Immunogenicity comparison of lactoferrin purified from Saudi Arabia camel clans milk

Abstract

Secretory lactoferrins play a crucial rolls at mucosal surfaces as not only antimicrobial molecules in primate as well as human, but as physiological protein. Its multiple functions extended to be one of immunogen could elicited autoimmune disorders. Purified camel lactoferrin (cLfs) from different Saudi camel clans were shown to be a potent immunogen when injected into rabbit. Four rabbit were subcutaneously immunized with different camel clans lactoferrin/Freunds adjuvant. Anti-cLfs potency titration was reach 1:32000 and did not significantly differences between different cLfs. The cross-reactivity level of different anti-Lfs were highly significant, specially between cLfs and bLf/hLf.

Keywords

Immunogenicity lactoferrin anti-lactoferrin polyclonal antibody milk camel lactoferrin

1. Introduction

Lactoferrin is an 80 kDa iron-binding glycoprotein consisting of 2 lobes that possess essentially identical structural folds and iron-binding sites, produced by neutrophils and it mostly secreted in mucus and milk of all animals and human as one of very interest immunological protective component. Lactoferrins from different host species vary in amino-acid sequence and in the location of the N-linked oligosaccharide side chains, but commonly possess a relatively high isoelectric point with positively charged amino acids clustered in the N-terminal region of the N-terminal lobe. Our previous studies showed that camel lactoferrin and its native and/or recombinant lobes (cLf) displayed significant antimicrobial activity [1, 2, 3, 4, 5, 6, 7, 8, 9, 10].

Food and Drug Administration (FDA) considered lactoferrin as a Generally Recognized as Safe (GRAS) substance [11], numerous clinical trials on the potential treatment of infectious diseases have been performed with intact molecule or artificial peptides derived from lactoferrin [12, 13]. The activated lactoferrin (activin™) is being used in food preservation, which could be sprayed onto carcasses or applied to beef surfaces prior to final packaging [11, 14, 15]. In addition, lactoferrin, as well as its peptides and a number of other natural peptides, are viewed as an important therapeutic alternative to small molecules [16].

In addition to its unique structure and differential antimicrobial potentials, camel lactoferrin was found to contain 5.6% carbohydrates in milk collected 15–30 days after parturition and 6.2% in colostral milk [17]. Is this high percentage of cLf carbohydrates moiety has structure variation? It’s still waiting explore. Whatever, in light of there are four Saudi camel clans (breeds), the question may release; is their purified lactoferrin have different immunogenicity in experimental animal, based on expected variations? The current study aim to produce rabbit antibody against cLf purified from four Saudi camel clans.

2. Materials and methods

The potential camel lactoferrin isolated from milk different of Saudi camel clans were analysis to induce antibodies in rabbit. Human and bovine lactoferrins and anti-bLf, hLf were all purchased from Sigma-Aldrich (Germany). The iron saturation of lactoferrins was at the level of $\sim$ 10% and $\sim$ 15% in the case of bLf and hLf, respectively as determined by spectrophotometry (A ${}_{280/466}$ ).

2.1 Collection and processing of camel milk

Camel milk was collected from four well-known camel clans (20 camels of each clan, total 80 animals) from Riyadh district. These four clans were Safra, Wadha, Majahem, and Hamra, and camel lactoferrins. Different lactoferrins protein were isolated from milk of corresponding animals were termed cLf1, cLf2, cLf3, and cLf4, respectively. The collected milk samples (1 to 1.5 kilograms collected from each female camel clan) were cool-stored until reaching the laboratory, where the milk was kept frozen at $-$ 20 ${}^{\circ}$ C until use. Camel milk was skimmed and casein was removed according to standard protocols as previously described [18, 19, 20, 21]. In short, a solution of sodium azide (0.2%) containing 5 mM PMSF and 5 mM EDTA was added to milk before defatting by centrifugation at 1000 $\times$ g for 30 minutes at 4 ${}^{\circ}$ C. The pH of the skimmed milk was then lowered to 4.6 with 1 M HCl to initiate casein precipitation. After centrifugation at 5000 $\times$ g for 20 minutes, the casein pellet was separated from the whey supernatant, and whey was used for the subsequent steps.

