Note: Descriptions are shown in the official language in which they were submitted.
10644ZS
The pre~ent invention relates to a process for obtalning
pure protein or protein mixture3 ll~ked by covalent bonds to
organic compounds and called proteln "con~ugate~".
Since the protein conjugate to be obtained differs from un-
desired con~ugation product~ and from the organic compounds
u~ed ~or conjugation in its electrophoretlcal properties, the
proce~s ~or obtaining the de~ired compositions i~ based on a
preparative electrophoresis of the abo~e mixture, carried out
after the con~ugation reaction~ and o~ a sub~equent separation
of the zone contalning the desired compo~ition~ from those
zones containing undesired protein con~ugates and free organic
compound~.
Thi~ i~ventlon e~pecially relates to a process for the
purification of proteln-dye~tuff conjugates, preferably of
antibody-containing immune sera or protein fraction~ that have
been reacted by con~ugation with dyes, especially with flu-
orescent dyes, for example fluore~cein isothiocyanate, by means
of preparative electrophoresis.
Thi8 inventlon further relates to protein conjugates that
have been purified according to this process and to their use
in the immunological diagnosis.
Due to the el~ctrophoretical properties of the starting
materisl and o~ the reaction products, the process of the in-
~ention permlt~ the ~epar~tion of the pure protein con~ugate
having a certain degree of reaction from unreacted protein and
~rom the organic molecule used for con~ugation, provided that
the~e three component~ have a different electrophoretic
mobillty.
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10644ZS
Protein-dyestu~f con~ugates, especially lmmune sera con-
tainlng antibodies and ~on3ugated with fluorescent dyes, are
particularly important in the so-called immunofluorescence
technlque, as will be shown in the ~ollowing without limiting
the invention to th~sa examples.
The immu~ofluorescence technlquet reported by A.H. Coons
in 1942, permlts a comb~ned in~e~tigation of serologic-immuno-
logic specificity and of the morphological characteristics of
; the material to be analyzed. This method i~ based on the so-
called ~ntigen-antibody reaction, in which one of the two
lmmunologic compon~nts - generally, the antibody - i8 con-
~ugated with a fluorescent dye~tu~f. The anti~gen-antibody
roactlon ~ompound i~ made vlsible in the microscopic preparation
by provoklng the visible fluorescence by means of rays in the
ultravlolet range.
The immunofluore~cence technique has acqulred great
signi~icance in the diagno8is for identlfying linked and free
antigen~ and antibodie~
According to the s~te of the art, fractions with a high
concentratlon of antibodie~ are reacted as so-called immune
sera with fluorescent dyes, preferably with fluorescein iso-
thlocyanate. The reaction product is then predominantly
purlfied by gel flltratlon and by ion exchange chromatography
- on dlethyl-aminoethyl (DEAE) cellulose, whereby the fluorescein-
conJugated immune globulins are i~olated.
Tho~e know~ methods have the drawback that the separation
of the dyestuff, which is not linked by a covalent bond, from
the fluorescein-con~ugated protein composition by means of gel
29 ~lltratlon or dlaly~is 1~ incomplete; in particular, a dyestuff
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OE 74/~ 011
1064425
which is linked to protein by adsorption or by a weak covalent ~-
bond that can relatively ea~ily be split up by hydrolysis, is
not eliminated. ~oreover, a dyestuff which reache~ a high
molecular weightD owing to autopolymerization or autocon-
den~ation, i8 also eliminated to an unsatisfactory extent only.
Compositions containing such free dyes lead to unspecific
fluorescence Coloratlons in the~microscopic preparation and
thus to false results.
The widely used prior art process for obtaining con~ugated
protein fraction containing antibodies by means of ion
exchange chromatography on DEAE cellulose generally provides
only the antlbody fractlon belonging to the IgG class. Under
the conditions applied for elution and separation, the an~i-
bod1es of th~ IgM and IgA cla~ses are separated only unsa~is-
factorily or are not separated at all, ~o that the preparation
obtained partly shows a substantial 108~ of the desired specific
antibodies. When modifying the elution conditions suitable
for the recovery of fluore~cein-con~ugated IgM and IgA anti-
bodios, so-called "overlabeled" ~-globulins as well a~ baso-
phllic proteins are also eluted. Owing to their strongly acid
nature, these "overlabeled" protein fraction~ lead, in the
immunologlc reaction, to an un~pecific linkage to a number of
basic proteins in the matorial to be examlned and thus again
to a misinterprotation of the ~luorescence results.
The proces~ of this inventlon avoids tho~e drawbacks. m e
eloctrophoretic purificatlon of dyestuff-con~ugated protein
preparatlons, preferably fluoresceln-con~ugated proteln pre-
paratlons, carrled out preferably ln an inert carrler material
29 aiter the con~ugation reaction, offers the following advantages:
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HOE 74/B 011
10644Z5
1. The process permit~ an optimum rec~very of "acGurate" zone~
containing antibodie~. Undesired protein fractions that are,
for example, free of ant$bodies can eas~ly be eliminated
together with the separation of the de~ired fraction.
