CH668649A5 - PROCESS FOR DETERMINING THE CONCENTRATION OF SPECIFIC IGG IN HUMAN SERUMS. - Google Patents

Description

DESCRIPTION

The present invention relates to a method for determining the concentration of specific IgG in human sera.

By "specific IgG" is meant the fraction of IgG molecules in the serum which identifies a specific antigen (or allergen).

Obtaining cell lines capable of continuously producing a specific antibody against a predefined immunogen was first described in 1975 (Koller & Mulstein, 1975). This method is based on the fusion between myematous cells and spleen cells from suitably immunized animals. The resulting hybrids can be selected so that they are the only cells that actively multiply. From these growing hybrids, clones secreting the desired antibody can be selected. These antibodies are therefore of monoclonal origin and can be maintained indefinitely.

This methodology has been used to prepare antibodies against a wide variety of antigens as natural products with biological activity, neuropeptides, peptide hormones, enzymes, Polysaccharides, glycoproteins, lypopolysaccharides, histocompatibility antigens, differentiation antigens and others cellular antigens, viruses, etc. They are also very useful in the field of diagnosis.

The present invention aims to provide a simple and reliable in vitro method avoiding in vivo methods while improving traditional in vitro methods. The method according to the invention is characterized by the characteristic clause of claim 1.

In the present invention, the method for evaluating the specific IgG is detailed. For this, antigens obtained by original techniques are absorbed or united in a covalent way to microtitulation plates or another plastic surface, nitrocellulose, etc. Then the serum sample is added and after a determined incubation period, the non-specific IgG is eliminated by successive washes. The IgG which remains in the wells (specifically linked to the allergens) is detected by adding the monoclonal antibody previously labeled with a radioactive isotope or an enzyme. After an incubation period, the excess anti-IgG is eliminated and the radioactivity or the enzymatic activity associated with the wells are measured. Monoclonal antibodies are therefore used, manufactured by the inventors, which specifically recognize serum IgG and do not recognize other immoglobulins or substances present in blood serum or plasma. The originality of the invention consists in that the monoclonal antibodies used can be considered as unique, in the sense that they did not previously exist in nature, since they are produced by a hybrid cell artificially prepared in the laboratory from of two natural cells. The products synthesized by the hybrids, monoclonal antibodies recognizing human IgG, would not survive in a natural environment and, therefore, cannot be approved for polyclonal antibodies produced in animals by standard techniques, although they retain some some of their properties.

In the evaluation of specific IgG in vitro, polyclonal antibodies have generally been used and the main limitation of these methods is their low sensitivity. Another problem in the use of polyclonal antibodies is that the specific fraction which recognizes the antigen is only 1-10% of the total y-globulin fraction. This limitation in the amount of specific immunoglobulin does not exist in monoclonal antibodies, since almost 100% of the protein is specific against the antigen. Therefore, if polyclonal antibodies are used, there are many possibilities for cross-reactions with non-specific substances.

Other advantages of monoclonal antibodies compared to polyclonal ones are: the ease of their purification and being able to have unlimited quantities of standardized reagents. It can be said that once a hybrid cell line (Hybridome) has been established, unlimited amounts of the monoclonal antibody can be prepared. This ensures the availability of reagents with permanent properties (homogeneity, specificity and affinity) and guarantees the achievement of reproducible and therefore comparable results between different laboratories.

When compared to known in vitro methods, the new invention has the additional advantage that there is no need to isolate and purify serum IgG (Galfre & Milstein), at least in the quantities or proportions required in the first. Much less time is required to obtain reliable results and there is no need to centrifuge the samples in the different phases of the analysis.

To measure the specific IgG in the serum, it is necessary to use extracts well characterized biological- and / or immunochemically (Carreira J. y col., Clin. Allergy, 14, 503,1984; Corbi y Carreira, Int. Archs Allergy Appi. Immun. 76,156, 1985).

The extracts used in this invention, although commercially accessible, are in all cases subject to a preliminary purification process to increase the efficiency of the union, the reproducibility of the method and the correlation with live tests. We measure live biological activity, their allergenic power in vitro and we identify and assess the relative activity of the different components of the extract (Stan-

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dardization of allergen extracts, Alergia e Inmunologi'a Abelló, S.A.).

Although the mechanism by which a non-covalent bond occurs between a protein in an aqueous solution and the plastic to which it is exposed is little known (Par-sons, Methods of Enzymology, 73, 224, 1981) the bond is extremely strong and what is more important, the biological activity of the sample is maintained. Most authors agree that the union of proteins with plastic surfaces occurs through non-specific hydrophobic interactions. It seems that the only reagent needed for the preparation of protein-coated plastics is the plastic itself. Several authors (Catt. Et al., Science 158, 1570, 1967; Crosignani et al., J. Clin Endocrinal Metab. 30, 153, 1970) have examined the effect of various parameters in the union of antibodies to plastic and its reached the conclusion that the temperature, pH, ionic strength do not influence beyond measure. In our experience, polystyrene, polyethylene, polypropylene or polyvinyl can be used almost without distinction. Plastic tubes, titration plates or individual wells of different shapes and sizes can be used.

