CS240831B1 - A method of preparing antibody-containing immunoreactions - Google Patents

Abstract

The invention relates to a method for preparing immunoreagents containing antibodies, which are universally applicable for qualitative and quantitative laboratory techniques for determining human plasma proteins in body fluids. The method for preparing immunoreagents containing antibodies is characterized in that soluble antigens are added to hyperimmune animal antiserum until ballast antibodies are saturated and the immunoglobulin fraction is subsequently isolated by repeated salting-out, ion exchange chromatography and subsequent dialysis into a low ionic strength environment.

Description

The present invention relates to a method for the preparation of immunoreactors containing antibodies specific to human plasma proteins and yet free of excess antigens, soluble immunocomplexes and lipoproteins and therefore suitable for qualitative and quantitative immunoassay diagnostic methods: immunoprecipitation techniques, radloimmunoassay techniques, enzyme immunoassays, enzyme immunoassays, enzymoimmunoassays turbidimetry, hletological examination ·

Generally, these antibody-containing immunoreactors can be prepared from crude animal antisera containing specific and ballast antibodies by removing unwanted antibodies with insoluble antigens usually bound to a suitable insoluble carrier. This procedure will ensure the absence of excess antigens used to remove ballast antibodies and soluble immunocomplexes. Lipoproteins are removed from the hyperimmune sera by either dellpidating agents or by isolation of immunoguline fractions from the suppressed anti-serum.

The preparation of insoluble antigens requires a high-quality carrier with a high cut to allow the antigen to bind not only to the surface of the particles! gel, but also inside particle1. These qualitative requirements are met by cyanogen bromide-activated Sepharose AB (cyanogen bromide-activated spherical agarose gel particle), which is, however, very expensive and thus difficult to access. Replacement of cyanogen bromide-activated Sepharose AB with inactivated Sepharose AB and its direct activation by a beetle is cheaper, but this work is associated with the risk of working with a highly toxic and highly volatile poison. Gels of Czechoslovak production Insolmer (synthetic polymer based on aromatic acid ester), Spherones (hydrophilic macroporous glycol methacrylate gels) do not have properties comparable to those of Sepharose AB in protein balancing, they bind antigens only on their surface and the efficiency of such bound antigen is antigens.

Removal of ballast antibodies by insolubile antigens from hyperimmune animal antisera is generally a lengthy process, carrier regeneration by washing and centrifugation (energy intensity) is required, and difficult for large-scale use. With this method of hyperimmune sera saturation, there is an increasing demand for equipping the washing machines with devices and equipment, increasing labor requirements, reducing production volume and increasing the time required to produce the preparations.

These disadvantages are avoided when using soluble antigens to remove ballast antibodies from hyperimmune animal antlers.

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SUMMARY OF THE INVENTION The present invention is based on a simple method for preparing antibody-containing immunoreactivities using solubilized antigens to remove ballast antibodies from animal hyperimmune antisera until saturation, followed by isolation of the immunoglobulin fraction by repeated salting-out, ion exchange chromatography and dialysis into a low ionic I environment. 02 ₀ Sol slew it you SYC cheers materials are inspected prior to their use imunoolektroforeticky the presence of antigens to the immunoglobulin G flax that when you SYC cheer procedure may not be introduced into antlséra in excess, but only equivalence antlgen IgG antibody IgG Antl. If, after removal of the other ballast antibodies in the antiserum, the anti-immunoglobulin G antibody still remains, it is removed by adding pure immunoglobulin G antigens to the aquivalence.

The aim of the above principles of saturation of ballast antibodies from hyperimmune antiserum is, besides the removal of unwanted antibodies, to prevent the introduction of excess immunoglobulin G antigens into the hyperimmune antiserum. Clogging of other excess antigens is not a problem. Analytical control of the antigens used and the processed hyperimmune antisera is preferably performed by immunoelectrophoresis.

Subsequent isolation of the immunoglobulin fraction from the saturated hyperimmune antiserum removes excess antigens used to saturate the ballast antibodies, lipoproteins, and soluble immunocomplexes, while maintaining high purity and yield of the immunoglobulin fraction and high avidity specific antibodies. Immunoglobulin fraction isolation from hyperimmune antiserum is a purification process where after filtration of saturated hyperimmune antiserum through a clarifying filter (e.g. Seitz K 5) by repeated salting, preferably ammonium sulfate, up to 40% saturation in pig antisera and up to 45% saturation from rabbit, goat , sheep, horse, bovine, guinea pig and mouse antisera) followed by lonthexchromatography (diethylaminetyl cellulose, preferably DEAJS cellulose DB 52) at pH · 5.7 for porcine and 7.0 for other antisera species, isolates the immunoglobulin fraction which is subsequently palliated into a low ionic strength medium (preferably against 0.003 M sodium hydrogen phosphate) and filtered through a dense fining filter (e.g. Seitz K 5).

