Folia Paychiatrica et Neurologica Japonica Vol. 4, No. 1 EXPERIMENTAL STUDIES ON COMPLEMENT FIXATION REACTION OF JAPANESE B ENCEPHALITIS VIRUS, NIIGATA STRAIN BY Tar6 dtake Department of Neurology and Psychiatry, Niigata Medical College, Niigata (Director ; Prof. T. Konritnuro) While it is a matter of course that diagnosis of a Japanese R encephalitis case should depend chiefly upon the clinical observations, a great progress has been made in the field of serology recently, consisting of the immunization reaction tests classifiable in Neutrali- zation Test and Complement Fixation Recction Test Thereby, DS H. encephalitis caused by various kind of neurotropic virus presents clinical data extremely similar in feature, aetiological diagnosis upon causative virus with rcutine prccrss is often rendered difficult or even impossible. Such situation makes sercdiagnosis all too important and necessary. Since 1933 when the technique of sero-neutralization test was devised by LPslie T. Webster and George L. Fitel’, the neutrali- zation antibody is proved to persist in blood comparatively long time, its removal being not yet successful until several years tbereafter sj, while the complement fixation aqtibody is known removable from the blood comparatively rapidly. The diagnostic value of the latter is so far higher and more significant than that of the neutralization. This is.especially the case here as the land is considered a thoroughly infected lccality of virus. The Comple- ment Fixation Test of Jap. B encephalits was so early studied as a specific antigen-antibody reaction in this country; in America technique diviced by /. Casals has been widely employed since 1941. 6, i~8) Toward the end of the recent world war, the technique was introduced in this country and has been eaamined at the National Epidemic Research Institute and the National Institute of Health, thereby certain novel techniques having been i nriounced?) 10) 11) 12 Notes on the Niigata Strain Japanese B encephalitis, starting in the last epidemic in Tokyo __ -. __ __ These studies have been prepared by the aid of the Academy Received for Publication, December 10,1949 Research Council Fund.
Transcript
Folia Paychiatrica e t Neurologica Japonica Vol. 4, No. 1
EXPERIMENTAL STUDIES ON COMPLEMENT FIXATION REACTION OF JAPANESE
B ENCEPHALITIS VIRUS, NIIGATA STRAIN
BY Tar6 dtake
Department of Neurology and Psychiatry, Niigata Medical College, Niigata (Director ; Prof. T. Konritnuro)
While it is a matter of course that diagnosis of a Japanese R encephalitis case should depend chiefly upon the clinical observations, a great progress has been made in the field of serology recently, consisting of the immunization reaction tests classifiable in Neutrali- zation Test and Complement Fixation Recction Test Thereby, D S
H. encephalitis caused by various kind of neurotropic virus presents clinical data extremely similar in feature, aetiological diagnosis upon causative virus wi th rcutine prccrss is often rendered difficult or even impossible. Such situation makes sercdiagnosis all too important and necessary.
Since 1933 when the technique of sero-neutralization test was devised by LPslie T. Webster and George L. Fi te l ’ , the neutrali- zation antibody is proved to persist in blood comparatively long time, its removal being not yet successful until several years tbereafter s j , while the complement fixation aqtibody is known removable from the blood comparatively rapidly. The diagnostic value of the latter is so fa r higher and more significant than that of the neutralization. This is.especially the case here a s the land is considered a thoroughly infected lccality of virus. The Comple- ment Fixation Test of Jap. B encephalits was so early studied a s a specific antigen-antibody reaction in this country; in America technique diviced by /. Casals has been widely employed since 1941. 6, i ~ 8 ) Toward the end of the recent world war, the technique was introduced in this country and has been eaamined a t the National Epidemic Research Institute and the National Institute of Health, thereby certain novel techniques having been i nriounced?) 10) 11) 12
Notes on the Niigata Strain Japanese B encephalitis, starting in the last epidemic in Tokyo __ -. __ __
These studies have been prepared by the aid of the Academy
Received f o r Publication, December 10,1949 Research Council Fund.
