DE3411472A1 - Method for determining the presence of insulin, antiinsulin antibody, method for determining the human insulin level and method for determining human insulin - Google Patents

Abstract

The invention relates to a method for determining the presence of insulin by means of cell lines which secrete antiinsulin antibodies and are selected from the group of the following cell lines: AE9D6, DB9G8, CC9C10, CG7C7, BE3F9 and CE9H9, and to an antiinsulin antibody which is secreted by a cell line which is employed for determining insulin and is selected from the group comprising AE9D6, DB9G8, CC9C10, CG7C7, BE3F9 and CE9H9, and additionally to methods for determining the human insulin level by employing combinations of monoclonal antibodies of particular specificity, the antibodies having been secreted by the cell lines according to Claim 1, in order to distinguish endogenous insulin secretion from exogenous insulin injection, and for determining the human insulin level by preparing monoclonal antiinsulin antibodies by contacting a conjugate of these antibodies with the insulin to be investigated; and determining the affinity of each antibody by an assay selected from radioimmunoassay, double binding assay or any other suitable biologically acceptable assay.

Description

THE UNITED STATES OF AMERICA, represented by the Secretary, United States Department of Commerce, Springfield, Virginia 22161, U.S.A.

Method for determining the presence of insulin, anti-insulin antibodies,. Method for determining the human Insulin levels and methods of determining human insulin

The invention relates to a method for determining the presence of human insulin, '- "Srrtiinsulinantikibe, a method of determining human insulin levels and methods for the determination of human insulin.

Cell lines secreting anti-insulin antibodies were obtained by hybridizing insulin-immune lymph node cells with of the myeloma cell line SP2 / O-Ag14. This was done

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BOEHMERT & ^: BOEHIViERT '■ -'.:.:. 3411472

to produce anti-insulin monoclonal antibodies of known specificity for studying and determining the immune response to insulin. The examination can be restricted to test sets. These antibodies are used as diagnostics
valuable to human insulin levels.

The invention can be applied to various types of insulin which are peculiar to animals or humans
are from which they are descended; e.g. for human -, -> ,, insulin / bovine insulin, pig insulin, mouse insulin,

Chicken insulin and rat, rabbit, fish, guinea pig and sheep insulin.

The insulin mAb antibodies in the cell lines which secrete these antibodies can be used to determine the circulating levels in the blood for various insulins, such as human, cattle, pigs, rats, chickens, fish guinea pigs, sheep and Mouse insulin. In addition, the mAb's according to the invention for the determination of
Use human blood levels of insulin as a diagnostic aid.

_ As state of the art, 'Moyce A. Schroer et al in

Journal of Immunology, Vol. 123 (2), Aug. 1979, pp
670-675 of the articles "H-2-Linked Ir Gene Control of
Antibody Responses to Insulin ". Also published by Joyce A. Schroer et al. Monoclonal Antibodies in Endocrine Research, Fellows and Eisenbarth (editors), Raven
Press, New York, 1981, pages 167-179.
The antibodies of this publication are a set of four antibodies produced by a cell line inferior to the cell line of the present invention.

Furthermore, from Joyce A. Schroer et al "Mapping Epitopes
Published on the Insulin Molecule Using Monoclonal Antibodies, European Journal of Immunology, 1983 (in press).

About the antibody target for

little is known about an antigenic determinant or protein molecule, particularly about the molecular basis of high affinity antigen antibody interactions on insulin. The present invention teaches the development and use of monoclonal antibodies (mAb) against antigenic structures on insulin. The antibodies according to the invention are anti-insulin antibodies and are secreted by cell lines produced by hybridizing insulin-immune lymph node cells and a myeloma cell line SP2 / O-Ag14. The cell lines and their corresponding monoclonal antibodies are designated AE9D6, 06908,009010, CGICI, BE3F9 and CE9H9. The mAbs secreted by these hybridomas are specific for certain insulin amino acid sequences and therefore act as diagnostic reagents for determining human insulin levels.

