FR2585838A1 - Process for determining the presence of a specific IgG in human sera - Google Patents

Abstract

The invention consists in a process for determining the presence of a specific IgG in human sera, characterised in that it comprises the following steps: binding of previously purified complex protein extracts upon a solid support, followed by incubation of the serum sample with the said bound protein extracts: washing non-specific proteins; incubation with anti-IgG monoclonal antibody labelled with <125>I or with an enzyme; subsequent washing of the solid support with the bound products and determination of the radioactivity or enzymatic activity incorporated into the solid support.

Description

METHOD FOR DETERMINING THE PRESENCE OF SPECIFIC IgG IN
HUMAN SERUMS
The disclosed invention relates to the determination of the concentration of specific immunoglobulin G in human sera, by a radioimmunoassay or solid phase enzymeimmunoassay method using monoclonal antibodies (mAbs).

Obtaining cell lines capable of permanently producing a specific antibody against a predefined immunogen was first described in 1975 (Paste & Milstein, 1975). This method is based on the fusion of myeloma cells and spleen cells from properly immunized animals. The resulting hybrids can be selected so that they are the only cells that are actively multiplying.

Among these growing hybrids, one can select clones which secrete the desired antibody. Such antibodies are therefore of monoclonal origin and can be stored indefinitely.

This methodology has been used to prepare antibodies against a wide variety of antigens, such as natural biologically active products, neuropeptides, peptide hormones, enzymes, polysaccharides, glycoproteins, lipopolysaccharides, thistocompatibility antigens, differentiation antigens and other cellular antigens, viruses, etc. They are also very useful in the field of diagnosis.

The present invention is characterized mainly by the fact that monoclonal antibodies are used in immunological techniques such as enzyme immunoanalysis (E.I.A.) or radioimmunoanalysis (RIA). Thus, two objectives are achieved: to provide a simple and reliable in vitro method which avoids in vivo methods, and on the other hand to substantially improve traditional in vitro methods.

In the present invention, the method for estimating the specific IgG is described in detail. For this, antigens obtained by original techniques are adsorbed or covalently linked to microtiter plates or other plastic surface, to nitrocellulose, etc.

Then, the serum sample is added and after a determined incubation period, the non-specific IgG is removed by successive washings. The IgG which remains in the wells (bound specifically 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 removed and the radioactivity or enzymatic activity associated with the wells is measured.

Monoclonal antibodies are therefore employed, produced by the Applicant, which specifically recognize serum IgG and do not recognize other immunoglobulins or substances present in serum or blood plasma. The originality of the invention lies in the fact that the monoclonal antibodies used can be considered as unique, in the sense that they did not previously exist in nature, given that they are produced by an artificially hybridized cell. prepared in the laboratory from two natural cells The products synthesized by hybrids, monoclonal antibodies which recognize human IgG, would not survive in a natural environment and therefore, cannot be assimilated to polyclonal antibodies produced in animals by standard techniques, although they retain some of their properties.

In the evaluation of specific IgG in vitro, polyclonal antibodies are generally used and the main limitation of these methods is their low sensitivity.

Another problem in using polyclonal antibodies is that the specific fraction which recognizes the antigen is only 1-10% of the total γ-globulin fraction. This limitation in the amount of specific immunoglobulin does not exist in monoclonal antibodies, since almost all of the protein is specific against the antigen. Therefore, if polyclonal antibodies are used, there is a great deal of possibility of cross-reactions with nonspecific substances.

Other advantages of monoclonal antibodies over polyclonal ones are the ease of their purification and the availability of unlimited quantities of standardized reagents. It can be said that once a hybrid cell line (hybridoma) has been established, unlimited quantities of the monoclonal antibody can be prepared. This ensures the availability of reagents having permanent properties (homogeneity, specificity and affinity) and guarantees the obtaining of reproducible results, and therefore comparable between different laboratories.

When compared to known in vitro methods, the new invention has the further advantage that it is not necessary to isolate and purify serum IgG (Galfre & Milstein), at least in the amounts or proportions required in those. A lot of time is needed to obtain reliable results, and it is not necessary to centrifuge the samples during the different phases of the analysis.

To assay the specific IgG in the serum, it is necessary to use extracts that are well characterized biologically and / or immunochemically (Carreira J. et sol.,
Clin Allergy, 14, 503, 1984; Corbi and Carreira, Int. Archs.

Allergy appln. Inmun. 76, 156, 1985).

The extracts used in this invention, although commercially accessible, are in any case subjected to a preliminary purification process to increase the efficiency of binding, the reproducibility of the method and the correlation with the in vivo assays. For these extracts, the biological activity is measured in vivo, the allergenic potency in vitro, and the different components of the extract are identified and their relative activity is evaluated (Standardization of allergen extracts, Alergia e Inmunologia Abello, S.A.).