63
City late in July 1948, i t soon spread over Kanto district, and suspicious cases appeared in Niigata Prefecture early in Augutit of the same yeir. 209 case^, mostly with serious clinical symptoms, have been recorded up to the middle part of September when the epidemic came to a standstill. The present strain was inoculated into the mouse brain from the autopsy material of a boy-patient who succumbed on 24th, August 1948 on the fifth day of ailment. Inoculation took place nine and half hours postmortem. The mice succumb now, in cases of intracerebral injection of brain emulsion with three, four days injection interval in four, five days regularly, in cases of nasal injection wih four, six day’s interval within seven, nine days.
The results of the neutralization tests upon recovering cases turned always poeitive, thus proving the strain the same with Nakayama strain in the light of the neutralization and complement fixation teat The injection of orderly dilution of virus suspension in the brain of 20 day old mouse brought LDm into lohom -&O (a -a I might nominate this virus the Japanese B Encephalitis, Nifgata Strain.
Complement Flxation Reaction
The effect of the neurotropic virus complement fixation test used to be always less specific compared with that of other types of viruses, firstly for the reason that no other antigen is brain- available, and secondly the techinique attaining specific reaction demands much skill. Since 1937, however, when Z.Casals and R. PaIaciosQ, CasaIsT, C. Es&ana and W . McD. HammonH), following B. Howit t ’9 improved preparation methbd of antigen and introduced suitable temperature in the blood serum inactivation, the procedure has grown facilitated in exact measurement of hemolysin, comple- ment and antigen and much more reliable.
As antigen material, though chicken embryos are sometimes employed, infected mouse brain proves highly useful, ita preparation relying usually on benzol procedure, high speed centrifugation, and freezing and thawing procedure?j’O~l*) of which I adopted the lastly mentioned one.
The nonspecific and anti-complement elements contained in antigen, i.c,infected mouse brain emulsion,are removed by repetition of freezing and thawing, the anti-complement elements in tbe test serum equally removed through keeping heated at a temperature proper to the animal species. Then the reaction between antigen and antibody gets apt to be carried out. Unit eelection of antigen and complement and the techniques were made largely b a d on Kifaoka’s method.
Preparation9
Washed Sheep Cel l Suspension ; Defibrinated sheep blood in being washed three times with 0.85 per cent saline adjusted into pH 7.2 in a centrifuge and subsequently a 3 per cent cell suspension is made of settled blood cell. This should be prepared at each instance of use as i t will be turned out unavailable for the next day ; defibrinated sheep blood, if completely aseptic and stored in an ice-box, is usable for several days.
Rabbit Anti-Slu?e&Cell Hemolysin ; While this is prepared by immunizing rabbit with washed sheep cell, there has not yet been any difinite method in the immunization. This time I extracted entire blood quantum when hemolysin value increased to more than 2ooo times, the separated sera was heated a t 65°C for 23 minutes, and then mixed with carbolic acid in a rate of 0.5 per cent and stored in an ice-box. In using i t should be diluted 90 that 3 units of hemolysin are contained in 0.25 cc.
Complement ; Blood mixture of more! than three guinea pig species is used as origin of complement sera, while baby-carrying specimens and those in bad nutrition are, owing to poor fixation value, generally unavailable. Seeming healthy specimens must be examined by the complement fixation antigen verification test, in- order to make i t sure that they carry antigen and not the anti- complement fixation in themselves. Before use, complement should be adjusted to contain 2 exact units in 0 . 5 ~ ~ By the way, a dry complement preparation has been produced here in Japan lately.
Immune Serum ; The procedure of preparing standard serum is as follows : 0.15 cx: of 10 per cent Jap. B encephalitis brain emulsion, previously centrifuged and clarified,is injected in the brain of guinea pig. the same amount being reinjected after ten days. After from 10 to 15 days, the entire blood quantum is extracted, sera separated. The sera, after being heated to 56OC for 20 minutes, is mixed with marzonin a t a rate of 1: loo00 and stored in an ice-box. This sera should give no complement fixation reaction to the brain antigen of uninfected mouse. The sera of B. encephalitis patients or of individuals vaccinated with Jap. B may also be available. Four units are employed for the measurement of the antigen value.
