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Bioehem Biophys

Indian Journal of Biochemistry & BiophysicsVol. 41, December 2004, pp. 299-304 .

Physico-chemical and antigenic characterization of unconventional heavy chainantibodies of Indian desert camel (Camelus dromedarius L.)

Sharvan Sehrawat and Ajit Singh*Immunology Section, Department of Veterinary Microbiology, Ch. Charan Singh Haryana Agricultural University,

Hisar 125004, India

Received 20 April 2004; revised 14 October 2:004

Heavy chain antibodies (HCAbs) of IgG2 and IgG3 subtypes were purified from the sera of Indian desert camel(Camelus dromedarius L.) by ammonium sulphate precipitation, followed by ion-exchange chromatography onOEAE-cellulose and affinity chromatography on protein A-sepharose and protein G-sepharose, and characterized bySOS-polyacrylamide gel electrophoresis, agar gel immunodiffusion (AGIO), counter-immunoelectrophoresis (CIEP),immunoelectrophoresis (IEP), ELISA and immunoblotting. IgG2 and IgG3 were found to have molecular mass 46.77 kOaand 43.65 kOa, respectively by SOS-PAGE under reducing conditions. They migrated in B-region in IEP and could bedetected in CIEP, because of being more negatively charged and smaller size. Anti-camel IgG3 cross-reacted in AGIO,ELISA and immunoblotting with IgGs of pig and ruminants (cattle, buffalo, sheep and goat), but not with immunoglobulinsfrom horse, dog, guinea pigs, mice, fish, poultry and human. The present findings suggest close antigenic relationship ofcamels with pigs and ruminants.

Keywords: Indian camel, heavy chain antibodies, purification, electrophoresis, antigenic relationship, immunoblots,Camelus dromedarius L.

IPC Code: C 07 K 16/06

Camel is uniquely well adapted in the extremeclimatic conditions of the tropical regions of Africa,Asia and Australia. Indian camel population is largelyconfined to the dry northern regions, comprisingRajasthan, Gujarat, Haryana and Punjab'. Aslivestock, it is an important factor in incomegeneration for the marginal farmers and landlesslabourers. The overall functioning of camelid immunesystem is far less understood than that of otherdomestic animal species. Isolation and characteri-zation of camel immunoglobulins (Izs) have,h b . d ,2-4 ~ . however, een carne OUt . rvecenr surge In t estudy of camel Igs is, however, due to the finding of

naturally occurring biologically-active anti-bodies ofIgG isotypes that are devoid of light chair; 2,iiJ CHI

domain in their structure and designated as heavychain antibodies (HCAbs)5. HCAbs of variousmembers of the family Camelidae have recently beenreported from other parts of the world'l". Theirusefulness as reagents in diagnostics, sero therapy,cancer therapy and biocatalysis has recently beenspeculated",

Although molecular features of HCAbs of camelids;idVC oeen understood by nucleotide and proteinsequence analysis", but the physico-chemical andantigenic chara ~::,r;7ati()n h~' ~prnl()gy is stillincomplete. Only a few preliminary studies itav': beenconducted on characterization of Igs from the Indiandesert camel'":", and isolation and characterization ofHCAbs has not been reported, so far. Therefore, thepresent study was undertaken to isolate and

. characterize HCAbs (IgG2 and IgG3) from the sera ofIndian desert camel (Camelus dromedarius L.).Antibodies were purified by using ammoniumsulphate precipitation, followed by ion-exchangechromatography and protein A- and proteinG-Sepharose affinity chromatography, and charac-

*Corresponding authorTel:.,9896176392 (Mob.); Fax: 01662-34613

vlf-ffiail: adjitprinik Gmsn.com or budhadjit59@rediffmail.comAbbreviations: AGIO, agar gel immunodiffusion; BSA, bovineserum albumin; CIEP, counter-immuno-electrophoresis; OEAE,diethylaminoethyl; ELISA, enzyme-linked immunosorbent assay;HCAbs, heavy chain antibodies; HEEO, high electroendosmosis;HIS, hyperimmune serum; HRPO, ~orseradish peroxidase; IEP,immunoelectrophoresis; Ig, Immunoglobulin; LC, light chain;PBST, phosphate buffered saline- Tween20; PEG, polyethyleneglycol; PVOF, polyvinyl difluoride; SOS-PAGE, sodium dodecylsulphate-polyacrylamide gel electrophoresis.