This procedure removes excess antigens entrapped in hyperimmune serum to remove ballast antibodies as well as soluble immunocomplexes and lipoproteins. The presently present excess immunoglobulin G antigen would not be removed by this step.

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The immunoglobulin fraction thus isolated from the hyperimmune antiserum was lyophilized and stored at + 4 ° C.

From the lyophilized immunoglobulin fraction, a sterile solution is prepared with a standard titer of specific antibodies, which is applicable to both classical immunoassay techniques and modern analytical methods: radioimmunoassay, enzymoimmunoassay, nephelometrile, turbldlmetrli, agglutination techniques, hietological examinations and the like.

The wide range of applicability of immunoreacts containing antibodies prepared by the method is determined by their properties, i.e., standard tltr specific antibodies, the absence of excess antigens used to remove ballast antibodies, the absence of soluble immunocomplexes, and 11-protein proteins.

The preparation of immunoreactors is illustrated by the following examples

Example 1.

Preparation of porcine immunoglobulin fractions against human immunoglobulin M.

The crude anti-human porcine anti-human immunoglobulin M anti-IgG contains, in addition to the anti-IgG antibody, also undesirable antibodies against alpha 2 macroglobullin (A ^ M) and Immunoglobulin G (IgG). Titrate the crude antiserum with a solution of alpha 2 macroglobulin antigens by adding an increasing amount of the solution of alpha 2 macroglobulin antigens to 1 ml of crude antiserum in each case up to 0,05 ml, 0,1 ml, 0,2 ml, etc. Tltrace se performed under immunoelectrophoretic controls. The amount of alpha 2 macroglobulin antigens is added to the crude hyperimmune antiserum until the anti-alpha 2 macroglobulin antibody has completely disappeared. Since the macroglobullin alpha 2 antigen solution always contains traces of other antigens, the anti-alpha 2 macroglobulin antibodies are introduced into the crude hyperimmune antiserum of the excess antigen when instructing the anti-alpha 2 macroglobulin antibodies.

Next, titrate the crude hyperimmune antiserum with an immunoglobulin G solution and determine the optimum amount of immunoglobulin G antigens to be added to the hyperimmune crude antiserum to clear the anti-immunoglobulin G antibody and avoid excess immunoglobulin G antigens being introduced into the antiserum. Titration is performed using immunoelectrophoresis. control.

After removal of ballast antibodies from the crude hyperlipidic antiserum, the saturated antiserum is filtered through a layer of Seitz K 5 and isolated therefrom.

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4m? Mogl granules, fraction three times by repeated precipitation · saturated ammonium sulphate solution to 40% saturation. The sediment containing the crude immunoglobulin fraction was dialyzed against 0.03 M acetate buffer pH

5.7 at + 4 ° C until the positive reaction to ammonium ions disappears. The dialysed immunoglobulin fraction was purified by DEAN cellulose chromatography DE 52 (10 g cellulose per 1 g protein) and the filtrate containing pure immunoglohulin fraction was dialyzed against 0.003 M sodium monohydrophosphate (NagHPO ^) for three to five days, changing the palliating solution daily. . The resulting precipitate was removed by filtration through a layer of Seitz K 5 and the filtrate was lyophilized.

Preparation of diagnostics:

From the lyophilisate obtained, a 5% solution of porcine immunoglobulin fraction against immunoglobulin M in saline pH -7.2 (PBS) with 0.1% sodium azide was prepared. Stanov! the titer of specific anti-immunoglobulin M antibodies by the simple radial immunodiffusion method (modified by Becker, W., Immunochem., 6, 539, 1969) and the protein concentration of the prepared solution.

The proportion of lyophilisate required to prepare the selected amount of reagent is calculated in proportion to the standard titer of specific antibodies, in the case of porcine immunoglobulin flax fraction against immunoglobulin M, this is 6,9 mg / ml. Filter the solution through a K 5 pad, measure the pH. If the pH is not in the range of 7.2-7.4, it is adjusted with either 1 N sodium hydroxide solution or 1 N hydrochloric acid solution.

Again, the titer of specific antibodies is checked by the Simple Radial Immunodefusion method, the resulting value being consistent with the previously performed calculation. If this is not the case, dilute the reagent and dilute or add another immunoglobulin lyophilisate fraction according to the latest calculation, repeat the pH measurement and control of the specific antibody titer until it reaches its desired value.

The immunoglobulin porcine anti-immunoglobulin M fraction solution is sterile filtered, preferably through a membrane filter (0.22 µ), and sterile filled in aliquots into glass vials.

Prepared in this way, it has a wide range of applications. It can detect immunoglobulin M in all kinds of qualitative immunoprecipitation tests used, for example Ouchterlony's double immunodiffusion, immunoelectrophoresis, upstream immunoelectrophoresis.

