DE2431719A1 - PROTEIN CONJUGATE PROCESS - Google Patents

Description

2431713

BEHRIHGTORRKE AKTIENGESELLSCHAFT, Marburg (Lahn) ^;

File number: Hoe 74 / B 011 - Ma I67

Datura: July 1, 1974

Process for obtaining protein conjugates

The invention relates to a method for obtaining proteins or protein mixtures covalently linked to organic compounds, so-called protein conjugates. Ea the protein conjugate to be obtained differs from undesired conjugation products and the organic compounds used for conjugation in terms of its electrophoretic properties, the "experienced r ..! | after conjugation the vorlie -enae ^Ger: sch a .α preparative elektrophoretisehen Vei'fshren unterv / orfen v.drd and the zone containing the desired preparations _r of which is separated with unwanted protein conjugates and the free organic compounds. will.

The invention relates in particular to a method for the purification of protein-dye Xon.jugaten, preferably of antikerperhaltigen Inmunsera or /. Protein fraction

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carried out conjugation reaction with dyes, in particular with fluorescent dyes, beisjjielsv.-ice with Flue re se einisotMoeyanat by means of preparative electrophoretic processes.

The invention further relates to what has been described Process of purified protein conjugates and their uses in immunological diagnostics.

The method according to the invention is made possible by the electrophoresis Properties of the starting materials and the reaction products enable the separation of the pure protein conjugate of a certain degree of implementation from the unreacted Protein and your organic used for conjugation Molecule provided that these three components have different electrophoretic mobility demonstrate.

Protein-dye conjugates, in particular with fluorescent protein-open have conjugated antibody-containing immune sera, ;; ie riHoIisteher.i is shown its special meaning in the immunofluorescence technique called EC.

Without inferring any restriction from this, should the essence of the inventive method on these representative Examples are highlighted.

The yon A.H. Coons 19 ^ -2 introduced technique of immunofluorescence allows the combined investigation of the serological immunological specificity and the morphological characteristics of the material to be analyzed. This method is based on the so-called antigen-antibody reaction which conjugates one of the two immunological partners - usually the antibody - with a fluorescent dye is. The visualization of the antigen-antibody

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Reaction connection takes place in the microscopic specimen by stimulating the visible fluorescence by means of radiation in the ultraviolet range.

Immunofluorescence has great diagnostic tools for identifying bound and free antigens and antibodies Noticed.

According to the prior art, antibody-rich fractions are preferred as so-called immune sera with fluorescent dye with fluorescein isothiocyanate - reacted. The further purification of the sales product takes place predominantly by means of gel filtration and by means of ion exchange chromatography on diethyl aminoethyl cellulose. The fluorescein conjugated immunoglobulins isolated.

DJ.ese procedures have the disadvantage that you can try them the removal of the non-covalently bound dye from the fluorescein-conjugated protein preparation by means of Gel filtration or dialysis, incomplete separation of the dye is observed, in particular the dye becomes adsorptive The dye bound to the protein or the dye which is relatively easily split off hydrolytically is not removed. In addition, there is a dye, which is the result of self-polymerization or self-condensation reached higher molecular weights, also only incompletely eliminated.

The use of such preparations containing free dye leads to unspecific fluorescence staining on the microscopic level Preparation and thus false statements.

In the case of the one predominantly used in accordance with the state of the art Process for obtaining the antibody-containing conjugated

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Protein fraction by means of ion exchange chromatography in DEAE cellulose, essentially only the antibody component that belongs to the IgG class is obtained. Among them applied elution and separation conditions become antibodies of the IgM and IgA classes only incompletely or at all not detached, so that the preparation obtained sometimes has a considerable loss of the desired specific antibodies having. When changing the elution condition for recovery Fluorescein conjugated IgM and IgA antibodies are so-called "over-labeled" ^ f-globulins as well as basophils Proteins also eluted. Due to their strong velvety character, these so-called "over-labeled" protein components lead in immunological reactions for unspecific binding with a number of basic proteins in the test material and thus also to misinterpretations of the fluorescence obtained.

