Note: Descriptions are shown in the official language in which they were submitted.
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sEHRINGwERKE AXTIENGESELLSCE~FT 92/~ 031 - Ma 940
Foreign text
Process for the puxification and concentrakion of rubella
virus
The invention relates to a proce~ ~or the purification
and concentration of rubella virus for diagno~tic and
therapeutic purposes by rever~ible binding to natural or
synthetic particles which carry receptor~ for the viru~.
Proce~ses have already been di~clo~ed for the i~olation
of, for exa~ple, rubella antigen from th~ supernatant
~rom infected animal cells, ~uch as, for example, the
i~olation by ultracentrifugation, ~hich tak~ place
either as pelleting or a~ di~ferential centrifugation by
mean8 of a density gradient (A. Paris-Hamelin et al.,
~. Virol. Meth. 10, 1985, 355 - 361). Other processe~ use
ultrafiltration through filter areas with different
eparation properties or precipitation proce~e~ ~uch as,
for example, those with polyethylene glycol (P~G)
(D.S. Bowden et al., J. gen. VirolO 65 (1984), 933 -
943)-
It ha~ also been di~closed how to co~centrate and purify20 influenzaviru~ using erythrocyte~ (B. Giesendorf et al.,
~iru~ Re earch 1, 1984, 655 - 667). Ths elimination takes
place in thi~ case enzymatic~lly with the aid o~ neur-
ami~ldase.
The known ~olutions for the purification of rubella
antigen have, however, inter alia the disadvantage that
the viru~ antigen obtained in this way can be contami-
nated to a greater or le~er extent with cellular pro-
teins and/or other cellular constituent~.
Furthermore, components o~ the ~etal calf serum (FCS~
used ~or the cell cultivation o~ten occur a~
co~tamination.
The object of the invention was therefore to find a
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~imple process in which the yield is improved and the
impurities are minimized.
It has been found, surprisingly, that human or ani~al
erythrocytes (which differ markedly fro~ viruse~ in their
density and their size) also under certain conditions
reversibly bind rubella virus, the re~ulting complex of
cell and virus can then be removed ea6ily by 8uitable
proce~ses from the medium in which the virus has been
repliaated, and then the virus can under the condition~
according to the in~ention be eliminated ~rom the
erythrocytes and, where appropriate, further concen-
tra~ed.
The process according to the invention can also be u~ed
for the puri~;.cation and concentration of those virus
constituents which reversibly bind to erythrocytes undsr
the condition~ according to the invention.
The term viru~ for the purpose o~ this invention also
includes these virus constituents.
The invention therefore relate~ to a proce B for the
puri~ication and concentration o~ rubella virus and virus
particle~ by re~arsible binding to natural or ~ynthetic
particles which carry receptors for this ~irus, where the
~iru~ particles are iæolated ~rom the u~pension, and the
~irus i5 detached from the particles and subsequently
isolated, where ~he binding of the ~irus takes place in
the pre~ence of doubly charged ions, and the release
takes place in thé pre~ence of a chelating agent ~or
doubly charged ions.
A pre~erred proce~s in thi~ connection is one in which
the doubly charged ions are Mg2+ and/or Ca2+.
The pre$erred proce~ is alRo one in which the chelating
agent is EDTA.
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An advantageous process is one in which the particle i~
an erythrocyte.
For the purification and concentration in thi~ case, for
example, the virus-containing supernatant from infected
cells i8 adjusted to a p~ of 4 ~ 10, pre~erably 6 - 8,
particularly preferably about 7. Methods suitable for
thi~ are known to the skilled worker. The temperature
during this i8 0C to 30C, preferably 0 - 10C~ very
preferably about 4C.
The salt concentration in the solution which contains the
viruse~ is moreo~er adjusted so that, at the time o~ the
binding of the viruses to the erythrocyte~, it contain~
in respect of Mg2+ 4 x 10-4 to 0.4, pre~erably 10-3 to 10-2,
particularly preferably 3-5 x 10-3 mol/l, and in re~pect
of Cà2~ 3 x 10-4 to 0.4, preferably 10-3 to 10-2, partiau-
larly preferably 2-4 x 10-3 mol/l.
Chlorides are preferably employed as compounds.
The proce 8 according to the invention can be carried out
with in each case one type o~ ion~.
Both ion~ are preférably employed, and very pr~ferably in
approximately the ~ame molar co~aentrations.
Erythrocyte able to bind rubellavirus are added to the
solution described abo~e. Erythrocytes ~rom a human,
! guinea pig, pigeon, wether, ~heep, rabbit or hor e are
used advantageously.
The concentration o~ th`e erythrocyte~ in~this case i~
0.04 - 4% by Yolume, preferably about 0.4% by ~olume.
The i~cubation is carried out ~or 1 min - 48 h, pre~er-
ably 20 min - 4 h, ~ery preerably about 1 h.
A~ a~vantageou process i~ also one in which, for reason~
o~ economy of work, incubation i9 carried out 'lo~er-
night".
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The complex of virus and cell is removed by ~ui~able
method~. Centrifugation and filtration proces~e~ known
per se to the skilled worker are particularly advanta-
geou~ for thi~.
To eliminate the virus, a complexing agent which com-
plexe~ doubly charged cations i6 addedO Complexing agent~
of this type are known per se to tha skilled worker and
pre~erably used, beside~ EDTA, are biodegradable complex-
ing agent as disclosed, for example, in EP 0 488 168.
