Since several years, the author has been studying on the complement fixation reaction with various materials containing various pathogenic agents as antigens. During the course of his studies, he discussed numerous aspects to be considered for serologic diagnosis; especially, he pointed out the necessity of the considerations on the following:
1) the purification of antigens,
2) presence or absence of inhibitory substance for antigen-antibody reaction in the antigens,
3) the normal components other than components of lipin series,
4) considerations on the mode of reaction.
The author and his co-workers investigated the field dimension of antigen-antibody by means of the two dimension method, therefromm they pointed out that, with the presently prevailing method, that is a testing technique on a point or along a line of a fixed antigen-antibody ratio, any case which should demonstrate a positive reaction may come out as negative as the result of the effect of the so-called zone phenomenon. They also claimed that the judgement on the grade of antibody production should be made at the optimal ratio of antigen and antibody, because an erroneous judgement might be given in case it was estimated by the antibody titer of the serum.

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Fundamental experiments on biological aspects and physico-chemical properties of hemagglutinin of vaccinia virus were carried out. Inhibition of hemagglutination by specific antibody was also investigated, with special reference to its reliability as a diagnostic aid in small-pox case. The results obtained are summarized as follows.
A. Production of V. H. in fertile hen's egg
1) From 6 to 14 day egg was inoculated with seed virus through various routes and every organ was examined after 72 hours for presence of V. H. The most luxurious production of V. H. was observed in chorioallantoic membrane when seed virus was inoculated via air sac. The most adequate age of egg at the time of inoculation was found to be from 11 to 12 days. V. H. was located just around the inoculated area of membrane, whereas other part of the membrane than that although showing as much infectivity, contained little amount of hemagglutinin.
2) Tne chorioallantoic membrane of 12 day egg inoculated with seed virus via air sac showed a little rise of V. H. titer after 24 hours, reached to its maximal titer after 48 hours and then retained that titer for 72 hours.
3) It is necessary for V. H. production to use seed virus of higher virulence than minimum V. H. -producible.
B. Physico-chemical properties of V. H.
1) V. H. was partly destroyed rapidly at lower pH than 6.0 or higher than 9.0.
2) The optimal pH range for hemagglutination was 7.0 to 8.0.
3) Monoiodium acetic acid, sodium cyanide, sodium azide, cystein hydrochloride, sublimat, Merzonin (Takeda), * formaldehyde, benzaldehyde, hydrogen dioxide, potassium permanganate, phenol, urea, sodium sulphite, sodium thiosulphate or sodium sulphate hardly destroyed V. H. at the concentration of 5×10^-3 to 5×10^-5 M or 0.5 to 0.05%, even after treated at 37°C for 2 hours. Hydroxylamine was a little effective in lessening V. H. titter.
4) Methyl-bis-β-chlorethylamine (nitrogen mustard) destroyed V. H. almost completely at the concentration of 10^-2 to 7.5×10^-3 after treated at 37°C for 2 hours, whereas methylbis-β-chloretylamine-sodium-oxide did not destroy it in the same conditions.
5) Petroleum ether, ethylether, chloroform, benzene, ethyl acetate, toluene or butylalcohol showed no destructive function on V. H. at -6°C after 30 minutes.
6) V. H. emulsion was treated with petroleum ether, ethylether or chloroform and then left overnight at 2°C. Ethylether and chloroform were effective in V. H. destruction, and petroleum ether was less effective.
7) V. H. could be preserved with little change of its titer when kept in ice box after treated with monoiodium acetic acid at the concentration of 5×10^-4 or heated at 50°C for 30 minutes.
C. Relation between V. H. and vaccinia virus particle
Vaccinia virus particle, thermostable and thermolable components of V. H. were separated from each other by means of either differential centrifugation or ultrafiltration. Thus, it was proved that their molecular sizes are different from each other.