The process according to the invention does not have the stated disadvantages e. Preferably in an inert carrier performed electrophoretic purification of dye-conjugated Protein preparations, preferably fluorescein-conjugated protein preparations, after the conjugation reaction offer the following advantages:

1. The process enables an optimal "true to size" antibody-containing zone extraction. Unwanted, for example antibody-free, ballast protein fractions can can be discarded without difficulty in eliminating the desired fraction. The yield of the antibody-containing dye-conjugated protein preparations is optimal. In the example used, the fluorescein-labeled TT-globulin fraction 70-90% of the amount used is recovered.

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2. As a result of conjugation of proteins with acidic organic Molecules, the isoelectric function changes in all that have come into reaction with organic molecules Proteins too low, with the result that they are more anodic under the conditions of electrophoresis migrate as unconjugated proteins. The removal of excessively acidic proteins as a result of a strong reaction with the molecule used for conjugation does not present any difficulties, since these migrate more strongly towards the anode. This result is particularly important for those with the acidic fluorescent dye fluorescein isothiocyanate (quinoid system with free carboxyl group) conjugated proteins.

3 · The optimal removal of the non (or adsorptively) bound acidic organic molecule, its presence in fluorescein-conjugated preparations lead to undesired, unspecific fluorescence staining is in the guaranteed method according to the invention. The reason for the easy removal of this dye portion is in that the acidic molecule itself migrates strongly towards the anode. The load from the electrical voltage under the conditions of preparative zone electrophoresis leads to a cleavage of relatively weak protein bound molecules and causes good quality electrophoretically obtained protein conjugates. in the In the case of proteins conjugated with fluorescent dye, the stability of such preparations is increased Storage in aqueous solution compared to those obtained according to the prior art, observed.

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4; If antibodies are used as proteins for the conjugation, Thus, the method according to the invention allows the recovery of all dye-conjugated antibody classes, Z-B. IgG, IgA, IgM. This has. especially when using antibodies from sera from humans, for example, Goat, sheep, horse meaning, in which in addition to the antibodies of the IgG class also the antibodies of other immunoglobulin classes a relatively high proportion of the total. Make up the amount of antibody. This factor also has a significant one economic interest, as sera containing antibodies are not available in unlimited quantities.

On the purification of conjugated with fluorescein dyes Transferring antibodies, leads to the invention

Procedure to increase the relation of specific Antibodies (Ab) to total protein (P), the so-called Ab / P quotient, which increases the desired causes specific fluorescence.

5. The method that is based on the use of expensive chromatographic Dispensed with auxiliary means, includes a substantial reduction in the price of the production of pure protein conjugates. "The inert carrier material used can be used repeatedly"

Carrier electrophoresis is used as an electrophoretic process preferred, especially electrophoresis, which contains the inert support in a horizontal tray. Using of, for example, polyvinyl chloride granules as the carrier material, after the separation of the antibody-containing fluorescein-conjugated protein fractions the desired preparation according to the visible bands cut out and then elute from the support with simple solvents. .

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If the conjugate is not colored / can the position of protein, Reactant and protein conjugate indirectly, for example can be detected via a paper copy, which is then colored according to the known measures.

Frequently used electrophoretic carrier materials are, in addition to polyvinyl chloride, polyacrylamide, cellulose, starch, Glass beads, sand, to name a few examples, but the invention does not have to be restricted to them.

According to the invention, however, electrophoresis arrangements can also be used Use in vertical design with and without carrier material. Very good cleaning effects are also achieved with continuous working electrophoresis equipment.

According to the present method, antibody preparations of any origin are separated, those with dyes acidic character have been covalently bound. A particularly suitable dye is fluorescein isothiocyanate.

However, non-fluorescent dyes can also be reacted with proteins and the proteins colored as a result of the conjugation reaction according to the present method be separated, provided that the dyes used have a different electrophoretic ^^ migration behavior.

Instead of antibodies, other proteins of animal, vegetable and microbial origin can also be used. if they are conjugates one of the starting protein and the for. Conjugation used organic molecule different have electrophoretic properties. Preferably used as organic molecules for. the conjugation of those compounds • used whose isoelectric points are in the acidic pH range 3.j.egen. ·

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The invention is to be based on exemplary embodiments are explained in more detail.