On the other hand, the forces that keep proteins united to nitrocellulose are little known, but it is very likely that hydrophobic interactions play a very important role.

However, despite the above, the absorbed protein can separate from the plastic during the incubation and washing processes. To avoid this, the antigens can be covalently united to the solid plastic phase by chemical modification of the latter.

The separation of that which is united to the plastic from that which remains free in solution can be carried out by simple suction or decantation. The reproducibility of the method increases if an unrelated protein, for example serum albumin and a detergent (Triton,

Tvveen, etc.).

The market for monoclonal antibodies with radioactive isotopes or with enzymes is carried out by conventional methods by selecting for example: 125I (Fraker & Speck, Biochem. Biophys. Res. Commun. 80, 849) or ß-galac-tosidase (O 'Sullivan and Marks. Methods Enzymol, 74, 147, 1978).

The methods for determining the radioactivity or the united enzyme are also carried out by conventional methods, using in the first case y-radiation counters, and in the second it is necessary to carry out an incubation with different enzyme substrates which will depend on the method of detection available.

The invention is illustrated below with the aid of two examples which can be considered explanatory and non-limiting thereof.

Example 1

Determination of specific IgG against antigens present in plant pollens (L. perennial).

a) Preparation of the monoclonal antibodies. 100 μg of purified IgG was intraperitoneally injected into mice with complete Freund's adjuvant on days -45 and -30. On day -3 they were injected with the same amount of IgG in 200 µl of phosphate buffered saline (PBS) intravenously. On day zero the mouse myeloma cells were fused with two spleen lymphocites from immunized animals using polyethylene glycol 50% The cells were distributed in six well plates and were cultured in a selective HAT medium.

b) Selection: After two weeks, the culture supernatants were collected and the possible specificity was analyzed.

against IgG. To avoid the selection of hybridomas secreting anticoprs with specificity against light chains or against idiotypic determinants, another different myeloma IgG was used. In the positive clones, the possible cross-reactivity with IgE, IgM or IgA was analyzed. Hybridomas which recognized IgG were cloned and subcloned into semi-solid agar. Those who recognized different epitopes in IgG were cultivated on a large scale to purify and mark them.

c) Labeling: After having purified the monoclonal antibodies by standard methods, they were labeled with 125 I in agreement with the method described by Fraker and Speck, 1978, Biochem. Biophys. Res. Common. 80. 849. In other cases they are labeled enzymatically with β-galactosidase by the method O'Sullivan and Marks, 1978, Methods in Enzymol. 74.147.

d) Determination of specific IgG: The analyzes were carried out without distinction on microtitulation plates of different sizes and plastic material, in plastic tubes, or in nitrocellulose discs.

1) The polystyrene plates were nitrated and then reduced in order to obtain stable polyaminostyrene plates. The wells of a modified plate were filled with 200 µl of 0.01% (w / v) antigen in the presence of 0.1% (w / v) EDC in 0.25 MES buffer M pH 6.0.

2) Unbound protein was removed by aspirating or decanting the contents of the wells and washing with saline which contained 1% bovine serum albumin (PBS-BSA).

3) To saturate the places of union which are still free, the wells were incubated with PBS-BSA for 1 hour at room temperature.

4) The wells were then washed three times with PBS in the presence of 0.1% Tween (PBS-Tween), carefully decanting the liquid from the wells at each step.

5) 50-200 µl of human serum was added to each well in successive dilutions.

6) In other wells were added similar volumes of standard sera previously calibrated in their specific IgG content.

7) The contents of these wells were incubated for 1-2 hours at room temperature.

8) The wells were washed three times with PBS-Tween and then incubated with 50-200 μl of one of the labeled monoclonal antibodies as indicated in point 7.

9) If the monoclonal antibody added at point 8 had been labeled with 125I at this point, the wells were cut to measure the radioactivity with the gamma radiation counter.

If the monoclonal antibody had been labeled enzymatically at this point, the enzyme substrate ONPG (3 mM ortho-nitrophönyl-ß-D-galactopyranoside) was added to PBS containing 10 mM MgCk and 0.1 mM 2-mercaptoethanol. Then the contents of the wells are incubated at 37 ° C for 2 h. At this time, the reaction was stopped by adding 100 µl of 1 M Na2CO3 and the absorbance was measured at 410 nm.

10) The counts per minute (cpm) or the absorbance obtained in the standard wells, has been represented on a diagram on which the ordinates represent cpm or Abs 410 nm and the abscissas the logarithm of the inverse of the dilution used. By this diagram, each sample was classified by comparing its cpm or Abs 410 to the values obtained with the standard sera.

