DD260132A1 - PROCESS FOR USING MONOCLONAL ANTIBODY AGAINST FITC - Google Patents

Abstract

The invention relates to a method for the use of monoclonal antibodies (MoAb) against fluorescein isothiocyanate (FITC) in immunological tests. The aim of the invention is the use of MoAk against FITC as a universal binding and thus detection reagents in a variety of immunological methods. The process according to the invention is characterized in that FITC is used as the uniform coupling substance for the labeling of the indicator reagents used in immunological detection methods. The detection or binding is then carried out by the use of MoAk against FITC, which are coupled with a marker used in highly sensitive immunoassays (radioisotope, enzyme, etc.) or solid messengers. For the detection reactions, the MoAk produced by the hybrid cell clones B13-AF9 and B13-DE1 were used against FITC, which were deposited in the Central Institute of Molecular Biology (list 1986) under the numbers DDR 0108 and DDR 0109 respectively. Fields of application of the invention are the most varied areas of the life sciences, including medicine.

Description

Field of application of the invention

The invention relates to a method for the use of monoclonal antibodies (MoAb) against fluorescein isothiocyanate (FITC) in immunological tests.

Fields of application of the invention are the most varied areas of the life sciences, including medicine.

Characteristic of the known technical solutions

Highly sensitive immunological detection methods are based on the specific recognition of the substance to be detected by antibodies. Prerequisite is the labeling of the indicator reagents with a substance that is still detectable in very low concentrations. The most important labeling substances for highly sensitive immunoassays are radioisotopes, enzymes, fluorescence markers or luminescence markers. The sensitivity of an immunoassay depends not only on the binding strength of the antibodies used but also on the effectiveness of the coupling of the labeling substances, which may be different for different protein molecules, including antibodies. For this reason, an optimal labeling method of the detection reagents must be developed for each immunoassay, and therefore for various immunoassays are also the most diverse labeled reagents.

Almost all proteins, including the antibodies, can be relatively easily labeled with the fluorescent dye fluorescein isothiocyanate (FITC) without seriously affecting the biological activity of the protein molecules. This property has long been used in immunohistology by the use of FITC-labeled antibodies and the evaluation of the tests under the fluorescence microscope. However, for a general use in highly sensitive fluoroimmunoassays FITC proved to be conditionally suitable.

The facile labeling of proteins with FITC and the possibility of producing antibodies to FITC were exploited in initial experiments for cell separation and immunohistological detection. Polyclonal (BARAN, M.M., et al., J. Immunol. Meth., 53, 311, 1982) and monoclonal anti-FITC antibodies (HAAIJMAN, J.J., et al., 84, 363, 1986) were used for cell separation.

Object of the invention

The aim of the invention is the use of monoclonal Anitkörpern against fluorescein isothiocyanate (FITC) as a universal binding and thus detection reagents in a variety of immunological methods.

Explanation of the essence of the invention

The invention has for its object to use monoclonal antibodies against FITC as detection reagents in a variety of immunological methods.

The inventive method for using monoclonal antibodies against FITC is characterized in that the indicator proteins used in an immunological test (antibodies, antigens, etc.) are labeled with FITC and that the FITC-labeled proteins after use in the corresponding tests with the monoclonal antibodies be detected against FITC, in turn, with a still detectable in low concentration marker substance such. As radioisotopes, enzymes, etc. are coupled. Since FITC can be easily coupled to different proteins, z. As radioisotope or enzyme-labeled monoclonal anti-FITC antibodies can be used as universal reagents for a variety of immunological tests.

The coupling with FITC and the use of anti-FITC antibodies can therefore be used for the detection of antigens and antibodies in solution, for immunohistological and cytological tests, as detection reactions in protein blotting, for nucleic acid hybridization, for cell recruitment, etc. Since the anti-FITC antibodies quench the fluorescence of the FITC, a test development is also possible on this basis

 The monoclonal anti-FITC antibodies used for the inventive use were from the hybrid cell clones

B13-DE1 and B13-AF9 / deposited at the Central Institute of Molecular Biology (ZIM list 1986) under the numbers DDR 0109 and DDR 0108 respectively.

