isolation of different classes of immunoglobulins from ...isolation of different classes of...

Egypt. J. Comp. Path. & Clinic. Path. Vol. 21 No. 4 (December) 2008; 246 - 257

246

Referred byReferred by Prof. Dr. Magdi Ahmed Ghoniem Professor of Biochemistry, Fac. Vet. Med.

Cairo University Prof. Dr. Wahied A. Mossa Professor of Parasitology, Fac. Vet. Med.,

Cairo University.

Isolation of different classes of immunoglobulins from blood and co-lostrum of buffaloes

By Ahmed, A. Ramadan*; Hany, M. Mohamed**; Mohamed, M. El-

loly*** and Manal, Bahaa Eldeen** * Animal Health Research Institute

**Animal Reproduction Research Institute ***National Research Center

SUMMARY

P ooled blood and colostrum samples were collected from a group of healthy female buffaloes immediately after parturi-

tion. Blood samples were used to separate IgG while colostrum was used to separate IgA and IgM. Using low concentrations of ammonium sulphate, high molecular weight proteins were removed from samples, then reaching to final concentration of 50% ammo-nium sulphate, immunoglobulin G (IgG) was precipitated and su-pernatant was chromatographed using Sepharose CL 4B column to separate immunoglobulins M and A (IgM and IgA) based on their relatively distant molecular weights. Purity of the isolated IgG was confirmed by Electrophoretic migration pattern, under non-reducing conditions, and by High Performance Liquid Chromatog-raphy for IgG, IgA, and IgM.

INTRODUCTION

T he use of serum antibodies for most applications requires

some degree of purity. It is gener-ally accepted that purities of 80% are required for many common ap-plications of these antibodies, for example as a coating reagent or label in an immunoassay or for the construction of an affinity column.

To achieve this degree of purity a variety of chromatographic and precipitation techniques have been developed for this purpose Tho-mas et al. (2002). The preferred method often involves ammonium sulphate precipitation (Gathumbi et al., 2001) and some forms of column chromatography such as i o n -e x c h a n g e o n a D E A E


247

(Milstein and Cuello, 1983), hy-droxyapatite chromatography (Larry et al., 1985), specific im-munoaffinity columns (Calabozo et al., 2001), or affinity chroma-tography on protein A (Jennifer et al., 2001), or G (David et al., 2001).

The growing importance of purified different classes of immu-noglobulins in therapeutic, diag-nostic, nutritional and industrial areas necessitates the isolation and purification of immunoglobulins from animals. Moreover, there are no commercially available immu-noglobulins isolated from buffa-loes; instead researchers depend on anti-cattle products which creates uncompromisable situation when we seek sensitivity and accuracy in diagnosis of diseases. Production of buffaloes IgG, IgM, and IgA will serve three main goals: a) help in sensitive and accurate diagnosis of buffalo's infectious diseases b) low-priced immunological tool that can be commercialize in Egypt and the developing countries where large populations of buffa-loes, c) rapid therapeutic tool for young calves.

MATERIAL AND METHODS

B lood and colostrum sam-ples: Healthy buffaloes (at

the Animal Reproduction Research Institute farm) served as colostrum and blood donors. Blood samples were collected in clean containers

and centrifuged at 2500 g for 30 minutes to separate serum which then pooled and kept at -40° C un-til IgG was depleted from it. Co-lostrum samples from different animals were pooled together and centrifuged at 7000 g to separate milk serum and to remove debris and clots and also kept at -40°C until IgA and IgM were depleted from it. Separation of immunoglobulin G: Separation was done according to Mohanty and Elazhary (1989) after slight modifications; precipi-tation was done on two steps. First step of precipitation using ammo-nium sulphate aimed to remove large size proteins and albumin. Blood or colostrum was diluted 3-flods with phosphate buffer saline, pH 7.2 then 0.5 volume of satu-rated ammonium sulphate solution (4.1 M solution pH 7-7.2) was added slowly with constant stirring for 30 minutes. The mixture was kept at 4° C overnight, then centri-fuged at 1000g for 30 minutes and pellet was discarded since it con-tained undesired large MW pro-teins. Supernatant was then col-lected and its amount was meas-ured, and another 0.5 volume of saturated ammonium sulphate so-lution was added slowly with con-stant stirring. The mixture was moved to refrigerator at 4° C over-night then centrifuged at 1000g for 30 minutes to separate pellet from supernatant. The pellet, containing