Although the mechanism by which a non-covalent bond occurs between a protein in aqueous solution and the plastic to which it is exposed is little known (Parsons, Methods of Enzymology, 73, 224, 1981) the bond is extremely strong, and thus most importantly, the biological activity of the sample is maintained. Most authors agree that protein binding to plastic surfaces occurs through non-specific hydrophobic interactions. It seems that the only reagent needed for making protein coated plastics is the plastic itself. Several authors (Catt et Coll, Science 158, 1570, 1967; Crosignani et Col., J. Lin Endocrinal Metabl. 30, 153, 1970) have examined the effect of various parameters on the binding of antibodies to a plastic, and came to the conclusion that neither temperature, pH nor ionic strength have any influence. According to the experience of the Applicant, polystyrene, polyethylene, polypropylene or a polyvinyl can be used almost indifferently. Plastic tubes, titration plates or individual containers of different shapes and sizes can be used.

Likewise, little is known about the forces that hold proteins bound to nitrocellulose, but it is very likely that hydrophobic interactions play a very important role.

However, despite what has just been said, the adsorbed protein can be released from the plastic during the incubation and washing processes. To avoid this, the atgenes can be covalently linked to the plastic solid phase, with chemical modification thereof.

The separation between what is bound to the plastic and what remains free in solution can be achieved by simple suction or decantation. The reproducibility of the process increases if an unrelated protein, for example serum albumin, and any detergent (Triton, Tween, etc.) are added to the wash liquid.

The labeling of monoclonal antibodies with radioactive isotopes or with enzymes is carried out by conventional methods; one chooses for example 125I (Fraker and Speck, Biochem. Biophys. Res. Commun 80, 849), or S-galactosidase (O'Sullivan and Marks. Methods Enzymol, 74, 147, 1978).

The methods of assaying the radioactivity or the bound enzyme are also carried out by conventional methods; in the first case, y radiation counters are used; in the second, it is necessary to carry out an incubation with different enzymatic substrates depending on the detection means available.

The invention is illustrated by means of the following examples, which should be considered as explanatory and not limiting thereof.

In these examples, reference is made to the single figure of the accompanying drawing, which is a graph of determination of specific IgG using monoclonal anti-IgG antibodies in polystyrene vessels, and on which the ordinate scale of left represents the specific binding of an enzymatically labeled MAb to an anti-Lolium perenne serum IgG, the right y-axis represents the specific binding of the same MAb when it is radioactively labeled with 125I, the broken line represents non-binding. specific, and the abscissa scale represents different dilutions of the serum on a logarithmic scale.

Example 1 Determination of the specific IgG against antigens present in plant pollens (L. Perenne).

A) Preparation of monoclonal antibodies
Mice were injected intraperitoneally with 100 µg of IgG purified in complete Freund's adjuvant, on days -45 and -30. On day -3, they are injected with the same amount of IgG and 200 µl of phosphate buffered saline (PBS) intravenously. On day zero, mouse myeloma cells are fused with spleen lymphocytes from immunized animals, using 50t polyethylene glycol.

Cells are aliquoted on six well plates, grown in HAT selective medium.

B) Selection
After two weeks, the culture supernatants were collected and analyzed for possible specificity against.
IgG. To avoid the selection of hybridomas which would secrete antibodies with specificity against the light chains or against idiotypic determinants, another
Different myeloma IgG. In positive clones, the possible cross-reactivity with IgE, IgM or IgA is analyzed.

Hybridomas which recognize IgG are cloned and subcloned on semi-solid agar. Those which recognize different epitopes in IgG are grown on a large scale for purification and labeling.

C) Marking
After the monoclonal antibodies have been purified by standard methods, they are labeled with I according to the method described by Fraker and Speck, 1978, Biochem.

Biophys. Common Res. 80, 849. In other cases, they are enzymatically labeled with S-galactosidase by the method O'Sullivan and Marks, 1978, Methods in Enzymol., 74, 147.

D) Determination of specific IgG
The analyzes are carried out without distinction on microtiter plates of different formats and plastic materials, in plastic tubes, or on nitrocellulose disks.

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

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

3) To saturate the binding sites which are still free, the wells are incubated with PBS-BSA for 1 h at room temperature.

4) Next, the wells are washed three times with PBS in the presence of 0.1% Tween (PBS-Tween), each time carefully decanting the liquid from the wells.

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

6) In other wells are added similar volumes of standard serum whose specific IgG content has previously been determined.

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

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

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

10) If the monoclonal antibody added at point 8 is labeled with 125I, in this point the wells are shared to measure the radioactivity with a gamma radiation counter.

If the monoclonal antibody is enzymatically labeled, in this point, the ONPG enzyme substrate (3 mM ortho-nitrophenyl-ss-D-galactopyranoside) is added in PBS containing 10 mM MgCl2 and 0.1 mM 2-mercaptoethanol.

Then, the contents of the wells are incubated at 370C for 2 h.

At this point, the reaction is stopped by adding 100 μl of
1 Na2CO3 and the absorbance is measured at 410 nm.

11) The number of counts per minute (cpm) or the absorbance, for the standard wells, is represented on a diagram on which the ordinates represent cpm or Abs 410 nm, and the abscissa, the logarithm of the reciprocal of the dilution employee.

