Production and purification of polyclonal antibody against attenuated and wild type Leishmania infantum in dogs

Abstract

Antibodies are still widely used in several programs including early research, imaging, Targeting drug delivery system, Affinity chromatography, flowcytometry technic, diagnosis and treatment. Purification of antibody is a standard approach for detection of infection agent in different species. The reservoir hosts for Leishmania infantum are Dogs and they have active role in the transmission of leishmania to humans by the bite of a sand fly belonging to genus Phlebotomus and Lutzomiya. Consequently, elimination of dogs in endemic areas and vaccination of dogs contributes to reduction of the human and canine VL cases. Serological antibody tests such as IFAT (Indirect Fluorescent Antbody Test), DFAT (Direct Fluorescent Antbody Test), ELISA (Enzyme–Linked Immunosorbent Assay), PCR (Polymerase chain Reaction Assay) have been extensively used to investigate canine infection with L. infantum. In this study we produced and purified polyclonal antibody against attenuated and wild type leishmania infantum in dogs. Anti-leishmania in dog serums precipitated with ammonium sulphate. The IgG recovered from ammonium sulphate precipitation was subject to ion exchange chromatography (IEC) and the purity of IgG was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reduced condition. The purity of proteins were above 95% and then purified IgG was conjugated with FITC. We determined optimum titer of dog IgG by observation parasites under fluorescent microscope. The optimum dilution of prepared FITC conjugated dog IgG was 1: 400. This polyclonal antibody can be used for other applications in research, diagnosis and clinic.

Keywords

Leishmania infantum purification Dog polyclonal antibody

1. Introduction

Visceral leishmaniasis (VL) is a vector-borne disease caused by protozoan parasite belonging to many species of Leishmania [1]. Hundred of new cases of human visceral leishmaniasis (HVL) have been reported in Northest endemic Areas of Brazil, Middle East, Iran and Mediterranea [2].

These diseases based on clinical symptom may be classified into three groups: visceral leishmaniasis, cutaneous leishmaniasis and mucocutaneous leishmaniasis. Visceral leishmaniasis is caused by parasite Leishmania donovani and Leishmania infantum (also known as Leishmania chagasi), which is the most severe form of leishmaniasis [3]. The reservoir hosts for leishmania infantum are Dogs and they have active role in the transmission of leishmania to humans by the bite of a sand fly belonging to genus Phlebotomus and Lutzomiya [4]. Consequently, elimination of dogs in endemic areas and vaccination of dogs contributes to reduction of the human and canine VL cases [5]. Dogs are the best experimental model to study of VL, since clinical signs in Canine visceral leishmania and HVL are the same [6]. Serological antibody tests such as IFAT (Indirect Fluorescent Antbody Test), DFAT (Direct Fluorescent Antbody Test), ELISA (Enzyme-Linked Immunosorbent Assay), PCR (Polymerase chain Reaction Assay) have been extensively used to investigate canine infection with L. infantum [7]. The susceptibility to canine VL (CVL) in dogs correlated with the development of TH2 (IL-4, IL-5, IL-10 and IL-13) immune responses and production of IgE, IgG, IgG1 and IgG2 antibodies to leishmania [8]. In this study, we produced polyclonal antibody against attenuated and wild type leishmania infantum in dogs and then purification of dog IgG by ion exchange chromatography and conjugation with FITC for detection of L. infantum under Fluorescent microscope.

2. Material and methods

2.1 Parasites

Promastigotes of L. infantum JPCM5 (MCAN/ES/ 98/LIM-877) H-line and WT, were cultivated in complete haemoflagellate minimal essential medium (HOMEM) (GIBCO) supplemented with 10% heat-inactivated fetal calf serum (HI-FCS) and gentamicin at 20 $\mu$ g/ml (Sigma). The Stationary phase promastigotes of the attenuated line were harvested after 48 subpassages and a suspension of cells was prepared in a concentration of 5 $\times$ 10 [8] cells/ml in PBS. These parasites incubated at 18–24 ${}^{\circ}$ C For 5–7 days. After this time, we observed parasites with invert microscope.