These anti-insulin antibodies also allow antigenic sites to be "mapped" onto insulin. Since the monoclonal Antibodies are cross reactive, they are also suitable for the treatment of diabetes. In particular, the Treatment of diabetes using both porcine and bovine insulin as injected into the bloodstream. the Test kits according to the invention are able to differentiate between these types of insulin and consequently are capable of the Human level. Insulins that are secreted by the body are differentiated from the amount of foreign insulin (Beef and pork) injected into the body,

to specify.

Six antiinsulin mAbs were developed. The immunological cross-reactivities of these mAbs for species-dependent / Variants of insulin or chemically modified insulin were determined. These mAbs bind to spe-

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specific amino acid sequences on the insulin molecule. Antibody AE9D6 recognizes the area around the amino acid sequence Thr-Ser-Ile of the Α chain of human insulin. The other five mAbs react similarly, although with exact sequence specificity has not yet been determined.

The body's immune system, especially the antibody-secreting white blood cells, act by reacting to foreign particles in the bloodstream. Some proteins, antibodies (or immunoglobulins) bind and thereby neutralize foreign proteins that enter the body. The foreign proteins, called antigens, direct the start of the production of a complementary antibody _; they combine to form an antigen-antibody complex. The antibody molecules contain binding sites that are specific and complementary to the structural features or epitopes of the antigen.

Insulin, which elicits an immune response that can be shown to be under MHC-linked IR gene control occurs is one of the physical and. chemically better characterized proteins. The insulin monomer is made up of two linked by interchain disulfide bonds Chains.

The Α-chain has two helical sections, which are separated by a loop area, which is cut by one between the chains disulfide bridge running between cysteines 7 and 11 is formed, are separated; the B-chain consists of sheet segments at the amino and carboxy ends, which by a central helical area that is intertwined with the carboxy-terminal helix area of the Α chain is connected.

Although insulin is not a classic antigen in the sense of a

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Is a foreign substance, insulin also has structural surface features, which allow insulin to bind other molecules. The anti-insulin antibodies according to the invention are the first antibodies designed to specifically adhere to an insulin surface feature. The dual binding capacity of these monoclonal antibodies is particularly useful for the precise determination of insulin levels important. An itiAb alone is not enough to achieve that To adequately determine insulin level; the use of two inAbs is preferred for the specificity test. Any Combination of the six monoclonal antibodies according to the invention can be paired in the double bond assay (described below). In this way, combinations of mAbs can induce endogenous insulin secretion Differentiate (by the body) from exogenous insulin (injected bovine or porcine insulin).

The method according to the invention can be carried out using any of the known cloning techniques. The following The method described is not intended to limit the invention by this description.

BALB / c mice are immunized with human or bovine insulin and either lymph node cells or the spleen cells are removed. The insulin immunized cells are then hybridized with myeloma cell line SP2 / O-Ag14. Hybrid cells are then placed in microtiter plates (wells) in HAT medium (hypoxanthine, aminopterin and thymidine) given to kill the unhybridized cells, i.e., the unfused cells. From the survivors Hybridomas will only produce a fraction of the desired antibody. The sources that promote positive cell growth show are examined by radioimmunoassay whether they bind to immunogen and then limited by Dilution with normal peritoneal macrophages

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cloned. Cloned anti-insulin antibodies producing Cell lines are then grown in tissue culture to to be frozen for preservation and later use, or as storage for cells which injected into pristane-treated mice to produce large quantities of antibodies or to contain antibodies To produce supernatants.