The invention will be explained below to 3 embodiments. ,

embodiments

1. Monoclonal Antibodies to Fluorescein isothiocyanate (FITC)

Specific monoclonal antibodies to FITC were used for the methods, which would be produced by clones B13-DE1 (ZIM List 1986 No. DDR 0109) and B13-AF9 (ZIM List 1986 No. DDR 0108). The antibodies were enriched from the ascitic fluid by ammonium sulfate precipitation or purified via hydroxyl apatite. As the labeling substance for the antibodies, ¹²⁵ J was used in the present examples, so that the evaluation was made on the basis of radioimmunoassays.

2. Labeling of the proteins used with FITC

The labeling with FITC is a standard method which in the present examples is as described by M.M. GANI, et al. (J. Immunol., Meth., 34, 133, 1980).

3. Applied detection methods a) Antibody detection

Sensitive radioimmunological detection of antibodies, including monoclonal antibodies, may be performed by solid phase radioimmunoassay according to the following incubation scheme:

Purified anti-immunoglobulin (Ig) antibodies are adsorbed to polyvinyl chloride (tablet blister), after washing under tap water incubation with the antibodies to be detected and (after further washing) in the last step with the corresponding ¹²⁵ J-labeled antigen (B. MICHEEL, et al., J. Immunol., Meth. 46, 41, 1981). When monoclonal antibodies to FITC are used, incubation with FITC-labeled antigen followed by ¹²⁵ I-labeled anti-FITC antibodies is performed instead of incubation with ^12S J-labeled antigen. A comparison of both methods is shown in Table 1.

Table 1: Detection of the reactivity of anti-HCG monoclonal antibodies ²¹ in two solid phase radioimmunoassays

Antibody dilution binding of radioactivity

on solid phase ³¹

10- ¹ ΙΟ " ² ΙΟ" ³ ΙΟ " ⁴ ΙΟ" ⁵ 10 " ^e 10- ⁷ 10" ³ ΙΟ ' ⁹ 10

Method 1 ⁴⁾ Method 2 ^5I 5906 5844 5682 6598 5934 6189 6112 5997 5209 5166 4089 5731 2363 2 046 1116 1318 754 937 700 793

1) HCG = human choriogonadotropin

2) The monoclonal antibodies produced by the hybrid cell clone B9-AB9 (ZIM list 1986, no. DDR 0107, WP 244564) are used 3 ^ value in c. p.m. (counts per minute)

4J Incubation order: goat anti-mouse Ig, MoAk vs HCG, ¹²⁵ J-HCG

5) Incubation sequence: goat anti-mouse Ig, MoAc against HCG, FITC-HCG, ¹²⁵ J-MoAk against FITC (to avoid unwanted binding, normal mouse serum was added to the dilution buffer of the last MoAk at a concentration of 1%).

Both methods show the same detection limit. The ¹²⁵ I-labeled anti-FITC antibodies could be used with the same result for a number of other antigen-antibody systems.

b) antigen detection

The detection of antigens in aqueous solutions was carried out on the basis of two-side binding tests (sandwich tests).

Such tests are based on binding an antibody to a solid phase followed by incubation with the sample to be tested and finally with labeled antibodies. The use of monoclonal anti-FITC antibodies was carried out in a test system for the detection of HCG in a standard solution. The following incubation sequence was carried out: adsorption of MoAk against HCG produced by hybrid cell clone B9-AB9, ZIM list 1986, no. DDR 0107, WP 244564 on polyvinyl chloride, HCG standard solution in different dilutions, FITC-labeled MoAk against HCG produced by clone B9- BA8, ZIM list 1986, No. DDR 0104, patent application WP 244565 ¹²⁵ J-marked MoAk against FITC.

The results are shown in Table 2.

