Process for producing secretory immunoglobulin A preparations

Secretory immunoglobulin A preparations substantially not containing virus are produced by a process wherein secretory immunoglobulin A which might be contaminated with viruses is (1) heated about 60.degree. C. for about 10 hours, or (2) subjected to the reaction with tri-n-butyl phosphate and a surfactant and the heating as mentioned above, as liquidized form in an aqueous medium, and then polymerized matters are precipitated from the resulting solution by adding polyethyleneglycol thereto.

FIELD OF THE INVENTION 
This invention relates to a process for producing secretory immunoglobulin 
A (hereinafter abbreviated as sIgA) and a preparation substantially not 
containing denatured sIgA can be obtained by this invention. 
BACKGROUND OF THE INVENTION 
Secretory immunoglobulin A is an immunoglobulin contained in secretion from 
exocrine gland, particularly in a large amount in foremilk and it has 
protective function against bacteria and viruses on mucosa surface. 
It is known that sIgA is effective in the cases for which supplementary 
local immunotherapy on mucosa can be applied as referred below; Primary 
immunodeficiency syndrome and diarrhea difficult to treat and aphthous 
stomatitis accompanied by secondary immunodeficiency (immunodeficiency by 
infection, nutritional disorders, drugs, etc.) (S. Matsumoto et al., Birth 
Defects 18, 229, 1983; Okino et al., Nihon Shonigakkai Shi, 84, 158, 
1990): 
Furthermore, it may be highly effective in treating recurrent upper 
respiratory inflammation (eg. otitis media) and treatment after operation 
of biliary obstruction (Kurono et al., Therapeutic Research 10, 4433, 
1989). 
It can not be denied that there is fear for the mixing of viruses such as 
hepatitis virus, AIDS virus cytomegalovirus, etc. into sIgA purified from 
foremilk. Accordingly, the inactivation of these viruses is indispensable 
on processing the sIgA to preparations thereof. However, there is no known 
process wherein the inactivation of the viruses are combined with the 
processes for purifying sIgA from foremilk. 
With regard to the inactivation of viruses, heating process in the form of 
a liquid (hereinafter abbreviated as "heating as liquidized form") has 
been applied to serum protein such as albumin etc. as the most reliable 
process for inactivating viruses which might be mixed therein, according 
to a report of Murray (The New York Academy of Medicine, 31, 341, 1955). 
This process has been widely employed for a long time. 
However, this process can be applied to only a heat-resistant material such 
as albumin, while most of protein are easily denatured by heat and liable 
to cause the decrease or disappearance of activity. Furthermore, this is 
not a process which can completely inactivate virus as far as hepatitis B 
virus is concerned. 
On the other hand, Prince, Horowitz and Brotman (The Lancet March, 29, 206, 
1986) disclosed that envelope containing viruses such as hepatitis B 
virus, AIDS virus, Non-A, Non-B Hepatitis Virus, etc. can be completely 
inactivated by treating at 24.degree. to 30.degree. C. for 6 hours in the 
presence of tri-n-butyl phosphate and a surfactant, and this process can 
be applied to many drugs originated from serum protein. However, this 
process seems to be ineffective to a group of viruses not having envelopes 
such as hepatitis A virus, etc. 
SUMMARY OF THE INVENTION 
The present invention intends to provide a process for producing sIgA 
preparations substantially not containing infectious viruses, 
substantially not containing denatured sIgA, and having high safety. 
Present inventors carried out researches along the above purpose, and, as 
the result, found that sIgA preparations having high safety and 
effectiveness is obtained by combining "heating as liquidized form" and 
fractionation with polyethyleneglycol (hereinafter abbreviated as PEG) and 
further found that sIgA preparations having higher safety are obtained by 
combining the treatment with tri-n-butyl phosphate and a surfactant with 
"heating as liquidized form" and PEG-fractionation. 
Present invention is (I) a process for producing a secretory immunoglobulin 
A preparation substantially not containing virus characterized in that 
secretory immunoglobulin A which might be contaminated with viruses is, as 
liquidized form in an aqueous medium, (1) heated at about 60.degree. C. 
for about 10 hours, or (2) subjected to the treatment of reacting with 
tri-n-butyl phosphate and a surfactant, and the heat-treatment as 
mentioned above, and the resulting polymers are removed by the addition of 
polyethyleneglycol; and (II) a process for producing a secretory 
immunoglobulin A preparation substantially not containing virus 
characterized in that secretory immunoglobulin A which might be 
contaminated with viruses is reacted with tri-n-butyl phosphate and a 
surfactant as liquidized form in an aqueous medium, heated at about 
60.degree. C. for about 10 hours, and polymerized matters formed in the 
resulting aqueous solution of said globulin A is removed by adding 
polyethyleneglycol thereto. 
