CS277597B6 - A method for the preparation of murine monoclonal antibodies against human proinsulin C-peptide - Google Patents

Abstract

This is a method for preparing murine monoclonal antibodies against the C-peptide of human proinsulin. The method includes a procedure for obtaining hyperimmunized mice, whose spleen cells are used to prepare hybridomas producing monoclonal antibodies suitable for use in quantitative determination of the concentration of human proinsulin C-peptide or for determination of human proinsulin.

Description

These monoclonal antibodies can be used mainly in the clinical diagnosis of diabetics for the analytical determination of human proinsulin C-peptide in the serum and urine of patients.

C-peptide is a component of proinsulin synthesized in pancreatic islet beta-cells. Still in the beta cell, most of the proinsulin (86 amino acids) is cleaved into insulin (51 amino acids), C-peptide (31 amino acids) and two dipeptides. Insulin and C-peptide are derived from beta cells in an equimolar ratio of 1: 1 and at the same time a small part of still uncleaved proinsulin (up to 20%).

Human insulin has the same immunoreactivity as animal insulins used to treat diabetics (1 amino acid mismatch for porcine insulin and 3 amino acids for bovine insulin). Therefore, in diabetics treated with insulin, insulin assay is not an indicator of secretion from the islets of Langerhans. In such diabetics, only the human proinsulin C-peptide test provides information on residual secretion.

. Antibodies to human proinsulin C-peptide can still be produced by repeatedly injecting C-peptide as an antigen to experimental animals, most often guinea pigs. The serum of the animals thus immunized, collected after a certain period of antigen action, serves as a source of antibodies used in particular for the quantitative determination of human proinsulin C-peptide in serum or urine by radioimmunoassay or enzyme-linked immunosorbent assay. This procedure, called conventional immunization, has several disadvantages. In the serum of immunized animals there is a heterogeneous mixture of antibodies, so-called polyclonal, whose spectrum is different and unique in each individual organism. Production batches of conventional sera are therefore difficult to standardize and are based on a wide range of quality.

The disadvantage of conventional sera against C-peptide is in principle eliminated by the use of hybridoma technology. The hybridoma product - a monoclonal antibody - is directed against a single antigenic determinant. This approach, i.e. the preparation of hybridomas producing monoclonal antibodies specifically recognizing human proinsulin C-peptide, has been used in the following publications: Madsen O.D., Cohen R.M. , Fitch F.W., Rubinstein A.H. , Steiner D.F .: The production and characterization of monoclonal antibodies specific for human proinsulin using a sensitive microdot assay procedure. Endocrinology 113: 2135-2144, 1983; Madsen O.D., Frank B.H., Steiner D.F .: Human proinsulin - specific antigenic determinants indentified by monoclonal antibodies. Diabetes 33: 1012-1016, 1984. Monoclonal antibodies specific for human proinsulin are currently used for research purposes only. Human proinsulin C-peptide monoclonal antibodies for immunohistochemistry only are listed in the Novo catalog (Denmark). These monoclonal antibodies against human proisulin C-peptide were not. so far prepared with such parameters (low association constant) that they can be used to build a diagnostic kit. C-peptide of human proinsulin has so far been determined with kits based on polyclonal antisera, eg Biodata-Serano or a kit introduced in 1989 by the Institute for Research, Production and Utilization of Radioisotopes, Prague.

Normal values of C-peptide in the serum or plasma of healthy people on an empty stomach are 0.3-0.9 μmol / l (ie 1-3 ng / ml), with an increase of 150-200% during stimulation. C-peptide is excreted in the urine at a quantum of 9-45 μmol / 24 hours, ie. in concentrations of 4-25 μmol / l. In particular, examination of C-peptide in urine by radioimmunoassay requires highly specific antibodies (due to the presence of many grafts of other proteins in urine). Polyclonal antibodies do not have sufficient specificity for universal use.

This lack of low specificity can be eliminated by using monoclonal antibodies obtained by the method of the invention, which is essential after immunization of female hybrids of BIO.A and BALB / c inbred mice by binding of human proinsulin C-peptide to protein carriers - bovine albumin or thyroglobulin. of these in adjuvant and subcutaneous administration, spleen cells from immunized mice are fused with Sp 2/0 - Agl4 myeloma cells and the resulting hybridomas are adapted to grow in vivo in the peritoneum of BALB / c inbred mice, followed by 1 ml of ascitic fluid produced hybridomas in the mouse peritoneum isolate 4 - 10 mg of monoclonal immunoglobulin against human proinsulin C-peptide with an association constant in the system ¹²⁵ I-Tyr-C-peptide / C-peptide in the range from K = 10 ⁷ 1 / mol to 10 ¹⁰ 1 / mol .

