DE4339533C2 - Detection method for hdm-2 specific antibodies - Google Patents

Description

The invention relates to a method for the detection of hdm-2 specific antibodies in body fluids. It also relates to a kit that can be used for this purpose.

The human genome includes one with hdm-2 (human double minute gene 2) designated gene. This gene shows great Homologies to the corresponding mdm-2 gene (murine double minute gene 2) the mouse on. The expression product of the hdm-2 gene is a protein whose primary structure is known and which has 491 amino acids (see Oliner, J.D. et al., Nature 358 (1992), 80-83). This protein will hereinafter referred to as hdm-2.

It is known that the hdm-2 gene is commonly found in sarcomas amplified form is present. Then there is also one increased expression of hdm-2. This can be done with the specific antibodies directed against hdm-2. The detection of such antibodies could therefore be used for diagnosis specific cancers can be used.

The present invention is therefore based on the object a method for the detection of hdm-2-specific anti to provide bodies.  

According to the invention, this is achieved in a process in which which one supports carrier-bound hdm-2 and / or carrier mat rial-bound, binding regions for hdm-2-specific anti body-containing fragments thereof with body fluids incubated and the specific to the hdm-2 and / or the Fragments bound antibody (a)

• - with marked, directed against the antibodies (a) Antibodies (b), or
• - with unlabelled antibodies (b) and the latter with marked, directed against the antibodies (b) Antibodies (c) can react.

The term "hdm-2" encompasses a wild-type hdm-2 protein Sequence. Such can consist of cells such as A-204 (human Rhabdomyosarcoma cell line, tumor bank, German cancer research center, Heidelberg). hdm-2 can also have a sequence different from the wild-type sequence. Such can be additions, deletions and / or Have substitutions of one or more amino acids. Hdm-2 may also be part of a fusion protein. On such as any other hdm-2 can be isolated from cells that express it after genetic engineering. Such Cells include prokaryotic and eukaryotic cells. Examples of the former are E. coli strains such as BL21 (cf. Studier, F.W. et al., Methods in Enzymology 185 (1990), 60-89), while the latter in particular mammalian, yeast and insect cells are to be mentioned. Under genetic engineering are common methods known from the prior art  understand with which nucleic acids of certain sequences produced, introduced into cells and expressed. Of the Specialist knows suitable materials for this, such as vectors, and conditions (see e.g. Maniatis, T. et al., Cold Spring Harbor Laboratory, 1982).

In the present case, an hdm-2 cDNA from A-204 cells (cf. above) reverse transcribed in the usual way and in amplified using a common PCR method. The cDNA is in their sequence with published data (e.g. EMBL gene bank) compared and inserted in the well-known vector pet 3d, whereby the recombinant vector pet 92/2 is obtained. This becomes the expression of hdm-2 in the bacterial strain BL21 (see above) transformed. An hdm-2 induction is achieved by adding IPTG. The bacteria will sedimented and after freezing and thawing a lysozyme and subjected to DNaseI treatment. The lysates are with Incubated urea solutions of various concentrations and after centrifugation released hdm-2 by a Polyacrylamide gel electrophoresis and subsequent Electroelution provided in pure form. The expert knows the above procedures and the necessary ones Materials and conditions.

According to the invention, hdm-2 fragments are also used Containing binding regions for hdm-2-specific antibodies. These fragments are described below with hdm-2-AKBR fragments designated. Preferably, the hdm-2 AKBR fragments comprise amino acids 1-284, 58-284 and 58-491 of hdm-2 (cf. Fig. 1). DNA sequences required for the above hdm-2-AKBR- Coding fragments can be found in FIG. 2.

Several can be used to produce hdm-2-AKBR fragments Procedures are used. A Ver proven driving, over the total length of hdm-2  distributed fragments constructed, with at least each two fragments have an overlapping area that React fragments with an hdm-2 specific antibody leaves and the overlapping, bound by the antibody Area identified and as hdm-2 AKBR fragment provides as well as this as the basis for a one or multiple repetition of the above cycle used.