300 INDIAN J. BIOCHEM. BIOPHYS., VOL. 41, DECEMBER 2004

terized by serological techniques, particularly todetermine their electrophoretic mobility by immuno-electrophoresis (IEP) , and counter-immunoelectro-phoresis (CIEP). Their antigenic relatedness with Igsof several other animal species was studied by agargel immunodiffusion (AGID), ELISA and immuno-blotting. The immunoblotting data on antigenic rela-tedness of camel IgG3 with Igs from various speciesacross different phyla has not been reported earlier.

Materials and MethodsImmunological reagents

Rabbit anti-camel whole serum and rabbit anti-camel IgG3 were raised as per the standardprocedure I 2. Anti-rabbit IgG affinity purifiedhorseradish peroxidase (HRPO) was purchased fromSigma Chemical Co., USA.

Collection of seraSera were collected from 50-100 ml of blood

collected aseptically from jugular vein of each of 20camels. Each serum sample was heated at 56°C for30 min to inactivate the complement and stored inaliquots at -20°C until further use.

Purification of camel IgsFor the purification of IgG SUbtypes, method of

Delvis13, with some modifications was followed.

Briefly, camel IgG subtypes i.e., IgG 1, IgG2 andIgG3 were purified using (NH4hS04 precipitation at .40% saturation, followed by ion-exchange chromato-graphy on DEAE-cellulose (Genei, India). The elutionof bound Igs was achieved using a linear gradient of0-300 mM NaCI and fractions of 3 ml were collected.Protein in various fractions was determined bytaking absorbance at 280 nm in Biophotometer®(Eppendorf, Germany).

Purification of IgG2 and IgG3Purification of HCAbs (IgG2 and IgG3) from

serum, (NH4hS04 precipitated Igs and ion-exchangepurified fractions was also done by protein A and/orprotein G-Sepharose chromatography as describedwith some modifications. Briefly, purification of IgG2and IgG3 directly from serum was also done byensuring their selective binding on protein A. For this,protein A was pre-equilibrated with 20 mM phosphatebuffer (PH 7.0). The eluted Igs were diluted withdistilled water and passed on protein G pre-equilibrated with 0.1 M acetate buffer (PH 5.0). Theelution was achieved with O.l.M citrate buffer(PH 3.0).

The (NH4)2S04 precipitated Igs were passed onprotein G and elution was achieved using 0.1 Macetate buffered 0.15 M NaCl, pH 4.5, 0.1 M acetatebuffered 0.15 M NaC!, pH 3.5 and 0.1 M glycinebuffer, pH 2.7. Unadsorbed fraction of Igs on proteinG-Sepharose was passed on protein A-Sepharosecolumn and eluted by 0.1 M acetate buffered 0.15M NaCl, pH 4.5 and 0.15 M glycine buffer, pH 2.7.

Ion-exchange purified fractions containing all IgGSUbtypes and IgG3 only were pooled separately,dialysed and concentrated against 20 mM phosphatebuffer, pH 7.0 and 20% PEG and the purification ofHCAbs was done from ion-exchange purifiedfractions containing all IgG SUbtypes as describedabove. Fraction containing IgG3 was passed onprotein G and elution was achieved using 0.1 Macetate buffered 0.15 M NaCl, pH 3.5. The pH in allfractions was neutralized immediately with 1 Mcarbonate-bicarbonate buffer, pH 9.8.

Protein in various Ig fractions was estimated bymicroplate method of Lowry" and direct spectro-photometric method based on A2so nm measurementsusing Biophotometer® (Eppen- dorf, Germany). BSAwas used as a standard for both the methods.