248

IgG, was washed once with 50% saturated ammonium sulphate so-lution and dissolved in minimum amount of PBS. In case of colos-trum, supernatant was saved to separate IgM and IgA. Chromatographic procedures for separation of IgM and IgA: Sepharose CL 4B gel was used to separate IgM and IgA from the pooled supernatant. A column with 50 ml X 1.5 ml dimensions was packed with Sepharose CL 4B gel (exclusion limit is 20 x 106 kDa) and standard procedures were taken to check the packing of the column. Phosphate buffer saline (0.15 M), pH 7.2-7.4, was used as a mobile phase through out the fractionation process. Small ali-quots (5-7 ml) of the pooled mix-ture was applied on the top of the gel and allowed to enter the gel pores then PBS was pumped in the column at 1 ml/min. IgM fractions appeared first followed by IgA fractions. Tubes representing each peak were pooled together. Protein concentrations were measured in each fraction, and saved at -70° C until assayed by RP-HPLC. Electrophoresis: Standard electro-phoresis procedures according to Lammeli (1970) were done to check the purity of the isolated IgG. Samples were done under non-reducing conditions i.e. no SDS or mercaptoethanol in the

sample buffer, running buffer, or in running gel. High Performance Liquid Chro-matography: A reverse phase High Performance Liquid Chroma-tography (RP-HPLC) system con-sists of 4-head gradient pump, UV monitor, and specific software (WinChrom3®) was used (CBG-England) to identify the purity of the isolated immunoglobulins in comparison to standards (The binding site, Birmingham, U.K.). A strong anion exchange column (HyperREZ XP polymeric SAX, 50 x 4.6 mm ID, 5 µ column) was used to identify the purity of the isolated subclasses of immu-noglobulins. The mobile phase used was as follow: solvent A: 0.1% trifluoroacetic acid (TFA) in 0.5 M NaCl + solvent B: 0.1% TFA in 80% acetonitrile. All chemicals used were HPLC-grade. A gradient flow program of 0-100% of solvent B in 30 minutes run time was set (25 minutes ac-tual run time and 5 minutes for washing the column). A volume of 10 µl was injected in the column using an HPLC syringe from each fraction of the immunoglobulins separately.

RESULTS

L iquid chromatographic separation of IgM and IgA

on Sepharose CL 4B: Figure 1 represents the elution

pattern of IgA and IgM fractions


249

from the sepharose CL 4B packed column. IgM started to elute from the column at fraction number 10 and ended at fraction number 24. IgA started at fraction number 40 and ended at fraction 56. Protein concentration was plotted on the Y axis against its representative frac-tion number on the X axis. Frac-tions of each immunoglobulin pooled together.

Electrophoretic pattern of IgG Figure 2 shows the purity of the isolated IgG from buffalo se-rum. The main purpose of this electrophoretic step was to check the purity of the isolated immu-noglobulin fraction before it was

run on RP-HPLC. RP-HPLC chromatographic pat-tern of IgG, IgA, and IgM Integrity and purity of IgG, IgA, and IgM was checked by using the anion exchange HyperREZ XP polymeric SAX column in associa-tion with gradient solvents used during the 25 minutes actual run time. Figures 4, 5, and 6 represent the elution patterns of IgG, IgA, and IgM from the anion exchange col-umn separately. The retention time of IgG was 1.258 minutes, for IgA was 15.433 minutes, and for IgM was 19.342 minutes.

Figure (1): Chromatogram of IgA and IgM elution pattern from the Sepharose CL 4B packed column (50 x 1.5 cm) using 0.15 M PBS (7.2-7.4 pH) as mobile phase.