In present invention, the starting material sIgA which might be 
contaminated with viruses is not limited particularly. It may be a sIgA 
fraction obtained from the milk of human or cattle, or a fraction of 
monoclonal antibody in sIgA type obtained from human or cattle, and it may 
be a fraction containing sIgA obtained from the foremilk of human or 
cattle, for example, a fraction containing sIgA obtained in each step of 
the following processes.

DETAILED DESCRIPTION OF THE INVENTION 
(I) Centrifuging Process 
The foremilk of human or cattle is centrifuged (e.g. 3,500 to 8,000 rpm., 
for 30 to 60 minutes), and then fat deposited as the upper layer or 
precipitates is removed. After filtration, the filtrate is adjusted to pH 
4.2 to 4.6 and stirred at room temperature for 30 to 60 minutes, followed 
by removing precipitated casein by centrifugation. 
(II) Process of Salting Out and Dialyzing 
The solution thus obtained is neutralized and subjected to salting out with 
ammonium sulfate (saturated to 50 %). The precipitated sIgA fraction is 
recovered by centrifuging, and dissolved in 0.01 M phosphate buffer 
solution (pH 6.5 to 7.5), followed by dialyzation employing same buffer 
solution. 
(III) Process of Treating with Anion Exchanger 
The salted out fraction of sIgA is sufficiently dialyzed and then charged 
on a column of anion exchanger, preferably DEAE-Toyopearl.RTM. 650C or 
DEAE-Sephacel.RTM., previously buffered with 0.01 M phosphate buffer 
solution (pH 6.5 to 7.5). The charging amount of the salted out sIgA 
fraction on the column is preferably 1 to 5 g as sIgA per 1 L of gel 
(filler) and the concentration of the protein is preferably 3 to 10 mg/ml. 
After washing the column with 5-fold column volume of the above buffer 
solution, sIgA is eluted from the column with 0.1 M phosphate buffer 
solution (pH 8.5 to 7.5) or a buffer solution prepared by adding 0.10 
to.0.15 M of sodium chloride to 0.01 M phosphate buffer solution (pH 6.5 
to 7.5). 
(IV) Process of Treating with Heparinoid-immobilizing Column 
A column immobilizing heparinoid such as heparin-Sepharose.RTM., 
heparin-Toyopearl.RTM. or heparin-Sulfate Cellulofine.RTM. is previously 
buffered with 0.1 M phosphate buffer solution (pH 6.5 to 7.5) or a buffer 
solution prepared by adding 0.1 to 0.15 M of sodium chloride to 0.01 M 
phosphate buffer solution (pH 6.5 to 7.5), and the above sIgA fraction 
eluted from the anion exchanger is charged on the buffered column. The 
charging amount of the sIgA fraction is preferably 10 to 50 g as sIgA per 
1 L of gel (filler) and the concentration of the protein is preferably 3 
to 10 mg/ml. 
By collecting fractions passed through the column without being adsorbed, 
sIgA fraction free of lactoferrin can be obtained. As the resin employed 
in this process, preferably employed in industry is Sulfate 
Cellulofin.RTM. not immobilizing high molecular substances thereon. This 
resin can be regenerated with 0.6 to 1.0 M sodium chloride. 
(V) Process of Precipitation with Polyethyleneglycol 
By adding PEG #4000 so as to make its concentration 20% w/v to the sIgA 
fraction subjected to the treating with the heparin-immobilizing column, 
sIgA fraction is precipitated. The precipitates are collected by 
centrifuging, dissolved in physiological saline to make about 20 mg/ml of 
sIgA solution. 
As the starting material of present preparations, any of the sIgA fractions 
obtained in the above "(II) Process of salting out and dialyzing" and the 
consecutive processes may be employed. However, in the case of employing 
the fraction obtained in "(II) Process of salting out" or "(III) Process 
of treating with anion exchanger" for carrying out the process of present 
invention, it is preferable to perform the above process (III) or (IV) and 
the consecutive processes, after the present process. 
In present invention, viruses are inactivated by heating as liquidized form 
in an aqueous medium at about 60.degree. C. for about 10 hours. 
SIgA can be dispersed in an aqueous solution of salts such as physiological 
saline and phosphate buffer solution to form a solution-like state and the 
heating is carried out in this state. 