Characterization of HCAbsMolecular mass

The molecular masses of IgG2 and IgG3 weredetermined by SOS-PAGE using discontinuous buffersystem of Laemmli 15. HCAbs were resolved in 10-15% gradient polyacrylamide gel.

Electrophoretic mobilityElectrophoretic mobility was determined by immu-

noelectrophoresis (IEP) and confirmed by counter-immunoelectrophoresis (CIEP) using anti-camelwhole serum and anti-camel IgG3 as described'<.

Antigenic relatedness with other animal speciesAGIO, ELiSA and immunoblotting were used to

demonstrate the antigenic relatedness of camel Igswith other animal species. AGIO was carried out asdescribed12

. Briefly, in 1% agarose gel on a standardglass slide, the central well contained anti-camel IgG3and peripheral wells contained affinity purified IgGfrom cattle, buffalo, sheep, goat and pig and wholeserum from different animal species viz., camel,cattle, buffalo, sheep, goat, pig, horse, dog, mouse,poultry, guinea pig, fish and human. Precipitationlines were allowed to develop during next 36 hr ofincubation at room temperature and the results wererecorded.

ssed on0.1 Macetateglycineprotein

epharose~ed 0.15H 2.7.~all IgGrparately,ihosphatecation ofpurified

describedassed on

0.1 M~H in alllth 1 M

y). BSA\

~G3 wereDUS buffered in 10-

by immu-counter-

nti-cameled".

e used tocamel Igsied out as

standardellgG3

rified IgGnd whole., camel,

g, mouse,·ecipitationt 36 hr ofsults were

SEHRAWAT AND SINGH: CHARACTERIZATION OF HEAVY CHAIN ANTIBODIES OF INDIAN CAMEL 301

For determining the antigenic cross-reactivity ofcamel Igs by blocking ELISA, the method describedpreviously" was used. Serial log dilutions (rangingfrom 1 to 6) of anti-camel IgG3 hyperimmune serum(HIS) were made in duplicate. The maximum dis-placement of A450was determined by blocking eachdilution of HIS with 1/lOth cone. of homologousantigen in 1I5th vol. Thereafter, using the dilution ofHIS that gave maximum per cent displacement withhomologous antigen, cross-reactivity was determinedin terms of per cent displacement of A450with a fixedcone. of heterologous antigens i.e., affinity purifiedIgG of cattle, goat and pig.

Immunoblotting was done essentially as reportedearlier". Briefly, sera samples from different speciesof animals viz., camel, cattle, buffalo, sheep, goat,pig, horse, dog, mouse, poultry, guinea pig, fish andhuman in dilution of 1:50 were treated 1:1 with 2xLaemmli's sample buffer in a boiling water bath forabout 5 min, cooled, loaded in the gel of 20 wells andresolved in 12.5% gel. After the run was complete,electroblotting was done. The blocking of polyvinyldifluoride (PVDF) membrane was done in phosphatebuffered saline- 0.05% Tween 20- 3% bovine serumalbumin (PBST-BSA) for about 1 hr at 25°C.Thereafter, the PVDF membrane was washed withPBST 3 times 'for 3 min each and was treated withrabbit anti-camel IgG3 (1: 10,000) for 2 hr at 25°C.After incubation, 3 washings each of 3 min weregiven in PBST. Then, goat anti-rabbit IgG-HRPOconjugate 1:5000 in PBST was used for 1·hr at 25°C.Washing of membrane was done for 5-times of 3 mineach and 20 ul of 30% H202 was added in 100 mlsubstrate solution, which was then immediatelypoured in a tray containing PVDF membrane. Themembrane was developed at room temperature withagitation until bands were suitably dark and thereaction was stopped with distilled water.

