Indian Journal of Dermatology
  Publication of IADVL, WB
  Official organ of AADV
Indexed with Science Citation Index (E) , Web of Science and PubMed
 
Users online: 873  
Home About  Editorial Board  Current Issue Archives Online Early Coming Soon Guidelines Subscriptions  e-Alerts    Login  
    Small font sizeDefault font sizeIncrease font size Print this page Email this page


 
Table of Contents 
BASIC RESEARCH
Year : 2012  |  Volume : 57  |  Issue : 1  |  Page : 15-19
Generation and characterization of chicken egg yolk antibodies against propionibacterium acnes for the prevention of acne vulgaris


Department of Microbiology, PSG College of Arts and Science, Coimbatore, India

Date of Web Publication10-Mar-2012

Correspondence Address:
Karthika Selvan
Department of Microbiology, PSG College of Arts and Science, Civil Aerodome PO, Coimbatore-641 039, Tamil Nadu
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-5154.92669

Rights and Permissions

   Abstract 

Introduction: Antigen-specific antibody has been widely used for immunological analysis in the field of diagnosis as well as in pure scientific research, where the IgY antibodies can be raised against P acnes antigen. Material and Methods: To produce IgY against Propionibacterium acnes, laying hens were immunized with P acnes (MTCC No: 1951) and subsequent booster injections were given. The antibodies produced were purified from the egg yolk of immunized chicken using the polyethylene glycol and ammonium sulfate precipitation method and, further, by Diethylaminoethyl (DEAE) cellulose ion-exchange column chromatography. The protein fraction of IgY was isolated from the egg yolk. The separation was rapid, and the success of each step was viewed on Sodium Dodecyl Sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The reactivity of anti-P acnes was evaluated by the Enzyme-linked immunosorbent assay (ELISA) test and the dot-immunoassay. Results: With ELISA, the highest titter of 1:10000 was observed on the 150 th day after vaccination. The results of dot-immunoassay suggested that anti-P acnes IgY developed a brown color as positive reaction, which showed the antigen-antibody binding even after a maximum dilution of 1/500. These results suggest that anti-acne IgY was produced and had strong specific antibody reactivity. Conclusion: The findings indicate that anti-acne IgY is worth utilizing as a preventive agent for acne vulgaris.


Keywords: Acne vulgaris, ELISA, IgY, Propionibacterium acnes, SDS-PAGE


How to cite this article:
Selvan K, Sentila R, Michael A. Generation and characterization of chicken egg yolk antibodies against propionibacterium acnes for the prevention of acne vulgaris. Indian J Dermatol 2012;57:15-9

How to cite this URL:
Selvan K, Sentila R, Michael A. Generation and characterization of chicken egg yolk antibodies against propionibacterium acnes for the prevention of acne vulgaris. Indian J Dermatol [serial online] 2012 [cited 2018 Dec 9];57:15-9. Available from: http://www.e-ijd.org/text.asp?2012/57/1/15/92669



   Introduction Top


The skin is the first line of defense for the body. The skin, which is the body's largest immunological organ, can neutralize many microscopic dangers. Acne vulgaris is one of the most common skin diseases, affecting approximately 50 million people in the world. The disease is important because it has significant morbidity and profound effects on patients' self-esteem. Although acne is a common disease, its pathogenesis is still uncertain. Multiple etiological factors, including follicular hyperkeratinization, sebum, Propionibacterium acnes, and inflammation, are thought to contribute. [1] Perhaps it is the multifactorial etiology of acne that makes it challenging to treat. P acnes remains the most frequently reported bacterial causative agent of acne vulgaris in humans. [2] There is a high unmet clinical need for new and better treatments, given the increase in the antibiotic-resistant strains of P acnes. [3] The treatment of acne is usually complex and may involve the use of topical antibiotics, hormones, isotretinoin, and bacterial vaccines. When skin pores become clogged with excess oil and dead skin cells, an anaerobic environment is created where P acnes can thrive. Limited antibacterial drug choices and the lack of safe and reliable vaccines that can induce protective immunity against acne vulgaris mean that adjunctive therapeutic strategies are needed. The use of specific antibodies as an adjunct to antibacterial drugs can be considered as one such alternative approach.

