HU202595B - Process for producing mouse monoclonal antibody connected with human alpha-1-foetoprotein - Google Patents

Abstract

The present invention relates to a method for producing a mouse monoclonal antibody or antibody pair associated with human alpha-1-fetoprotein. The method involves immunizing the AxDBAz Fj hybrid mouse strain with cyclic alternating antigen intake for five cycles over 10 to 12 weeks. The invention also relates to the preparation of a monoclonal antibody pair suitable for human alpha-1-fetoprotein enzyme immunoassay. EN 202 595 A Scope of the description: 4 pages without drawing -1-

Description

This invention relates to a human monoclonal antibody to human alpha-1-foetoprotein (AFP).

Monoclonal antibodies are highly specific, high purity serological reagents, the method of preparation of which was first described by Kohler and Milstein (1975, Natwe, 256,495).

An important novelty in the production of monoclonal antibodies is the use of conventional, so-called. against polyclonal antibody production, it is a non-living animal process; hybrid cells produced for this purpose produce the antibody. In polyclonal antibody production, the antibody-inducing substance (protein molecule, microorganism, blood cell, etc.), that is, the antibody mixture, is formed which reacts with several, sometimes hundreds, specific sites in the antigen. Monoclonal procedures solve the individual production of potential antibodies.

Fetal alpha (AFP) is a major component of serum in fetal life, such as albumin in adulthood. Its role is unclear and may play a role in suppressing the maternal immune response to the fetus (Peck, A.B., et al., J. Immunol., 128, 1134 (1982) and Littmann Β. M. et al., Cell. Immunol., 30, 35 (1977)]. AFP is a 7072,000 molecular weight glycoprotein of the alpha globulins. Its appearance and concentration change in the fetal serum is accompanied by a proportional change in the AFP levels of the amniotic fluid and maternal serum.

A congenital malformation of the central nervous system is a neural tube closure disorder, accompanied by abnormal levels of AFP, so that the blood serum of AFP in a mother with such an abnormal fetus is elevated relative to normal. The difference in pregnancy is 16-18. between weeks of age and significant and characteristic of the fetal pathology.

Most of these fetuses are stillborn or, in less severe cases, die within 5 years, severely mentally handicapped and physically challenged. Early diagnosis and medical intervention are therefore essential.

Polyclonal and monoclonal antibodies can be used to determine AFP. By their nature, cross-reactions of polyclonal antibodies are significantly more frequent, and albumin in particular may interfere with the assay, which can only be eliminated by complicated and costly purification steps.

AFP antibody binding is the basis for the specificity of the assay method. In addition, it is necessary, in a series of rapid methods, to detect antigen-antibody binding in a measurable manner. The two methods commonly used are immunoreactions with radioactive material or coupling the immune reaction with an enzymatic reaction. The latter is more appropriate in cases where the use of the determination method is decentralized and the conditions and instruments of the isotope technique cannot be expected in all cases.

The modem mode for linking the immune response to an enzymatic reaction is the so-called modem. sandwich enzyme immunoassay The essence of this is that two specific but different antibodies reacting with the target substance are required, which react in part with different antigenic determinants of the target substance and each have different side-functions. One antibody binds well to plastic surfaces and retains its immunoaffinity when attached, the other is well coupled to the detection system enzyme and does not lose the antigen binding ability of the antibody in the antibody-enzyme complex. In the assay, the first antibody is bound to a plastic sheet, which reacts with one of the determinants of the antigen, and then detects the bound antigen with the other antibody, which, in response to the other determinant of the antigen, enters the system. By adding a substrate to the enzyme, the enzymatic reaction takes place and the resulting colored product can be accurately determined photometrically.

AFP has been shown to have multiple sites (seven or six) of antigenic determinants, and accordingly, monoclonal techniques can produce multiple antibodies of different specificity, any of which is capable of detecting and quantifying AFP with certainty. A suitably selected pair of antibodies of different specificity may serve for the sensitive and rapid detection of a specific reaction.

AFP-associated monoclonal antibody is described by Stenman [J. Immun. Meth., 46, 33 (1981)] and Uotila (Mol. Immunol., 77, 791 (1980)]. Antibody-producing hybrid cells were obtained after hyperimmunization of BALB / C mice with AFP by fusing animal spleen cells with myeloma cells (SP2 / 0-Agl4). Of the resulting hybridoma cells, the so-called positive cells producing the desired specificity were up to 1-2 cells per increment, with a completely unsuccessful experiment with this antigen.