250

Figure (2): Electrophoretic pattern of IgG isolated from blood of buffaloes after its precipitation using ammonium sulphate solution.

Figure (3): Diagram shows the elution of the IgG, IgA, and IgM from the anion exchange HyperREZ XP polymeric SAX column in relation to change in gradients of the solvents (A and B) using 0.1% trifluoroacetic acid (TFA) in 0.5 M NaCl (solvent A) and 0.1% TFA in 80% acetonitrile (solvent B).


251

Figure (4) Chromatogram represents the separation of IgG from colos-trum of buffaloes using anion exchange HyperREZ XP poly-meric SAX column.

Figure (5) Chromatogram represents the separation of IgA from colostrum of buffaloes using anion exchange HyperREZ XP polymeric SAX column.


252

Figure (6) Chromatogram represents the separation of IgM from colostrum of buffaloes using anion exchange HyperREZ XP polymeric SAX column. DISCUSSION

B uffaloes are widely distrib-uted in the world mainly in

the third world regions. Buffaloes are known to have their unique physiological, reproductive, and even infectious traits. Although cows and buffaloes are closely re-lated genetically, there is some dif-ference between them especially in immune response genes (Ir genes). Singh et al. (2009) noticed that during a serodiagnosis of babesia bigemina from endemic zones in India, the antibodies found in 56.11% of cows and 23.33% in buffaloes. Also, Ravindran et al. (2008) reported lower incidence of Linguatula serrata Frohlich, an endoparasite of ruminants, in buf-

faloes (8%) than in cows (19%). Khalifa et al. (2008) reported lower incidence of infection with three Sarcocystis species in Sohag, Egypt, in buffaloes (28%) com-pared with cows (84%). Moreover, Maroudam et al. (2008) noticed a delayed-onset and sub-clinical signs of experimental foot and mouth disease type O in buffaloes than in cows. There may be anti-genic similarity in immunoglobu-lins of cows and buffaloes but dif-ference in some epitops exists. These differences can decrease the sensitivity and accuracy of diagno-sis buffalo's infections using cows' products. Regarding the gene ex-pression of a single cytokine, Min-gala et al. (2007) concluded that a


253

small difference in the cytokine structures originated from genetic differences between swamp buf-falo compared with other bubaline breeds, might be the reason behind the existence of disease resistance in swamp buffalo. Molina and Skerratt (2005) reported differ-ences in the T cell response be-tween cattle and buffaloes against Fasciola gigantica experimental infection. They attributed the variation between the 2 species to their differences in resistance and resilience to infection with F. gi-gantica. Also, Helmy (2000) re-ported the lowest incidence of in-fection with Borrelia spirochetes in buffaloes than other species tested (man, camel, sheep, goat, and cow) during a one-year survey study. Finally, Zaghawa (1998) reported the prevalence of lowest neutraliz-ing antibodies against Bovine Vi-ral Diarrhoea (BVD) in buffalo compared with cattle, sheep, camel, and goat in a surveillance study.

It seems that buffalo is resis-tant to some infectious diseases or have minor clinical signs in com-parison with other animals spe-cially cows. There is a great body of evidences pointing to the varia-tion between cows and buffalo re-garding their differences in im-mune response against viral, para-sitic and bacterial infections. This variation increases our need for specific immune kits prepared

from buffalo immunoglobulins to study and diagnose buffalo infec-tious diseases. It is well known that there are no specific immune kits for buffalo per se. For exam-ple, diagnose of foot and mouth disease or rift valley fever in buf-faloes depends on the use of kits that are specific for cows. There is a shortage in anti-buffaloes anti-bodies in the international market. Diagnosis of buffalo diseases re-quires specific, sensitive, and ac-curate tools to evaluate their exact immune response as a first step to-ward solving their infectious prob-lems. Absence of specific immune kits for buffalo hinders efforts of controlling diseases affecting buf-faloes in Egypt.