By heating at about 60.degree. C. for about 10 hours, a considerable amount 
of sIgA is denatured, although sIgA still remains in the solution. 
However, it was found that the denaturation can be suppressed by the 
addition of a stabilizer (Ref. to Experiment 1). 
As the stabilizer, there may be used sugar alcohols such as sorbitol, 
disaccharides such as cane sugar, and amino acids such as glycine. One or 
more of these stabilizers may be used. When one kind of the stabilizer is 
used, preferable is a sugar alcohol such as sorbitol. A disaccharide and 
an amino acid are preferably used jointly with each other. 
After the heat-treatment, the solution contains polymerized sIgA dissolved 
therein, however, sIgA polymer and monomer can be separated from each 
other, because the polymer precipitates by adding PEG to the solution to 
make PEG concentration 5 to 10%, preferably 7 to 8%, whereas sIgA monomer 
begins to precipitate from 10% of PEG concentration, and most of the 
monomer precipitates at 15% and almost completely precipitates at 20 to 
25% of PEG. 
As PEG, there may be exemplified PEG #4000 (average molecular weight; 
3,000), PEG #2000 (average molecular weight; 2,000), PEG #6000 (average 
molecular weight; 7,500), etc. Preferable pH in the fractionation with PEG 
is 6 to 8. 
By the above heat-treatment, most of viruses are inactivated, however, when 
contamination with a heat-resistant virus such as hepatitis B virus, etc. 
is considered, it is desirable to carry out the treatment by reacting with 
tri-n-butyl phosphate and a surfactant. 
Accordingly, the possibility of combining the above heat-treatment with the 
treatment with tri-n-butyl phosphate and a surfactant was investigated. As 
the result, it was found that the combination itself is possible, however, 
sIgA is polymerized and coagulated to some extent even in the treatment 
with tri-n-butyl phosphate and a surfactant. Therefore, in order to remove 
efficiently the coagulated polymer, it is convenient to carry out the 
heat-treating after the treatment with tri-n-butyl phosphate and a 
surfactant and then fractionation by PEG. 
In the treatment with tri-n-butyl phosphate and a surfactant, the examples 
of the surfactant include nonionic surfactants such as Tween 80 and 
anionic surfactants such as sodium cholate. As to the concentration of 
tri-n-butyl phosphate and a surfactant in the treatment, the former may be 
0.2 to 0.4% and the latter may be any concentration as far as it is not 
more than 1%. These agents may be reacted generally at 20.degree. to 
30.degree. C. for 5 to 7 hours. 
In an embodiment, 0.3% of tri-n-butyl phosphate and of Tween 80, or 0.3% of 
tri-n-butyl phosphate and 0.2% of sodium cholate are added as respective 
final concentrations to a solution containing sIgA, and the mixed 
solutions are treated at 24.degree. C. and 30.degree. C. for 6 hours, 
respectively. 
Tri-n-butyl phosphate and the surfactant in the sIgA solution which has 
been subjected to the above treatment can be by adding PEG to the solution 
so as to make PEG concentration 20 to 25% causing sIgA to precipitate, and 
then centrifuging. The resultant sIgA precipitates are dissolved by adding 
a medium such as physiological saline thereto, and the solution is heated 
at about 60.degree. C. for about 10 hours, preferably under the addition 
of stabilizer(s) mentioned above. Consecutively, the solution is subjected 
to the above PEG fractionation, thereby sIgA treated by the two steps of 
virus-inactivation is obtained. 
In other embodiment, sIgA was precipitated from a sIgA solution which had 
been subjected to the first step of virus inactivation, by adding PEG to 
the concentration of 20% thereto, and tri-n-butyl phosphate and the 
surfactant were removed from the precipitates by centrifuging. The 
resultant sIgA was dissolved by adding physiological saline thereto so as 
to make 1% w/v solution (pH 7). As stabilizers, 50% w/v sorbitol-2 M 
glycine were added to the solution, and the mixture was subjected to the 
above "heating as liquidized form" (60.degree. C. for 10 hours) and the 
fractionation with 8-23% PEG. 
Respective yields in the embodiments were 63 and 60%, which were lower to 
some extent than that of treating by heating alone. 