Table 2: Detection of HCG in a standard solution in a two-site binding assay with MoAk against HCG and a MoAk against FITC

Dilution of Binding of radioactivity Standard solution ¹ ' on the solid phase ²¹ 1:10 13747 ± 4294 1:20 10 696 + 1497 1:40 5896 ± 653 1:80 3 599 ± 272 1: 160 1965+ 195 1: 320 1542+ 158. , 1: 640 1240 ± 81 1: 1280 1119 ± 78 1: 2560 1094 ± 52 ' 1: 5120 1116 ± 25 1) Standard solution contained 1 IU / ml 2) values in cp.m. (counts per minute), means and standard deviations of quadruplicate determinations

As can be seen from the results, the sensitivity limit of the test is below 10 mlU / ml (which would correspond to the dilution level 1: 100). The test thus complies with the international standard for highly sensitive HCG detection methods. The ¹²⁵ I-labeled anti-FITC antibodies could be used with similar results for other antigen determinations, c) use in immunocytology

The detection of antigens on cells was performed on the basis of an indirect radioimmunoassay. Such assays rely on incubating cells with an antigen-specific antibody and, in a second step, detecting binding of the antibody to the cells by a labeled anti-antibody. In conventional assays, the detection of the bound antigen-specific antibody employs an anti-immunoglobulin antibody of another species. The use of anti-FITC antibodies was carried out in a test system for the detection of carcinoembryonic antigen (CEA) on cells. The effectiveness of detection by ¹²⁵ I-labeled anti-FITC antibodies was compared to the conventional detection method by ¹²⁵ I-labeled rabbit anti-mouse immunoglobulin antibodies.

The following experimental set-up was chosen: Method 1: Cells of the colon carcinoma cell line LS 174 incubated with FITC-labeled MoAk against CEA, which were produced by the hybrid cell clone D14-HD11 (ZIM list 1986, No. DDR 0103), then incubation with ¹²⁵ I labeled

Rabbit anti-mouse immunoglobulin antibodies

Method 2: Cells of colon carcinoma cell line LS 174 incubated with FITC-labeled MoAk against CEA produced by hybrid cell clone D14-HD11 (ZIM List 1986, No. DDR 0103), then incubation with ¹²⁵ I-labeled MoAb

against FITC

 The results are shown in Table 3.

Table 3: Detection of CEA on cells of colon carcinoma cell line LS 174 in the radioimmunoassay using MoAk against CEA and ¹²⁵ J-labeled rabbit anti-mouse immunoglobulin antibodies or ¹²⁵ J-labeled MoAk against FITC

Dilution of the FITC-labeled MoAk against CEA

Method 1 Method 2 ( ¹²⁵ J anti-mouse ( ¹²⁵ J-MoAk Immunoglobulin) against FITC) 3120 ¹¹ 12163 2925 8699 2461 5451 1105 2 040 450 1010 325 850

control

1) values in cp.m. (counts per minute).

The results show that the detection limit is comparable with both methods. It is at the antibody dilution 1: 51200. The quantitative differences in the readings are probably due to differences in the ¹²⁵ J mark.

The method could also be transferred to other test systems. It offers particular advantages in work that leads to undesirable cross-reactions in the conventional experimental approach (such as in autologous or allogeneic systems).

Claims (2)

1. A method for the use of monoclonal antibodies (MoAk) against fluorescein isothiocyanate (FITC) for immunological detection methods, characterized in that the for such - Method indicator reagents are coupled with FITC and that after use of the FITC-coupled reagents, a detection or binding by MoAk against FITC takes place, which in turn are coupled to a marker or solid support or by the deletion of the FITC fluorescence a measurable Cause effect. 2. A method for the use of monoclonal antibodies (MoAk) against fluorescein isothiocyanate (FITC) for immunological detection method according to claim 1, characterized in that the MoAk against FlTC of the hybrid cell clones B13-AF9 (ZIM list 1986: DDR No. 0108) and B13- DE1 (ZIM list 1986: DDR No. 0109) are produced.