Due to several requirements for antibody production, such as high affinity antibody, a variant that binds to the plastic surface and another that retains its immunoreactivity in its complex with the enzyme, even positive hybrids obtained after hybridization need to be sorted to produce a cell line pair suitable for production. If the number of hits in a preparatory step is low, it disproportionately diminishes the possibility of selection to the appropriate cell line pair, and thus ultimately the quality of the cell line pair that can be produced.

Surprisingly, we have found that the FI generation obtained after crossing two mouse strains produces high specificity and high affinity antibody upon immunization. Unexpectedly, hybridoma cell lines derived from the spleen cells of such animals retain the ability to produce high levels of highly specific and high affinity material.

The efficiency of antibody production can be further enhanced by changing the antigen dosing regimen during immunization prior to spleen cell extraction and building the process from immunization cycles instead of the usual repeat immunization.

The present invention relates to a process for the production of an antibody or pair of antibodies suitable for the AFP enzyme immunoassay. In accordance with the present invention, AxDB A ₂ Fi hybrid mouse strain is used to produce the antibody (s). The present invention also relates to a novel immunization process; immunizing the F, hybrid mouse strain by cyclically alternating AFP antigen delivery route.

Specific antibody producers are selected from clones of spleen cells hybridized with tumor cells. Monoclonal antibodies were obtained with single clones

EN 202 595 Characterized in a matrix-based competition study. The desired antibody pair combination is selected from antibody pairs that bind to different antigenic determinants so that they do not lose their immunaffinity when bound to one plastic surface and bound to the other detection enzyme.

Using the method of the invention, a greater number of colonies of hybrid cells show positivity, and the number of colonies producing the specific antibody may be as high as 20 increments. From a limited number of experiments, the cell line that best serves production can be selected by selection. From a limited number of experiments, a pair of antibodies can be highlighted, one for surface binding and one for enzymatic detection.

In accordance with the present invention, prior to selecting antibodies that bind to the surface or enzyme without loss of immunoaffinity, a competition assay will search for pairs of antibodies that bind safely to different antigenic determinants, thereby minimizing interference between each other.

Positive cell lines obtained as described generate antibodies and react in pairs in a matrix system with AFP antigen. Where the two antibodies bind to different antigenic determinants, the second reaction shows a low level of competence with little inhibition. Where the binding sites of the two antibodies are identical or close, the first reaction is competent with the second, with a high percentage of inhibition.

General characteristics of the antibodies produced by the method of the invention are:

lg isotype IgG 1 and 1, respectively. IgG 2a

Titer of 10 ³ -10 ⁵

Cross reaction with HSA 0-10%.

The process of the invention is described in detail in the following example.

Example

AxDBA ₂ won by F! mice are occasionally immunized with 10 gg of antigen, complete with Freund's adjuvant for the first two cycles, 3, 4,

In cycle 5, the antigen is introduced with incomplete Freund's adjuvant. The last inoculation of the antigen takes place in aqueous solution. Methods and dates of vaccination according to the following cyclic schedule:

/. spreadsheet

Day Intake Input method The volume of the solution is ml 1st cycle 0 AFP-CFA soles or soles if skin 0.1 and. 0.1 2. AFP-CFA foot pads or. if skin 0.1 and. 0.1 4. AFP-CFA soles or soles if skin 0.1 and. 0.1 2nd cycle 10. AFP-CFA if skin 0.2 12. AFP-CFA if skin 0.2 14. AFP-CFA if skin 0.2 3rd cycle 40. AFP-IFA soles or soles if skin 0.1 and. 0.1 42. AFP-IFA soles or soles if skin 0.1 and. 0.1 44. AFP-IFA soles or soles if skin 0.1 and. 0.1

Continue Table

Day Intake Input method The volume of the solution is ml 4th cycle 50. AFP-IFA if skin 0.2 52. AFP-IFA if skin 0.2 54. AFP-IFA if skin 0.2 5th cycle 80. AFP Aqueous Solution 84 except for spleen abdominal cavity 0.2

CFA = Compl Freund Adjuvant

IFA = incomplete Freund adjuvant.

Spleen cells are fused with mouse myeloma cells Sp2 / oAg-14 in a known manner. [1st Andó et al. (1984); Tissue Culture and Rés., P. Röhlich and E. Bácsi editors, Academic Press Hungary, Elsevier Press, Holland, p. 241] Hybrid cells are selected in a known manner (Köhler, G., Milstein, C., Eur. J. Immunol. 6, 511 (1976) and Hengartner H., Luzzati AL, Schreier M. Curr. Topies in Microbiol. and Immunol., 81, 92 (1978)], and the antigen producers thereof are determined as follows:

Microtiter plate wells 100 to 100 gl amount of 3 gg / ml AFP solution was pipetted to 16 hours at 4 ° C the plates are incubated in antigen 15 mM Na ₂ CO _3, 35 mM NaHCO ₃ (pH 9.6) buffer diluted.