The neutralizing antibody potency of the sIgA prepared by this invention is 
as follows: 
(1) Process of "Heating as Liquidized Form"--PEG Fractionation 
SIgA prepared by the above process of "heating as liquidized form" and PEG 
fractionation, and sIgA prepared from same material by a process omitting 
the heat-treatment process from the above process were tested by direct 
agglutination employing Escherichia coli (NHI/J strain etc.) and the 
neutralizing antibody potency of these sIgAs to viruses such as rotavirus, 
echovirus, etc. were determined. By comparing the results obtained with 
both sIgAs, no difference was found between them in the direct 
agglutination with Escherichia coli as well as in the neutralizing 
antibody potency to the viruses, so that it was confirmed that the 
activity of sIgA is not impaired by this process (Ref. to Test Examples 1 
and 2). 
(2) Process of tri-n-butyl phosphate - surfactant - "heating as liquidized 
form" - PEG fractionation 
The direct agglutination employing Escherichia coli (NHI/J strain etc.) and 
the neutralizing antibody potency of sIgA prepared by this process was 
compared with those of sIgA prepared from same material by a process 
omitting the above steps. As the results, no large difference was observed 
between them in the direct agglutination of Escherichia coli as well as in 
the neutralizing antibody potency of viruses, so that the usefulness of 
present invention was found as in the above (1) (Ref. to Test Examples 3 
and 4). 
From the above results, it was also found that sIgA preparations prepared 
by present invention do not contain denatured sIgA. 
Furthermore, according to a model experiment about the inactivation of 
viruses, it was clarified in high possibility that citomegalovirus and 
hepatitis A virus which are heat-resistant to the same extent as 
poliovirus as well as AIDS virus which is weaker in heat-resistance than 
the above viruses are inactivated by present heat-treating, and also even 
viruses which are highly heat-resistant such as hepatitis B or non-A non-B 
virus are inactivated by employing the treatment with tri-n-butyl 
phosphate and a surfactant jointly with the heat-treating (Ref. to Test 
Example 2). 
The sIgA solution obtained by present invention may be dialyzed to 
physiological saline, etc. and then sterilized by membrane filtration, 
etc. The resulting solution may be administered as it is, or freeze-dried 
and processed to capsules to administer as an oral preparation. The form 
of the preparation varies according to the usage. In processing to the 
preparations, enteric coating may be applied, although this coating in not 
required particularly, because sIgA is a chemically and enzymatically 
stable substance. Present sIgA is employed as an agent for supplementary 
local immunotherapy on mucosa, hence oral administration and instillation 
in nose or eyes are considered in the administration, and intravenous 
injection is not necessarily meaningful, however, the mode of 
administration is not limited particularly. 
Present invention is explained further by exemplifying Experiments, 
Examples and Test Examples in the following: 
Experiment 1 
Tests of Heat-stabilizing Agent and PEG Fractionation for sIgA 
Experiments of heating a solution containing sIgA at 60.degree. C. for 10 
hours as a liquidized form in the presence or absence of heat-stabilizers 
were carried out, and the degree of polymerization-coagulation of sIgA was 
analyzed to screen best stabilizer as well as to investigate the optimal 
PEG concentration for removing the polymerized matters. 
Generally, the heat-denature of protein is accompanied by a phenomenon that 
the protein is coagulated by polymerization and come to insolubilyze in 
water, and the reaction proceeds unreversibly. It is important in knowing 
the denature of protein to grasp the degree of polymerization-coagulation. 
Accordingly, in the first place, the solution of the above sIgA in 1% w/v 
physiological saline was treated as it is or under the addition of a 
stabilizer mentioned below at 60.degree. C. for 10 hours. And then, the 
recovered amount of sIgA was analyzed by employing Superose.RTM. 8 
(produced by Pharmacia Co.) column. The results are shown in Table 1. 
TABLE 1 
______________________________________ 
Provisional experiment of heat-treating sIgA solution 
"as liquidized form" 
Recovery 
Appearance after 
ratio of 
Stabilizer heat-treating sIgA 
______________________________________ 
(1) not added whitely turbid 
19% 
(2) 2M glycine slightly, 66% 
whitely turbid 
(3) 50 w/v % sucrose 
almost clear 68% 
(4) 50 w/v % sorbitol 
almost clear 77% 
(5) 50 w/v % sucrose- 
almost clear 78% 
2M glycine 
(6) 50 w/v % sorbitol- 
almost clear 80% 
2M glycine 
______________________________________ 
The sIgA samples obtained in Table 1 was diluted, PEG was added thereto to 
a concentration of 5 to 10%, preferably 7 to 8%, and the mixture was 
allowed to stand at 4.degree. C. for 1 hour and then centrifuged. The 
amount of polymerized matters in the supernatant was investigated. As the 
result, it was found that most of the polymerized matters precipitates at 
the above PEG concentration in any of the cases. In order to know the 
yield of sIgA after removing sIgA polymer, PEG was further added to the 
supernatant of each sample containing 8% of PEG to make the final 
concentration 23%, whereby sIgA was completely precipitated and recovered. 