Results

Purification of camel IgG subtypes

By ion-exchange chromatographyThe IgG started eluting at 220 mM NaCI and

extended up to 280 mM NaCl. The major peakfraction was obtained at 250 mM NaCl cone. In initialfractions (no. 33-37), all IgG SUbtypes i.e., IgG 1,IgG2, IgG3 were eluted. Estimated M, of IgG 1 heavyand light chains were 52.48 kDa and 25 kDa,respectively and of heavy ,ch~ns of IgG2 and IgG3were 46.77 kDa and 43.65 kDa, respectively. In later

fractions (no. 38-42), only IgG3 was eluted at 260mM NaCI and no bands corresponding to 52.48 kDa,46.77 kDa and 25 kDa were visible in Coomassiestained gel. In still later fractions (no. 43-45), bandsof IgG3 along with a minor band of albumin (67 kDa)were present while in last fractions (46-52), onlyalbumin was present. The pattern of elution indicatedthat IgG3 molecules had considerable chargeheterogeneity.

By protein A and protein G-Sepharose affinity chromatographyA band of 46.77 kDa was detected in the unbound

fraction from the serum applied on protein G-Sepharose, while bound fraction eluted with 0.1 Mcitrate buffer, pH 3.0 contained a band of 43.65 kDa,as detected under reducing conditions and a band ofapprox. 112 kDa in each fraction under non-reducingconditions in SDS-PAGE (Fig. 1).

1 2 543

kDa205

97.4

66

45

29

20.1

14.36.5

Fig. I-SDS-PAGE of protein A- and protein G-Sepharosepurified HCAbs from camel serum [Purified IgG2 and IgG3 wereresolved in 10-15% polyacrylamide gradient gel byelectrophoresis under reducing and non-reducing conditions inorder to determine their molecular size and assess their purity.Purity of IgG2 and IgG3 was evident from the Coommassiebrilliant blue stained gel and Mr of 46.77 kDa and 43.65 kDa ofheavy chains of IgG2 and IgG3. respectively was determined byconstructing a standard curve of Rj values vs. log Mr of themarkers. Lane 1. Mol. mass markers (97.4. 67. 45. 29. 20.1 and14.3 kDa ); lane 2. IgG3 (protein A--> protein G purified fraction)(eluate at pH 3.0); lane 3. IgG2 (un adsorbed fraction on proteinG); lane 4. IgG3 (protein A-+ protein G purified fraction) (eluateat pH 3.0) (reduced); and lane 5. IgG2 (unadsorbed fraction onprotein G) (reduced)]

302 INDIAN J. BIOCHEM. BIOPHYS .• VOL. 41. DECEMBER 2004

IgG3 was eluted from (N~hS04 precipitated Igswith 0.1 M acetate buffered 0.15 M NaCl, pH 4.5,0.1M acetate buffered 0.15 M NaCl, pH 3.5 and 0.15 Mglycine, pH 2.7, as revealed by 43.65 kDa band inSOS-PAGE under reducing conditions. From proteinA, a major portion of IgG2 along with some amountof IgG3 eluted sequentially with 0.1 M acetatebuffered 0.15 M NaCl, pH 4.5 and 0.1 M glycinebuffer, pH 2.7, as revealed by the presence of bandsof 46.77 kDa and 43.65 kDa under reducingconditions in SOS-PAGE. Similar results wereobtained on further purification of ion-exchangepurified Igs by protein A and protein G-Sepharose.No band was detected in unadsorbed fractions whenion-exchange purified IgG3 was passed on proteinG-Sepharose, whereas a band of 43.65 kDa wasobserved in eluate.

Characterization of HCAbsElectrophoretic mobility

In IEP, anti-camel IgG3 formed a singleprecipitation arc in f3-region with purified camel IgG3and two arcs in y- and f3-regions merging with eachother, with camel whole serum. The migration patternof camel IgG3 shows the presence of higher netnegative charge, as compared to on IgG 1 (Fig. 2). IEPof camel whole serum using anti-camel whole serumas usual detected arcs corresponding to that of camelserum albumin in a-region, IgM and HCAbs inf3-region, and conventional camelid IgG in y-region.

Smaller size and higher net negative charge ofIgG3 resulted in anodal migration and hence theprecipitation arc between the wells containing IgG3and anti-camel IgG3 (cathodal drift in HEEO agarose)appeared in CIEP. No visible arcs were presentbetween the pairs of wells containing (N~hS04precipitated camel Igs and those containing camelwhole serum and anti-camel IgG3.