The starting point for this method is one of A-204 cells hdm-2 cDNA obtained (see above). Be from this DNA fragments distributed over the entire length of the cDNA in amplified using a common PCR method, with each at least two fragments overlap exhibit. The amplified DNA fragments are as described above, inserted in pet 3d and after Transformation and IPTG induction in the bacterial strain BL21 expressed. The hdm-2 fragments obtained are as described above for hdm-2, isolated and one Subjected to polyacrylamide gel electrophoresis. This happens a Western blot analysis, where labeled, generally available hdm-2 specific antibodies, e.g. B. JF2, for Binding to the hdm-2 fragments can be used. By Binding one of these antibodies to two overlapping The binding region becomes the region of hdm-2 fragments of the antibody is assigned to the overlapping area. This The area is called a fragment labeled hdm-2-AKBR provided. For this, z. B. a common one PCR method applied. With the hdm-2-AKBR fragment the above cycle can be repeated one or more times to the binding region of the hdm-2-specific antibody narrow down further. This binding region can be up to few amino acids can be limited. The u. U. necessary short hdm-2 fragments can be synthetic getting produced. Those skilled in the art are the above Procedures and those necessary for their implementation Materials and conditions known.  

According to the invention, hdm-2 and / or hdm-2 AKBR fragments bound to a carrier material. Of course you can too a single hdm-2-AKBR fragment or this together with hdm-2 bound. Any support for Binding of proteins suitable material, in particular Microtiter plates, tubes, microspheres and slides, be used. For very small hdm-2 AKBR fragments, especially peptides, it is advisable to bind to that Carrier material via a conventional carrier, e.g. B. BSA, to be done. Such a bond as well as one without Carrier, can be achieved by conventional methods. Present Microtiter plates are used as the carrier material. For this purpose, hdm-2 and / or hdm-2 AKBR fragments in Carbonate buffer or urea-phosphate buffer added, diluted differently and into the holes one Microtiter plate given. After overnight incubation at 4 ° C follow several washing steps in physiolo Buffer. The binding of hdm-2 and / or the hdm-2-AKBR fragments are stable.

According to the invention, bound hdm-2 and / or bound incubated hdm-2-AKBR fragments with body fluids. As such are meant all liquids that come from a animal body, especially a mammal and whole especially a human being can be preserved. The rivers liquids preferably include serum, lymph, saliva and Urine. They also include liquids that spill out  solid tissues, such as lungs, brain and bone marrow, as well Tumors such as colorectal carcinoma and hepatocell carcinoma and sarcomas can be isolated. The incubation takes place according to usual procedures. Sera from Patients diluted differently and bound hdm-2 and / or bound hdm-2-AKBR fragments in the microtiter plate admitted. Follow for 1 hour incubation at 37 ° C several washing steps in physiological buffer. The connection of specific anti-hdm-2 antibodies is stable.

According to the invention, such bound antibodies (hereinafter referred to as antibody (a))

• - with marked, against the antibodies (a) directed antibodies (b), or
• - with unlabelled antibodies (b) and the latter with marked, directed against the antibodies (b) Antibodies (c) implemented.

The label can be radioactive or non-radioactive. In the latter case, other common markers are used. Particularly suitable are fluorescent dyes, such as. B. Fluorescein isothiocyanate, and enzymes such as alkaline Phosphatase or peroxidase. A can be used as an amplifier system Biotin / streptavidin complex can be used. The markers are generally available. The conjugation with the Antibodies (b) or (c) are carried out according to the regulations of Manufacturer. Also labeled are antibodies (b) and (c) generally available.

The choice of the appropriate antibody (b), whether labeled or unmarked depends on which animal or which Animal type the body fluid used comes from. Act it for example a liquid from a human, so are used as antibodies (b) those against human immunoglobulin. In corresponding  If there are additional antibodies (c) be set, this regarding the animal or the animal species selected from which the antibodies (b) originate. The choice ge suitable antibody is known to the person skilled in the art and can be used without further be carried out.

