primary binding is more sensitive than the secondary

binding test.

The purpose of this study is to produce polyclonal

antibody against the ES product of F. gigantica

Lombok isolate using rabbit and to determine the

antibody response of rabbit to the ES product using

the ELISA test, and Western blotting. The success in

producing specific antibodies against specific

antigens will increase sensitivity and specificity of

immunodiagnostic test to perform early detection of

Fasciolosis in livestock.

2 MATERIALS AND METHODS

2.1 Research Design

Two local rabbits (4 months old) were placed in a

special cage and fed with pellets, fresh vegetables,

and drinking water (ad-libitum). The rabbits were

then immunized with 0.5ml (300μg / ml) of

emulsified ES product with 0.5ml Freund's complete

adjuvant (FCA). Sera were harvested and tested with

the ELISA before initial immunization and after

booster. The ELISA results were then confirmed by

Western blot analysis.

2.2 Immunization and Serum

Collection

Animal Ethics approval (Protocol No.

235/UN18.8/ETIK/2017) had been obtained for

conducting this study. Immunization was carried out

by subcutaneous injection at multiple sites (no more

than 5) behind the neck and was dispersed under the

skin by gentle rubbing. Four weeks after initial

immunization with the ES product emulsified with

the FCA, the rabbits were then immunized twice

subcutaneously with 0.5ml (300 μg / ml) of the ES

product mixed with 0.5ml Freund's incomplete

adjuvant (FICA) at 1-month interval.

Blood sampling was performed by taking blood

from the auricularis vein of the rabbits using a 3ml

syringe. Blood were placed on a sterile tube and were

then incubated at room temperature for 3 hours. After

incubation, the serum can be obtained by

centrifugation at 5000rpm at 4

C for 15 min. The sera

were then aliquoted into several eppendorf tubes and

then stored at -20°C until further assays.

2.3 Measurement of Humoral Immune

Response

Humoral immune response of each rabbit to the ES

product was measured by ELISA technique, and then

confirmed by Western blot. ELISA was performed

according to Sriasih’s procedure (Sriasih et al., 2005).

The ELISA plates (96 wells) were coated with 50 μl

of the ES product and incubated for 1 hour at room

temperature. After incubation, the ES product was

removed and the wells were washed 5 times using

phosphate buffer saline (PBS) containing 0.05%

Tween 20. Blocking agent (100 μl PBS containing

bovine serum albumin (BSA) or skim milk) or were

added into each well and were then incubated for 1

hour at room temperature. Following incubation, the

plate was washed again (5 times washing cycling).

Fifty microliters serum that had been diluted a

hundred times were added into each well and then

incubated for 1 hour at room temperature. After 5

times washing, 50μl anti-rabbit IgG horse-radish

peroxidase conjugated were added and then incubated

for 1 hour. One hundred microliters of substrate

(ABTS in 100ml of citrate buffer) were added into the

wells and incubated for 15 min room temperature.

Optical density (OD) was then measured at 405nm

wavelength using an ELISA reader machine.

2.4 Western Blotting Analysis

After electrophoresis, gels were equilibrated in

transfer buffer for at least 10 min. Polyvinylidine

difluoride (PVDF) membrane was pre-incubated in

100% methanol for 1 min then rinsed with several

changes of water. After rinsing, the membrane and

filter papers were also allowed to equilibrate for at

least 15 min in transfer buffer. The transfer of

proteins from the gel to the membrane was carried out

at a constant voltage of 15 Volts for 35-40 minutes

using a Trans-Blot® Semi-Dry (SD) electrophoretic

transfer cell as per the manufacturer’s instructions.

Production of Rabbit Anti-Excretory/Secretory Product of Fasciola gigantica Lombok Isolate Antibody

579

For immunostaining, the membranes were

washed multiple times in distilled water and

immediately blocked with blocking buffer (Tris

buffer saline pH 7.4 [20 mM Tris-HCl, 100 mM

NaCl] containing 5% [w/v] skim milk and 0.1% [v/v]

Tween-20) at room temperature for 1 hour. After

washing twice in washing buffer (Tris buffer saline

pH 7.4 [20 mM Tris-HCl, 100 mM NaCl] containing

0.1% [v/v] Tween-20), the membrane was cut into

strips and further incubation of each strip was carried

out in individual reservoirs. Each strip was incubated

with 10 ml of diluted rabbit sera (1:500) for 1 hour at

room temperature. Each strip was then washed for

five 5-10 min cycles with washing buffer. After

washing, diluted anti-rabbit IgG HRP (1:8000) was

applied and incubated for 1 hour at room temperature.

Strips were then washed as previously described.

Immunodetection was developed by addition of TMB

substrate.

2.5 Data Analysis

Humoral immune responses of each rabbit were

recorded and analyzed using a simple statistical

calculation (Mean ± Standard Deviation).

3 RESULTS AND DISCUSSION

The ES product used for rabbit immunization in this

study was derived from the Lombok isolate of

F.gigantica (Sriasih et al., 2013). The ES products

were injected into the rabbits as a bioreactor to

produce the ES antibodies because they have a distant

genetic relationship with cattle, are easy to handle and

easy to maintain. Rabbits are also laboratory animals

that are widely used in various studies for the

production of antibodies, tumorigenesis, nutrition,

genetics, radiation research and anaphylactic

research. The immune system of the rabbit will

recognize and react to the antigen. The lymphocyte

cells exposed as part of the body's defense system

then will multiply and develop into plasma cells that

produce antibodies. The antibodies formed are

polyclonal antibodies with varied composition in

serum, either as a result of repeated immunization, or

due to variations that occur during an immune

reaction (Tizard, 2004).