In particular, the immunization procedure described to obtain hyperimmune mice is the most important step in the preparation of a monoclonal antibody against human proinsulin C-peptide. This procedure was chosen as optimal and from many tested.

a) Mouse immunization procedure

Female hybrids of inbred mouse strains BIO.A and BALB / c were immunized several times with C-peptide of human proinsulin bound to bovine thyroglobulin and bovine albumin. The protein-bound antigen was emulsified in complete or incomplete adjuvant (Bacto adjuvant incomplete / complete-Freund, Difco Laboratories). These immunogenic forms of the antigen were administered subcutaneously to the abdomen and paws of the mice. In hybrids (BIO.A x BALB / c), mice respond better to human proinsulin C-peptide than homozygotes.

b) Preparation of hybridomas producing monoclonal antibodies against human proinsulin C-peptide

Hybridomas were obtained on a principle known from the literature (Fazekas de St. Groth, S., Shceidigger, D .: Production of monoclonal antibodies: Strategy and tactics, J. Immunol. Meth. 35: 1-21, 1980; Galfré, G., Howe, SC, Milstein, C., Butcher, GW, Howard, JC: Antibodies to major histocompatibility antigens produced by hybrid cell lines, Nature 266: 550, 1977). The positivity of specific antibodies was monitored in the sera of mice immunized with C-peptide. Spleens were removed from mice whose sera were positive at a dilution of 1: 500 (determined by radioimmunoassay) and used to fuse with Sp2 / 0-Agl4 myeloma mouse cells. Cells from 1 spleen were plated in eight 96-well flat-bottom plates after fusion. From 3 fusions, 3 hybridomas producing antibodies to human proinsulin C-peptide were obtained. The stability of the hybridomas was checked by triplicate cloning.

c) Adaptation of hybridomas to growth in vivo in BALB / c 'inbred mice

To propagate hybrid cells in BALB / c mice, hybridoma cells resulting from the fusion of a heterozygous hybrid hybrid mouse spleen (BIO.A x BALB / c) had to be adapted for growth in vivo. Arched monoclonal antibody-producing hybridoma cells were applied in an amount of 5 x 10 6 cells to the peritoneal cavity of hybrid mice (BIO.A x BALB / c). To better adhere the applied cells, the mouse was treated with Landing (2,6,10,14-Tetramethylpentadecane, Sigma, USA) (0.3 ml intraperitoneally) 7 days before transferring the hybridoma cells. After 20 days of growth of hybridomas in the peritoneal cavity, the mouse was sacrificed and the cells (5 x 10 ⁶ ) were transferred to the peritoneal cavity of thymic-free mice (nu-nu). Recipients were again given the Přistaň hybridoma 7 days before cell transfer. After 20 days of growth of hybridomas in the peritoneum, the mouse was eradicated and the cells (10) were transferred to the peritoneal cavity of BALB / c mice pretreated with Landing. In most mice, the BALB / c hybridoma strain grew. Adapted hybridomas for growth in the BALB / c mouse strain were frozen and stored in liquid nitrogen after propagation in tissue culture.

d) Method of biosynthesis of monoclonal antibodies in vivo

Adapted hybridoma cells injected into the peritoneal cavity of BALB / c mice are able to produce ascitic fluid that contains a large amount of monoclonal antibody under economically advantageous conditions. It is possible to isolate the monoclonal immunoglobulin from the ascitic fluid, for example, by several times by reprecipitation with 50% saturation with ammonium sulfate. The advantage of a monoclonal antibody over a polyclonal antibody (see introduction) is mainly that the monoclonal antibody reacts with a certain site (one determinant) on the human proinsulin C-peptide molecule. This reaction is highly specific. Hybridomas producing monoclonal antibodies to human proinsulin C-peptide can be cultured in vitro in media suitable for animal cells and are adapted for growth in vivo in the peritoneal cavity of BALB / c mice. From cans stored in liquid nitrogen, antibody production can be ensured without the need for additional antigen. Monoclonal antibodies react with human proinsulin C-peptide and there is no need to get rid of ballast antibodies. In this way, monoclonal antibodies with a high association constant can be obtained, and therefore they can be used for the analytical determination of human proinsulin c-peptide in the serum and urine of patients by the RIA method. The application area is therefore clinical diagnostics in diabetics. In addition, monoclonal antibodies are useful for the determination of human proinsulin by immunoradiometric (IRMA) or ELISA. The areas of application here are the diagnosis of tumors based on islet beta-cells (nesidioma - insulinoma), familial hyperproinsulinemia and the study of insulin and proinsulin secretion in other pathological conditions. The use of monoclonal antibodies against human proinsulin C-peptide for immunohistochemical and immunocytochemical analyzes, including electromicroscopy, can be envisaged to study the biosynthesis of proinsulin and proinsulin under normal and pathological conditions and to address the issue of proinsulin internalization. The monoclonal antibody can be used during the biosynthetic production of human insulin, including quality control of the final product.

