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KONU$PAYEVA ET AL

Tobie2. P»*mił'nl«slc^nn*r-i nnlk •xA'iA’i/>K-!implm;: <l» yi^n |iy r*vt«"«i

	Winter	Spring	Siiiiurp-r	AuUiniti
A In pity	1	2		1
Atyrau		2	2	2
Ar>il-k	a
S)iymk«<nt	i	2	1
T^liil			17

(pH 8.6) was passed through a 5-mL HiTrap heparin colunm (Amersham Biosciences) and eąuilibrated in the same buffer. Elution was performed at 2 mL/min over a

0    to 1 M NaCI gradient <60 mL) using HFLC eąuipinent (pump 120, detector 430; Knntron Instruments, St-Quentin-en-Yvelines, France). Lactoferrin eluted as a single peak at 0.3 SI KaCI. Purity was checked by poly-acrylamide gel electrophoresis < 12.5# acrylamidel with or witliout denatunng agent? (SDS and mercaptoetha-nol with heating for 5 min in a boiling water bath).

Polyclonal Antibodies

Rabbits wcrc iinmunizcd at monthly intcrvals by multiplc intradermal injcctions of untigcii-adjuvnnt inixture (Levieux ct al., 2005) prcpared by cmulsifyiug

1    vol of salinę coutaining 0.5 to 1 mg of purified protein/ mL and 1 vol of complete 'first injection) or incomplete (booster injections* FYeutuTs a<buvant Bach rabbit re-ceived 2 mL of the emulsion. Animals were bied 7 d after each booster injection and the sera were analyzed for antibody aciivity and spedficity by immunoelectro-phoresis 'Scheidegger, 1955) and single radial iinmuno-diffusion assay • SH1D; Mandniet al., 1965). Theimmu-nogens used were purified IgG and Lf.

Immunochemicat Assay of Proteins

Concentrations of IgG and Lf in individual colostrum and miik samples were deternnned by the SR1D assay nsing 1.9-mm-thick agar plates containing 1.2# agar ni 0.005 A/ barbital buffer łpll 7.3>and suitable ąuanti-lies of each 6pecific antiserum. as described by Levieux < 1£>91 > and adapted for Lf deternnnation according to Levieux et al. «2005). Circular wells 11.5-mm diameter) were punchod out of thogel and filled willi 3-pL aliąuots of adequalely diluted samples or 3 pL of purified pro* teius of known conccntration as standard*. The purified proleins and samples were diluted in the barintal buffer containing 1# nonimmunized rabbit serum and 1 mg of sodium azidc/iuL Plates were ineubatod in a moist box at 37°C for 15 to 20 h, and the diameter of the ring-shaped prccipitates was measured using a inagnifying video camera system (Levieux, 1991). Standard curves wereconstructed by plotting the diameter of the procipi-taling ring vs. the sąiuirc root of the protein conoenlra* tion. With the diffusion time used, a liuear regression was always obtained. Samples and standard* were plated in dupticate. The coefficients of variation of the assays were 3 to 5#.

Statlstical Analysls

A linear model was tested, with IgG and Lf conoentra-tions as dependent vnriables. The tested variation fac-tors were region, species, season, and tJieir interactions. The significance level for the variance analysis was set at 0.05. The reaults are presented as mean r standard error. Because the variances were not homogeneoiis, data were log-transformed to attain a norinal distribu-tion of value5. The correlation between Lf and IgG was tested using a Pearson correlation. To identify the fac-tors linked to Iow and high values for IgG and Lf simul-taneously. the samples were divided into IgGl and Lfl • concentrations below the median value6>and lgG2 and Lf2 (concentrations above the median values), giving 4 groups of samples «IgGl/Lfl. IgG2/Lfl, IgGl/Lf2, and IgG*2/LP2>. The partitioniug into tliosc groups by spe-cics. scason, and region was cvaluatcd by a V test. Separale analyses were condueted for raw cainefs tnilk. colostrum. and shubat. The R software was used for all statistical analyses (Ihaka and Gentleman, 1996).

RESULTS

Camel Raw Mllk Samples

LfConłenL The mean Lf concentration in raw cam-cVn milk was 0.229 ±0.135 mg/inL. According lo geo-graphie factors, the Lf contcnt was higliur, on avcragt\ in the Shymkent region (Table 3), but the diffcreuces were not significant (Table 4). AJsu.nosignificant differ-cnces were found among species in spite of the higher mean Lf contcnt in hybrids (Tables 3 and 4). The AN-OVA showed a significant seasonal effect (Table 4) only for Lf <P < 0.05>, with the Lf oontent l)eing liiglier in the spring *0.271 + 0.161 mg/mL) tJian in the autumn •0.176 t 0.083 mg/mL; Table 4).

Because the seasonal effect was significant, the monthly changes in Lf concentration are sliown • Figurę 1). Peak Lf concentrations were elearly observed in April <0.29*1 ± 0.152 mg/mU and in June (0.312 r 0.19*1 mg/niL; P < 0.05). The lowest Lf concentrations were observed from July to October (0.190 = 0.072 nig/niL).

IgG Conłenł. The mean IgG concentration in raw camels milk was 0.718 i0.330 mg/mL. The IgG content was. on avcrngc, higher in the Anilsk region 'Table 3). but likc Lf. the diffcrenoes were not significant (Table 4'. Also, no significant difference was found for species

Jourrwil oł D;ury Sr.ior>oo Vd 90 No 1 ?O0/