I

Fig. 2-Immunoelectrophoresis (IEP) of camel IgG3 (an HCAb)using anti-camel IgG3 [HCAbs show electrophoretic migration inthe ~-range, whereas full length IgGI migrates in the y-range.WellI, camel whole serum; 2, purified camel IgG3; and slot. anti-camel IgG3]

Antigenic relatedness of camel IgG3 with IgGs from otherspeciesByAGID

Anti-camel IgG3 cross-reacted with IgG of cattle,sheep, pig, buffalo and goat in AGIO but not withsera samples from horse, dog, chicken, mouse, guineapig, fish and human. Precipitation arcs of IgGs fromcattle and pig formed spurs with camel IgG3 giving aline of partial identity. In other species, precipitationarcs made by IgGs in adjacent wells merged with eachother giving a line of identity, with respect to eachother (Fig. 3).

By ELISAMaximum per cent ~50 displacement (33.3%) by

1/1Oth cone. of homologous antigen (camel IgG3) wasfound at log 4 dilution of rabbit anti-camel IgG3.Using the same dilution of rabbit anti-camel IgG3 andcamel IgG3. per cent ~50 displacement by purifiedbovine, goat and pig IgG, was found to be 7%. 13.7%and 14.0%, respectively (Fig. 4). The result showedthat the strength of cross-reaction of anti-camel IgG3increased from bovine to goat to pig IgGs.

By immunoblottingThe immunoblot obtained was interpreted on the

basis of noise to signal ratio (Fig. 5). Anti-camel IgG3gave the highest signal with heavy chain of camelIgG3, IgG2 and IgG 1. A band of about 35 kDa in lanecontaining (N~)2S04 precipitated camel Igs andcamel whole serum was also detected with a moderatesignal. The signals in decreasing order of intensitywere also obtained with heavy chains of IgGs of pig

Fig. 3--AGID of camel IgG3 and IgGs from other animal speciesusing anti-camel IgG3 [Precipitation arcs of IgGs from cattle andpig show spurs with that of camel IgG3. indicating their partial

'identity. Precipitation arcs of IgGs from buffalo, sheep and goatshow line of identity with each other. cattle and pig. therebyindicating partial identity with camel IgG3. Central well. anti-camel IgG3; well I, camel IgG3; 2. cattle IgG; 3, buffalo IgG3; 4.sheep IgG; 5. goat IgG; and 6. pig IgG]

(t(r~(

(

)

Ic1f\

(J

(

l

It(

"art

SEHRAWAT AND SINGH: CHARACTERIZATION OF HEAVY CHAIN ANTIBODIES OF INDIAN CAMEL 303

from other

of cattle,not with 0.8

se, guineagGs from

E3 giving a " 0.'

xipitation !with each:t to each 0.4

0.2

3.3%) by:gG3) wasel IgG3.IgG3 and

¥ purified%,13.7%It showed

dIgG3

O+-----_r----~~----~----_r----~1 2 4 53

log dilution of HIS

Fig. 4-Dilution and displacement curves of rabbit anti-camelIgG3 hyperimrnune serum (HIS) with camel IgG3 as the coatingantigen in blocking ELISA [Downward displacement of ~50 nmat log 4 dilution of HIS by one-tenth cone. of camel IgG3(homologous antigen) and equivalent cone. of IgGs from bovine,goat and pig shows that camel IgG3 is closer to pig IgG than togoat IgG and is least closely related to cattle IgG]

ed on theelIgG3

of camelDa in lane

Igs andmoderate

. intensity"Gsof pig

(53.70 kDa), cattle (56.23 kDa), sheep (50.12 kDa),buffalo (52.48 kOa), goat (50.12 kDa) and human(52.48 kDa). Signals of intensity slightly higher thannoise were also obtained with heavy chain of IgGs ofguinea pig (56.23 kOa) and dog (53.70 kDa). Faintdiffused bands were also observed with light chain(-25 kDa) of IgGs of cattle, pig, sheep, and buffalo.