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Vaccinia virus preparations agglutinate chicken erythrocytes and this hemagglutination is specifically inhibited by antivaccinial sera. Since the inhibition of helnagglutinanoit by antisera may be one of the most sensitive reactions as a method for detecting the specific antibody, it seems probable that this reaction may be applied to the diagnosis for variola.
In the present study, we, first, investigated various factors having relations to the hemagglutination by vaccinia virus materials and its inhibition by specific antisera in order to look for the optimal conditions deciding the standard procedure, and then, searched the inhibiting antibodies in the sera of vaccinated persons or variola patients for the purpose of determining whether the hemagglutination inhibition is worthy of use for variola diagnosis.
The results were as follows:
I. Investigations of the factors relating to the hemagglutination and the inhibition
1) The diluting medium was one of the most important factors affecting the regularity of the reaction. The findings showed that 0.1% normal chicken plasma in physiological saline might be one of the most suitable diluting media to expect a regular reaction constantly.
2) Either testicular lapine preparations or suspensions of chorioallantoic membranes of developing chicken embryos infected with vaccinia virus were available as the hemagglutinin materials. The hemagglutinin titers of these preparations were about 5×10⁷-5×10⁹.
Cow lymph could not agglutinate chicken red cells.
When the virus preparations were stored in a refrigerator, their hemagglutinin titers remained essentially unchanged for a month, and when inactivated by treating with 0.05% formalin, 0.05% phenol, 0.01% merzonin (sodium ethylmercurithiosalicylate) or by ultraviolet irradiation, the titers were reduced only in a small quantity.
3) The differences in susceptibility to agglutinating effects of vaccinia virus materials were observed amongst individual chicken red cells. The optimal quantity of red cells was 0.125% in the final concentration.
4) The hemagglutination by vaccinia virus preparations was best shown at 37°C. The results could be read 1 1/2-2 hours after the incubation.
5) The available procedure of hemagglutination was carried out as follows. Serial dilutions of serum and 8 agglutinating doses of virus preparation were mixed, incubated at 37°C for 30 to 60 minutes, and 0.25% suspension of red cells being subsequently added. Then, the mixtures were again incubated at 37°C for 1 1/2-2 hours before being read.

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We studied the inhibitor gradient for Mumps-NDV-Influenza group by heat-stable mucoprotein-like substance (M-substance) from ox-bile. The M-substance is a sort of competitive inhibitor which is digested by various receptor destroying enzymes.
The inhibitory titer of this substance for hemagglutination of untreated MIN group viruses is highest for mumps virus (Enders strain), and lowest for NDV and when these viruses are heated at 52°, 30′, its inhibitory activity is increased for influenza virus, but not for Enders strain and NDV. When the viruses inactivated by ultraviolet irradiation, formalin or merthiolate, are used, the hemagglatination inhibitory titer of this substance is the same titer as the when the active viruses are used. By such treatments the viruses can not be indicator viruses.
These treated viruses have the same biological properties as active viruses, except that the infectivity are lost. After digestion of M substance by influenza viruses, NDV and mumps virus, the inhibitory activity of this substance against heated PR 8, is completely removed except that by mumps virus. In the same way, the inhibitory activity against untreated or heaten mumps do not be removed.
It is doubtful that mumps virus has the enzymic action for the M substance. It is suggested from these results that M substance is useful as an accessory method in differentiation of MNI group, but it will be necessary to do further investigations for the method mentioned to be applied to the practical purposes.

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Studies were made on the laboratory diagnosis of influenza and mumps, and the following results were obtained:
1) Serological diagnosis of influenza was made by hemagglutination inhibition test. There was observed a considerable difference in the antigenic specificity of each influenza strain, and so, it is always necessary to isolate viruses in each influenza epidemic.
2) Serological examination of mumps was also carried out by the hemagglutination inhibition tests. Mumps virus combined with its antibody extremely slowly as compared with the influenza viruses: It is necessary, therefore, that before adding to the suspension of red cells, the mixture of antigen and antiserum must be kept at 4°C overnight or at 37°C for one hour.
3) Hemagglutination inhibition test and complement fixation test were made on 18 pairs of acute and convalescent sera of mumps patients. Four folds or greater hemagglutinin titer was observed on 88.8 per cent of them. Furthermore was observed remarkable correlation of antibody titers between hemagglutination inhibition test and complement-fixation test.
4) The hemagglutination inhibition test and the complement-fixation test were performed several times on the sera of 4 mumps cases during the period of one year. Same tests were made on 158 samples of sera obtained from mumps cases. It was found that the presence of complement-fixation antibodies indicates the recent infection of mumps.
5) As an antigen for complement-fixation test, V-antigen (virus-bound antigen) and S-antigen (soluble-chorioallantoic membrane antigen) were used. However, it was not observed that S-antibody did considerably faster decrease than V-antibody from the blood stream, as reported by Henle and others.

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The hemagglutination inhibition test has been performed against the Japanese B encephalitis virus on sera collected not only from the patient (31 cases, 37 test samples) but also from the normal habitants (28 cases) in both Tokyo and northern part of Hokkaido and from the poliomyelitis cases (10 cases) in order to estimate its diagnostic value. The virus which was used as the antigen had been isolated from the case in the summer 1951 and was transferred brain to brain within the 10th generation.
The test was found to be specific for the Japanese B encephalities if more than 1:160 in the inhibition tier was read as positive, because the serum from the normal persons and the poliomyelitis cases showed the titer essthan 1:80. There is a certain correlation between the daily occurrence of the inhibition titer and that of the complement fixing antibody. The titer of the inhibition test became, however, positive at the end of the first week after the onset of disease, reached the maximum in the 3 weeks and began to lower in the 2 months. At any rate it becomes positive at the earlier stage of disease when the complement-fixation test proves still negative. In other words it seems very likely that the hemagglutination inhibition test is more sensitive than the complementfixation test.