Affinity determinations

The affinity of each antibody for its causing insulin was determined by cold inhibition of the binding of the antibody to 125-iodine insulin in a modified radioimmunoassay (RIA). Binding curves were obtained with constant dilution of the Iinmunsera, although variations in the concentrations of labeled insulin or a mixture of unlabeled and labeled insulin were identical. This ensures that 125 I-insulin is indistinguishable from non-labeled insulin in this determination method. 20 μl of a dilution (diluent 1% BSA, 0.02% sodium azide in PBS) of hybridoma ascites that bound 27 to 75% labeled insulin were _{mixed with 20 μl of unlabeled} insulin at a quarter log 10 concentration (6 equal samples for any concentration) mixed over a 100-fold concentration range and incubated for 1 hour at 37 ° C. 20 µl containing 10 to 15 x 10 cpm of 125 I-insulin was added. The samples were mixed and equilibrated at 4 ° C overnight. Immune complexes were precipitated by adding 50 μl of 27% strength polyethylene glycol containing 80 μl / ml of human AB serum as a carrier. The samples were centrifuged at 1000 × G for 1 hour. The supernatants were aspirated and the precipitate counted in a gamma counter. The maximum recoverable 125 I-insulin counts

were determined for each assay using a highly concentrated sample of antibody - subsurface binding

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BOEHMERT & BÖ £ HfrERT - '■ ".: -; - 3411472 -: Q

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(5 to 15%) was determined using three blanks: Diluent Only, NMS i ™ 1: 5 Dilution, and P3-X63-Ag 8 ascites at 1: 5 dilution. The percent binding at each concentration of cold insulin

were used to calculate the levels of bound insulin, and then that of bound insulin The affinities were determined from the negative slopes using a linear regression program Lines determined.

Species dependent displacement assays were performed exactly as above, except that different insulins were used to compete for binding of the mAb to iodinated immunogen. Each concentration of variant insulin was tested for binding inhibition over a million-fold concentration range in 10-fold increments. The cold species variant, insulin, τ25 I-insulin and the antibody dilution mixture were only incubated for 1 hour at 2 ° C instead of overnight at 4 ° C. Samples were then processed as described above ₍ processed and data expressed as percent binding versus concentration of inhibitor (100 % binding was equated to binding in the absence of inhibitor). The 50% inhibition points for each curve were determined and used to calculate I ₅ - Calculate values by dividing the concentration of species variant insulin required to obtain 50% binding inhibition by the concentration of cold immunogen which gave 50% inhibition.

Double binding assays were performed by allowing either 100 µl of an affinity-adjusted mAb or 2 x 40% (NH ₄ ) ₂ SO.-iTeils ascites fluid at 1: 100 dilution to bind to a polyvinyl plate for 4 to 16 hours. the

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The plate is then washed three times with PBS / TW20, followed by saturation of all non-specific binding sites on the plate with a KLH solution (0.1% KLH in PBS / TW20) for 30 minutes at 22 ° C. KLH vmrde as the protein selected for the saturation of non-specific binding sites, as it causes the lowest combustion background. 100, ul Bovine insulin was added at 1.0 µg / ml to saturate all binding sites. Preliminary experiments showed that 100 ng / ml insulin was sufficient to saturate all antibody binding sites, with a 10-fold Excess resulted in equivalent bonds and was used in all subsequent assays. Not bound Insulin was removed by washing 5 times with PBS / TW20. The second mAb, either biotinylated for ELISA, or iodized for RIA, was in 100, ul in 1: 1000 fold Dilution added (83 ng antibody per sample) for biotinylated antibodies (preliminary experiments showed that all dilutions from 1: 1000 to 1: 5000 had easily detectable ELISA values compared to background blanks where the insulin produced by KLH

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was set) or 7 X 10 - 1.2 X 10 cpm (7 to 112 ng Antibodies per plate) for labeled antibodies, and allows overnight binding. The plates were washed 5 times with PBS / TW20 washed and then counted in the gamma counter

or developed for ELISA. Since the biotinylated mAbs - ■

sometimes showed high underground binding when KLH insulin Replaced in the double bond assay, blanks containing high insulin concentrations (100-fold excess) were common with the biotinylated second mAb for each antibody pair added to each assay, included and only the ELISA values that changed with the addition of excess insulin were considered suitable for double bond. Any pair of antibodies was tested 2 to 5 times with RIA and 2 to 5 times with ELISA. Merely RIA values that are more than that Twice the background was considered significant and a binding of 0.2 to 27 ng

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125 I antibody (2 to 75 X 10 cpm) was detected. The ELISA values at 492 nm ranged between 0.1 up to 1.9 optical density above the subsurface. Double bond data are given in Table 1.