As the result, the recovery ratio of sIgA was 10% in the case that no 
stabilizer was added thereto, whereas respective yields of sIgA were 88, 
87 and 73% in the cases that sorbitol, sucrose-glycine and 
sorbitol-glycine were added thereto as stabilizers, showing larger 
recovery ratios than in the above case. 
SIgA monomer fraction begins to precipitate from 10% of PEG concentration, 
the most part of the monomer precipitates at 15% and almost completely 
precipitates at 20 to 30%. 
From these results, it was found that, in the case of adding no stabilizer, 
the coagulation and precipitation of sIgA is observed making the solution 
turbid whirely by heating as liquidized form at 60.degree. C. for 10 
hours, however it is considerably suppressed by the addition of 
stabilizers. 
Experiment 2 
Test of Virus Activity after Treating of Inactivation 
In Experiment 1, it was confirmed that the denature of sIgA in 
heat-treatment at 60.degree. C. for 10 hours in the form of aqueous 
solution is suppressed by the presence of a stabilizer. However, this fact 
suggests a possibility that virus is not completely inactivated because 
there is a possibility that the protein constructing virus particles is 
also not denatured. 
Accordingly, in order to prove that the treatment according to present 
invention is effective only to inactivate viruses, there were carried out 
experiments of virus inactivation by "heating as liquidized form" in the 
presence and absence of 50% w/v sorbitol - 2 M glycine as stabilizer, 
employing poliovirus (type 1) and vesicular stomatitis virus (VSV). 
Furthermore, since it is considered that protein itself may possibly be a 
stabilizer for virus, there was carried out experiments of "heating as 
liquidized form" in the presence of 2% or 50% w/v sorbitol - 2 M glycine 
to 60.degree. C. for 10 hours, employing human albumin as a model protein. 
The results are shown in Table 2. 
TABLE 2 
______________________________________ 
Virus inactivation by "heating as liquidized form" 
Heat-treated "as 
liquidized form" at 
Virus Stabilizer 60.degree. C. for 10 hours 
Not treated 
______________________________________ 
Poliovirus 
None &lt;3.1 5.6 .times. 10.sup.4 
Presence &lt;3.1 5.6 .times. 10.sup.4 
HSA/Stabilizer 
&lt;3.1 10.0 .times. 10.sup.4 
VSV None &lt;3.1 10.0 .times. 10.sup.4 
Presence &lt;3.1 17.8 .times. 10.sup.4 
HSA/Stabilizer 
&lt;3.1 31.6 .times. 10.sup.4 
______________________________________ 
(In the Table, numerals are TCID.sub.50) 
The results in Table 2 suggested high possibility that AIDS virus which is 
less heat-resistant than poliovirus, and cytomegalovirus and hepatitis A 
virus which are heat-resistant to same extent as poliovirus are 
inactivated. From the facts that any of poliovirus and VSV are inactivated 
by this treatment, it is considered that the inactivation has no relation 
to whether a virus has an envelope or not. However, it is not necessarily 
assured that hepatitis A and non-A non-B viruses which are highly 
heat-resistant can be inactivated only by this treatment. 
Accordingly, there were carried out virus-inactivation experiments by 
treating with tri-n-butyl phosphate-surfactant which is considered 
effective against a virus having envelope such as hepatitis B or non-A 
non-B virus, or the like. Employing VSV having an envelope and poliovirus 
not having an envelope as viruses to be tested, virus-inactivation 
experiments were carried out, wherein respective viruses were treated at 
24.degree. C. and 30.degree. C. for 6 hours under the addition of 0.3% 
tri-n-butyl phosphate -1% Tween, a surfactant, or 0.3% tri-n-butyl 
phosphate-0.2% sodium cholate. In consideration of the possibility that 
protein itself may be a stabilizer for viruses, experiments of treating by 
the above treatment under adding 2% of human albumin (HSA) as a model 
protein to the above agents were carried out simultaneously. The results 
are shown in Table 3. 