Antigen; 0.03 cc of the brain emulsion of about 2 days old mouse infected with Niigata or Nakayama strain is extracted after f u l l development of typical symptoms of the disease(al1owing from 4 to 5 days for it8 development),exactly measured and pounded thoroughly with mortar. 10 per cent emulsion is made, by mixing 0.35 per cent hypotonic saline added with previously inactivated serum of normal healthy guinea pig at a rate of two per cent. The emdsion is left standing one night or from 14 to 20 hours in an icebox of 2" to 4'C and centrifuged at a speed of 2500 rounds per minute for 30 minutes on the following morning. The supernatant
thawed in water of 37OC. After repetition of freezing and thawing five times, i t is centrifuged one hour a t a speed of 4500 rounds per minute. The supernatant thus obtained is almost transparent and red brown in color. Tbis original liquid antigen is preparatorily tested and, by use, should be diluted so wide as to contain 2 units in 0.25 w. Before storing in En ice-box, 05 per cent carbolic acid is to be added thereto. Any antigen portion more than 15 days of standing is to be discarded. On the other hand, in order to keep a control, an antigen is prepared according to the same procedure mentioned above out of the unadjusted mouse brain. Though a mixture of dry-ice and alcohol ordinarily used as a suitable medium for low temperature, as, in my instance, as i t was out of question to get sufficient amount of necessary material, I was able to attain the aim by fixing a simple device upon carbonic acid bomb such as used in laboratory for fropen sections. This proves that for preparing antigen and proceed in complement fixation test, dry-ice i s by no means an indispensable material so far.
Reading of the Degree of Hemolysia
Hesults of the test can bg -read readily, but the degree of hemolysis in the test-tube will be represented by means of numerals us follows.
4 ............... No hemolysis 3 ............... 25 per cent hemolysis 2 ............... 50 per cent hemolysis 1 ............... 75 per cent hemolysis T- ............... Hemolysis stain (J ............... Complete hemoly si s .
Among t k e . the grades from 4 to 2 am to present positive complement fixation reaction return, that is to say. the grades above 2 are estimated the end mark of positivenws.
Preliminary Test
Hemol ysin test : In order to measure the capacity of bemolysin, from a 100 times stock dilution with physiological saline are prepared various dilution grades as 1': 1O00, 1 : 1500, 1 : ZOOO, 1 : 2500,l: 3000, 1: 3590 and 1: 4000. Each portion of 0.25 cc is shaken up with addition of 0.25 cc of 3 per cent cell suspension and left standing for ten minutes in room temperature. As the next turn, 05 cc of 30 times dilution of complement and 0.5 cc of physiological saline are poured into each of the test tubes, while tube with hemolysin and serum suspension, tube with complement and serum suspension and tube with serum suspension and physiological sziIineare provided for control. Reading is made of the tubes as they stood in water
66
of 37°C for 30 minutes. (Table I ) The maximum dilution a t which hemolysis takes place completely is chosen a s 1 unit ; for subsequent use a 3 units dilution has praved appropriate.
Hemolyt ic System; Hemolytic system consists afi usual of a mixture of 3 units hemolysin dilution and 3 per cent sheep cell suspension of the same amount, shaken thoroughly and left standing in room temperature for 30 minutes.
complement fixation antigen is arrang2d as in Table 11. Only those which present no diverse Complement value in systems with resp. without antigen, a re employable as complement serum. (Table 11) Thirty times dilution of complement serum taken from more than three guinea pig specimens is put into a series of test tubes. each containing amounts of the dilution beginning with 0.1 up to 0.26 cc in gradation of 0.02 CC, thereupon filled with saline to 1 CC. After addition of 05 cc of hemolytic system, they are shaken up and left standing in water of 37°C for 30 minutes before reading. The minimum complement amount to cause complete hemolysis makes exact un i t , and dilution containing two exact units in 0.5 cc has been employed in later teats (Table 111). therefore, 2 full units employed in CasaCs' principle his surplus of 0.02 cc for each one exact unit of ours.