DiscussionHCAbs, IgG2 and IgG3 were purified from sera of

Indian desert camel. Elution pattern of IgG3 in ion-exchange chromatography indicated its chargeheterogeneity which alongwith high net charge wasfurther reflected in immunoelectrophoresis results,wherein IgG3 migrated in ~-region as detected byanti-camel IgG3. In view of higher net negativecharge and smaller size, IgG3 was tested in CIEPusing anti-camel IgG3 in the opposite (anodal) well.Distinct precipitation arcs were obtained in the gelbetween pairs of wells containing anti-camel IgG3(anodal) and purified IgG3 (cathodal), however, inwhole serum and (NRthS04 precipitated Igs, no sucharcs were observed, possibly due to the operation ofprotein-protein non-specific interactions. Also, it maybe noted that IgG3 obtained from later fractions of

• aI speciescattle and

their partialp and goatig, therebywell, anti-10 IgG3; 4,

1 2 .1 4 5 • 7"' • 10 1112 13

20.1

14.3

• Fig. 5--Westem blotting of camel Igs and sera from differentanimal species using anti-camel IgG3 [Strength of reaction ofanti-camel IgG3 was maximum against heavy chains of camelIgGI, IgG2 and IgG3, followed by that against IgG heavy chainsof pig, sheep, cattle, buffalo and goat in decreasing order. Weakor no reaction was visible against IgGs of dog, horse, human,mouse, guinea pig, chicken and fish. Lane 1, Mol. mass markers;2, protein G-Sepharose purified camel IgG3; 3, ion-exchangepurified camel IgG3; 4, ion-exchange purified camel IgGs; 5,(NRthS04 precipitated camel Igs; 6, camel serum; 7, cattleserum; 8, buffalo serum; 9, sheep serum: 10, goat serum; 11, pigserum; 12, horse Igs; and 13, dog serum]

ion-exchange chromatography was of relativelyhigher negative charge, which was used afterconcentration in CIEP. IgG3 in whole serum and(NRthS04 precipitated Igs is present as aheterogeneous molecule in less amount than in thepurified IgG3 preparation.

Subtypes of HCAbs, varying in molecular size,exist in different members of the familyCamelidae5,18,19. In the present study, IgG2 (46.77kDa heavy chain) bound only to protein A, whereasIgG3 (43.65 kDa heavy chain) bound to both proteinA- and protein G-Sepharose. These findings weresimilar to those reported earlier' for dromedary.However, in another study" using SOS-PAGE and.differences in binding to protein A-and protein G-Sepharose column, molecular masses of 52 kDa and56 kDa, respectively were reported for IgG2 and IgG3heavy chains, in llama. Non-reduced HCAbs showedslower migration in the gel used in the present studyand gave higher molecular size as interpolated fromthe standard curves of log M, vs. Rf values (Fig. 1).

Antigenic characterization of purified HCAbs wasdone by three different methods viz., AGIO, ELISAand immunoblotting in order to know their relatedness

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Cft

inbynustrcoof

304 INDIAN J. BIOCHEM. BIOPHYS., VOL. 41, DECEMBER 2004IIV

with the Igs from different animal species. In AGIO,precipitation arcs appearing between anti-camel IgG3and affinity purified IgG from cattle, buffalo, sheep,goat and pig are suggestive of evolutionary closenessof camel with these species. Earlier, purified IgGsfrom these species were not used for antigenicrelatedness 10,20.Spur-formation of anti-camel IgG3with cattle and pig, and lines of identity among IgGsof other species suggested that camel IgG3 has someuncommon epitopes, which are not found in IgGs ofother species. A close antigenic relationship betweenIgs of the Indian buffalo, cow, sheep and goat anddistant relationship between Igs of man and horsewere shown by using rabbit anti-buffalo Ig serum andrabbit anti-cow IgG, IgA and IgM sera in agar gelimmunodiffusion and immunoelectrophoretic

hni 21tee mques .In blocking ELISA, the per cent decrease in A450

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the Departments of Vet. Microbiology and Vet.Parasitology for their kind permission for use ofvarious equipments during the study.

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