TABLE 3 
______________________________________ 
Inactivation of viruses by treatment with tri-n-butyl 
phosphate (TNBP) - surfactant 
TNBP/Tween 80 
TNBP/sodium cholate 
Not not 
Virus HSA Treated treated Treated 
treated 
______________________________________ 
Polio- 
Non 5.6 .times. 10.sup.5 
3.2 .times. 10.sup.5 
5.6 .times. 10.sup.4 
1.8 .times. 10.sup.4 
virus Presence 3.2 .times. 10.sup.4 
5.6 .times. 10.sup.4 
3.2 .times. 10.sup.4 
5.6 .times. 10.sup.4 
VSV Non 31 3.2 .times. 10.sup.5 
31 5.6 .times. 10.sup.5 
Presence 31 1,8 .times. 10.sup.6 
31 1.0 .times. 10.sup.5 
______________________________________ 
(In the Table, numerals are TCID.sub.50) 
The results in Table 3 show that the treatment with tri-n-butyl phosphate - 
surfactant inactivates only a virus having an envelope and completely 
ineffective against a virus not having an envelope. Namely, it is 
considered that hepatitis B or non-A non-B virus, AIDS virus, etc. are 
inactivated, and hepatitis A virus, etc. can not be inactivated. 
From these results, it is apparent that "heating as liquidized form" can 
inactivate most of pathogenic viruses, however more complete inactivation 
is required to a virus such as hepatitis B virus which can not be 
inactivated by "heating as liquidized form" at 60.degree. C. for 10 hours, 
and hence it is desirable to perform the heat-treating together with 
treating with tri-n-butyl phosphate-surfactant. 
EXAMPLE 1 
After removing fat from 2 L of human foremilk by centrifugation, the 
supernatant was adjusted to pH 4.5 and Casein was removed therefrom by 
centrifugation. The supernatant thus obtained was salted out by adding 
ammonium sulfate thereto to 50% saturation. The resulting precipitates 
were collected and sufficiently dialyzed with 0.01 M phosphate buffer (pH 
7.4). The dialyzate remained inside of the membrane was charged on 1L of 
the column of DEAm-Sephacel.RTM. (produced by Pharmacia Co.) previously 
sufficiently buffered with the same buffer as above. After the column was 
sufficiently washed with the same buffer, sIgA was eluted therefrom with 
0.01M phosphate buffer solution - 0.1 M sodium chloride, pH 7.4 (PBS). 
Consecutively, the eluate was charged on 100 ml of the column of 
heparin-Shephrose.RTM. (produced by Pharmacia Co.) buffered sufficiently 
with PBS, and the unadsorbed portion was collected. By adding polyethylene 
glycol (PEG) #4000 (produced by Wako Pure Chemical, Ltd.) to the portion, 
sIgA was precipitated, the precipitates were collected and dissolved by 
adding physiological saline (produced by Otsuka Pharmaceutical Co., Ltd.) 
thereto to prepare 2% sIgA solution. To per ml of the solution, 0.5 g of 
sorbitol (produced by Wako Pure Chemical, Ltd.) was added to dissolve 
therein, and the resulting solution was poured dividedly into hard glass 
bottles and sealed. These bottles were sunk into hot water of 60.degree. 
C. and heated for 10 hours. After the heating, the solution was diluted to 
5-fold with physiological saline, PEG #4000 was added thereto so as to 
make its concentration 8% w/v, the mixed solution was allowed to stand at 
4.degree. C. for 2 hours, and the resulting precipitates were removed by 
centrifugation. To the solution thus obtained, PEG #4000 was added so as 
to make its final concentration 23% and the solution was allowed to stand 
at 4.degree. C. for 8 hours. The resulting precipitates were collected by 
centrifugation and dissolved by adding physiological saline to make 2% 
sIgA solution. This solution was dialyzed employing physiological saline 
and sterilized by membrane filtration. The yield of sIgA from the foremilk 
was 40% (Lot 1/T). Furthermore, the abovementioned precipitates at 20% of 
PEG, which had not been heated, were dissolved with physiological saline 
to make 2% sIgA solution. After dialyzing with physiological saline, the 
solution was sterilized by membrane filtration to make a preparation (Lot 
1/N). 
EXAMPLE 2 
A sIgA preparation was prepared from human foremilk by the same manner as 
in Example 1. The yield of sIgA from the foremilk was 40% (Lot 2/T). 
Another preparation was also prepared by dissolving the precipitates which 
were obtained at 20% of PEG and had not been treated by heating into 
physiological saline to make 2% sIgA solution, dializing the solution with 
physiological saline and sterilizing by membrane filtration (Lot 2/N). 