The plates were washed three times with PBS-Tween solution containing 137 mM NaCl, 1.47 mM KHjPa, 8 mM Na ₂ HPO ₄ , 2H ₂ 0.0.05% Tween, pH 7.2. 100-100 g of the hybridoma supernatants to be tested are then incubated in the wells for 2 hours at 37 ° C.

The above wash was repeated three times and 100100 μΐ of diluted rabbit anti-mouse Ig-HRPO was incubated on the plates at 37 ° C for 1 hour (HRPO = horseradish root peroxidase).

The plates are washed again and finally 200 μΐ of substrate solution is added to the wells of the plate. (Composition of the substrate solution: 333 µg / ml o-Phenylenediamine, 0.005% H /) ₂ , 63 mM Na ₂ HPO ₄ .2H ₂ 0.26.2 mM citric acid, pH 6.0). Incubation was carried out at 37 ° C for 30 minutes. The reaction was terminated μΐ weighed into 50 / well of 4N H ₂ SO ₄ on the results evaluated by spectrophotometry at 492 nm.

Antibody-producing hybridoma cells are cloned and, for example, 24 antibody-producing clones are raised during processing of spleen cells from a mouse.

Qualification of Cell Antibody Production: Cells producing specific antibody are inoculated at 10x cells / ml into the mouse abdominal cavity (1 ml / mouse).

Tumor cells produce ascites fluid with a high immunoglobulin content (5-10 mg specific Ig per mouse). The working dilution of ascites fluids is defined as the dilution whereby the color product of the enzymatic reaction is EL = 1.0 in the enzyme-linked immunoassay.

In the assay, 3 µg / ml AFP was used as antigen by dilution of the test antibody in series followed by an enzyme-linked, detection peroxidase-labeled anti-AFP antibody.

HU 202 595 A

The working dilutions of the antibodies prepared as described are 5 x 10 ⁴ , 1 χ 10 ³ and 1 χ 10 ^{4 respectively} .

(In the order of AFP / 1, AFP / 14, AFP / 33).

Human serum albumin was tested for non-specific, so-called antibodies. cross-activity. In the peroxide-detected assay, 3gg / ml AFP and gg / ml HSA are measured side by side. Taking the value for AFP at 100%, express the extent of the undesirable cross-reaction with HSA. 10

For the three antibodies selected, this value is 0%; 10% effect

The competition assay between the antibodies is performed by preparing peroxidase-labeled antibody-enzyme complexes from the test antibodies.

[Wilson, Μ. B. and Nakane, PK (1978), Immunofluorescens and Related Technignes, Knapp W., Holubar K. and Wiek G. edited by Elsevier North Holland, Amsterdam, p. 215], on the other hand, 100 µg / ml of unlabeled antibodies. solutions.

In the presence of gPg / ml AFP, all unlabeled antibodies in the matrix system are contacted with each of the labeled antibodies. After reading the color formation, calculate the degree of competition: 25

E ⁴⁹² without competitor

- x 100 =% reduction.

In the presence of E ⁴⁹² competitors

Antibody binding to a different determinant is considered to be a pair of antibodies where the degree of suppression is less than 25%.

first reactive antibody second AFP / 1 AFP / 14 AFP / 33 reactive antibody Competency%

AFP / 1

AFP / 14

AFP / 33

78.0 ± 4.4 69.7 ± 6.0 1.8 ± 3.6

78.5 ± 9.4 78.6 ± 9.6 20.6 ± 13.2

9.7 ± 8.2 11.7 ± 10.6 63.1 ± 7.5

Suitable antibody pairs for the test may be AFP / 1 and AFP / 33 or AFP / 14 and AFP / 33.

Claims (3)

PATENT CLAIMS A method for producing a murine monoclonal antibody or antibody pair bound to human alpha-1-foetoprotein by hybridoma technique, wherein the immunization procedure is carried out on an AxDBA ₂ hybrid mouse strain for five cycles of 10-12 weeks with a cyclic antigen delivery route. 2. The method of claim 1, wherein the immunization is carried out by the antigenic route of Table I. 3. A method for producing a pair of monoclonal antibodies suitable for human alpha-1-foetoporotein enzyme immunoassay, characterized by selecting specific antibody producers from clones of tumor cell hybridized spleen cells from an immunized mouse strain, monoclonal antibodies obtained from each clone, of the binding antibody pairs, the desired antibody pair is determined such that one does not lose its immunoaffinity when bound to one plastic surface and the other bound to the detection enzyme.