Separation of immunoglobu-lins from blood and colostrum could be achieved using several methods and techniques. Separa-tion of IgG from colostrum and blood has been successfully ac-complished by precipitation using saturated ammonium sulphate so-lution (Harlow and Lane, 1988 and Ajaib and Sat Pal, 1993), or by combining caprylic acid treat-ment and ammonium sulphate pre-cipitation (Mohanty and Elaz-hary 1989). In the current study, ammo-nium sulphate at 50% concentra-tion successfully precipitated IgG from blood and colostrum. Immu-noglobulins in solutions form hy-drogen bond with water through


254

their exposed polar and ionic groups. Enough concentration of small and highly charged ions such as ammonium sulphate [(NH4)2SO4] when added to protein solu-tion compete with proteins for binding to water resulting in re-moval of water molecules from the proteins, decreases their solubility, and ends with their precipitation. This process can be reversed con-veniently by adding water to the precipitated proteins. Many factors affect the concentration at which a particular protein will precipitate (Harlow and Lane, 1988) such as number and position of polar groups of the protein, molecular weight of the protein, the pH of the solution, and the temperature at which the precipitation process oc-curs. Preliminary experiments in this study showed that the optimal concentration of ammonium sul-phate at which buffalo blood and colostrum will precipitate IgG is between 45% and 50%. Also, it is worthwhile to use 2 steps for pre-cipitation of IgG to eliminate un-desired large aggregates of pro-teins. The first precipitation step was successful in removing large molecular weight protein aggre-gates and any proteins that may precipitate with low concentrations of ammonium sulphate. The sec-ond precipitation step reached the final concentration of ammonium sulphate to 50% which was quite enough to precipitate IgG.

Sepharose CL 4B gel was then employed to separate IgA (≈ 170 kDa) from IgM (≈ 900 kDa) since both immunoglobulins are distant from each other (Klaus, 2001). The supernatant remained after the second step of precipita-tion of IgG from colostrum con-tained IgA and IgM. Sepharose CL 4B with exclusion limit of 20 x 106 kDa was optimal for separation of the two immunoglobulins from each other based on their molecu-lar sizes. The mobile phase con-sisted of phosphate buffer saline (PBS) 0.15 M, (pH 7.2-7.4). IgM eluted first from the column fol-lowed by IgA. Electrophoretic pro-cedures done under non-reducing conditions (without mercaptoetha-nol and SDS in the sample buffer), to keep the integrity of the immu-noglobulins, clearly indicated the purity of each of them. Also the chromatographic analysis using HPLC confirmed the purity of the isolated immunoglobulin classes.

I n conclusion, this study aimed to isolate polyclonal immu-

noglobulins of buffaloes in pure form from a rich sources, blood and colostrum. The used protocol for separation of IgG, IgA, and IgM from serum and colostrum re-vealed high purity. It is our inten-tion to use these purified immu-noglobulins in diagnostic purposes after conjugation with horseradish peroxidase or alkaline phos-phatase.


255

REFERENCES

Ajaib Singh and Sat Pal Ahujas (1993): “Individual variation in the composition of colos-trum and absorption of co-lostral antibodies by the pre-colostral buffalo calf”. J Dairy Sci. 76: 1148-1156.

Calabozo, O.; Duffort, J.; Car-pizo, A.; Barber D. and Polo F. (2001): “Monoclonal antibodies against the major allergen of Plantago lanceo-lata pollen, Pla l 1: affinity chromatography purification of the allergen and develop-ment of an ELISA method for Pla l 1 measurement”. Al-lergy, 56 (5): 429-435B.

David M.; White, Mark A.; Jen-sen, Xin Shi, Zhi-xaing Qu, and Barry G. (2001): “Des-ign and expression of poly-meric immunoglobulin fusion proteins: A strategy for tar-geting low-affinity Fcγ recep-tors protein expression and purification:. 21 (3): 446-455.

Gathumbi, J.; Usleber, E. and MaÈrtlbauer, E. (2001): “Production of ultrasensitive antibodies against aflatoxin B1”. Letters in Applied Mi-crobiology. 32(5):349-351.

Harlow, Ed. and Lane, David (1988): “Antibodies: a labo-ratory manual”. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY (USA).