Using this diagram, each sample is classified by comparing its cpm or Abs 410 with the values obtained for the standard sera.

Example 2
Determination of specific IgG against antigens present in plant pollens XL.Perennel.

A) Preparation of monoclonal antibodies
100 µg of purified IgG are injected intraperitonally into mice in complete Freund's adjuvant on days -45 and -30. On day -3, they are injected with the same amount of IgG (s, K) in 200 µl of phosphate buffered saline (PBS) intravenously. On day zero, mouse myeloma cells are fused with spleen lymphocytes from immunized animals using 50% polyethylene glycol. The cells are partitioned into aliquots on six well plates and grown in HAT selective medium.

B) Selection
After two weeks, the culture supernatants were collected and analyzed for possible specificity against.
IgG. To avoid the selection of hybridomas which would secrete antibodies with specificity against the light chains or against idiotypic determinants, we use another
Different myelone IgG (e, N). In positive clones, the possible cross-reactivity with IgE, IgM or IgA is analyzed.

Hybridomas which recognize IgG are cloned and subcloned on semi-solid agar. Those which recognize different epitopes in IgG are grown on a large scale for purification and labeling.

C) Marking
After having purified the monoclonal antibodies by 125 standard methods, they are labeled with I according to the method described by Fraker and Speck, 1978, Biochem
Biophys.Res. Common. 80, 849. In other cases, they are enzymatically labeled with S-galactosidase by the method of O'Sullivan and Marks, 1978, Methods in Enzymol, 74, 147.

D) Determination of specific IgG
The analyzes are carried out indifferently on microtiter plates of different formats and plastic material, in plastic tubes, or on nitrocellulose disks.

1) To each of the wells, tubes or discs, a volume (50 to 200 μl) of a solution of protein extracts prepared according to the methods described above is added and it is left to incubate overnight at 40C.

2) Unadsorbed protein is removed by aspirating or decanting the contents of the wells and washing them with saline which contains 1t bovine serum albumin (PBS-BSA).

3) To saturate the binding sites which are still free, the wells are incubated with PBS / BSA for 1 h at room temperature.

4) Next, the wells are washed three times with PBS in the presence of 0.1% Tween (PBS-Tween), carefully decanting the liquid from the wells on each passage.

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

6) In other wells, are added similar volumes of standard sera, the content of which has previously been determined.
Specific IgG.

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

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

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

10) If the monoclonal antibody added at point 8 is labeled with 125I, in this point the wells are shared to measure the radioactivity with a gamma radiation counter.

If the monoclonal antibody is enzymatically labeled, in this point the enzyme substrate is added
ONPG (3 mM ortho-nitrophenyl-BD-galactopyranoside) in
PBS containing 10 mM MgCl2 and 0.1 mM 2-mercaptoethanol.

Then, the contents of the wells are incubated at 370C for 2 h.

At this time, the reaction is stopped by adding 100 µl of 1 M NaziO3 and the absorbance is measured at 410 nm.

11) The number of counts per minute (cpm) or the absorbance obtained for the standard wells is represented on a diagram (see graph 1) on which the ordinates represent cpm or Abs 410 nm, and the abscissa, the logarithm of the inverse of the dilution used. Using this diagram, each sample is classified by comparing its cpm or Abs 410 to the values obtained with the standard sera.

Claims (9)

1. Method for determining the presence of specific IgG in human sera, characterized in that it comprises the following steps: fixing complex protein extracts, purified beforehand, to a solid support, followed by incubation of the. serum sample with said fixed protein extracts: washing of non-specific proteins; incubation with the anti-IgG monoclonal antibody labeled with 125I or with an enzyme; subsequent washing of the solid support with the fixed products, and determination of the radioactivity or the enzymatic activity, incorporated in the solid support. 2. Method for determining the presence of specific IgG in human sera, according to claim 1, characterized in that the monoclonal antibody has been labeled with an enzyme (for example: B-galactosidase). 3. Method for determining the presence of specific IgG in human sera, according to claims 1 and 2, characterized in that a sugar is attached to a plastic as a solid support. 4. Method for determining the presence of specific IgG in human sera, according to any one of claims 1, 2 and 3, characterized in that a glycoprotein is linked to the solid support. 5. Method for determining the presence of specific IgG in human sera, according to claims 1, 2, 3 and 4, characterized in that one binds, to nitrocellulose as solid support, a glycoprotein, a sugar or a protein. . 6. Method for determining the presence of specific IgG in human sera, according to any one of the preceding claims, characterized in that the values obtained are compared semi-quantitatively with a standard serum. 7. Method for determining the presence of specific IgG in human sera, according to any one of the preceding claims, characterized in that the binding of the protein extract to the solid support is covalent. 8. A method for determining the presence of specific IgG in human sera, according to any one of claims 1-6, characterized in that the binding of the protein extract to the solid support is not covalent. 9. Method for determining the presence of specific IgG in human sera, according to the preceding claims, characterized in that the solid support is the same container in which the test is carried out.