Implementation of bound antibodies (a) with labeled Antibodies (b) or with unlabelled antibodies (b) and then with labeled antibodies (c) can in the usual way respectively. In the present case, the implementation with the antibody takes place pern (b) in both alternatives within 1 h at 37 ° C instead of. After several washes, the first age native a substrate solution corresponding to the marker for ent added to the detection reaction. This is done according to the manufacturer's instructions. In the second alternative the antibodies (c) are added after the washing processes. Their implementation and the development of the detection reaction take place in a corresponding manner.

The method according to the invention has a high sensitivity on. This is particularly high, even if the antibodies (c) be used. In addition, the present procedure can be carried out quickly in any laboratory. Special security no arrangements are necessary for this. The fiction Moderate procedure is therefore particularly suitable for implementation major screening.

According to the invention, a kit is also provided which is used for Carrying out the above procedures is suitable. This kit contains

• - Carrier-bound hdm-2 and / or carrier mat rial-bound hdm-2-AKBR fragments and labeled  Antibodies (b) and customary washing buffers and, if appropriate a substrate corresponding to the marking or
• - Support material-bound hdm-2, and / or support material rial-bound hdm-2-AKBR fragments, unlabeled anti body (b) and labeled antibodies (c) as well as usual Wash buffer and possibly one of the markings corresponding substrate.

For marking as well as the other components of the kit the above apply to the method according to the invention made executions.

Brief explanation of the drawing

1 shows the amino acid sequences of the invention used hdm-2-AKBR fragments,

FIG. 2 shows the DNA sequences of those listed in FIG. 1 hdm-2 AKBR fragments.

The present invention is illustrated by the examples.

example 1 Expression of hdm-2 and His-hdm-2 fusion protein A) Expression of hdm-2

From the cell line A-204 (see above) an hdm-2- cDNA reverse transcribed with a "hexamer random primer" and by means of the oligoprimers "Fhdm-2-B" and "Rhdm-2" in one common PCR methods amplified (sequence "Fhdm-2-B": CGC GGA TCC ATG TGC AAT ACC AAC ATG TCT G; "Rhdm2": CGC GGA TCC TCA GGG GAA ATA AGT TAG CAC AAT C;) The amplified Sequence was published with data (EMBL gene bank)  compared and via the restriction enzyme site BamHI in two PCR primers while maintaining the entire coding Area in the vector pet3d in the bacterial strain HMS 174 cloned. The expression obtained was used to express hdm-2 Vector pet 93/9 in the bacterial strain BL21 (see above) transformed. After culturing the bacteria at 37 ° C to to an optical density of 0.6 (600 nm) in 100 µg / ml Ampicillin and 30 µg / ml chloramphenicol containing LB medium was induction of hdm-2 at 30 ° C for 3 hours performed with 2 mM IPTG.

(B) Expression of His-hdm-2 fusion protein

From the cell line A-204 (see above) an hdm-2- cDNA reverse transcribed and by means of the oligoprimer "Fhdm-2-B" and "Rhdm-2" (see above) by PCR amplified. The amplified sequence was over BamHI interfaces in both PCR primers in the known, cloned with BamHI expression vector pQE-8. The Expression of this at the N-terminal end with 6 histidines linked hdm-2 protein occurred in the E. coli bacterial strain SG13009 (cf. Gottesmann, S. et al., J. Bacteriol. 148, (1981) 265-273). After culturing the bacteria at 37 ° C up to an optical density of 0.6 in LB medium with 100 µg / ml ampicillin and 25 µg / ml kanamycin became one 6-hour induction of His-hdm-2 fusion protein at 37 ° C performed with 1 mM IPTG.  