2.2 Dogs

All dogs were examined for clinical signs of the disease and tested for the presence of specific anti-Leishmania antibody by an immunofluorescence assay (IFA). The dogs had already been vaccinated against canine parvovirus and rabies and have also been treated with anti-cancer medication such as praziquantel and pyrantel. The age of dogs was 12 months and the animal cods was V20 and V33. They were divided into two groups: 2 dogs in vaccinated groups and 2 dogs in unvaccinated groups. The dogs in vaccinated groups were injected subcutaneously with 100 $\mu$ l of the suspension of stationary phase promastigotes of H-line and WT L. infantum. After three mounth, specific anti-Leishmania total IgG antibody was measured by IFA. The unvaccinated dogs or control groups were injected subcutaneously with 100 $\mu$ l of PBS. Blood samples were collected from infected dogs with leishmania infantum H-line and WT [9]. The protocol for vaccination of the dogs had been reviewed and approved by the Medical Ethics and Animal and Use Care Committee of the Kerman Medical University (study protocol number KA/89/15), in accordance with the Guide for the Care and Use of Laboratory Animals Eighth Edition.

2.3 Ammonium sulphate precipitation

Equal volume of diluted serum and saturated 50.00% ammonium sulphate (Merk) were mixed by slow addition of the ammonium sulphate solution during stirring. Blood sample clarified by centrifugation (3500 g, 15 min) and diluted with 1:1 phosphate-buffered saline (PBS, PH: 7.2, Merk). Next day this material washed twice with 50.00% ammonium sulphate. The precipitate was dissolved in PBS, PH: 7.2 (1:1), and then dyalised against phosphate- buffered saline (PBS, PH: 7.2) [10, 11].

2.4 Ion exchange chromatography

In order to purified Dog IgG against attenuated and wild type Leishmania infantum, we used Ion exchange chromatography. Vaccinated Dog serum was collected and precipitated by ammonium sulphate (Merk). After diyalysis, samples were transferred into a Ion exchange chromatography. It was performed with anion exchange column (50 mm $\times$ 5 mm LD, Pharmacia) packed with diethyl amino ethyl (DEAE)-sepharose. Column equilibrated with Tris-phosphate (PH:8.28, Merk) and the column elution was performed in two steps, in the first step, the column eluted with tris-phosphate buffer (Merk), and in the second step, the column eluted with tris-phosphate buffer containing 50 mM of NaCl (Merk). The collected protein were assassed by using of UV spectrophotometer (set on a 280 nm absorbance, Bio-Rad Laboratories, Redmond, USA). The eluted proteins were collected in 3 mL fractions and analyzed by sodium dodecyl sulfate polyacryl amide gel electrophoresis SDS-PAGE [12].

2.5 SDS-PAGE analysis

The purity of IgG was confirmed by sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE) at reduced condition. The final concentration of polyacrylamide solution was 10%. Samples were boiled with 2% SDS for 10 min and were loaded on the electrophoresis gel. After separation, the proteins were stained with Coomassie ${}^{\text{\textregistered}}$ Brilliant Blue R-350 (Sigma).

2.6 Conjugation of dog igG with FITC

Purified polyclonal antibody dialysed against 500 ml FITC labling buffer (Merk) at 4 ${}^{\circ}$ C For 48 hours. 20 $\mu$ l of 5 mg/ml FITC (Sigma) in DMSO (Sigma) was added for each milligram of antibody and incubated 2 h at room temperature and removed unbound FITC antibody with dialysis against 500 ml final dialysis buffer (Merk). Next day, labeling of anti leishmania infantum with FITC was added to leishmania infantum parasites on slide and then, it observed under fluorescent microscope [13].

3. Results

3.1 Evaluation of anti-leishmania in dogs

Two unvaccinated dogs as a control groups were analyzed for presence of specific anti-Leishmania IgG antibody. The titer of specific anti-Leishmania IgG antibody in dogs was ( $\geqslant$ 1:100) by IFA. Two vaccinated dogs were tested for determination of specific anti-Leishmania IgG antibody. The titer of these specific antibody by IFA was ( $<$ 1:1600).

3.2 Purification of dog IgG antibody against attenuated and wild type leishmania infantum

The purification of polyclonal antibody from vaccinated dogs with attenuated and wild type Leishmania infantum followed by ammonium sulphate precipitation and then, DEAE ion-exchange chromatography. The protein content of the dog sera were 53 mg after precipitation by ammonium sulphate. Purification by Ion exchange chromatography yielded about 15 mg of IgG antibody which was about one third of the primary protein content. After that, we used Tris-phosphate buffer (PH: 8.28) containing 50 mMNacl at the same PH with flowrate 1 ml/min. Finally we washed column by the use of 1 M Nacl with the same buffer. We obtained Optical Density (OD) of collected fractions by spectrophotometer and it is shown in Fig. 1.