Example ι · '

A 3% antibody-containing serum protein solution from the goat is reacted with fluorescein isothiocyanate in a known manner in an alkaline medium. The conjugated antiserum is carried out in a horizontal electrophoresis apparatus containing 3.6 l of PVC powder (Geon X 427 from Serva, Heidelberg) (Dimensions: 1 = 65.0 cm, w = 76.0 cm, h = 1.2 cm) at pH value 8.1, a field strength of 6 V per cm, with a current strength of 0.6 A for a running time of 15 hours separated. The antibody-based

^ -Globulin fraction, which usually remains in the application groove or even migrates cathodically without conjugation to fluorescein isothiocyanate, has clearly migrated in the direction of the anode. The light yellow to greenish coloration of the antibody-containing - ^ - globulin fraction is clearly distinguished from the other protein fractions and from the free dye parts, which are dark yellow to reddish-brown in color. The desired antibody-containing ^ "- globulin fraction ^m -'- t optimal F / P quotient, which incidentally can be determined in bands immediately after separation, is cut out as a PVC zone and washed out of the carrier using 0.9% saline solution. The eluate is concentrated to about 1 g % protein by ultrafiltration, »

Then the final setting and quality control can take place.

When using 3.6g of a fluorescein-conjugated antibody-containing Protein solution (120 ml of antiserum) are 0.5 g of a fluorescein-conjugated antibody-containing ^ -globulin fraction won.

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If antibody-containing serum proteins from rabbits are used, in this way, when using 8 g of a fluorescein conjugate, one obtains Antibody-containing anti-se'rums (120 ml) 1.0 g of a fluorese-conjugated antibody-containing T-GlobuD.in fraction.

Similar yields are also obtained when antibodies are obtained from Huraan sera. Taking into account that the Proportion of the; p-globulin fraction in an antiserum approx. 15 to 18%, the yields, based on this ^ "- globulin fraction, to be described as optimal.

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A 3% human transferrin solution is reacted with fluorescein isothiocyanate in a known manner in an alkaline environment. The conjugated protein is placed in a horizontal electrophoresis apparatus (dimensions : 1 = 65.0 ch, b = 7.6 cm, h = 1.2 cm) at pH 8.1, a field strength of 6 V per cm, at a current strength of 0.6 A during a Runtime separated by 15 hours. The transferrin conjugate has clearly migrated towards the anode. The yellow-brown coloration of the transferrin-containing zone is clearly evident from the free dye components, which are differently colored and have migrated further towards the anode. The transferrin conjugate with the optimal F / P quotient, which, by the way, can be determined in bands immediately after separation, is cut out as a PVC zone and washed out of the carrier using 0.9-6% saline. The egg is concentrated to about 1 g % protein by ultrafiltration.

Then the final setting and quality control can take place.

When using 3.6 g of IIuman-Transferrin approx. 3.0 g of des corresponding fluorescein conjugated protein obtained.

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Claims (8)

Ka IG7 Pat ent entitlement 1. Methods for Purification of Protein Conjugates, ciie compared to the organic compounds used for conjugation in their electrophoretic properties differentiate, characterized in that the Kohkonjugat for the separation of unwanted by-products and not converted conjugation partners is subjected to preparative electrophoresis. 2. The method according to claim 1, characterized in that antibodies conjugated with dyes contain cammaglobulin fractions cleaned v / earth. 3. The method according to claim 1, characterized in that conjugated with fluorescent dyes antibody-containing Gamma globulin fractions are purified. 4. The method according to claims 1-3, characterized in, that electrophoretic processes with inert carriers are used. 5 · According to the · claims 1-4 purified protein con jugate. 6. According to Verfahrm dar · Claims 1 - A purified protein-dye-con jugate. · 7 · Use of the protein conjugates according to claim 5 in the immunological diagnostics. 8. Use of the protein-dye conjugates according to claim in immunological diagnostics. 4/1172