The complexing agent concentration required for the
eli~ination i~ advantageously about 3 x 10-2 to 3 x 10'3
mol/l and very particularly ad~antageously about 7 x 10-3
mol/l. The elimination and elution advantageously take~
place at a pH of 6 - 12, particularly ad~antageously at
a pH of about 9.
Since erythrocyte~ and virus di~er greatly in ~ize and
density, they can subæequently easil.y be separated ~rom
one another by suitable methods known per ~e to the
skilled wor~er, and thi~ ~eparation advantageously takea
place by low-speed centrifugation, in which ca~e ~he
erythrocytes ediment and the virus remain~ in the
~upernatant. The op~imal centri~uga~ion conditions can
easily be determined experi~entally where appropriate.
It i~ al~o ad~antageous to employ the process in a column
- 25 proceR~ which includes the following ~tep~:
a3 preparation of a colum~ with ~uitable erythrocyte
b) addition of the viru~-containing ~olution taking
into account the required ionic conditions,
whereupon the binding to the erythrocytes takes
place, -
c) washing of the colu~n and ~ub~equently
d) elution o~ the viru. or of the ~iru~ constituents by
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addition of the elution bu~er.
After the disclosure o~ the process according to the
invention it is easy for the skilled worker to optimiza
a column process of this type.
Variations which are traceable to the pre~ent inventi~e
principle are conceivable for the ~aid proce~s step
Thus, for example, the cellular virus receptor can be
used after isolation, for example ~rom the erythrocytes.
The virus receptor can in this case be coupled to sedi-
mentable plastic particles or magnetizable carriers.
A viru~ receptor o~ this type can also be prepared in an
alternative way, ~or example by genetic manipulation,
The advantage o the present prccess i~ that the ~irus i8
concentrated and, at the same time, unwa~ted contaminan 8
are remo~ed with simple means.
The ~ollowing example is i~tended to illu~trate the
i~vention but not to restrict it.
~xample:
Material required
- 10 l of crude rubella viru~ ~uspen~ion
- immobilize~ wether erythrocytes
- 2 N HCl
- 2 l PBS, pH 7.0 with
O . 1 g/l MgCl2 ~ X 6 H2O and
0.132 g/l of CaCla x 2 H2O
- 0.2 1 PBS, pEI 7.5
- 0.5 g EDTA
Ad~orptio~
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10 1 of crude rubella virus 6u~pension were cooled to 4C
and adjusted to pH 7 with 2 N HCl. 160 ml of erythrocyte
~uæpension (25% by volume) were added; this correspond~
to a final concentration of 0.4% by volume. The mixture
was ætirred with a stirrer at 4C ~or l - 16 h. The
suspension was centrifuged in 1 l centrifuge cups in a
Cryofuge 8000 (Heraeus, Germany) at 4C and 2700 rpm for
1 0 ~rli~ .
After the centrifugation, the erythrocyte pellet-was
resu~pended in 1000 ml of PBS, pH 7Ø A second
centri~ugation wa~ carried out undex the abovementioned
conditions, and the ~upernatant was decanted off.
Preparation of the elution buffer
0-25% EDTA (ClOHl~N2O8Na2 2H2O; SERVA, Order No. 11280)
was added to P~S p~ 7.5 without Mg2~/Ca2~ and completely
dis~ol~ed. The ~olutio~ was ~ub~equently titrated to pH 9
with 2 N NaOH and cooled to 4C in a cold room.
The elution buffer i8 prepared fre~hly each time where
poesible.
The erythrocyte pellet obtained by the aboveme~tioned
proce~ wa cautiou~ly u~pended in 1~100 of the volume
o~ the crude viru~ suspension u~ed (10 1 ~ 100 ml) of
elution buffer and gently ~tirred at 4C for 15 min.
The su~pen~ion waæ centri~uged in a Cryofuge 8000 at 4C
and 3000 rpm for 10 min. The supernatant contained the
large~t part of the ~irus employed and wa~ called "eluate
1". Eluate 1 was centri~uged once more to clarify ~rom
remaining erythrocyte~: 4C, 3000 rpm, 10 min.
- The erythrocyte pellet was eluted once more under
identical conditions~ this re~ulted in "eluate 2".
The eluates can be pooled or further processed
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separately.
The purificatlon was checked inter alia by employi~g an
immunoblot quantification. This entailed the proteins
being fractionated in a reducing SDS polyacrylamide gel
(Laem~li, U.K., Nature 227 ~1970), pages 680 - 685), th~
proteins being transferred to nitrocellulo~e (sowen~ B .
et al., Nucleic Acids Re~. 8 (1980~, page~ 1 - 20) and
the protein band being detected by the following detec-
tion system which i~ know~ per se to the skilled worker,
employing rubella i~munoglobulin, biotinylated anti-human
immu~oglobulin and peroxida~e-labeled strepta~idin.
The quantification of an immunoblot of this tXpe using a
~canner i~ depicted in Figures 1 and 2, where 1, ~2 and
C identify structural protein of rubella virus. PEGAg
(Fig. 1) i~ material processed according to the prior art
(Bowden et al. (1984)) and EryAg (Fig. 2) i~ matsrial
purified by the process according to the invention.
A 2D-lD video den~itometer (Biotech/F:ischer Software) wa~
employed for the sca~ning (peak hsight: 5, i~tegration
20 resolution: 100%, peak width: 2, graph length: 15).
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De~criptio~ of the igure~
Figure l: Immunoblot quantification of a rubella antigen
obtained by PEG precipitation.
Figure 2~ Immunoblot quantification of a rubella antigen
i~olated by the process according to the inven-
tio~.
El, E2, C: Structural proteins of rubella virus
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