2.2 Purification of camel lactoferrins

Camel milk whey was used as a main source for lactoferrin purification. The lactoferrin was purified using a dual column separation method that includes chromatography on the CM-Sephadex and the Heparin-Sepharose fast flow columns. One-liter CM-Sephadex (5 gram of swollen powder) equilibrated with 0.1 M phosphate buffer saline, at pH 7.5 and containing 150 mM NaCl was mixed overnight with one liter of whey milk at 4 ${}^{\circ}$ C. Next day, the solution was stilled, and the unbound whey protein was collected as flow through. The column containing CM-Sephadex bound protein was thoroughly washed with 0.01 M PBS and 150 mM NaCl until the protein content reached the plateau, where the elution was step started. The elution buffer (0.01 M PBS and 150 to 500 mM NaCl) was gradually added. Three milliliter fractions were collected and their protein content was checked. The fractions containing protein were checked with SDS-PAGE. The lactoferrin was eluted from the CM-Sephadex at about 350–400 mM ionic strengths, as previously elucidated. The fractions containing lactoferrin were collected, mixed, concentrated, and then dialyzed against 150 mM NaCl. The resulting solution was loaded into the Heparin-Sepharose CL-6B affinity chromatography column and mixed end-over-end overnight, and was then washed with PBS. The bounded lactoferrin was eluted from the Heparin-Sepharose column with 0–1 mM NaCl/PBS (pH 7.5) buffer. Lactoferrin was specifically bound to the Heparin-Sepharose affinity column and dissociated from it at NaCl concentration of about 250 mM, without co-purification of other milk proteins. The iron saturation of four cLfs, checked by spectrophotometry (A ${}_{280/466}$ ), was of approximately 35% in all of them (partially iron-saturated) and were founded at the level of $\sim$ 15% [1, 22]. The purity of purified lactoferrins was evaluated with Gel electrophoreses (12% SDS-PAGE) according to standard protocols [1, 23, 24] using denatured samples under reducing conditions. Protein bands were visualized by Coomassie blue staining. All kinds of protein types were measured by Bradford protein assay Kit (BioRad, USA), as previously described [24].

2.3 Rabbit anti-cLf antibody preparation and evaluation of its reactivity

In this work, all immunological methods were carried out in accordance with the relevant guidelines and regulations, and all related experimental protocols were approved by the Kind Fahd Center for Medical Research and the King Abdulaziz University. Four rabbits were subcutaneously (SC) primary immunized with cLf1, cLf2, cLf3, or cLf4 at concentration of 3 mg/ml/each emulsified in Freunds Complete adjuvant at day 0 at 3–4 sites on the rabbit dorsal sides, then secondary immunized at day 28 with 1.5 mg/ml/each emulsified in Freund’s Incomplete Adjuvant [23, 24, 25]. The booster dose was given after 7 days. About 10 ml blood samples were harvested from those rabbits before immunization via ear vein bleeding and used as unimmunized rabbit samples. Three days after last immunization, about 20–25 blood samples were drawn from each rabbit in Falcon 50 ml tubes, and the sera was collected after centrifugation, then aliquoted (ml/tube) and stored at $-$ 80 ${}^{\circ}$ C.

Figure 1.

Antibody against lactoferrins titration. Rabbit anti-camel lactoferrin (cLf1, cLf2, cLf3, and cLf4) as well as anti-human lactoferrin and mouse monoclonal antibody against lactoferrin was titrated respectively, using sandwich ELISA. All results represented as main $\pm$ SD, and the SD represented as vertical bar on each column.