Helmy, N. (2000): “Seasonal abu-

ndance of Ornithodoros (O.) savignyi and prevalence of infection with Borrelia spiro-chetes in Egypt”. J. Egypt. Soc. Parasitol., 30 (2): 607-619.

Jennifer A. Waters C. O'Neill, Ali A., Robert G., Peter K., and John M. (2001) : “Purification of woodchuck hepatitis surface antigen us-ing a monoclonal antibody raised against the antigen”. Journal of Virological Meth-ods. 93 (1-2): 97-103.

Khalifa, R.; El-Nadi, N.; Sayed, F. and Omran, E. J. (2008): “Comparative morphological studies on three Sarcocystis species in Sohag, Egypt”. Egypt. Soc. Parasitol., 38(2):599-608.

Klaus, D.E. (2001): “Immunol-ogy: Understanding the Im-mune System”. Academic Press. 1st ed. pp 65.

Lammali, U.K. (1970): “Cleavage of structural proteins during the assembly of the head of bacteriophage T4”. Nature. 227(5259): 680. 685

Larry H., Martin V. and Hector J. (1985): “One-step purifica-tion of mouse monoclonal antibodies from ascites fluid by hydroxylapatite chroma-tography”. Journal of Immu-nological Methods. 76(1): 157-169.

Maroudam, V.; Nagendrakum-


256

ar, S.; Madhanmohan, M.; Santhakumar, P.; Thiaga-rajan, D. and Srinivasan, V. (2008): “Experimental trans-mission of foot-and-mouth disease among Indian buffalo (Bubalus bubalis) and from buffalo to cattle”. J. Comp. Pathol., 139 (2-3): 81-85.

Milstein, A. and Cuello, C. (1989): “Hybrid hybridomas and their use in immunohisto-chemistry. The improved lytic function and in vivo ef-ficacy of monovalent mono-clonal CD3 antibodies.” European Journal of Immu-nology, 19(2): 381-388.

Mingala, C.; Odbileg, R.; Konn-ai, S.; Ohashi, K. and Onu-ma, M. (2007): “Molecular cloning, sequencing and phy-logenetic analysis of inflam-matory cytokines of swamp type buffalo contrasting with other bubaline breeds”. Comp. Immunol. Microbiol. Infect. Dis., 30 (2): 119-131.

Mohanty, J. and Elazhary, Y. (1989): “Purification of IgG from serum with caprylic acid and ammonium sulphate precipitation is not superior to ammonium sulphate pre-cipitation alone”. Comp. Im-munol. Microbiol. Infect. Dis., 12(4):153-160.

Molina, E. and Skerratt, L. (2005): “Cellular and humo-ral responses in liver of cattle

and buffaloes infected with a single dose of Fasciola gi-gantica.” Vet. Parasitol., 15; 131 (1-2): 157-163.

Ravindran, R.; Lakshmanan, B.; Ravishankar, C. and Subr-amanian, H. (2008): “Pre-valence of Linguatula serrata in domestic ruminants in South India”. Southeast Asian J. Trop. Med. Public Health, 39 (5): 808-812.

Singh, H.; Mishra, A.; Rao, J. and Tewari, A. (2009): “Comparison of indirect fluo-rescent antibody test (IFAT) and slide enzyme linked im-munosorbent assay (SELISA) for diagnosis of Babesia bigemina infection in bo-vines”. Trop. Anim. Health Prod., 41(2):153-159.

Thomas, T.; Shave, E.; Bate, I.; Gee, S.; Franklin, S. and Rylatt, D. (2002): “Prepara-tive electrophoresis: a general method for the purification of polyclonal antibodies”. Jour-nal of Chromatography A, 944: 161–168.

Zaghawa, A. (1998): “Prevalence of antibodies to bovine viral diarrhoea virus and/or border disease virus in domestic ru-minants”. Zentralbl Veteri-narmed B., 45(6):345-351.

isolation of different classes of immunoglobulins from ...isolation of different classes of...

Documents