Immunotherapy can be used in the place of antibiotics which are used traditionally against pathogens. Antibodies also have biochemical properties that make them strikingly effective for local skin care. They neither activate mammalian complement nor interact with mammalian Fc receptors that could mediate inflammatory responses in the skin or in the human body, which makes antibodies very safe and reliable. Antigen-specific antibody has been widely used for immunological analysis in the field of diagnosis as well as in pure scientific research, where the IgY antibodies can be raised against P acnes antigen.


   Materials and Methods Top


Growth and maintenance of Propionibacterium acnes

P acnes (MTCC 1951) was obtained from the Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh (India). It was cultured anaerobically on blood agar containing 0.1% Tween-80 for 48 h at 37°C. A suspension in phosphate-buffered saline (PBS) was prepared from this culture. Two milliliters of this suspension was transferred aseptically to 300 ml of freshly prepared 1% peptone, 0.5% yeast extract, and 0.1% glucose (PYG) medium, supplemented with 0.004 M magnesium, manganese, iron salts, 0.01% cysteine, and 0.1% Tween-80, and incubated for 72 h at 37°C in an anaerobic atmosphere. [4] The log-phase bacterial culture was centrifuged at 5000 g at 4°C for 20 min and the culture supernatant (CS) was removed, filtered, and stored at −20°C. The bacterial pellet was washed three times in chilled 100 ml PBS and finally suspended in 10 ml of the same. The P acnes suspension was incubated at 80°C for 30 min to heat kill the bacteria. The heat-killed P acnes (PA) suspension was adjusted to the concentration of 1 Χ 10 8 CFU/ml and stored at 4°C until used. The molecular weight of the proteins resolved was estimated in comparison to the molecular weight markers.

Animals

Five-month-old white leghorn chickens were obtained from the conventional poultry housing, Namakkal, Tamilnadu, India. The chickens were maintained in an animal facility and housed in disinfected cages under controlled hygienic conditions. The chickens were fed non-medicated food and provided water ad libitum. The institutional committee for animal care approved all the described procedures.

Immunization of chickens

P acnes was mixed and emulsified with Freund's complete adjuvant (FCA) in a 1:1 ratio to form the vaccine. [5] Hens were immunized by injection of 0.5 ml of the vaccine to each of the breast muscles. Two weeks after the initial immunization a booster injection with P acnes antigen emulsified with FCA was given in the same manner, with a second booster dose in the fourth week. Test bleedings were made and checked for anti-acne serum antibodies. Hens were kept in individual cages and their eggs were collected when required, marked for identification, and kept at 4°C until assessment. The egg yolk was isolated, mixed thoroughly with a glass rod, and then stored at 4°C. [6]

Purification of antibodies from egg yolks

The antibodies were extracted from egg yolk using polyethylene glycol (PEG) [7] and precipitated by ammonium sulfate. The partially purified antibody suspension was subjected to dialysis. The IgY was further purified using DEAE cellulose ion-exchange column chromatography. The IgY fraction was then concentrated with polyvinylpyrrolidone (PVP) at room temperature. The concentration of total protein was estimated using Lowry's method [8] and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [9] and the lipid content extracted from the protein purified from eggyolk was determined gravimetrically.

Titration of antibodies by dot-ELISA

Two microliters of the crude cell lysate of P acnes was spotted on nitrocellulose paper and air-dried for 30 min. The NCP was blocked with 1% bovine serum albumin (BSA) in Tween-20 Tris-buffered saline [T/TBS; 0.05% Tween-20, 10 mM Tris, and 150 mM NaCl (pH 7.5)] for 1 h, and subsequently washed three times with T/TBS. The NCP was then kept on wet filter paper in a moist chamber, and undiluted serum (2 μl/spot) was added to the antigen spots. After 1 h of incubation at 37°C, the NCP was washed and incubated with anti-chicken IgG horseradish peroxidase conjugate (1:500) for 1 h at 37°C. Finally, the NCP was developed using TMB solution, with H 2 O 2 (Genei Pvt. Ltd, Bangalore) as a chromogenic substrate. The development of well-defined dark brown spots was considered a positive reaction. [4]

Determination of antibody titer by indirect ELISA

ELISA plates were coated with antigen at a concentration of 1 μg/100 μl/well using coating buffer (0.05 M carbonate bicarbonate buffer; pH 9.6) and incubated overnight at 4°C for binding of antigens. After coating, unbound antigens in the wells were removed by washing thrice with PBS containing 0.05% Tween-20 (PBST). The empty sites were blocked by adding 200 μl per well of 1% BSA in PBS and the plates were incubated at 37°C for 1 h. Plates were subsequently washed with PBST and incubated with 100 μl of either polyclonal chicken antibodies or egg yolk antibodies (IgY) at appropriate dilutions. Control wells had PBST and preimmune sera served as respective controls. Plates were incubated for 1 h at 37°C and subsequently washed with PBST.