EXAMPLE 3 
Human foremilk was subjected to the removal of fat and casein and then to 
dialysis in the same manner as in Example 1. The resulting solution was 
charged on 7 L column of DEAE-Toyopearl.RTM. (produced by Tohso Corp.) 
previously sufficiently buffered with 0.01 M phosphate buffer (pH 7), and 
then the column was washed sufficiently with the same buffer, followed by 
eluting sIgA with 0.1 M phosphate buffer (pH 7). Consecutively, the eluate 
was charged on 600 ml column of Sulfate-Cellulofine.RTM. (produced by 
Seikagaku Kogyo Co.) previously sufficiently buffered with 0.1 M phosphate 
buffer (pH 7), the unadsorbed portion was collected, and PEG #4000 was 
added thereto so as to make the concentration of PEG 20% w/v to 
precipitate sIgA. The resultant precipitates were collected and dissolved 
by adding physiological saline thereto to make 2% sIgA solution. To per ml 
of the solution, 0.5 g of sorbitol and 0.15 g of glycine were added and 
dissolved therein and the mixed solution was treated at 60.degree. C. for 
10 hours. 
After the above "heating as liquidized form", the solution was diluted to 
5-fold with physiological saline, PEG #4000 was added to the diluted 
solution so as to make 7% w/v of PEG concentration, the solution thus 
obtained was allowed to stand at 4.degree. C. for 1 hour, and then the 
resultant precipitates were removed by centrifugation. To the resulting 
solution, PEG #4000 was added to make a final concentration 20%, the mixed 
solution was allowed to stand at 4.degree. C. for 6 hours, and the 
resulting precipitates were collected by centrifuging and dissolved by 
adding physiological saline to make 2% sIgA solution. The solution thus 
obtained was dialyzed with physiological saline and sterilized by membrane 
filtration. The yield of sIgA was 48% (Lot 3/T). Another preparation was 
also prepared by dissolving the precipitates which were obtained at 20% 
PEG and had not been treated by heating into physiological saline to make 
2% sIgA solution, dializing the solution with physiological saline and 
sterilizing by membrane filtration. 
EXAMPLE 4 
In the same manner as in Example 3, 2% sIgA solution was prepared by 
subjecting 10 L of human foremilk to the removal of fat and casein, 
dialysis, column chromatography with DEAE-Toyopearl.RTM. and 
Sulfate-Cellulofine.RTM., collecting sIgA fraction, precipitating sIgA by 
the addition of PEG #4000 to the fraction to make 20% w/v of PEG 
concentration, collecting the resulting precipitates and dissolving the 
precipitates with physiological saline. 
The sIgA solution thus prepared was divided to three aliquot portions. To 
one of the portions, tri-n-butyl phosphate was added so as to make 0.3% 
and Tween, a surfactant, 80 to make 1%. To one other portion, tri-n-butyl 
phosphate was added so as to make 0.3% and sodium cholate to make 0.2%. 
After the former portion was treated at 24.degree. C., and the latter at 
30.degree. C., for 6 hours respectively, PEG #4000 was added to the both 
portions so as to make 20% w/v to precipitate sIgA. The precipitates 
resulting from each of the both portions were collected and dissolved by 
adding physiological saline thereto to obtain both 2% sIgA solutions. To 
per ml of these solutions each, 0.5 g of sorbitol and 0.15 g of glycine 
were added and dissolved therein, and both of the mixed solutions were 
treated at 60.degree. C. for 10 hours. Each of the solutions treated by 
"heating as liquidized form" as mentioned above were diluted with 
physiological saline to 5-fold solution, PEG #4000 was added thereto so as 
to make 7% v/w, and then allowed to stand at 4.degree. C. for 1 hour, 
followed by removing resultant precipitates by centrifuging. To each of 
the solutions thus obtained, PEG #4000 was added so as to make final 
concentration 20% and allowed to stand at 4.degree. C. for 8 hours, the 
resultant precipitates were collected by centrifuging and dissolved by 
adding physiological saline thereto to make 2% sIgA solution, and the 
solution was dialyzed with physiological saline, followed by sterilization 
by membrane filtration. The preparation obtained by employing a surfactant 
with Tween 80 in the virus inactivation was designated Lot 4/T, and 
another one obtained by employing sodium cholate was designated Lot 4/C. 
One other portion remained of the 3-divided portions was dialyzed with 
physiological saline, sterilized by membrane filtration and designated as 
non-treated lot (Lot 4/N). The yields of sIgA from foremilk were 40% in 
Lot 4/N, 38% in Lot 4/N. 