Antigen Test ; Now the first vital thing is the proof that there is no anti-complement action contained in antigen obtained, thus, as is to be shown later in Table VI, the results of two series of complement dilution, one containing and the other lacking of normal antigen, should come out just the same. The test is now carried out between two series of gradatory dilution of the positive serum whose validity of positiveness is still unknown, in one side and of the just approved antigen on the other. A regular reciprocity prevailing between them makes up " field *' of complement fixation reaction. One example of the results is reproduced on Table IV : the gradation of dilution of antigen p r s c d s by 1 : 1, 1 : 2, 1 : 4, 1 : 8, 1 : 16, 1 : 32, that of the positive serum of unsettled positiveness by 1 : 2, 1: 4, 1: 8, 1 : 16, 1: 32, 1: a, 1: 128. Now,O.Ecc of both dilution series is mixed together reciprocally, to which two exact units of complement, each in accordance to the procedure of complement fixation test. added (Table V). They are then brought in water of 37OC for 30 minutes, left a while in room temperature, once again exposed 30 minutes to 37OC with addition of 0.5cc of the hemolytic system, until the final reading. Hereby, a s a matter of course, examples of the normal antigen plus positive serum of unknown validity as well a s the serum with no additicn should be prepared as control, each exactly adjusted accxding to the formula.
I t is from this "field " that the unit of antigen validity is settled. First of all, the positive end mark of serum is found out on the
Complement Test; The procedure of the guinea pig senim '
m
series of 1 : 1 antigen pxtion, and the dilution grade on the mark is denoplinated as one unit to the given immune serum. Next, by tracing the series upward to dilution of multiple equivalent with 4 units of the e e m and then following the series downward, the positive end mark of the antigen is reached. The mark corresponds to one unit of antigen, where here two units are used practically.
From this combination, the antigen validity of antigen example and the antibody validity of serum will now become known factors, while serum of unknown validity will be employed as a standard serum in tests for other antigen.
Material Sera ; Material sera are necessarily inactivated previously, that is to say, 60°C for sera of human being, mouse and horse, 65‘C far rabbit and dog, 56OC for guinea pig, adequate t ime being 20 minuts for each instance. A s is already known, anti- complement action is rather often-found in a turbit or hemolytic sera.
Complement Fixation Test
Seven test-tubes are prepared for each one material serum, and the procedure is carried out as in Table V.
(1) To the 1st test-tube is poured 0.75 cc of physiological saline of pH 7.2, and from the 4th to the 7th is poured 025 cc of the same.
To the 1st test-tube is added 0.25 cc of the inactivated, tested serum, dividend portion of 0.25 cc is poured now into the 2nd, 3rd and 4th tube respectively and successively. As the next step, 0.25 cc out of the 4th tube is transferred into the 5th tube. The same continues until the 7th tube gets likewise diluted twofold, while the 055 cc out of the last tube is thrown away.
Through the above, dilution grade of the series is so adjusted as:Is t to3rd tube 1 : 4 ; 4 t h l : 8 ; 5 t h l : 1 6 ; 6 t h l : 32;7thl: 64,each containing 0.25 cc serum portion, where the 1st and the 2nd atand as the control serum and the control normal antigen respectively, beside the control serum containing 1.0 cc saline.
(3) To each tube is added now 05 cc of complement of 2 exact units under thorough mixing.
(4) 0.25 cc of Jap. B antigen containing 2 units is added to each tube, from the 3rd to the 7th, equal dosis of saline to the 1st (control serum) and the same of normal antigen to the 2nd (control normal antigen).
(5) Af te r mixing the tube is left in water of 37OC for %minutes. (In Casals’ original, the tubes stand 18 b u m in ice-box inatead of 37°C water. where interference of the anti-cornplement action tends sometimes to be observed. I t aecm~ that our metbod is more &- cient in saving time and effective.)
(2)
(6) Lastly, 0.5 cc of hemolytic system is added and well shaken up. After repeating the 37'C-30 minutes procedure the reading is made instantly.
In parallel with the above. secondary complement test is carried out in order to exclude anti-complement action in antigen, i f existent. A s is to be seen in Table V I , to two series of ten test tubes, theref ore, from the 1st to the 5th. and from the 6th t o the 10th. are poured 0.15 cc, 0.2 cc, 025 cc. 0.3 cc and 0.4 cc of the complement each, while to the first series is added now 0.25 cc of employed antigen each containing 2 units. Then all tubes of two series are filled with physiological saline up to 1 CC, naturally with three of contrd tubes each Containing employed antigen Jap. B, normal antigen and sheep cell suspension respectively. Reading must agree in both series with and without antigen.
Regularly, the control antigen should read 0, and the mntrol serum 4 and the minimum complement dosis for complete hemolysis should exactly correspond to one tube of 0.25 cc of conlplement dosis.
