FI80801B - KJJUGAT AV EN MONOCLONAL ANTIKROPP OCH ANVAENDNING. ELLER ETT FRAGMENT DAERAV OCH ETT KELATERANDE AEMNE, DESS FRAMSTAELLNING - Google Patents

Description

80801

This invention relates to a fluorescently labeled conjugate of a monoclonal antibody or fragment thereof and a chelating agent for in vitro diagnostics.

Immunological methods have become standard methods in clinical chemistry, especially for the determination of biologically active compounds at low concentrations. Radioimmunoassays are the most widely used assay methods. Alternatives to radiolabels have been sought due to their health disadvantage and, in many cases, short shelf life. EP applications 0184899 and 0186947 relate to paramagnetic contrast agents for in vivo diagnostic, e.g. NMR methods, which according to one embodiment of the invention may be a paramagnetic metal bound via a chelating agent to a polymeric or polymerized carbohydrate or polymerized sugar alcohol or a derivative thereof. One alternative to radioactive labels is a fluorescent label. Some lanthanide ions, especially Eu3 + and Tb3 +, form highly fluorescent chelates as appropriate. . with organic multi-toothed ligands e.g. ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminopentaacetic acid (DTPA).

WO 84/03698 describes Eu3 + chelating DTPA derivatives that can be coupled to antibodies for in vitro diagnostics. The chelating compounds, e.g. The isomers are separated by HPLC and the synthetic sequence is continued with pure isomers, and Eu3 + is chelated to the third step of the synthetic sequence (I Hemmilä et al., Anal. Biochem. 197 (1984) 335), also in several steps.

The Eu3 + chelate of the isocyanate derivative of DTTA is reacted with the antibody. The reaction is slow (overnight, 4 ° C) and the degree of conjugation is poor, about 10%, which means that a large excess of reagent must be used (chelate / antibody = 60: 1). For this reason, unbound chelate must be removed by gel filtration. In this ratio, the conjugate contains 7 Eu3 + / IgG.

U.S. Patent 4,454,106 relates to the incorporation of DTPA into an antibody by a mixed anhydride method and the labeling of the resulting conjugate e.g. with a fluorescent metal, e.g. Eu3 +. The method described in the patent gives about 1.5 DTPA / IgG. This binding ratio has been obtained by testing with the H 2 In • label. However, the patent does not provide an example of the use of Eu3 + as a label. There is also no literature on Eu3 + labeling; example for DTPA anhydrides, so labeling cannot be imagined performed in any of the previously described ways. In addition, e.g., TR-FIA's sensitive. . capacity is not yet sufficient at a ratio of 1.5 Eu3 + / lgG, but within-assay and between-assay variations are large.

DTPA has also been attached to proteins using DTPA · 3 80801 cyclic anhydride (cDTPAA) (I) of formula 0 0 0 yH-CH2 ch28oh O N-CH2Ch2-n-CH2CH2-N O (i) C-CH ^ \ h, - /

H S

(Hnatowich et al., Int. J. Appl. Radiat. Isot. 33 (1982) 327). U.S. Patent No. 4,479,930 (Hnatowich) relates to the use of dicyclic anhydride as a chelating agent for a radioactive metal. The aim is to provide stable radiolabeled biological molecules for in vivo analyzes. There are many benefits to using cDTPAA. It is a substance that can be easily synthesized in one step. It is also commercially available (Sigma, Aldrich). It is an unambiguous dry stable substance unlike mixed anhydride (U.S. Pat. No. 4,454,106), which must always be prepared just before use. The reproducibility of the method is poor due to instability, and there is no reliable analytical method for the resulting anhydride.

On the other hand, cDTPAA is a sparingly soluble compound. Hnatowich has used a chloroform suspension for the administration of small amounts of cDTPAA. Dosing from an inhomogeneous mixture is not very accurate by this method.

The conjugate of this invention is characterized in that the chelating agent is mono- or bicyclic DTPA.

Time-resolved fluoroimmunoassay (TR-FIA) takes advantage of the difference between the fluorescence lifetimes of a specific signal and a non-specific background, resulting in a signal-. . the noise ratio improves. Almost all published methods use an Eu3 + -labeled antibody, of which 4 80801 bound Eu3 + is quantified after the development of dissociative fluorescence. Wallac-LKB has patented the DELFIA method (Dissociative Eu Fluorescence Enhancement FIA) (EP 64484).

The present invention simplifies the method described in WO 84/03698, in which the Eu3 + -DTTA chelate obtained by several synthetic steps is coupled to an antibody with a reaction time of 12 hours at + 4 ° C. In the present invention, the protein is first formed into a DTPA conjugate with a mono- or bicyclic anhydride. Bicyclic anhydride is either a commercial product or prepared according to Eckelman (Eckelman et al., J. Pharm. Sci. 64 (1975) 704). The monocyclic anhydride has been prepared according to Halpern (Radioimmunoimaging and Radiimmunotherapy, Ed. S W Burchiel & B A Rhodes, Elsevier 1983, pp. 198-9). Typically, when making a derivative, the mono- or bicyclic anhydride is dissolved in dry DMSO, unlike Hnatowich, where complete pipetting has good pipetting accuracy due to complete dissolution. An appropriate amount of this homogeneous solution is pipetted into the bicarbonate solution of the antibody. The reaction time is 1 h at room temperature. The DTPA derivative of the antibody thus obtained can be used for various diagnostic purposes. in vitro and in vivo.