Antibody-producing secreting cell lines See Table 2 for the description of the cell lines used according to the invention.

Test sets

Test sets for the use of cell lines and their mAb products can easily be prepared in the laboratory using conventional techniques. You can for cross-specificity or insulin level quantification and for the general purpose of the invention, the presence of a specific type of insulin to determine, to be interpreted.

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Table 1

Results of the double bond assay

1 H H H H M. L. 2 L. - - - L. 3 H L. - - H 4th H L. L. M. H 5 L. L. L. H H 6th L. L. L. M.

H = high,> 10xbkg RIA and;> 1, 0 optical density at 492nm.

M = medium, either> 10 χ bkg RIA and 0.1 to 1.0 optical Density at 492 nm or> 1.0 optical density at 492 nm unc 2 to 10 χ bkg RIA.

L = Low, 2 to 10 χ bkg RIA, or 0.1 to 1.0 optical density at 492 nm.

- = then no detectable binding, if that

second mAb is chemically bound, but if this mAb is first applied to the plate, binding is observed.

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Table 2

Characterization of 18 antiinsulin hybridoma antibodies

Cell line a
Immunogen Isotype Affinity constant Specificity mAbs AE9D6 Human IgGl Ko (M ~ 1) a chain key 1 DB9G8 Bovine IgGl 3x10 ° (0.98) ^b ? 2 CC9C10 Bovine 'IgG2a 5x10 ⁸ (0.95) 7th 3 CG7C7 Human IgGl 2x10 ⁷ (0.73) 7th 4th BE3F9 Bovine IgG2a 7x 10 ⁷ (0.99) 7th 5 CE9H9 Human IgGl 3x10 ⁷ (0.97) 7th 6th 2x1O ⁷ (0.96)

Myeloma cells were used for the infusion, somatic cells were BALB / c
up to 14 days primary immune lymph node cells.

Correlation coefficient of Scatchard plots.

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Claims (10)

Expectations 1. Method of determining the presence of insulin by means of Cell lines that donate anti-insulin antibodies, selected from the group of the following cell lines: AE9D6, DB9G8, CC9C10, CG7C7, BE3F9 and CE9H9. - 2. Method for determining the Α chain of insulin, marked by using an anti-insulin antibody secreting cell line designated AE9D6. 3. The method according to claim 1, characterized in that the cell line is DB9G8. 4. The method according to claim 1, characterized in that the cell line is CC9C10. 5. The method according to claim 1, characterized in that the cell line is CG7C7. 6. The method according to claim 1, characterized in that the cell line is BE3F9. 7. The method according to claim 1, characterized in that the cell line is CE9H9. 8. Anti-insulin antibody selected from a cell line used to determine insulin from the group consisting of AE9D6, DB9G8, CC9C10, CG7C7, COPY BOEHMERT & BOEHME-RT - 2 - BE3F9 and CE9H9 / is secreted. 9. Method of Determination of Human Insulin Levels characterized by employing combinations of monoclonal Antibodies of certain specificity, which antibodies have been secreted from the cell lines according to claim 1 to distinguish endogenous insulin secretion from exogenous insulin injection. 10. method for determining the human insulin level, characterized by preparing anti-insulin monoclonal antibodies according to claim 1; Bringing a couple of these into contact Antibodies with the insulin to be tested; and determining the affinity of each antibody by an assay selected from radioimmunoassay, double bond assay, or any other suitable biologically acceptable assay. COPY