Reading of Resultn
1) The results read positive, when hemolysis fails in the Jay. B antigen tube and is complete in control normal antigen and controI sera tubes. Reading falls decidedly negative, when complete hemo- lysis (0) is observed in control tubes as well as in all the tubes from the 3rd to the 7th.
2) The antlbody validity is read by the maximum dilution grade of sera, thereby carrying amounts more than 2 in reading.
3) When sera reading falls 1 or t in the 3rd tube and 0 in the control tube, the whole must be retested, because here the used antigen is suspectable as positive
4j Certain sera falling positive with syphilis reaction may eventually fall also positive with this test
5) By diagnosis of Jap. B encephalitis, i t will beatlvisable to reestablish an increase in the antibody value through retesting of the same patient more than twice, preferably at a n interval of a t least 7 to 10 days.
Observation 1
I made survey tracing the positive return recorded in the romplement fixation test. utillizing centrifuged mouse brain emulsion as well as Jap. B encephalitis vaccine upon the test personnel with negative neutralization test and negative complement fixation test. Thereby place injection of 0.03 cc of the three weeks old mouse brain emulsion treated with Jap. B encephalitis Niigata strain was used. A-
69
foregoing, a 10 per Cent physiological saline brain emulsion was prepared from 3 weeks old moue manifesting typical encephalitic symptoms an3 injected subcutaneously or intpncutaneously. I also injected Jap. B enceplalitis vaccine jnto persons under the same pbysical conditions.
Virns Brain Emulsion Injection Cases; (Fig. 1, Fig. 2) Twenty four Cases make up the subjects of observation, of four which were discarded on the way and five were set apart for the special purpoae.
0.5 cc of virus brain emulsion was injected twice each five days to m e half of the rases subcutaneously on the brachium. and to the rest half, intracutaneously on the inside of upper forearm. Three of the latter cases got injection only once. The results fell out now as follows: of all. no positive case was recorded until five days after the first injec.tion:..hereafter, the date starts always from that of the first injection. whereas it .turned out positive almost all within ten days. Generally speaking, the intracutaneous cases turned J ositi\-e always one or two days later than subcutaneous ones, while the fornier reached in from twelve to twenty days and the latter in from 13 to 30 days theii highest antibody value each, thus acquit- ing of the highest antibody value differs according to application modus of sen The durability of positivity thereby starts cow to fall slowly in from 20 to 40 days, there being hardly any difference in duration and fading of the highest antibody value between both modi either. Four cases turned negative in from 55 to e0 days, two of which, when itemized, belonged to subcutaneous cases and the other two to intracutaneous ones in two settings. In other thirteen cases, the decline curve rose once again, thus marking valley of from 50 to 65 d a y s Still, the slope of risf stood more gentle than the original one, and the tip f a r below the previous one.
The decline of the height o f positivity. regardless of valley, pursued its downward course slowly to reach negativity at last in frdm 90 to 100 days. Every case was retested fortnightly and twice upon negativity, but always getting indicated for negative. The intraaitaneous cases in one setting turned all positive in some 100 days. Four subcuta~?eous cases in Fig. 1 and five intracutaneous cases in Fig. 2 are demonstrated with the trend.
Besides. in five cases injections went in four settings once a week with 06 cc, 0.5 cc. 1.0 cc and 1.0 cc of the virus brain emulsion sub- resp. intracutaneously. in order to ascertain the duration period. The positive phase fell as in the previous test, only with such even- tuality that in three cases there appeared suddenly a pit in the downward curve a t the fourth injection, only to go over to a sudden rise again. In one instance the rise went up so high that i t exceeded the highest of t te ultimate curve, though it slo~ed down pretty readily.
Generally sreaking. slope-down gces on so extremely slowly, that ther: was no case returning lower positivity than 1 : 8 dilution grade on the 100th day. Since that date, however, the fall was rather rapid a s in four cases i t turned negative in from 120 to 140 days, and a s the average i t was given 139 days.
Jap. B cnce$haliiis Vaccine Cases ; (Fig. 3) Ten cases came into consideration. Injection proceeded with 0.5 cc vaccine each five days in two settings. Positivity succeeded in from 6 to 9 days, therefore, quicker than in the previous cases, where of the highest
7 1
degree of antibody value was attained in from 10 to 20 days. Parallel-going with this, the negative turn took place in from 40 to 6.1 days, in one exceptional case in 35 days.