Figure 1.

chromatographic pattern of purified anti leishmania in the nature of dog IgG by ion exchange chromatography with Tris-phosphate buffer, PH:8.28 (first peak) and 50 mM Nacl elution (second peaqk). Fraction 1, tris-phosphate pH: 8.28, Fraction 2, tris-phosphate + NaCl 50 mM pH: 8.28. Sample: IgG dog, column: DEAE-sepharose, flowrate:1 ml/min.

3.3 SDS-PAGE analysis

In order to confirmed the purity of Dog IgG, we used sodium dodecyl sulfate polyacryl amide gel electrophoresis (SDS-PAGE). The results of reduced SDS-PAGE has been shown in Fig. 2. Sera from dogs with attenuated and wild type Leishmania infantum presented bands towards 50 KDa which shows dog IgG heavy chain. Also diffused bands between molecular weights of 20–30 KDa represented dog IgG light chains. In this study, we obtained a protein with purity above 95%.

Figure 2.

SDS-PAGE analysis of anti leishmania antibodies produced in dog by ion exchange chromatography in 10% polyacrylamid gel under reduced condition and stained with comassie brilliant blue R-350. Lane 1: 50 mMNacl, lane 2: LMW marker, lane 3, 4, 5, 6, 7, diffirent fraction of sample in first peak.

3.4 Conjugation of dog IgG with FITC

In order to see attenuated and wild type Leishmania infantum parasites under microscope, we conjugated dog IgG with FITC. The result of titration of FITC conjugated anti leishmania indicated that FITC-IgG applied at 1/400 dilution, which is display a better ability to generate true positive result. These resulte determined by flourcent microscope (Fig. 3).

Figure 3.

leishmania is shown with 1/400 titer of FITC conjugated anti-leishmania infantum.

4. Discussion

Visceral leishmaniasis (VL), also known as kala-azar, black fever, and Dumdum fever is the most severe form of leishmaniasis. Leishmaniasis is a disease caused by protozoan parasites of the Leishmania genus. This disease is the second-largest parasitic killer in the world. The parasite migrates to the internal organs such as the liver, spleen and bone marrow, and, if left untreated, will almost always result in the death of the host. In this study, we try to use simple and inexpensive techniques to detect parasites [14]. The sources of our antibodies were vaccinated dog with L. infantum H. line and WT. Dogs were used as a host animal for polyclonal antibody production because Leishmania infantum is the most important causes of canine leishmaniasis in the worldwide [15]. Dog polyclonal IgG was purified by Ion exchange chromatography. This method is used commonly in protein purification due to its high binding capacity and cost effective. The isolation of proteins from Ion-exchange chromatography depends on factors such as buffer type and pH, properties of the proteins, flow rate of the mobile phase, dynamic capacity, characteristics of isoelectric point of the proteins, charged ligand bound as stationary phase and ionic strength. The strength of protein binding during Ion exchange chromatography depends on PH and it decreased with increasing ionic strength of the buffer solution [16]. Obtained polyclonal antibody could be used for many immunoassay tests such as enzyme-linked immunosorbent assay (ELISA), Western blot tests, flow cytometry and etc. In this technique, we obtained the high purity of dog IgG and these produced polyclonal antibody with higher titer had less cross reactivity with other species [17]. These result indicated that the purification of Dog IgG in DEAE-sepharose gel with starting buffer contain Tris-phosphate buffer have high purity. When we used proteins in salt step with high concentration of salt, the purity of antibodies were moderate [18]. In this experiment, we conjugated Dog polyclonal IgG with FITC (fluorescein isitiocyanate) because coupling phycobiliproteins such as phycoerythin (PE) and allophycocyanin (APC) to antibody is more difficult. In addition, FITC, biotin, Texas Red can be efficiently conjugated to small amount (1 mg) of purified antibody. We observed parasites under fluorescent microscope with greater clarity, since the polyclonal antibody can bond to more and different epitopes, resulting in better sensitivity. In other words, this product is highly economical and suitable ways toward self-sufficiency. These methods are reliable and inexpensive for detection dog diseases including, parasitic and inflammation disease, etc.

Footnotes

Acknowledgments

This work was financially supported by Kerman University of Medical Sciences and Departmant of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences. The manuscript was written based on a dataset of a Master’s thesis registered in Kerman University of Medical Sciences.

Conflict of interest

The authors report no conflicts of interest in this work.

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