Diagnostics prepared as described above can be used fully in quantitative immunoprecipitation tests such as the simple radial immunodiffusion method: 240 831

In the filtrate-veronal oxalate buffer pH 8 8.6, the ionic strength I 0,0 0.05 ee boils agarose to 1% concentration and after cooling to 50 ° C ee adds 0.10 ml porcine immunoglobulin fractions against the immunoglobulin M for 30 ml gel · Mix and pour horizontally onto a 8.5 x 18 cm slide glass ·

After solidifying the gel (about 30 minutes) with a ground transfusion needle, 2 mm diameter starter wells are excised and treated with standard solutions and test samples (5x diluted) in human sera · After 48 hours at room temperature in a humid chamber, rinse the plate. saline, dried, stained and evaluated. The immunoglobulin M content in the samples to be examined is read from a calibration curve constructed as a plot of the square of the precipitation ring diameters on the concentration of the standard samples. In a variant of the simple radial immunodiffusion method, which does not dilute the examined human sera, one immunodiffusion plate of

8.5 x 18 cm, i.e., 30 ml of gel, use 0.25 ml of porcine immunoglobulin fraction.

Using the diagnostics prepared according to this method, it is possible to quantify immunoglobulin M in human body fluids by, for example, the nephelometric method:

The porcine immunogl granule fraction against Immunogl granule Use is diluted 100x with a 4% solution of polyenthylene glycol 6000 and filtered through a clarifying layer to e.g.

Seitz E 5 • Dispense the prepared solution into 1 ml nephelometer cuvettes and add standard or investigated human serum to the individual amounts of reagent.

Mix the solution mixtures and measure the light scattering on the laser nephelometer after 30 minutes of standing. A calibration curve is plotted from the measured scattering values of the standard samples to read the immunoglobulin M content in the human sera to be examined.

The detection of the content of Immunogl obulin M can also be performed by the method of terbidimetry:

The porcine immunogl granule fraction against the immunogl granul M is diluted with a 20x 4% polyethylene glycol 6000 solution and filtered through a clarifying pad on eg Seltz E 5. Dispense the prepared solution into 1 ml spectrophotometer cuvettes and add the standard or test reagent to the individual amounts. human serum.

The individual solution mixtures are mixed and allowed to stand for 30 minutes. Measure the absorbance at 340 nm on a UV spectrophotometer.

The porcine immunoglobulin fraction against immunoglobulin M can be used to quantitate the immunoglobulin by immunoenzyme and radioimmunoassay methods. The porcine immunoglobulin fraction against immunoglobulin II is diluted 1000-fold

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0.05 M bicarbonate buffer pH-9.6 and applied ee in 0.1 µl wells to the microtiter plates. After the incubation period (typically 60 minutes), the wells are washed three times with wash solution and the standard and test samples are applied. After an incubation period of 30 to 60 minutes, the wells are washed three more times with wash solution and the labeled antibody (enzyme or radioisotope) is applied. After incubation for 15 minutes, the color reaction of the enzyme is induced and the abaorbance is then measured or, in the case of radioisotopic labeling, the radioactivity is measured directly.

From the measured values of the standard samples, a calibration curve is constructed from which the values of the measured samples are read.

In principle, in the same manner as described in Example 1, other animal immunoglobulin fractions against human serum proteins can be prepared for e.g. immunoglobulin G, immunoglobulin A, immunoglobulin A, C3 and Ch component of human complement, alpha 2 macroglobulin, transferrin, heavy fragment immunoglobulin G chains, immunoglobulin G light chain fragment, albumin, orosomucoid, ceruloplasmin, prealbumin, alpha 1 antitrypsin, hemopexin, haptoglobin, secretory component, fibrinogen, fetoprotein, ferrltin and others.

The above examples of preparation and use reveal a wide range of applicability of immunoreacts containing antibodies prepared as described above for the detection and quantitation of various types of plasma proteins. The broad spectrum of applicability is due to the properties of the reagents prepared, i.e. the standard titer of specific antibodies, the absence of excess antigens, immunocomplexes and lipoproteins.

Claims (4)

SUBJECT OF THE INVENTION 240 831 A method for the preparation of inamoreagenic antibodies comprising antibodies suitable for immuno-precipitation techniques, radiolunoassays, immunoenzymoanalytic techniques, agglutination techniques, nephelometrics, turbidimetry, and hietological examination, characterized in that ee adds soluble antigens to the hyperinune animal to the baffles and then to the baffles. The immunogenic granule fraction is isolated by salting out, then by lantern chromatography and then by dialysis into a low ionic strength medium of 0.005-0.02. 2. A method for preparing immunoglobulins comprising the antibodies of claim 1, wherein the anti-globulin G immunoglobulin present in the hyperimmune animal antiserum is removed by adding an equivalent amount of pure immunoglobulin G, 3. A method for preparing immunoglobulins containing antibodies according to claim 1, characterized in that a crude porcine antiserum is used to prepare immunoglobulins, 4. A process for the preparation of immunoreactivities comprising the antibodies of claims 1 to 3, wherein the dialysis is carried out into an ionic strength environment.