Example 2

Determination of specific IgG against antigens present in plant pollens (L. perennial). a) Preparation of the monoclonal antibodies:

100 (ig of IgG pu ri fied with

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mice in a complete Freund's adjuvant on days -45 and -30. On day -3, they were injected with the same amount of IgG (e, K) in 200 (il of phosphate buffered saline (PBS) intravenously. On day zero, the mouse myeloma cells were fused with the lymphocytes animals immunized using 50% polyethylene glycol, cells were distributed into six well plates and cultured in HAT selective medium.

b) Selection: After two weeks the culture supernatants were collected and the possible specificity against IgG was analyzed. To avoid the selection of hybridomas secreting antibodies with specificity against light chains or against idiotypic determinants, another IgG with different myeloma (e, A.) is used. In the positive clones, the possible cross-reactivity with IgE, IgM or IgA was analyzed. The hybri-domes that recognized IgG were cloned and subcloned into semi-solid agar. Those who recognized different epi-topes in IgG were cultivated on a large scale to purify and mark them.

c) Marking: Once the monoclonal antibodies have been purified by standard methods, they are marked with 125 I in agreement with the method described by Fraker and Speck, 1978, Biochem, Biophys. Res. Common. 80, 849. In others they were labeled enzymatically with β-galactosidase by the method of O'Sullivan and Marks 1978, Methods in Enzymol. 74.147.

d) Determination of specific IgG:

The analyzes were carried out without distinction on microtitulation plates of different sizes and plastic material, in plastic tubes, or in nitrocellulose discs.

1) To each of the wells, tubes or disks was added a volume (50 to 200 jil) of a solution with protein extracts prepared according to the methods previously described and they were left to incubate overnight at 4 ° C.

2) The unabsorbed protein is withdrawn by aspirating or decanting the contents of the wells and washing with a saline solution containing 1% bovine serum albumin (PBS-BSA).

3) To saturate the places of union still free, the wells were incubated with PBS-BSA for 1 h at room temperature.

4) Then the wells were washed three times with PBS in the presence of

Tween 0.1% (PBS-Tween), carefully decanting the liquid from the wells at each step.

5) 50-200 μl of human serum were added to each well in successive dilutions.

s 6) In other wells are added similar volumes of standard sera previously calibrated in their specific IgG content.

7) The contents of these wells were incubated for 1-2 h at room temperature.

8) The wells were washed three times with PBS-Tween and then incubated with 50-200 μl of labeled monoclonal antibodies as indicated under c.

9) The contents of the wells were incubated as indicated in point 7.

10) If the monoclonal antibody added at point 8 had been labeled with '-5I at this point, the wells were cut to measure the radioactivity on a gamma radiation counter.

If the monoclonal antibody had been labeled enzymatically at this point, the enzyme substrate ONPG (3 mM ortho-nitrophenyl-P-D-galactopyranoside) was added in PBS containing 10 mM MgCh and 0.1 mM 2-mercaptoethanol. Then the contents of the wells are incubated at 37 ° C for 2 h. At this time, the reaction was stopped by adding 25 100 μl of 1MNa 2 CO 3 and the absorbance was measured at 410 nm. 11) The counts per minute (cpm) or the absorbance obtained on the standard wells has been represented on a diagram (see graph 1) in which the ordinates represent cpm or Abs 410 nm and the abscissas the logarithm of the inverse of the dilution used. By this diagram, each sample was classified by comparing its cpm or Abs 410 to the values obtained with the standard sera.

Translation of the legends of graph 1 corresponding to the standard curve for the specific IgG.

Determination of specific IgG using anti-IgG monoclonal antibodies (mAbs) in polystyrene wells.

The ordinate scale on the left represents the specific union of an enzymatically labeled mAb with an anti-Lolium perenne serum IgG. The ordinate scale on the right 40 represents the specific union of the same mAb when it has been radioactively marked with 125I. The broken line represents different dilutions of the serum on a logarithmic scale.

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1 sheet of drawings

Claims (9)

668649 2 1. Method for determining the concentration of specific IgG in human sera, characterized in that a human anti-IgG monoclonal antibody is used and that it comprises the following steps: - Attachment of antigens to a solid support followed by incubation of the serum sample with these attached antigens; washing the support to remove non-specific proteins from the antigen; incubation of the support with the anti-IgG monoclonal antibody labeled with 1251 or with an enzyme; washing of the solid support and determination of the inherent radioactivity, or of the enzymatic activity. 2. The method of claim 1, wherein the monoclonal antibody has been labeled with an enzyme, for example: ß-galactosidase. 3. Method according to claims 1 and 2 wherein the sugar is fixed to a plastic, as a solid support. 4. Method according to one of claims 1, 2 and 3, in which a glycoprotein has been joined to the solid support. 5. Method according to claims 1,2,3 and 4, in which nitrocellulose has been combined as a solid support, a glycoprotein, a sugar or a protein. 6. Method according to one of claims 1 to 5, in which the values obtained are compared semiquantitatively with a standard serum. 7. Method according to one of claims 1 to 6, wherein the union of the antigen with the solid support occurs covalently. 8. Method according to one of claims 1 to 6, wherein the union of the antigen with the solid support occurs in a non-covalent manner. 9. Method according to one of claims 1 to 8, in which the solid support is the container itself in which the process is carried out.