1 !? IS
Fig. 3. Jap. B virus vaccine (Injected subcutaneously)
From the above results, it may be: maintained that the complement fixation anti-body formation effected through brain emulsion injection of Niigata Strain virus by means of vaccine procedure may be a little delayed in completion, but it persists SO long as from 3 months t o 4 mouths. The vaccinated caw complained nothing but injection pa in Notes : The tendency towad positive t u r n is here r a t h e r definitive than in t h e animal cases,U),U'."),") par t ly because here t h e conditioning of norm was more s t r i c t and partly because t h e procedure was more exacted than in t h e la t te r cases. Besides, t h e r e in human cases has been laid much weight upon check-up of t h e clinical data. The fact t h a t there has been no f r i s t for t h e negative t u r n ascertainable subsequently, seems t o speak for a ra ther long persistence period of immunization.
Observation 11
I sucmeded in the summer of 1949, in ac.quiring sera from 28 cases among inhabitants in Niigata Prefecture and one case from Y d g a t a Prefecture and in testing upon Niigata Strain complement fixation besides certain discarded serum examples with respect to hemolysis r e p , anti-complement action.
Date of the completed complement fixation test from the onset of disease reads as follows :
1) Of 11 cases tested in from the 2nd to the 5th sick day ; 7 cases, fell negative, eacb 1 case positive in 1 : 8 dilution and in 1 : 16
72
dilution. 2 cases positive in 1 : 32 dilution 2) Of 4 cases tested in from the 6th to the 9th day : 3 cases
positive i n 1 : 8 dilution. one cases positive in 1 : 32 dilution. 3) Of 7 cases tested in from the 10th to the 15th day : 3 cases
positive in 1 : 8, 2 cases in 1 : 16 and 1 case positive in 1 : 64 dilution. 4) Of 4 cases tested in from the 16th to the 20th day : 3 cases
positive i n I : 32 and 1 case positive in 1: 64 dilution. In these cases the same sera came into use repeatedly. And
the above data tells that the complement fixation reaction remaim negative until the 5th sick day regardless of clinical development (Fig. 4', while in two cases it turned positive a few days later, thus the serum series, a s a whole, showing :i tendency to get accentuated in antibody value with the time.
By the way, i t should be noted that here the Nakayama strain as well as t h e Niigata strain were employed, the results having proved all the same in both media.
N e u t r a 1 i z a t ion test was made upon two 1 : 8 positive cases on the sixth sick day and of one 1 : 32 case on the seventh sick day in one and same setting, the results p r o v i n g n e g a t i v e . Thereby the comple- ment fixation anti-
Fig. 4. Complement flxation tes t
Jap. B encephalitis
body value kept rising afterwards still higher. N o t e s : The above data seem to conform with the clinical reports t o a certain grade.ln.IC13 Whether this is really the case or not, will be decided through retesting in some other day.
Observation 111
While sera of syphilitic should tend to fall positive in the Jap. B encephalitis complement fixation test, I came across to interesting fact in serum of general paresis cases.
By chance of the said test upon the in-patients of this clinic. in four among the five general paresis cases treated with malaria therapy the test fell positive (Table VII!. whereas, the test UPOR all of ten general paresis out-patient cases fell negative, seven
73
of which, bowever, tumed positive subsequent to malaria therapy (Table IX); That positive turn took place after the second fever attack in one caae and in the rest after the fifth attack. The turn entered in some casw ten to twenty-five days after recoveryfrom ten, to twdve attacks and persisted still positive six months after- wards.
In another instance, a trial of the said test upon people with history of malaria of tropical, of quartan and of tertian type proved all negative Again, in a trial of ten attacks upon cases with oompletely negative Jap. B encephalitis domplement fixation -ion, i t remained always negative through every stage of the test, though the ultimate outcome is still euetained. (Table VIII)
Judging f m the above. one can maintain that malaria fever, once encountered. prevents positive turn of the Jap. Benaphalitis compkrnent fixation test, while genera 1 paresis cases, hitherto negative, turns positive through malaria attack pretty readily. By the way, i t is deairable that retestings be repeated for a longer time before an ultimate judgment is set. Notes: Though there h e bem no report of the kind until now. the facts stand established t h t sera of general paresis cases with negative return t o our previous test turned positive subsequent t o malarial therapy against the f*t that thooe of malaria-infested non-syphilitics behave always negative toward the test. This may highly probably be of certain significance upon the said therapy just in connection with general paresis.