For titration and reactivity evaluation of anti-cLf1, anti-cLf2, anti-cLf3, anti-cLf4, and mouse monoclonal antibodies against hLf and bLf, coated ELISA plates with cLf1, cLf2, cLf3, cLf4, hLf or bLf (at 1 $\mu$ g/ml) in carbonate/bicarbonate buffer were prepared overnight, then blocked for 60 min with (3% gelatin, 0.1% Tween 20, 0.1 M PBS), then follow as previously described in details [25, 26, 27, 28]. The unimmunized serum was used as negative control. The corresponding antibody at different dilutions then anti-rabbit IgG, anti-mouse IgG and anti-human IgGperoxidase-labeled were added. Plates were developed with TMP substrate. The plates OD were read at 490 nm, the data were presented as mean $\pm$ SD of triplicate. Cross reactivity evaluation was done similarly but each antibody was incubated with all lactoferrins tested.

3. Statistical analysis

All experiments were repeated two to three times and the results are expressed as mean $\pm$ SD. Comparison of the data was per-formed using the Student’s $t$ -test. Significance was defined as a $P$ value of ${}^{*}<$ 0.05, ${}^{***}<$ 0.001.

4. Results and discussion

Increasing the interest with the camel milk for its multiple health benefits [29, 30, 31], need to identify its ingredients in depth one by one. One of most important protein molecule in the camel milk is the lactoferrin [32], so our main aim is preparing a local and specific antibody against camel milk lactoferrin. The lactoferrin, as it one of mucosal immunological molecules, it implicated in elicit autoimmune disorder via working as immunogen in several autoimmune diseases [33, 34, 35, 36].

4.1 Purification lactoferrins

The dual column separation method for Lf purification that includes chromatography on the CM-Sephadex and the Heparin-Sepharose fast flow column utilized in this study was very efficient. In fact, the CM-Sephadex step generated 50–70% purity, whereas the Heparin-Sepharose step produced lactoferrin samples with a purity of about 99% (data not shown).

4.2 Rabbit immunization

By the end of immunization schedule, it seem the rabbit were healthy and idi not revealed any kind of superficial ulcers in injection area from using the Freund’s Adjuvant system during immunization. Three days after last immunization about 20–25 blood sample was withdrawn from each rabbit in Falcon 50 ml tubes and the sera was collected after centrifugation.

Figure 2.

Anti-lactoferrin antibodies cross-reactivates. All 6 ant-lactoferrin were cross react each together using ELSA as described under material and methods. All results represented as main $\pm$ SD, and the SD represented as vertical bar on each column. $P$ value of ${}^{*}<$ 0.05, ${}^{***}<$ 0.001.

4.3 Activity and cross-reactivity of anti-lactoferrin antibodies

Figure 1 represents the anti-Lf titration shows that the anti-cLf titer reaches 1:30,000 to 1:33,000, whereas anti-hLf and anti-bLf antibodies were more reactive and showed higher affinity, 1:64,000 to 1:120,000 against their corresponding antigen, likely because they were purchased from a commercial source (Sigma-Aldrich, Germany). It seem that the depicted results (Fig. 1) did not revealed any significance differences between the immunogenicity lactoferrin (cLf1, cLf2, cLf3 and cLf4) purified from different camel clans (Hamra, Wedha, Safra and Majahem). Is this indicated to that there is/are no structural differences between those lactoferrin? It need furthermore clarification based on glycosylation and mass spectroscopy analysis.

Figure 2 shows the results of the cross-reactivity analysis of anti-Lf antibodies and illustrate that the anti-cLf antibodies (against the cLfs from four camel clans) highly cross-react with each other, while being less cross-reactive against human and bovine lactoferrins. It is seem also that the camel lactoferrins is a potent immunogen. However, its immunogenicity are similar independent its source.

On the other hand, anti-bLf and anti-hLf antibodies seem to be more cross-reactive with each other than with any of the anti-cLf antibodies. These results confirm the previous conclusions that amino acid sequences of human and bovine lactoferrins share higher identity levels than camel lactoferrin [32].

Footnotes

Acknowledgments

This work was supported by the King Abdulaziz City for Science and Technology General Directorate of Research Grants Programs, under grant No. LGP – 35–84.

Conflict of interest

The authors have no competing interests as defined by Nature Research, or other interests that might be perceived to influence the results and/or discussion reported in this paper.

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