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I. Properties of the hemagglutinin:
Experiments on the hemagglutination reaction by Japanese B encephalitis virus, described by A. B. Sabin et al, in 1950, were performed.
The results are summarized as follows.
1) All of six strains of newly isolated virus agglutinated erythrocytes of chicks from 24 hours to 3-4 weeks after hatching.
2) Optimal conditions for the reaction as to the method of extraction, the nature of the diluent, pH, temperature, the nature and age of erythrocytes, etc. were determined.
3) The relationship between infecting virus particles and hemagglutinating agent was studied by means of absorption, filtration through Seitz discs, high-speed centrifugation, chemicals, methanal precipitation, etc. There seemed to be no constant relation between hemagglutinating agent and LD50 of the infecting agent. It was assumed that the hemagglutionating agent was not the virus particle itself.
II. The hemagglutination inhibition tests with human sera and immune guinea pig sera.
1) One hundred and seven human sera of suspected encephalitis cases and 153 samples of immune guinea-pig sera were tested by means of hemagglutination inhibition test and complement-fixation reaction. The correlation index of these two reactions was 0.60 and 0.80, respectively.
2) Hemagglutination inhibition was hardly to be recognized during the first week after the onset of disease; the correlation with complement fixation became higher after the second week. H. I. test may be utilized as supplemental method for the diagnosis of was Japanese B encephalitis.
3) Removal of normal inhibitor by treatment of sera with chloroform as reported by earlier worker was not confirmed.
4) Small scale experiments with immune guinea-pig sera did not show close parallelism between H. I. test and virus neutralization reaction.

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[in Japanese]

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I tried the hemagglutination reaction with Nakayama, G. I. and two other strains of virus of Japanese B encephalitis according to the method of Sabin and Buesher (1950) and found the virus agglutinated the chick red cells in proper conditions which were strictly limited specially in pH and temperature.
The non-specific inhibitory action of normal animal serum to hemagglutination was highly potent. I found amylalcohol-carbon tetrachloride was effective in removing this inhibitory action. No inhibitory action was found in the spinal fluids of normal persons and patients. In cases where Japanese encephalitis was present the inhibitory action was pronounced.
Specific inhibitory substance to hemagglutination appear in the serum and spinal fluids of Japanese encephalitis patients, though the titre of the spinal fluid was found to be lower. The inhibition titre increases during the hospitalization of the patient.

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1) Guinea pigs have been generally used for poducing positive sera for use with complement fixing antigen for Japanese B encephalitis. But cats are superior to guinea pigs for this purpose, particularly in point of view of large volume of sera and of ease in breeding.
2) Japanese B encephalitis virus has been isolated from the brain of Toyokawa who died from Japanese B encephalitis in 1951. This virus is not different from the virus of Nakagawa strain of Japanese B encephalitis in the complement fixation reaction.
3) The patients of Japaness B encephalitis, whose sera are positive in the complement fixation reaction, are very few in 1951 and the dates of the onset of the disease are later in 1951 than in 1949 and 1950.
4) In 1951, in Kyoto Prefecture, the dissemination of Japanese B encephalitis virus in human beings and in guinea pigs is much rougher than in 1949 and in 1950.
5) High titers of neutralizing antibodies are demonstrated in the sera of many normal human beings in 1951 in which no complement fixing antibodies are demonstrated.
6) All titers of complement fixing antibodies of sera of patients, who falled in Japanese B encephalitis in 1949, are 4 to lower than 2, when they are bled and tested in 1951.

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Much higher spinal cord concentration of poliomyelitis virus can be obtained by using intraspinal inoculation technique of Habel and Li than by intracerebral technique. A complement fixing antigen was developed, using CNS tissue obtained from adult mice infected with this technique. This antigen produced a specific reaction with the sera of mice immunized with Lansing strain of poliomyelitis virus.
The antigen and the immune serum of Lansing strain showed no cross reaction in complement fixation test against the antigens and the immune sera of Theiler's encephalomyelitis (TO strain). Japanese encephalitis (Nakayama strain), St. Louis encephalitis, Negishi strain, and lymphocytic choriomeningitis viruses. Furthermore, Lansing strain showed specific reaction in neutralization test with the immune serum of Lansing strain only, without reacting with the immune sera of any other neurotropic viruses.
Two sera obtained from the cases suspected of nervous diseases fixed complement in the presence of Lansing strain antigen. Besides, their convalescent sera exhibited, following the elapses of time, an increase in the titer of neutralizing antibody against Lansing strain. As to whether or not the serological result and the clinical diagnosis for poliomyelitis coincide each other, or if coincide, to what grade, will be discussed with further investigations.

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