Example 2 Isolation and cleaning of hdm-2 and His-hdm-2 fusion protein (A) Isolation and cleaning of hdm-2

400 ml of bacterial culture from Example 1, (A) were after the hdm-2 induction by centrifugation at 500 g for 10 minutes sedimented, in 50 mM Tris-HCl, pH 8.0, 100 mM NaCl wash and centrifuge again. After freezing and opening the sediment was thawed in 1.6 ml of a resus lysis buffer pending (50 mM Tris-HCl, pH 8.0, 10% sucrose). Afterwards other were lysozyme (final conc. 2 mg / ml) and 0.5 M EDTA (Final conc. 50 mM) added. After 20 minutes of incubation MgCl₂ (final conc. 80 mM) and DNAse I (250 µg) added, followed by a 10 minute incubation at Room temperature (final volume 6.5 ml). For this, 10 µl 0.1 M PMSF was added and the mixture was stirred for 15 minutes 10,000 g, centrifuged at 4 ° C. This centrifugation step was repeated three times, each time after resuspen dation of the sediment and 10 minutes incubation in 1 M, 3 M or 7 M urea with PMSF (0.1 M, 10 µl) The supernatant the last centrifugation step continued as follows purified: after gel electrophoresis in a 10% polyacrylic amide gel, cutting out the desired protein band and elec Troelution overnight at 4 ° C in 25 mM Tris-HCl, 0.2 M Gly cin, 0.5% SDS, pH 8.8 in a commonly available bio trap elution chamber was obtained in pure form hdm-2.  

(B) Isolation and purification of His-hdm-2 fusion protein

250 ml of bacteria culture from Example 1, (B) were after the Induction of the His-hdm-2 fusion protein sedimented and a times washed in 40 ml PBS. 1 g of bacterial pellet was added in 3 ml of solution A sonicated for 1 min and then at RT 8-12h at medium speed on a magnetic stirrer touched. The suspension obtained was again at RT for 30 min sedimented at 15,000 rpm. 2-6 ml of the resultant Supernatants were made on a commonly known nickel chelate Chromatography column (Ni-NTA resin) given. The pillar mat rial was previously with 3 column volumes of solution A (see next hend). The column was lifted after the Bacterial extract washed successively with solutions A to F, with 2-3 column volumes of the corresponding solution, until no more protein could be eluted. Of the Protein content of the collected fractions was determined by Absorbance measurement at 280 nm determined photometrically, whereby 1 O.D. corresponded to approx. 1 mg protein / ml. The His-hdm-2 fusion protein was in the fractions after elution with solution D or E included, usually only a very small proportion bacterial proteins was included. To this share too remove, the eluted protein was again attached to the Ni-NTA resin column bound by the pH of the ver agreed fractions with the main amount of to be cleaned Protein was adjusted to pH 8.0 and then still were placed on the column. The subsequent elution was carried out with solutions A to F as described above. The Purity and quality of the eluted His-hdm-2 fusion proteins was checked by SDS-PAGE.  

Composition of the solutions used:
Solution A:

6 M guanidinium hydrochloride, 0.1 M NaH₂PO₄, 10 mM β-mercaptoethanol, 0.01 M TrisHCl, pH 8.0
Solution B:
8 M urea, 0.1 M NaH₂PO₄, 0.01 M TrisHCl, pH 8.0
Solution C:
8 M urea, 0.1 M NaH₂PO₄, 0.01 M TrisHCl, pH 6.3
Solution D:
8 M urea, 0.1 M NaH₂PO₄, 0.01 M TrisHCl, pH 5.9
Solution E:
8 M urea, 0.1 M NaH₂PO₄, 0.01 M TrisHCl, pH 4.5
Solution F:
6 M guanidinium hydrochloride, 0.2 M acetic acid
The above method of (B) is characterized by an extremely fast and efficient purification of the expressed protein.