TEST EXAMPLE 1 
There were prepared solutions each containing a 4 mg/ml of each lot of the 
sIgA preparations prepared in Examples 2 and 3, and each solution was 
mixed with 2.times.10.sup.7 of Escherichia coli strain NIH/J or strain 
b/8M. The agglutination images at 3 minutes after the mixing were compared 
each other. As the results, all of Lots 1/T, 2/T and 3/T showed the same 
agglutination images as those shown with Lots 1/N, 2/N and 3/N. While, in 
the case of a preparation which was made separately by subjecting to 
"heating as liquidized form" under adding no stabilizer at all and not 
subjecting to PEG fractionation, nearly 90% of the sIgA was denatured, and 
hence the sIgA solution showed no agglutination image with the 2 strains 
of Escherichia coli similarly to the contrast consisting of physiological 
saline only. 
TEST EXAMPLE 2 
Each Lot of sIgA preparations prepared in Examples 1 and 2 was subjected to 
determining neutralizing antibody potency to human rotavirus, poliovirus 
I, coxsakie B3 or A16 virus and echovirus. The results are shown in Table 
4. 
In the results, the Lots 1/T, 2/T and 3/T of present sIgA preparation 
exhibited the same neutralizing antibody valencies to these viruses as 
Lots 1/N, 2/N and 3/N which were not treated by heat. 
TABLE 4 
______________________________________ 
Neutralizing antibody potencies of 
sIgA preparations to each virus: 
sIgA* 
pre- Neutralizing antibody potency to virus** 
paration Polio Coxsakie 
Coxsakie 
Echo 
Lot No. Rota I B3 A16 6 
______________________________________ 
1/N 4 10 76 22 19 
1/T 5 10 56 27 19 
2/N 19 5 2 7 4 
2/T 19 4 2 7 6 
3/N 726 2 8 6 7 
3/T 670 2 8 7 7 
______________________________________ 
*Each sIgA preparation was used as 5 mg/ml solution made by diluting the 
preparation with a serumfree culture. 
**Neutralizing antibody potency was evaluated by maximum dilution rate 
necessary to neutralize 50% of 100TCID.sub.50 in each virus. 
TEST EXAMPLE 3 
Each solution containing 4 mg/ml of each Lot of sIgA preparations prepared 
in Example 4 was prepared and mixed with 2.times.10.sup.7 /ml of 
Escherichia coli strain NIH/J or B/8M-1. After 3 minutes form the mixing, 
agglutination images were compared with each other. As the results, both 
Lots 4/T and 4/C exhibited the same agglutination images as non-treated 
Lot 4/N. 
TEST EXAMPLE 4 
Each Lot of preparations made in Example 4 was subjected to the 
determination of neutralizing antibody potency to human rotavirus, polio I 
virus, coxsakie B3 or A16 virus and echovirus. The results are shown in 
Table 5. 
In the results, there was not observed large difference btween neutralizing 
antibody potencies of present sIgA preparation Lot 4/C and $/T and those 
of non-treated Lot 4/N. 
TABLE 5 
______________________________________ 
Neutralizing antibody potencies of 
sIgA A preparations to each virus: 
sIgA* 
pre- Neutralizing antibody potency to virus** 
paration Polio Coxsakie 
Coxsakie 
Echo 
Lot No. Rota I B3 A16 6 
______________________________________ 
4/C 203 1 33 1 1 
4/T 241 1 40 1 1 
4/N 298 1 48 1 1 
______________________________________ 
*Each sIgA preparation was used as 5 mg/ml solution made by diluting the 
preparation with a serumfree culture. 
**Neutralizing antibody potency was evaluated by maximum dilution rate 
necessary to neutralize 50% of 100TCID.sub.50 in each virus. 
Method of the Determination 
The determination of sIgA was carried out by a developed sandwich ELISA 
method wherein anti-secretory-component-antibody (produced by Igaku 
Seibutsugaku Kenkyusho Co., Ltd.) is employed as a solid phase and 
peroxydase-labeled purified anti-alpha-chain-antibody prepared by the 
method of Hashida S. et al. (J. Appl. Biochem. 6 56 (1984)) using 
anti-alpha-chain-antibody (produced by Bio-yeda; Daiichi Kagaku Yakuhin 
Co,) is employed. 
Infection valence of virus and neutralizing antibody potency to virus were 
calculated observing CPE caused with virus on a microplate employing the 
established cell line of MA104 originated from the kidney of rhesus monkey 
as cells for growing virus (Virus Jikkengaku Souron, Chapter 13, 1973; 
published by Marujen Co.).