The ELISA test results of humoral rabbit response

before immunization (baseline), 4 weeks after the

initial immunization (FCA), 4 weeks after the second

immunization (FICA1) and 2 weeks after the third

immunization (FICA2) using two blocking agents,

BSA and non-fat dry milk are presented in Table 1.

Table 1: The humoral response of the rabbits to the ES

antigen on ELISA test.

Data on Table 1 depict that only rabbit #2 showed

a good immune response. The baseline accounted for

an optical density value of 0.459 in the ELISA with

BSA blocking and 0.548 with non-fat dry milk

blocking. After immunization with the emulsified ES

antigen with FCA, the optical density value increased

to 1.184 (BSA) and 1.392 (non-fat dry milk). This

may occur as a result of activation of the B cells

memory whose work is stimulated by T cells to

produce antibodies in large quantities (Goldsby et al.,

2000; Abbas et al., 2007).

Figure 1: Polyclonal immune response of rabbit#2 anti ES

antibody blocked with different blocking agents.

The use of different blocking agents in this study

(Figure 1) did not have a significant effect on the

optical density values in the ELISA test. Both BSA

and non-fat dry milk are regularly used blocking

agents in the ELISA test. Milk contains a number of

different proteins, and one of them is casein

phosphoprotein. This phosphoprotein can cause a

high background value due to the non-specific

reaction to the phospho structure.

The Western blotting result (Figure 2) also

corroborated the ELISA assay results and showed that

the ES protein bands were only recognized by the

rabbit serum (rabbit #2) after immunization. Cruse

and Lewis (2002) suggest that the addition of

adjuvants to the injected isolate serves to enhance the

immunogenicity of the isolate. The presence of

Mycobacterium sp in Freund’s complete adjuvant in

early immunization will stimulate B cells and T cells

to produce an immune response. The primary

immune response of B cells is activated to proliferate

Rabbit #1 Rabbit #2 Rabbit #1 Rabbit #2

Baseline 1.866±0.002 0.459±0.013 1.628±0.01 0.548±0.007

FCA 2.06±0.012 1.184±0.011 1.729±0.008 1.392±0.001

FICA1 2.135±0.005 1.928±0.007 1.772±0.003 1.901±0.002

FICA2 2.165±0.006 1.867±0.002 1.783±0.002 1.864±0.002

BSA Blocking

Non-fat dry milk blocking

Baseline FCA FICA1 FICA2

Humoral immune response of rabbit# 2

BSA Non-fat dry milk

ICPS 2018 - 2nd International Conference Postgraduate School

580

and differentiate within antibody secretion cells and

memory cells. Some antibody cells migrate and

survive in the bone marrow for long periods. The

second immunization (booster 1) and the third

immunization (booster 2) with Freund’s incomplete

adjuvant will produce secondary immune response

with higher concentration than the first immunization.

Figure 2: Western blot analysis of rabbit anti-ES polyclonal

antibodies. MW = standard protein marker; 1 = rabbit pre-

immune sera; 2 = rabbit sera collected after second booster.

The formation of rabbit antibodies is influenced

by the antigenicity of the injected ES proteins. The

principal feature of a substance or compound is

determined by physicochemical limitation and degree

of foreignness (Tizard, 2004). The physicochemical

limitation of a substance or compound is that the size

of the antigen molecule must be large, rigid and has a

complex chemical structure (Kuby, 2007). The

chemical structure of the ES proteins derived from the

F. gigantica Lombok isolate that are large and

complex will produce faster antibodies.

The results of this study indicate that rabbit can be

used as a manufacturer to produce the ES antibodies.

Antibodies formed 4 weeks post immunization,

followed by increased humoral response of the rabbits

after booster 1. Setyaningsih (2004) in her study using

rabbits to produce polyclonal antibody against ES

proteins isolated from F.gigantica buffalo isolate and

goat isolate reported that antibodies anti-ES of F.

gigantica goat isolate are formed faster than that of

buffalo isolate. Antibodies anti-ES of F. gigantica

goat isolate were formed at 4 weeks while antibodies

anti-ES of F. gigantica buffalo isolate formed at 12

weeks. The time difference to elicit antibody-forming

responses to the host may vary and is dependent on

the immunogenicity, the form and stability of

stimulants, animal species, injection routes, and the

sensitivity of tests used to detect antibodies formation

(Tizard, 2004). The host's response to the

immunogens given is not only determined by the

immunogenic physical properties but is also

determined by host-related factors such as genetics,

age, nutritional status and secondary effects derived

from a disease.

4 CONCLUSIONS

Anti-ES polyclonal antibody specific to the ES

antigens of F. gigantica Lombok isolate has been

successfully produced in rabbit. The availability of

the antibody will certainly facilitate the development

of a better immunodiagnostic tests for controlling

Fasciolosis.

ACKNOWLEDGEMENT

This study was funded by the Ministry of Research,

Technology and Higher Education. The authors also

would like to thank Postgraduate study program of

Mataram University for travel grant given.

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