The Characteristic of Antitetanus Immunoglobulin Y Isolated from Eggs to substitute Horse Antitetanus Serum
Abstract
Laying hens are highly profitable producers of polyclonal antibodies in comparison with mammals as antibodies can be purified from egg yolks. The cost for producing IgY is lower than for mammalian antibodies since chicken housing is cheap and isolation process is economical, high yielding, uncomplicated and fast. The purpose of the study was to explore the opportunity of using antitetanus IgY from egg yolks to substitute the production of antitetanus serum from horses. The eggs were collected from adult Isa brown hens which have been immunized by tetanus toxoid. The immunization was applied intra venously with an initial dose of 15 Lf. The immunization was repeated three times with gradual dose of 100, 200, and 300 Lf with an interval of one week intra muscularly. The first immunization was tetanus toxoid mixed with Freund adjuvant complete and subsequently mixed with Freund adjuvant incomplete. Antitetanus IgY was extracted from egg yolks by means of PEG–Chloroform and purified using fast protein liquid chromatography. The purity of antitetanus IgY was determined by Bradford method (l = 595 nm) and Agar Gel Precipitation test. The molecular weight of purified antitetanus IgY was determined with Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis. The antitetanus IgY were treated by pH, heat, and enzyme. The biological activities of treated antitetanus IgY was determined by enzyme linked immunosorbent assay. Neutralization potency test of antitetanus IgY was determined by Spearman-Karber method. The antitetanus IgY highest titer of egg yolks was 80.16 ± 33.55 IU/ml reached at seven weeks after starting immunization schedule and the lowest was 1.69 ± 0.63 IU/ml. Protein concentration (IgY) after extraction and purification were 0.652 ± 0.041 mg/ml and 1.644 ± 0.424 mg/ml respectively . Biological activity of antitetanus IgY decreased significantly (p<0.01) at of pH 2, pH 3, and at 72.5 oC, and lost its activity at 90 oC after 20 minutes. Addition of 25% sucrose solution was able to maintain antitetanus IgY activity until 50 % at 75 oC for 5 minutes, while addition of 50% glucose solution increase antitetanus IgY activity at 75 oC and 80 oC for 5 minutes. The effect of pepsin, trypsin, and protease enzyme s decreased the activities of antitetanus IgY. Spearman-Karber value of potency of antitetanus IgY was 35 IU/ml. The injection of 0.2 and 0.4 IU antitetanus IgY per mice respectively, protect all mice from the letal dose tetanus toxin. The doses of 0.8 IU and 1 IU anti tetanus IgY per mice protected twice dose of letal tetanus toxin. Antitetanus IgY given orally on adult animal was ineffective as passive immunotherapy. This research concluded that the hens were capable of produc ing antitetanus in egg yolks and can be used as a resource of antitetanus serum to substitute horse antitetanus serum.
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