Notes on Devices Used
Dry-ice has long been looked upon as indispensable in preparing axitigenP)7)@1W11)=) but I found that through u ~ e of carbonic acid bomb it is eaey to produce freezing temperature a t right time and place.
By the reacdon reading of antigen-antibody complement fixation, I could attain by use of a constant thermo bath-tub in shorter hours results of lesser error under more constant conditions than by the overnight method ueing ice-box.
Conclusion
With regard to the present complement fixation test with Jap. B encephalitis virus Niigata strain as antigen, I reached to the following conclusion :
1) By the use of carbonic acid bomb in the case of antigen preparation through f r e a i n g and thawing procedure, an adequate and necessary freezing can be attained, consequently tbe teat can becarried out even where dry-ice is unobtainable
74
2) To carry out the complement fixation teat correctly and to get reliable results, it is imperative that the unit of complement, antigen or immunization sera must be precisely measured as well as exactly manipulated.
3) Cautions must always be given to outbreak of the anti- complement action under way. This can only be avoided by making preparatory tests or by arranging ample controls.
In Observation I, in which a freah brain emulsion deeply infested with Jap. B encephalitis virus was carried into 29 cases and the Jap. B encephalitis vaccine in substance into 10 cases, purporting to detect the complement fixation test in the sera of tbese cases :
The outbreak to positivity is not observable until the 5th day, but observable onward from the 6th to 10th day.
Positivity tends to be detected one or two days earlier by the subcutaneous virus injection cases than by the intracutaneous one
In the virus injection cases, serum antibody is seen persistent from 90 to 120 days long post injectionem, and in the Jap. B encephalitis vaccine cases from 40 to 54 days long.
4)
5)
6)
In Observation 11, in which the complement fixation teat was carried out with sera in early epidemic period, 1949 in Niigata Pref eciure :
In some rases the test remained negative until the fifth sick day.
In all the cases test turned to be positive after the sixth sick
7)
8) day, the antibody value going increasingly high.
In Observation 111, in which effect of malaria treatment on general paresis cases, especially onwhom previously negative upon Jap. B encephalitis complement fixation test, was considered, with ample controls :
9) The sera of general paresis cases turn to the paeitive bet- ween the 2nd and the 5th fever attack.
lo) Such sera present positive reaction after termination of malaria therapy continuously.
These facts seem to suggest that there exists certain agent causing in general paresis, syphilitic in nature, to present positivity toward Jap. B encephalitis complement fixation test specifically conditioned through fever-attack
Reference B
1) L. T. Webster & G. L. F i t e : Experimental studies on encephalitis. 1V. Specific inactivation of virus by sera from persons exposed
t o encephalitis, St.Louis type, 1993.1J. Exp. Med., Vol. 61, 411. 1935. 2)' S. Kasohara & others : Expertmental s t u d y on encephalitis. Tokyo
Iji-Shinshi. No. -. 669. 1937. 3). M.Malswmofo : Quantitative s tudy upon Neutralization reaction.
Jikken Igaku, Vol. 28, No. 1, 1944. 4) H. Fukumi : Measurement of neutralization exDonent. NiDDOn Saikin- _ _
gaku Zasshi, Vol. 2, No. 1. 1947. F.S.Salafrunca & L Esfi'rilu : Repor t on t h e presence of Jap. B encephalitis neutralizing antibody among Filipinos and certain Phi- lippine ani ,ds . Am. J. of Trop. Med.. Vol. 2F. No. 2. 1949 J.Casals & R. Palacios : Complement fixation t e s t in t h e diagnosis of virua infections of t h e central nervous system. J. Exp. Med. Vol.. 74: 409-426,1941. Complement-fixation in encephalitis and Rabies virus infection. Science. Vol. 93 No. 2047, 163, 1441. J.Casals : T h e technique and practical application of t h e comple- ment fixation wi th encephalitis viruses. J. Bact., Vol. 50. No. 1, 1-5, 1%. W. McD. Hammon & C. Espana : A simple method of producing control guinea pig immune sera f o r use with complement fixing antigens f o r ar throped-borne virus encephalitis. Proc. SOC. Exo. Biol.. Vol. 86. 113-115, 1947.
