豚における Immunoglobulins IgG,IgA および IgM の分離精製について

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This paper deals with a procedure of purification of three classes of immunoglobulinfrom porcine serum and sows colostrum. The procedure was summarized as follows(Charts 1 to 3).I. For the purification of immunoglobulins IgG and IgM, pooled porcine serumwas treated with (NH4)2804 to a concentration of ? saturation. Then precipitation wascarried out with a DEAF,-cellulose column (anion-exchange chromatography). The step-wise elution entailed tlte use of seven buffers: (J) 0.01 M NaH:P0+ adjusted to pH 7.6with 0.01 M NaOII, (2) 0.02 M NaH.P0. adjusted to pH 6.3 with 0.02 M Na0H, (3) 0.05 MN1H204, (4) 0.1 M N3lH2PO4, (5) 0.15 M NlH2P04, (6) 0.3 M NlH2P04, and (7) 0.4 MNaH,.P0+. IgG was seen in an elution obtained with buffers (l) and (2). This IgG waspurified by chromatography on CM-cellulose (cation-exchange chromatography) and getfiltration on Sephadex G-200. IgM could be obtained with buffers (4) to (6). The proteincontaining IgM was also fractionated by repetition at get filtration three times onSpehadex G-200 using 0.2 M Tris-HCI and 0.15 M Nail buffer (pH 8.1) for purification,From 100 ml of pooled porcine serum, about 675 rug of IgG was collected, and IgMwas estimated to be 117 mg.2. Colostrum was collected from sows for the purification of IgA. Fat was removedfrom the colostrum, which was then subjected to decaseination and delipoproteinization.The whey was fractionated with Sephadex G-200 against 0.2 M Tris-HCI saline buffer(pH 8.1) which had been used for elution. The resultant fraction was then applied to acolumn of DEAF,-cellulose by stepwise elution. The protein that was assumed to be IgAwas eluted with 0.125 M and 0.15 M Tris-HCI buffer (pH 7.4). This crude IgA was fractionated by repetition of get filtration three times on Sephadex G-200 with Tris-HCIsaline buffer (pH 8.1).From TOO ml of colostrum, about 192 mg of IgA was finally collected.3. Antisera were prepared by injection of rabbits with the three classes of immuno-globulin in Freunds complete adjuvant. Alpha- or beta-globulin was seen in ant In recent years, some trials for separating the plasma protein components have beenput in practice by the author33=3) to explain the behavior of transaminases in fowl bloodplasma and their physicochemical properties. As a result, it was pointed out that several components obtained by subfractionationof albumin37) and beta-globulins36) were regarded satisfactorily as carriers of reverse (R)-aspartate aminotransferase (GOT), R-alanine aminotransferase (GPT), and reversibleGPT. Several physicochemical determinations laave already been performed to clarifythe properties of these components36?37).On the contrary, it is known that the fairly effective purification of ant active material?on1y for forward (F)-GOT substrate was accomplished as fraction IV-73?35). Moreover, aprotein component with an active fragment of F-GOT has frequently been found in acertain globulin group3?35). Notwithstanding, that fraction has not yet been subjectedto a highly effective purification, because the problem of how to separate and purify itremains unsettled. In order to obtain a basic solution for this problem, it is necessary to?devise a method for systematic separation of blood plasma globulins.The present report deals with a method developed and introduced into practical,application by the author.The results of experiments with this method are summarized as follows.I. On the basis of the relations among several quantities listed in some tables, acomponent distinguishable by disc electrophoresis that had migrated to position 5-b wasobtained by subfractionation from the original fraction, IV-4, as fraction IV-4(d). It wasfound to be F-GOT. Moreover, one of the R-GPT samples existed among the conjugatedprotein components with the relative position, 6-a to b. This conjugated component wasobtained from the original fraction, IV-l, as fraction IV-1(f).2. These components were found to be globulin with the same relative position asgamma.-globulins by the use of cellulose acetate electrophoresis"9?33). This result was thesame as obtained from components 4-b and c36).These data lend support to the conclusion that a few gammax-globulins are constantlypresent in fowl blood plasma and that their fractionation can be carried out. It is im-portant to test t available are carried only by the GOT group. It became evident that components 5-band 4-c were derixzed from the reaction of transamination with F-GOT and R-GOT,respectively.If relations among several isozymes of R-GPT or CRT-ase36?37) are examined, it willbe noted that the carriers of these isozymes are evenly distributed among several compo-nents obtained from albumin and globulins. This does not necessarily mean, however,all the isozymes show the same distribution.As mentioned above, on certain reverse transaminases, especially those of the R-GPTgroup, these isozymes seem to have an important action. This considration is ttseful,especially when the physiological function of the GPT group is studied.For this reason, it is interesting to obtain more detailed information on this aspectfrom future investigation.
社団法人日本獣医学会の論文
1973-06-25

豚における Immunoglobulins IgG,IgA および IgM の分離精製について

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