For the chicken antibodies, 100 μl of diluted (1:1000) rabbit anti-chicken immunoglobulin coupled to horseradish peroxidase (Genei Pvt. Ltd, Bangalore) was added, and the plates were incubated for 1 h at 37°C. After incubation the plates were washed with PBST and enzyme activity was determined by adding 100 μl of TMB solution with H 2 O 2 (Genei Pvt. Ltd, Bangalore). The plates were allowed to stand at room temperature in the dark for 20 min. The reaction was stopped by adding 50 μl of 4N H 2 SO 4 and the plates were read at 490 nm in an ELISA reader. All samples were tested in triplicate. [10]


   Results Top


Protein profile of P acnes by SDS-PAGE

Protein profile of the crude cell lysate of P acnes was analyzed by SDS-PAGE and was visualized by Coomassie brilliant blue staining. The banding pattern revealed ~25 bands, with molecular weight ranging from 29 to 205 kDa. A standard protein marker was also run in parallel along with the antigen sample [Figure 1].
Figure 1: SDS-polyacrylamide gel electrophoresis of antigen: protein molecular weight (lane 1) and crude cell lysate of Propionibacterium acnes used as an antigen (lane 2)

Click here to view


Generation of antibodies against P acnes in chickens

The white leghorn chickens were immunized intramuscularly with heat-killed P acnes to generate antibodies. Preimmune and hyperimmune sera were collected at specified time intervals during and after the immunization schedule. Similarly, the eggs were collected before and after the immunization. The IgY antibodies were obtained from the chicken egg yolk using the PEG method and precipitated by ammonium sulfate. The precipitate was desalted by dialysis to remove ammonium sulfate and further purified by DEAE cellulose ion-exchange column chromatography. The antibody level was monitored in both chicken serum and egg yolk. The amount of IgY per milliliter of yolk was significantly higher when the chickens received booster injections at regular intervals [Figure 2].
Figure 2: Results of ELISA demonstrating the antibody response in sera and egg yolk from hen immunized with P acnes

Click here to view


Protein profile of purified IgY by SDS-PAGE

The purity of chicken egg yolk antibodies and its molecular weight were determined by SDS-PAGE using 10% gel. [9] The high-molecular-weight protein (180 kDa) showed the purity of IgY. Protein profile of IgY antibodies from different steps of purification were analyzed and visualized by Coomassie brilliant blue staining. A standard protein marker was also run in parallel along with the antibody sample [Figure 3].
Figure 3: SDS-PAGE of IgY: diluted egg yolk (lane 1); IgY fractions purified by PEG precipitation (lane 2); salted-out fraction (lane 3); and column-purified fraction of IgY (lane 4)

Click here to view


Total protein estimation of IgY fractions

Protein content of the water-soluble fraction (WSF) obtained by the PEG method, ammonium sulfate-precipitated fraction, dialyzed fraction, and column-purified antibody fraction were estimated using Folin-Ciocalteau reagent. [8] The IgY concentration in egg yolk was increased during the immunization period and reached 4.0 mg/ml after immunization.

Estimation of total lipids of IgY fractions

The total lipids present in the WSF were extracted [11] using chloroform-methanol (2:1 v/v) and weighed. The PEG method-purified water-soluble fraction had very low amount of lipids - about 4.29 mg/ml, which is 1.5% of the total lipid content in egg yolk. The very low concentration of lipids may be due to the usage of PEG in the purification steps, which makes this method standard for delipidation of yolk.