Example 3 Manufacture of hdm-2-AKBR and His-hdm-2- AKBR fragments (A) Preparation of an hdm-2-AKBR fragment

From the cDNA obtained from the cell line A-204 (see above), two overlapping DNA sequences were combined in one using the oligoprimer pairs "Fhdm2-B" / "Rhdm2-284" and "Fhdm2-58 / Rhdm2-284" common PCR procedures amplified. One DNA sequence encoded an hdm-2 fragment of amino acids 1-284, while the other encoded an hdm-2 fragment of amino acids 58-284. The sequences of the oligoprimers used were as follows:
"Fhdm-2-B" (see above); "Rhdm-2-284": CGC GGA TCC TCA CCC TGC CTG ATA CAC ACT AAC; "Fhdm-2-58": CGC GGA TCC GGC CAG TAT ATT ATG ACT AAA C.

The amplified sequences were published data (EMBL library) and compared via the restriction enzyme put BamHI in the vector pet 3 d (see above) loo kidney. The sequences were expressed according to Transforma tion and IPTG induction in the bacterial strain BL21 (cf. hend). The expressed sequences (hdm-2 fragments) were isolated and as described in Example 2 (A) for hdm-2 subjected to polyacrylamide gel electrophoresis. This follow te a conventional Western blot analysis, in which a general Available hdm-2 specific antibody for binding to the hdm-2 fragments was used. This antibody showed binding to both overlapping hdm-2 Fragments. The overlapping area, i.e. H. the amino acids 58-284, was considered the binding region of the antibody.

(B) Preparation of a His-hdm-2-AKBR fragment

From that obtained from cell line A-204 (see above) cDNA were two overlapping DNA sequences using the Oligoprimer pairs "Fhdm-2-B" / "Rhdm-2-284" and "Fhdm-2-58" / "Rhdm-2-284" amplified in a common PCR method.

The amplified sequences were published data (EMBL gene bank) compared and about the restrictions BamHI enzyme site in the vector pQE-8 (see above) cloned. The sequences were expressed after  Transformation and IPTG induction in the E. coli bacterial strain SG 13009 (see above). The sequences expressed (His-hdm-2 fragments) were made as in Example 2 (B) for His-hdm-2 fusion protein described, isolated and one Subjected to polyacrylamide gel electrophoresis. This was followed by one usual Western blot analysis, in which a general Available hdm-2 specific antibody for binding to the His-hdm-2 fragments was used. This antibody showed binding to both overlapping hdm-2 Fragments. As in (A) above, the overlapping Area, d. H. amino acids 58-284, as the binding region of the Antibody viewed.

Example 4 Detection of specific hdm-2 antibodies in the Serum from patients through hdm-2

To carry out an ELISA, hdm-2 from Example 2, (B) in urea buffer (3 M urea, 0.1 M NaH₂PO₄, pH 8.0) added. For coating a 96-hole plate were 100 µl per hole with 20 ng and 8 ng antigen as well as 1% BSA as an empty control. After Incubations overnight at 4 ° C were followed by 3 short ones Wash steps with PBS. Then the Blocking free binding sites of the polymeric carrier by incubation for 1 hour with 1% BSA in PBS at 37 ° C. A serum to be tested from a patient with a small cellular bronchial carcinoma was diluted 1: 100 (PBS, 1% BSA, 0.05% Tween 20) for 1 hour at 37 ° C on the Plate incubated ("checkerboard titration"). After 8 washes steps with PBS (0.05% Tween 20) became a general Available peroxidase-coupled goat anti-human Antibody (dilution according to the manufacturer) added  give. After 30 minutes of incubation at 37 ° C, 8 followed Wash steps and then the peroxidase detection reaction with TMB developing solution (50 mM sodium acetate, 0.4 mM 3.3 ′, 5,5′-tetramethyl-benzidine dihydrochloride, 4.4 mM H₂O₂) within 30 minutes at room temperature. After stopping the reaction with 2 M HCl, the Color intensity determined photometrically at 450 nm. Absorb were more than double the BSA control rated as a positive reaction.