. In TR-FIA, the Eu3 + chelate must be sufficiently stable but easily dissociable under acidic conditions. The more toothed the complexing agent, the more stable the complex it forms. Dissociation from such complexes is also slow. However, it has been found (I Hemmilä, Lanthanides as Probes for Time-resolved Fluorometric Immunoassays, Thesis 1986) that asymmetric N'-benzyl-DTTA containing four chelating acid groups dissociates Eu3 + faster (1 min) than the corresponding from symmetric N2-benzyl-DTTA (20 min).

li 5 80801 The use of a cyclic anhydride of DTPA in the preparation of a DTPA derivative of an antibody gives an asymmetric conjugate (II) containing four chelating acid groups, the structure of which is similar to that of the N'-benzyl-DTTA conjugate (III). Only the protein-binding group and bond of DTTA are different

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j) H 2 CH 2 COOH y CH 2 COOH

Prot-NH-C-Nft / OVcH2 "N-CH2CH2-N-CH2CH2N (III) - CH2cooh

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p ch2 ch2cooh ch2cooh Prot-NH-C-H-CH2CH2H1-CH2CH2 “1i ch2cooh (II) Thus, a DTPA derivative of an antibody prepared with a cyclic anhydride is comparable to an N-benzyl-DTTA derivative for use in TR-FIA. Working with cyclic anhydride is simpler and very much faster. DTPA anhydride, commercially available or prepared in one easy synthetic step, is reacted with the antibody for 1 h at room temperature.

. . Excess DTPA is removed by dialysis while changing the buffer to suit labeling. The chelating agent is added as a commercially available chloride. EuCl3 does not need to be used in excess but in a suitable amount tested, so gel filtration does not need to be performed. Instead, the reagent described in WO 84/03698 is laboriously prepared by four synthetic steps. Two isomers are formed in the synthesis, only one of which forms a rapidly dissociating Eu chelate. It follows that the yield of useful chelate after separation of the isomers is poor. In addition, due to the poor degree of conjugation of β 80801, the consumption of laboriously synthesized reagent is high. In addition, since the metal to be chelated is incorporated in the final step of this invention, the same antibody DTPA conjugate can be used for different diagnostic purposes, both in vitro and in vivo, by selecting the metal as appropriate for each purpose.

The purified F (ab ') 2 fragment of the PAP (Prostatic Acid Phosphatase) monoclonal antibody and the purified whole antibody and the purified monoclonal antibody of SHBG (Sex Hormone Binding Globulin) have been used in the derivation. Bicyclic DTPA has been manufactured by Sigma. We have made the mono cDTPA ourselves. Dimethyl sulfoxide (DMSO) is manufactured by SNEA or Aldrich.

Example 1 Coupling of DTPA to a monoclonal antibody 1) Protein treatment

The purified PAP monoclonal antibody is used as such or cleaved with pepsin to F (ab ') 2, which is purified using protein A-Sepharose-4B affinity chromatography. The resulting F (ab ') 2 fraction (0.02 M in PBS buffer, pH 8.0) or whole monoclonal antibody (IgG) is concentrated on an Amicon concentrator and dialyzed against NaHCO3 buffer, pH 8.0. The dialyzed sample is lyophilized and frozen. F (ab ') 2 is dissolved in water just before use so that the protein concentrate; the concentration becomes 10 mg / ml and the NaHCO 3 concentration becomes 0.1 M. The SHBG antibody has been used in its entirety (IgG).

li 7 80801 2) Derivatization A. cDTPA / F (ab ') 2 derivative / IgG derivative

For the conjugation reaction, cDTPAA is dissolved in dry DMSO, from which the desired amount is pipetted into the protein solution. The solution is stirred for 30 s-1 min and incubated at room temperature for 1 hour. The pH of the solution is checked. The sample is dialyzed against 0.1 M Na citrate buffer, pH 5 to remove free DTPA. For example, when making cDTPA / F (ab ') 2 / IgG = 5, a cDTPA-DMSO solution (17.85 mg / ml) is pipetted into a 5 μl protein solution with 5 mg F (ab') 2 or IgG / 0.5 ml .

When adding DMSO to the protein solution, it must be taken into account that the amount of DMSO does not exceed 5% of the volume of the protein solution.

B. mono cDTPA / F (ab1) 2 derivative / IgG derivative

Made in the same way as a cyclic derivative. Mono cDTPAA in DMSO solution is added to the protein solution.