The yield of the dye~tu~f-conJugated protein preparations
containing antlbodies i~ optimum. In an exRmplified case
of a fluorescein-labeled ~-globulin fraction, 70 to 90 % of
the amount used are reco~ered.
2. The con~ugatlon reaction of proteins with acidic organic
molecules reduce~ the equipstential point~ in all the proteins
reacted wlth the organic molecules, 80 that under the electro-
phoresis condition~, the reaction products migrate toward
the anode at a higher speed than non-con~ugated proteins.
There i~ no difficulty in eliminating proteins which have
become too acidic owing to an intensive reaction with the
molecule used for conJugation, since these migrate toward
the anode at a higher speed. This ~inding iq especially
significant with the proteins that are con~ugated with
fluorescein isothiocyanate a_ the acidic fluorescent dyestuff
(qulnoid sy tem with a ~ree carboxylic group).
3. The proces~ of the lnvention ensures the optimum separation
of the acidic organic molecule, which is not linked (or
llnked by ad~orption) and which, if present in the fluores-
celn-con~ugated compositions, lead~ to undesired, unspecific
fluorescence colorations. The reason for thiq easy elimin-
ation of thi~ dyestu~f portion is that the acidic molecule
itself has a ~trong tendency to migrate toward the anode.
The electric potential established under the conditions of
; 29 the preparatlve zone electrophore~is cau~es the molecules
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1064425
which are relatively loosely bound to protein to be split of~
and enhànces the quality of the protein con~ugate~ recovered
by electrophoresi~. In the case of protein~ conjugated with
fluorescent dyos, the stabllity of ~uch preparation~, when
~tore~ in an aqueou~ ~olution, i8 lncreased in comparl~on with
those obtained according to the state of the art.
4. When antibodie3 are used as proteins for the con~ugation,
the process of the inventlon allows all the dyestuff-con-
~ugated antibody classe~ to be obtained, for example IgA,
IgG, IgM. This is of importance especially for the use of
antibodies obtained from sera, for example of man, goat,
sheep and hor~e, in which-besides the antibodies of the
IgG class-al~o antibodies of other immunoglobulin classes
account for a relatively hlgh percentage of the tdal anti-
body amount. This factor 18 also of a substantial economic
interest since antlbody-containlng sera are available to
only a limited extent.
- U~ed for the purificatlon of antibodie~ con~ugated with
fluorescent dyes, the proce6s of the invention brings about
~, 20 an increa~e in the relationship between specific antibodies
(Ab) and total protein (P), the so-called Ab/P quotient,
which again intonsifies the desired specific fluore~cence
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of~ct.
5. Tho proce~s, which ¢an be operated without using expensive
chromatographical ad~uvents, results in a substantial cost
reduction for the manufacture of pure protein con~ugate~.
~; The inert carrier material used may be employed ~everal
^ times.
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10644Z5
The preferable electrophoretic method used is the carrier
electrophore~is, in particular the electrophoresis u~ing the
inert carrier material in a horizontal container. When, for
example, polyvlnyl chloride granules are used as the carrier
material, it i8 possible after the ~eparatlon of the fluores-
celn-con~ugated proteln fractions containing the antibodies
to cut out the desired preparation in accordance with the
visible bands and then elute it from the carrier by means of
slmple solvents.
When the con~ugate i8 uncolored, the positions of protein,
reaction component and proteln con~ugate can be established in-
directly, for example by means of a paper dabblng which is then
colored in ~nown manner.
In additlon to polyvinyl chlorlde, for example polyacryl-
amldo, cellulo~e, starch, glass beads, sand and the like are
frequently used as electrophoretic carrler materials.
Electrophoresls equlpment used accordlng to the lnvention
also include vertical arrangements uslng carrier material or
not. Very good puriflcatlon effects are also obtained using
contlnuously operatlng equipment.
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The process of thi~ invention ~eparate~ antibody preparations
of any origin, whlch have been linked by covalent bond to dyes
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o~ acidic nature, especially to fluorescein lsothioc~snato.
It is, however, also possible to react non fluorescent
~ 25 dye~ wlth proteln~ and to separate the proteins colored by the
; conJugation reaction accordlng to the proces~ of the invention,
provlded the dyes u~ed 8~0W different electrophoretlc migration
properties.
29 In~tead of antibodies, other proteins o~ animal, vegetable
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HOE 74/B 011
10644Z5
and microbial origln may also be used, if they exhibit a~ con-
~ugates an electrophoretical property that differs from that
of the starting protein and of t~e organic molecule used for
the conJugation. Organic molecules preferably used for the
con~ugation are compoundæ having their equ~potential points
within the acld pH-range.
