Actividades en la escena del crimen La recogida de vestigios

Th e rabb i t hyper immune s e rum was prod u ced in adult

laboratory rabb i t s accord ing to the method o f Tan (1 9 7 0 ) .

D ay 1 - A s even day cul tu r e grown in l iqu id JS was

heat - f ix e d for t h i r ty minutes at 5 6 °C . The cul ture should c ontain not l e s s than 1 0 8

lepto s p i r e s per m l and four m l of the c u l ture were ino culated in to the rabb it through the e ar vein .

D ay 6 - The above procedure was r epeated .

D ay 1 2 - The rabb i t s wer e ino c u l ated with a s even -day

o l d , l ive cultu r e o f not l e s s than 1 0 8 lepto s pi r e s per m l through the ear ve in .

D ay 1 8 - The rabb i ts were anae s theti s ed with e ther and

then b l ed by card i ac punc tu re . The b l ood was let to c lot and the se rum s e parated by

centri fugat ion . The s erum was then s tored in

AP PEND I X V I

P RODU CT I ON O F THE I MMU NOGL OBU L I N FRACT I ONS

The an t ibody a c t i v i ty �n an t i s e r a � s predominan t l y c on t a ined in the gammagl obu l in ( I gG ) frac t ion and for th i s rea s on , th e I gG f r a c t ion wa s s e l e c t e d to b e u s e d as th e ant ibody

c omponent of the rad io immunoa s s ay . The me thod of extrac t ing the f r a c t ions i s by Wha tmans Advanc e d I on Exchange

Ce l lu l o s e s ( d i e thy l amino e th y l - 5 2 ) . *

a ) o f the DE- 5 2 c o l umn

Wh atrnans pre - swo l len d i ethylam i noe thy l c e l lulose ( D E - 5 2 )

i s chosen for the c o l umn . E i gh t y grams of DE- 5 2

c e l lulose and 5 0 0 m l o f 0 . 0 2M p h o sphate buf fer o f p H 8

wer e mixed . Th i s m i x ture has a pH of 1 0 and it i s

lowered to p H 8 by add ing 0 . 2M NaH 2 Po4 s o l u tion . I t i s

s t irred s lowly for f i f teen minute s . The c e l lulo s e i s s lowly poured i nto the c o l umn a n d then degas sed w i t h the

a i d of a vacuum devi c e . The c o l umn is run overn i g h t

w i th 0 . 0 2M phosphate buffer s o l ut ion . * *

b ) E l u tion of t he F r a c t i o n s

*

The co lumn i s f i r s t ly s tandard i s ed by runn i ng 0 . 0 2M pho sphate bu f fer s o l u t ion ( pH 8 ) through the column .

The samp l e ( 2 ml ) o f rabb i t hyper immune s erum to

s er ovar hardjo was c e n t r i f uged t o c l ear any debr i s a nd d i a lysed i n 0 . 0 2M pho sphate bu f fer s o lution overnight

be fore being run through the c o l umn . The f i r s t

f r a c t ion e lu ted wi th 0 .0 2M phosphate b u f f e r solution i s IgG . The other f r a c t io n s we r e obtained by elution w i t h 0 . 0 3M , 0 . 0 5M and O .lM pho s phate bu f fer solutions . * * T h e fract i o n s were p o o l ed and c oncentrated with the a i d o f po lye thy l ene g l y co l . O n c omp l e t ion , the co lumn was w a s hed with I M NaC l and f i nal l y wi th 0 . 0 2 M pho sphate buf fer s o l u t ion .

Appendix I I * * App end ix I I I

1 3 0 .

APPEND I X VI CONTINUED

c ) Conc entrating the F r a c t i on s

I nd iv i dual frac tions wer e poo l ed i n to d i a ly s i ng tubes

and p l ac ed i n a tray . They were then covered with

po l y e t hyl ene g lycol for about two hour s . Th e amou nt of

eac h f rac tion requ ired w a s approximate ly two to three ml .

d ) Exam i n at ion the Fra c t i ons for

Immun oe l ectrophores i s o n a gar ge l s was emp l oyed to exam i n e the immunog lobu l i n fr actions for any contamin­ ati n g o r exce s s prote i n . Each frac tion was p laced

separa te ly i n we l l s i n t h e gel , wh i l s t goat anti -rabb i t s er um ( M i l e s Y e d a ) * * wa s p l aced i n the troug h s . The goat a n t i -rabb i t serum ac ted as the ant ibody to the f ra c t i ons . On e l e c trophor e s i s , the frac t i on s and the ant i bo dy d i f fu s ed into the g e l f o rming pr e c i p i tin l i nes whe r e t he two met . The I gG fra c t i o n produced only one di s t i n c t l i ne of pre c ip i ta t ion wh i c h i nd i c a ted that

the f r a c tion was pure I g G and there fore c o n s idered sui t a b l e for u se as the antibody component of the rad i o i mmuno s say .

e ) Prot e i n Concentration o f the F r a c ti ons

* * * * *

The p r o tein c on centration o f the g ammag lobu l i n fraction wa s m e a sured u s i ng a spe c t ropho tome ter ( UN I CAM ) . * * *

The gamm_{ag lobu l i n f r a c t i o n was f ound to contain 1 0 mg}

of p r o t e i n per ml .

App e n d i x I I Appe nd i x I