saaplee extracted free rabbit reticulocyte Tracks 1 ani t M a rieftn A chain treated ANA incubated with and withaut aniline respectively. Traete a and 4 shows felon in treated IMA inofteted with and without oniline raspactivsly. Tracks ■ and • ahewo felon in 8-IT treated ANA with ond without aniline respectively, doaanotrotlnp the ANA aodification activity of the folonin has boon retained after dori vet lest ion.
15«
The method (described in Section 2:11) has been employed to
demonstrate that concentrations of conjugates shown to be toxic in the
cell*free assay (Section 4:8:1) can produce the diagnostic RNA modification
associated with gelonin activity (Figure 4:12). Tracks 1 and 3 from Figure
4:12 demonstrate the production of the diagnostic fragment (indicated by
the arrow) when ribosomes are treated with r i cin A chain and gelonin
respectively followed by treatment with aniline. Without the subsequent
aniline treatment, RNA from ribosome treated wi t h either rlcln A chain
(track 2) or gelonin (track 4) does not show the characteristic breakdown
fragment. This result strongly indicates that ricin A chain and gelonin
exhibit identical RNA modification activity, a n d that the cleavage of the
fragment from the modified RN A is dependent u p o n subsequent incubation with
a n i l i n e .
Gelonin after derivatisation with 2-IT also exhibits an Identical RNA
modification activity (track 5) which is not apparent without aniline
treatment (track 6). Conjugate material at a concentration equivalent to
the 1CS 0 (determined in Section 4:8:1) from fraction 51 of the blue
sepharose step (track 7), and from fraction 79 o f the chromatofocusing step
(track 8), also produced the diagnostic RNA fragment after aniline
treatment of toxin exposed ribosomes. This d ata shows that the inhibition
of protein synthesis observed with these fractions is concomitant with
specific modification of 28S rRNA which in turn, is associated with RIP
activity.
* ;8 ;3 A n a lY fl» of f l u a l i s t a u binding a c t i v i t y
a trifiln . B - « lo n ln
The binding activity of the various conjugate preparations were
analysed in a modification of the standard aslalofetuln radioimmunoassay
160
Mg/well of asialofetuln and Chen blocked vlch a 1% "marvel" solution in PBS
Co prevent non-specific binding. The wells w ere extensively washed with
PBS and the conjugates or ricln B chain then applied to defined wells.
After 2 hours at 25°C the wells were washed w ith PBS and t hen probed with
anti-ricin B cha i n antibodies which were detected with [ia*IJ radlolabelled
protein A. The extent of binding was determined by comparing bound [,a#IJ
cpm from a standard curve created using defined quantities o f ricln B chain
(Vitetta, 1986).
Since the major contaminant in the conjugate preparations was
unreacted B chain, the lectin activity of the conjugate cou l d not be
analysed using anti-ricin B chain antibodies. Instead, b o und samples were
probed with anti-gelonln antibodies which were shown to have insignificant
cross reactivity with rlcin B chain. Thus the binding assay was modified
to probe specifically for conjugate (B chain-gelonln) as it was assumed
that gelonin w o u l d only be able to remain b o u n d to the w e lls if coupled
with ricin B chain. Prior to binding, lactose was removed from the samples
b y dialysis. E ach sample was assayed in triplicate and counted three times
using a Mini Instruments type 6-20 7 counter to obtain a steady state
reading. The results are shown in Table 4:1.
Rlcin B c h a i n (control 1) probed with anti-gelonin antibodies and the
protein A shows that these antibodies do not react with r i cin B chain.
Therefore any counts detected when these antibodies are u s e d to probe any
conjugate fraction must be the result of binding to the gelonin component.
The use of anti-gelonin antibodies is therefore a valid means of detecting
bound conjugate, as it has been assumed that the gelonin component can only
bind to the asialofetuln coated wells when conjugated to rlcin B chain.
The second control comprises ricln B cha i n in which the sample has
been probed w i t h antl-B chain antibodies, b u t without the subsequent
protein A step. This reveals the level of background counts detected. As
161
correct for these counts. This was achieved by omitting the antibody step
(either a ricin B chain or a gelonin) and then determining bound counts.
With all three fractions, w hen the antibody step w a s omitted a background
count of about 8S0 cpm was detected.
W h e n an anti-gelonin antibody step followed b y [>9*I] protein A was
included, significant counts above background were detected in all three
samples tested, indicating that some gelonin (and therefore conjugate) was
bound to the wells. W h e n anti-ricin B chain antibodies were u sed as the
probe, the counts detected in chromatofocused fractions 78 and 79 were
approximately 1 0 fold higher than the counts obtained when using anti-
gelonin antibodies. This reflects ricin B chain contamination apparent
upon SDS - PAGE analysis of the chromatofocused conjugate (Figure 4:8).
After affinity chromatography using blue sepharose C L 6B the counts
detected when material f rom fraction 51 is probed w i t h anti-ricin B chain
antibodies is significantly reduced compared to the chromatofocused
fractions. This may indicate an increased conjugate purity after this step
(not apparent by SDS-PAGE analysis. Figure 4:10).
Analysis of this data has to be confined to a qualitative analysis of
conjugate binding. This is because it proved Impossible to produce a
quantitative curve of galactose binding activity directly since no
absolutely pure ricin B chain-gelonin conjugate was available. Points that
can be made from this data a r e :-
1. In this assay system, anti-gelonin antibodies did not significantly
cross-react with r i cin B chain and are therefore a valid probe for
detecting conjugate (i.e. gelonin S-S linked to B chain which in turn
162