9)" M. Ashida : Study upon Jap. encephalitis complement fixation reac- tion. Nippon Seikin-gaku Zasehi, Vol. 2, No. 1, 1947.
lo)* M. Mafsumoto : Serological diagnosis of Japanese encephalitis. Kiso t o Rinshoh, Vol. 3, 63-68. 1949.
11)" M. KifaoRa : Serological diagnosis of Japanese encephaiitis. Saishin Igaku, Vol. 4. No. 8, 1949.
12). Technique of Jap. B Encephalitis Complement Fixation Reaction, by National Inst i tude of Health (mimeographed), 1948.
13). K . Uchigomo : Clinical observat ions of Jap. encephlitis.1948. Rinshoh Ihoh, Vol. 3, NO. 2, 23-24, 1949.
14)" I. Nishirra : Complement fixation reaction of Jap. epidemic ence- phalitis. Selikal Zasshi Vol. 58, No. 7, 1161-1182, 1939. Encephalitis virus of St. Louis type. i6id., Vol. 58, No. 8, 1352-1368, 1939.
15) B. F. H m Y t : T h e complement fixation reaction In experimental equine encephalitis, lymphocytic choriomeningitis and t h e St. Louis t y p e of encephalitis. Jour. of Immunol.. Vol. 33, 235-250, 1937.
* Published in Japanese
76
TABLE I H E M O L Y S I N TEST
I 2
3 4
5 6
7 _ _ _ . .
Hemolysin Complement Sheep Cell Suspension
05cc If
n /I
c
N
1.0 0.75
1.25
0
0 0 2
3 4
4
4
4
4
* 1 U n i t = I : B M 3 Units 11: 666 (March 11, '49)
TABLE I 1 C O M P L E M E N T T E S T
Tube No. Dilution of Jap. B 0.85 per Sensitized Judg- Guinea Pig Antigen cent Seep cell ment Sera( 1 : 30) Saline Suspension A B
I 2
3
4
5 6
7 8
. _ _
Control Sheep Cells
0.1 CC. 0.25 CC. 0.65 cc. I .{
0.2 055 8 1 0.25
0.1 0 0.15 0.85 ; 0.2 I
0.25 0.75
0. I5 I, 0.6 I E ,f
- -- 0.9 I ,
I P
0 0 1 .O
2 4
1 4 0 4
0 4
2 2 I 1
o c 0 0
4 4
Judgment ; A......Good for the Complement B......Unfit (April 16, '49)
n
I .
9 t;; s o 5 O 2 0 L.10 l a
c1
TABLE I11 U N I T OF T H E COMPLEMENT
Tube NO. Complement 0.E per cent HLolyt ic Judgment (1: 30) W i n e System
I 0.1 cc. 0.9 cc. 0.5 c c - 1 4 2 0.12 0.88 I/
3 0.14 0.86 I,
0 exact unit- 0 full unit
4
4 0.16 0.84 I
5 0.18 0.82 I
6 0.20 0.80 R
7 0.22 0.78 v
8 0.24 476 , 9 026
Control Sheep 0 Cello
0.74 -. .-
I,
.-
05
* In this case, an ezost unit hae met with in the 4th tube. In the following Jap. B encephalitis complement fixation tests, 2 exuct w n i t s per 0.5 cc. complement are used.
(August 20, '49)
TABLE IV S E R 0- ANT ICE N F I E L D
Dilution Control - _ _ _ _ - .
Normal 1 : 2 I : 4 I : 8 I : 16 I : 32 I : 64 I : 128 Sera Antigen
![INDEX [ ] ?· INDEX Diesel Engine, Propulsion ... Niigata Power Systems Co., Ltd. PIELSTICK, NIIGATA…](https://static.documents.pub/doc/80x56/5b6f21287f8b9af12d8be4dd/index-index-diesel-engine-propulsion-niigata-power-systems-co-ltd.jpg)