The essential feature of this invention reside~ in applying
preparative electrophoretic methods for the manufacture o~
pure protein con~ugates, especlally of gamma-globulin fractions
reacted with fluorescent dyes. The reaction of ~-globulin
fractlons with eonventional fluorescent dyes is not expected
to yield an electrophoretically homogeneous product, since the
structure~ of the immunoglobulin classes used for the reaction
are known to be diiferent and rather have a different number of
amino groups available for the reaction. Although electro-
phoretlcal methods are known to provide relatively uniformly
mlgratlng zones of unroacted ~-globulina, the uniform migration
of immunoglobulins reacted with dyes and belonging to different
classes has to be regarded as a surprise. Moreover, the dyes
linked to protein molecule~ by adsorptlon could not be expected
to be ~eparated from the protein in an electric field. Finally,
owing to the known intense linkage of fluorescent dyes to ion
exchange material aa used in the state of the art, it could not
be foreseen that the free dyestuff used in an electrophoretic
method? especially together with polyvinyl chloride as a carrier
material, migrates over a particularly long di3tance as com-
pared to that of the protein con~ugates.
The known proce~ of eleGtrophoresls yleld~ a product of
29 aub~tantially hlgher purity than chromatographlcal methods do.
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10644Z5
The product obtained exhibits, as a surpri~ing technlcal effect,
a higher ~peciflclty when used as an immunological reagent than
the product obtalned accordlng to the state of the art.
The ~ollowlng Example~ illustrate the invention.
E X A M P L E 1:
A 3 % antibody-containing ~erum protein ~olution (goat) was
reacted ln known manner with fluore~cein~ i~othiocyanate in an
alkallne medium. In a horizontal electrophoresl~ equipment
(size: 65.0 cm ln length, 76.0 cm in width, and 1.2 cm in
helght), which contalned 3.6 l of PVC powder (Geon X 427, trade
;~ mark of MeQ~r~. Serva, Heidolberg, W.aermany), the con~ugated
antiserum wa~ soparated at pH 8.1, at a field potential of 6 V
per cm, and at an amperage of 0.6 A, over a period of 15 hours.
The ~-globulin fraction containing the antlbodies, whlch
generally remains unmoved at the application point or even
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mlgrates toward the cathode unle~s conJugated to fluore~cein
:i'! i~othiocyanate, migrated clearly toward the anode. The light
yellow to greeniJh coloration Or the ~-globulin fractlon con-
talnlng the sntlbodle~ stood clearly out against the rest Or
prot~ln fractlon~ and free dye~tuff portlon~ wlth their dark
yellow to reddl~h brown color. The deslred ~-globulln fractlon
containing the antibodles and havlng the optlmum F/P quotlent -
which can also be determined bandwi~e l~mediately after separa-
tion - was cut out as a PVC zone and washed clear of the
i 25 carrier material by mean~ of a 0.9 X sodium chloride solution.
The eluate wa~ concentrated to about 1 g % of proteln by ultra-
flltratlon.
Thls last operation was followed by the final standardization
29 and quallty control of the product.
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644~5
From 3~6 g o~ a fluorescein-con~ugated protein solutlon
containing antlbodies (120 ml of antiserum), 0.5 g of a fluore~-
celn-conJugated ~-globulin ~raction containing antibodies was
obtained.
When ~erum proteins of rabbits containing antibodies were
u~ed in an amou~t of 8 g of a fluore~cein-con~ugated antiserum
containing antibodie~ (120 ml), 1.0 g of a fluore~cein-con-
~ugated ~-globulin ~ractlon containing antibodies was obtained.
Analogou~ yields were obtained by recovering antibodies
from human Qera. Considering that the proportion of the
~-globulin fraction in an anti~erum accounts for about 15 to
18 %, these yield~, calculated on thi~ ~-globulin fraction,
c~n be regarded a~ optimum.
E X A M P L E 2:
A ~ % human tr~n~ferri~ solution was reacted in known
manner with fluorescein isothiocyanate in an alkaline medium.
In a horizontal electrophoresis equipment (size: 65.0 cm long,
76~0 cm wide9 and 1.2 cm high) containing 3.6 l of a PVC powder
(Geon X 427, trade mark o~ Messrs. Serva, Heidelberg, W.Germany),
a con~ugated protein was ~eparated at pH 8.1, at a field
potential o~ 6 V per cm and at an amperage of 0.6 A, over a
perlod of 15 hourQ. The transferrin con~ugate clearly migrated
toward the ancde. The yellow-brown coloration of the trans-
ferrin-containing zone stood clearly out again~t the different
colors of the ~ree dyestuff portions which had migrated still
further toward the anode. The transferrin con~ugate having
the optimum F/P quotient - which can be determined bandwise
immadlately after separation - was cut out a~ a PVC zone and
29 wa~hed clear of the carri~r material by means of a 0.9 ~ sodium
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HOE 74/B 011
10644~:5
chloride ~olution. The eluate was concentrated to about 1 g %
of protein by ultra-filtration.
This operation W&~ followed by the final standardization
and qual1ty control of the product.
From 3.6 g of human transferrin, about 3.0 g of the cor-
re~ponding ~luore~cein-con~ugated protein were obtained,
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