Example 2 Labeling of a DTPA conjugate of a PAP antibody or its F (ab ') 2 fragment with EuCl3

Labeling was performed using 10 M Eu relative to the antibody-DTPA conjugate. When labeling with the Eu-DTPA derivative chelate described in WO 84/03698, it is used in an excess of 160 M relative to the antibody.

EuCl 3 (Fluka 46130) was dissolved in 0.1 M sodium citrate buffer, pH 5-6, 2.75 mg EuCl 3 / ml, 15 min before use. the DTPA conjugate of the antibody or fragment thereof dissolved in 0.1 M sodium citrate buffer (10 mg conjugate 80801 gate / ml, pH 5-6) was mixed with an appropriate amount of EuCl 3 sodium citrate solution and incubated for 1 h at room temperature. The% staining is 98.

Example 3 Labeling of SHBG antibody DTPA conjugate with EuCl3

The DTPA derivative of the antibody was prepared using a ratio of DTPA / IgG = 20: 1. EuCl 3 (Fluka 46130) was dissolved in 0.1 M sodium citrate buffer at pH 6. Labeling was performed using Eu at about 10 M relative to the IgG-DTPA derivative at room temperature for one hour.

Example 4

For comparison, the SHBG antibody was labeled with the Eu chelate described in WO 84/03698. In this case, an excess of 160 M Eu chelate was used relative to the counterpart. material. Other labeling conditions were: 50 mM K 2 HPO 4, pH = 9.4, overnight incubation at + 4eC. Separation of labeled antibody was performed by gel filtration to remove excess: chelate.

After incubation, the reaction mixture was poured onto a gel filtration column (Sepharose 6B 1.5 cm x 30 cm, top Sephadex 6-25, 1.5 cm x 5 cm). Elution was performed using 0.05 M Tris-HCl, 0.9% NaCl, 0.1% NaN 3 (pH 7.75, 25 ° C) as elution buffer.

Fluorescence was determined by taking a 10 μl sample in 200 μl of Delfia Enhancement solution. After shaking, fluorescence was measured with an Arcus fluorometer. European Union-; : concentration was calculated using 0.2 pmol Eu = 106cps as a reference.

9 80801

Comparative results: Using the EuCL3 label, the following number of Eu molecules were attached to the antibodies:

Eu molecules / protein molecules cDTPA / F (ab ') 2 (5: 1) 1.9-2.0 CDTPA / F (ab') 2 (10: 1) 2.7 cDTPA / IgG (10: 1) 2 , 4 cDTPA / IgG (20: 1) 3.7 (e.g. 3) CDTPA / IgG (50: 1) 6.9 m-CDTPA / IgG (300: 1) 11.0

In the example of WO 84/03698, labeling with an excess of 60 M Eu chelate gives 7 Eu molecules / IgG and increasing the ratio of 160-500 M excess Eu chelate does not increase the ratio above 10 Eu molecules / IgG. An excess of 160 M Eu chelate gave 9.3 Eu molecules per antibody molecule (e.g. 4).

The labels of Examples 3 and 4 were tested by the SHBG Delfia kit method using approximately the same protein concentration at dilution of both labels. Briefly, wells of microtiter plates were coated with polyclonal anti-SHBG antibodies. control results and labels were pipetted into wells and incubated, washed and measured using Delfia Enhancement.

80801 SHBG reference solutions EuCl3 labeling Eu chelate labeling nmol / l (e.g. 3) cps (e.g. 4), cps 0 3197 4281 6.25 43535 81940 12.5 77255 147983 25 146026 263334 50 238831 442793 100 389177 841566 200 787518 1579440 SHBGtn TR-FIA worked similarly on both labels and had equally good resolution with respect to different standards.

In one embodiment, the invention also relates to a method for determining the degree of conjugate between a chelating agent and a biological molecule. The degree of conjugate refers to the molar ratio of chelating agent / biological molecule. The degree of conjugate is usually determined by attaching a radioactive isotope to the chelating groups of the conjugate. However, this assay is cumbersome and inaccurate due to the short half-life of the isotopes. Instead, as in the present invention, by attaching Eu to the chelating groups and measuring the fluorescence, the degree of conjugate can be determined very accurately and simply.

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Claims (6)

1. With a fluorescent substance labeled conjugate of a monoclonal antibody or fragment thereof, and a chelating agent for in vitro diagnostics, the chelating agent is mono- or bicyclic DTPA. 2. Conjugate according to claim 1, characterized in that the monoclonal antibody or its fragment is antibody to the SHBG antigen or its fragment. The conjugate of claim 1, characterized in that the monoclonal antibody or its fragment is antibody to the PAP antigen or its fragment. 4. Conjugate according to claims 1-3, characterized in that the fluorescent substance is Eu. Process for the preparation of a conjugate according to claims 1-4, characterized in that the biological molecule and the chelating agent are first coupled together and the conjugate is labeled with a fluorescent substance. 6. In vitro diagnostic immunological method, characterized in that a conjugate according to claims 1-4 is used for the determination of an analgesic antigen. li