The table shows the data accordingly performed ELISA compared to determining specific Antibodies using immunoblot. The almost complete The anti-hdm-2 ELISA shows agreement between the two methods as a suitable method for clinical series testing Material. Furthermore, the ELISA shows a greater sensitivity tivity than the immunoblot, which makes the suitability of the ELISA is underlined as an initial examination procedure.  

table

Comparison of the detection of hdm-2 antibodies in sera from patients with bronchial carcinoma by immunoblot and ELISA with recombinant hdm-2

The table shows that the results of Immunblot and ELISA match, the greater sensitivity of the ELISA indicates suitability as an initial examination method.

Claims (11)

1. A method for the detection of hdm-2-specific antibodies in body fluids, characterized in that one contains carrier material-bound nes hdm-2 and / or carrier material-bound, binding regions for hdm-2-specific antibodies fragments thereof, one such Fragment is obtainable by a method in which fragments distributed over the total length of hdm-2 are constructed in the usual manner, with at least 2 fragments each having an overlapping area, the fragments reacting with an hdm-2-specific antibody and the overlapping, bound by the antibody and identified as a fragment with binding region for hdm-2-specific antibodies in the usual way, incubated with body fluids and the specific, to the hdm-2 and / or fragments with binding region for hdm-2 -specific antibody-bound antibody (a)

• - with labeled antibodies (b) directed against the antibodies (a), or
• - Can react with unlabelled antibodies (b) and the latter with labeled antibodies directed against the antibodies (b) (c).

2. The method according to claim 1, characterized in that the fragment with binding region for hdm-2-specific antibodies as the basis for a one or more repetitions of the cycle according to claim I ver is applied. 3. The method according to claim 1 or 2, characterized in that the  Body fluids serum, lymph, saliva and urine as well as from solid Tissues and tumors include fluids obtained. 4. The method according to claim 1 or 3, characterized in that the hdm-2 is obtained by expressing a cDNA sequence. 5. The method according to any one of claims 1 to 3, characterized in that the hdm-2 fragments are amino acids 1-284, 58-284 and 58-491 of hdm-2 include. 6. The method according to any one of claims 1 to 5, characterized in that the carrier material microtiter plates, tubes, microspheres and Slides included. 7. The method according to any one of claims 1 to 6, characterized in that the antibodies (b) in the first alternative and the antibodies (c) in the second alternative are labeled with an enzyme. 8. The method according to any one of claims 1 to 6, characterized in that that the antibodies (b) in the first alternative and the antibodies (c) in the second alternative are marked with a fluorescent dye. 9. The method according to any one of claims 1 to 6, characterized in that the antibodies (b) in the first alternative and the antibodies (c) in the second alternative are radioactively marked. 10. Kit containing

• - carrier-bound hdm-2 and / or carrier-bound fragments thereof, binding regions for hdm-2-specific antibodies,
  such a fragment being obtainable by a process in which fragments distributed over the entire length of hdm-2 are constructed in a conventional manner, with at least 2 fragments each having an overlapping region, the fragments reacting with an hdm-2-specific antibody and the overlapping area identified by the antibody and provided in the usual way as a fragment with a binding region for hdm-2-specific antibodies,
  and labeled antibodies (b) according to claim 1 as well as usual washing buffers and optionally a substrate corresponding to the labeling or
• - carrier-bound hdm-2 and / or carrier-bound fragments thereof, binding regions for hdm-2-specific antibodies,
  such a fragment being obtainable by a process in which fragments distributed over the entire length of hdm-2 are constructed in a conventional manner, with at least 2 fragments each having an overlapping region, the fragments reacting with an hdm-2-specific antibody and the overlapping area identified by the antibody and provided in the usual way as a fragment with a binding region for hdm-2-specific antibodies,
  and unlabeled antibodies (b) and labeled antibodies (c) according to claim 1 as well as customary washing buffers and optionally a substrate corresponding to the labeling.

11. Kit according to claim 10, characterized in that the fragment with Binding region for hdm-2-specific antibodies as the basis for a single or multiple repetition of the cycle according to claim 10 used becomes.