Estimation of antibody titer by ELISA

The antibody titrations were carried out using Nunc polyvinyl microtiter plates. [10] Indirect antibody capture assay (IACA) showed that antibodies are generated in chickens against the injected P acnes antigens. The optimum titer for the activity of column-purified egg yolk antibodies was found to be 1:10000 dilutions against the P acnes antigen [Figure 4]. The titers were significantly increased after immunization with P acnes, with the highest titer reading of 0.893 at 450 nm was observed.
Figure 4: ELISA demonstrating the antibody response in egg yolk from hen immunized with P acnes: diluted egg yolk (lane 1); IgY fractions purified by PEG precipitation (lane 2); salted-out fraction (lane 3); and column-purified fraction of IgY (lane 4)

Click here to view


Specificity of antibodies by dot-ELISA

The specificity of chicken egg yolk antibodies (IgY) against P acnes was assessed by dot-ELISA. The column-purified egg yolk antibodies bound specifically with the components of antigen. The binding pattern of IgY was visualized by color development. Positive reaction was observed at 1/500 dilution by the development of a brown color. Dot-blot analysis indicated that the IgY antibodies were specific to the P acnes antigen. When preimmune IgY (control) was used, no bands were observed [Figure 5].
Figure 5: Results of dot-ELISA demonstrating the antibody response in egg yolk from hen immunized with P acnes

Click here to view



   Discussion Top


Acne is the most common of all skin diseases. Statistics show that 85% of all people between the ages of 12 and 25 years have some type of acne. The microbiology of acne vulgaris and its immunologic ramifications are the focus of present research that is trying to elucidate the pathogenesis of the inflammatory acne lesion. The microbial flora plays an important part in the pathogenesis of acne; the most likely culprit is P acnes, a strict anaerobe. P acnes is overwhelmingly the predominant microorganism in the normal pilosebaceous follicle as well as in the acne state. It has been divided into two serotypes and five biotypes. Up to 10 [7] viable P acnes have been isolated from a single sebaceous unit. P acnes is not pathogenic by normal standards because there is no correlation between the number of bacteria and the severity and type of acne. Nevertheless, P acnes appear to be the target of oral and topical antibiotics, and the reduction in numbers of P acnes is a just parameter of the therapeutic effectiveness of an antibiotic. [12]

The present investigation was carried out to raise specific polyclonal hyperimmune antibodies in chicken against P acnes. Chickens, as host for the production of egg yolk antibodies against P acnes, show a remarkable ability to rapidly and efficiently generate an abundant supply of high-titer antibody that is able to bind to P acnes. The advantage of chicken IgY is the ease of the production and the large gains of approximately 100 mg IgY per egg yolk. The crude IgY fraction showed only 36% purity, indicating that the rest of the fraction was contaminated with livetins, lipoproteins, and fatty acid molecules, which could reduce the sensitivity of ELISA. The DEAE cellulose column-purified samples were analyzed for their purity, reactivity, and specificity by SDS-PAGE and immunoblotting techniques, respectively. In SDS-PAGE, high-molecular-weight proteins (180 kDa) were detected by using Coomassie brilliant blue stain. This shows the purity of the egg yolk antibodies. Dot assay studies of P acnes (IgY) showed the specific binding of the P acnes with antibodies. A peak titer of 1/500 dilution was observed. The blot treated with normal antibodies (negative control) did not show any positive reaction. Immunoblots therefore confirmed the presence of specific antibody against P acnes.

The increase in the specific antibody concentration of egg yolks from immunized hens over the course of the immunization period was monitored. The antibodies against P acnes first appeared in serum on the seventh day after starting the immunization schedule. The antibodies were detected in egg yolk after a week. The concentration of antibodies increased in the egg yolk with subsequent booster doses, with an average yield of 80 mg per egg yolk on the 150 th day after start of immunization. The activity of egg yolk antibodies, determined by ELISA, showed the presence of P acnes-specific antibodies. Normal antibodies obtained from unimmunized chickens did not show any activity against any P acnes antigen. ELISA is a sensitive technique to find out the antibody titers during the immunization period and also to determine antibody activity during different purification processes. With ELISA, the highest titer of 1:10000 was observed during the 150 th day of observation. The chicken egg yolk antibodies are present in the egg for up to 100 days after the immunization. [13] There is a report showing significant correlation between the ELISA result and the anti-acne potency tested in vitro against P acnes. [14] Good correlation between ELISA and the purified fractions of the antigen used for ELISA has been reported earlier. [4]

Immunoglobulins are the powerful antibodies produced in healthy humans and other animals to fight infections and prevent disease. A laying hen can produce approximately 300 eggs annually and the volume of one egg yolk is approximately 15 ml. Each egg contains approximately 3-5 mg IgY per ml of yolk, which means a total 70-100 mg IgY antibody per egg, depending upon the size of the egg. This could supply close to 100 g of antibody per hen per year. A single egg contains as much as an average bleed from a rabbit. In addition, egg collection is simple and noninvasive. Chicken require only a 1-month immunization regimen to produce high titers of polyclonal antibody, and they are very useful for producing antibodies against conserved mammalian protein. [15] IgY technology should be considered as a good alternative for acne therapy. The acne sufferer may have finally found an effective cure in this anti-acne antibody.