Example 1

Monoclonal antibodies against human proinsulin C-peptide are produced by hybrid cells resulting from the fusion of Sp2 / O-Agl4 mouse myeloma cells and cells obtained from the spleen F1 of BIO.A x BALB / c mouse strains immunized with human proinsulin C-peptide according to the scheme described in the following table:

Table

Scheme of immunization of mouse F ^ (BIO.A x BALB / c) human proinsulin C-peptide

dose day of administration method of administration and amount form per 1 dose of antigen ¹ 1. 2. 3. 4. -5. 6.-7. Day 0 Day 21 Day 35 50th and 51st day 52nd and 53rd day s.c .; 50 μg / 0.2 ml, abdomen s.c .; 50 μg / 0.2 ml, paws s.c .; 50 μg / 0.2 ml, abdomen i.v. and 2x 15 μg / 0.2 ml; tail vein i.p. and 2x 30 μg / 0.2 ml compl. adj uvans n nekompl. adg uvans ⁴⁴ , 9 compl. ad ^ uvans solution in physiol. solution solution in physiol. solution

The C-peptide bound to bovine thyroglobulin was used at doses 1, 2 and 3. The C-peptide bound to bovine albumin was used at 4-7. dose.

^ Bacto adjuvant incomplete (complete) Friend, Difco Laboratorie.

Human, synthetic 1-Tyr-C-peptide, which was prepared in cooperation with the Institute of Organic Chemistry and Biochemistry of the Czechoslovak Academy of Sciences and the Research Institute of Endocrinology in Prague (B. Bendlová, M. Lebl, P, Štolba, L. Stárka) was attached to bovine thyroglobulin or albumin on the principle of a one-step glutaraldehyde reaction (Avrameas, S., Ternynck, T .: The crosslinking of proteins with glutaraldehyde and its use for the preparation of immunosorbents, Immunochemistry 6: 53-66, 1969). The albumin: C-peptide-glutaraldehyde reaction ratio was 1: 10: 100. The quality of the conjugate was monitored by determining the immunoreactivity in RIA, attaching ¹²⁵ I-Tyr-C-peptide and attaching Tyr-C-peptide-Ile followed by amino acid analysis. Thus, it was determined that about 7 mol of C-peptide was bound per 1 mol of albumin. and about 1 mole of C-peptide per mole of thyroglobulin.

Example 2

Hybridoma cells producing monoclonal antibodies to human proinsulin C-peptide are grown in vitro as semi-suspension cultures. The basic culture medium is Eagle's minimal essential medium with Hanks' salt mixture supplemented with non-essential amino acids, L-glutamine (3 mM), sodium pyruvate (1 mM). This medium (referred to as RPMI, Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (0.05 mM), Hepes buffer (N- (2-hydroxyethyl) piperazine-N'-2 acid) for hybridoma culture. ethanesulfonic acid) (10 mM) and inactivated bovine serum (Bioveta, Ivanovice na Hané, 10%). Hybridomas are cultured at 37 ° C.

Example 3.

In order to propagate the adapted hybridoma cells for growth in vivo, 5 x 10 ⁶ cells were applied to the peritoneal cavity of BALB / c mice. To better adhere the applied cells, the mouse was treated with paraffin oil (0.5 ml intraperitoneally) 10 days before transferring the hybridoma cells. After 10 days of hybridoma growth in the peritoneal cavity, the mouse was eradicated and the ascitic fluid produced was collected. In the case of hybridomas producing monoclonal antibody C-PEP-01, a total of 2 ml of ascitic fluid containing 9 mg / ml of immunoglobulin was obtained. The antibody produced is a monoclonal immunoglobulin of the IgG1 subclass with kappa light chains, its isoelectric point is in the range of pH 6.0-6.7.

Example 4

Examination of the C-peptide by radioimmunoassay requires antibodies with the highest possible specificity and the highest possible affinity. Monoclonal antibodies C-PEP-01, C-PEP-02, C-PEP-03 react specifically with C-peptide of human proinsulin and human proinsulin (preparation from Lilly, USA). They do not react with human and porcine insulin, human glucagon and porcine proinsulin (preparation from Novo, Denmark), human somatostatin (preparation from Serono, Italy). The highest association constant of the three tested monoclonal antibodies is the monoclonal antibody C-PEP-01, which has an association constant in the bright ¹² I-Tyr-C-peptide / C-peptide: K = 1.1. 10 ⁹ 1 / mol. The two remaining monoclonal antibodies C-PEP-02 and C-PEP-03 have a K = 10 ⁷ 1 / mol.

Claims (1)

Process for the preparation of higher monoclonal antibodies against human proinsulin C-peptide, characterized in that after immunization of female hybrids of inbred mice of strains BIO.A and BALB / c by binding human proinsulin C-peptide to protein carriers - bovine albumin or thyroglobulin, emulsifying these in adjuvant and subcutaneously, spleen cells from immunized mice are fused with Sp 2/0 - Agl4 myeloma cells and the resulting hybridomas are adapted to grow in vivo in the peritoneum of BALB / c inbred mice, followed by 1 ml of ascitic fluid produced by hybridomas in the mouse peritoneum. isolates 4-10 mg of monoclonal immunoglobulin against human proinsulin C-peptide with an association constant in the ¹²⁵ I-Tyr-C-peptide / C-peptide system ranging from K = 10 ⁷ 1 / mol to 10 ¹⁰ 1 / mol.