 
   References Top

1.Thiboutot DM, Gollnick HP. Treatment considerations for inflammatory acne: Clinical evidence for adapalene 0.1% in combination therapies. J Drugs Dermatol 2006;5:785-94.  Back to cited text no. 1
[PUBMED]    
2.Perry AL, Lambert PA. Propionibacterium acnes. Lett Appl Microbiol 2006;42:185-8.  Back to cited text no. 2
[PUBMED]  [FULLTEXT]  
3.Kim J. Acne vaccines: Therapeutic options for the treatment of acne vulgaris?. J Invest Dermatol 2008;128:2353-4.  Back to cited text no. 3
[PUBMED]  [FULLTEXT]  
4.Basal E, Jain A, Kaushal, GP. Antibody response to crude cell lysate of Propionibacterium acnes and induction of pro-inflammatory cytokines in patients with acne and normal healthy subjects. J Microbiol 2004;42:117-25.  Back to cited text no. 4
    
5.Herbert WJ. Methods for the preparation of water-in-oil and multiple, emulsions for use as antigen adjuvants; and notes on their use in imminization procedures. In: Weir DM, editor. Handbook of Experimental Immunology. 3 rd ed. Oxford: Blackwell Scientific Publishers; 1967. p. 1207-14.  Back to cited text no. 5
    
6.Woolley JA, Landon B. Acne comparison of antibody production to human interleukin-6 (IL-6) by sheep and chickens. J Immunol Methods 1995;178:253-65.  Back to cited text no. 6
    
7.Polson A, Von Wechmar MB, van Regenmortel MH. Isolation of viral IgY antibodies from yolks of immunized hens. Immunol Commun 1980;9:475-93.  Back to cited text no. 7
[PUBMED]    
8.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75.  Back to cited text no. 8
[PUBMED]  [FULLTEXT]  
9.Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5.  Back to cited text no. 9
[PUBMED]    
10.Voller A, Bartlett DE, Bidwell DE, Clark MF, Adams AN. The detection of viruses by enzyme-linked immunosorbent assay (ELISA). J Gen Virol 1976;33:165-7.  Back to cited text no. 10
    
11.Folch J, Lees M, Stanley GH. A simple method for the isolation and purification of total lipids from animal tissue. J Biol Chem 1957;226:497-509.  Back to cited text no. 11
[PUBMED]  [FULLTEXT]  
12.Burkhart G, Burkhart N, Lehmann F. Acne: A review of immunologic and microbiologic factors. Postgrad Med J 1999;75:328-31.  Back to cited text no. 12
    
13.Devi CM, Bai MV, Lal AV, Umashankar PR, Krishnan LK. An improved method for isolation of anti-viper venom antibodies from chicken egg yolk. J Biochem Biophys Methods 2002;51:129-38.  Back to cited text no. 13
[PUBMED]  [FULLTEXT]  
14.Jung YS, Matasumoto S, Yamashita M, Tomimatsu K, Teruya K, Katakura Y, et al. Propionibacterium acnes acts as an adjuvant in in vitro immunization of human peripheral blood mononuclear cells. Biotechnol Biochem 2007;71:1963-9.  Back to cited text no. 14
    
15.IgY lifesciences.com [Internet]. A new beginning boosting the immune system The key to better health. Available from: http://igylifesciences.com/technology/ [Last accessed on 2011 June 07].  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

Top
Print this article  Email this article
 
 
  Search
 
  
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Article in PDF (831 KB)
    Citation Manager
    Access Statistics
    Reader Comments
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
    References
    Article Figures

 Article Access Statistics
    Viewed4145    
    Printed155    
    Emailed0    
    PDF Downloaded78    
    Comments [Add]    

Recommend this journal