DD301844A9 - Monoclonal enzyme immunoassay for the quantitative determination of CEA - Google Patents

Abstract

The monoclonal enzyme immunoassay (EIA) optionally contains the monoclonal antibodies D11-A / B10, D11-D / G2, B4-A / H11, D11-A / D11, D11-C / C11, and B4-F / C3. The method of its compilation is characterized by the selection of CEA-specific antibodies by means of immunohistological studies on paraffin sections of normal and neoplastic tissues, the characterization of peroxidase-labeled CEA antibody with the modification of a per se known method, the combination of the most suitable "catcher" - and "Tracer" antibodies, and in particular the exploitation of a synergistic effect when using multiple antibodies, wherein the examination of the appropriate antibody combination in a direct two-way binding test on a larger number of serum samples with special attention to the quality criteria of specificity, sensitivity, recovery, linearity and precision. In one embodiment, the effective combination of six monoclonal antibodies is demonstrated in such a bilateral binding assay. Areas of application of the invention are the clinical diagnostics and the follow-up of patients with tumor diseases. {Monoclonal antibodies, CEA determination, immunohistology, selection regime, synergism, peroxidase labeling, antibody combination}

Description

Field of application of the invention

The invention relates to an enzyme immunoassay (EIA) based on monoclonal antibodies, which is suitable for the quantitative determination of Cprcinoembryonalen antigen in microtiter plates. Furthermore, the invention relates to a method for the selection of monoclonal antibodies with regard to their suitability as components of a test kit for the quantitative determination of CEA.

Characteristic of the known technical solutions

The CEA is a glycoprotein with a molecular weight of about 180000 Dalton, which is formed and secreted by many carcinomas, especially of tumors of the digestive tract and thus in a detectable amount as a marker protein in the blood (S. v. Kleist, Das Karzinoembryonale Antigen (CEA) - Biological Basis and Clinical Application, Stuttgart-New York, 1983; GTRogers, Biochim., Biophys., Acta 1983, 665, 227). The development of a homoeopathic CEA assay is problematic in that this tumor-associated antigen has antibody binding sites (epitopes) that also occur on several cross-reacting (CEA-related) antigens. Among the most important cross-reacting antigens are NCA-1 (JP Mach and G. Pusztaszeri, Immunochemistry 1972, 9, 311; S. v. Kleist et al., Proc. Natl. Acad., U.S., 1972, 69, 2492; Occurrence in granulocytes, pulmonocytes and, for example, also in mammary carcinomas), the NCA-2 (P.Burtin et al., J. Immunol., 1973, 11, 19, 1926, in meconium, normal colon), BGP-I (T.Svenberg, Int J. Cancer 1976, 17, 588; gall bladder, bile ducts) and NFA (Y. Matsuoka et al., Cancer Res. 1982, 42, 2012, Faeces). The cited CEA-related antigens may be present in the blood but have no significance as tumor markers. Therefore, a possible reaction in the test antibodies to be used with these cross-reacting antigens, especially with NCA-1 and BGP-1, must be clarified.

Nucleic acid analyzes performed in the last two years have shown that the genetic code for the CEA consists of three largely identical domains in addition to a so-called leader sequence (S. Oikawa et al., Bioch. Biophys. Res., Comm., 1987, 142, 511; Oikawa et al., Bioch. Biophys. Res. Comm., 1987, 144, 644, Zimmermann, W., et al., Proc. Natl. Acad., U.S.A., 1987, 48, 2960; JAThompson et al., Proc. Natl. Acad. See, U.S.A., 1987, 1965; Beauchemin et al., Mol. Cell. Biol., 1987, 7, 3221). The basic structure dco NCA is based on the leader sequence as well as the first of these three homologous gene regions, from which its close structural and immunological relationship to CEA is derived (Neumair M. et al., J. Biol. Chem. 1988, 323, 3202). The well-known heterogeneity of CEA and related molecules (reviewed in Rogers 1983, supra). to which essentially the standardization problems for CEA tests are attributed, evidently results from different glycosylations. The antibody binding sites (epitopes) present on the respective molecules may consist of amino acids, carbohydrates or a combination of both. Na : b According to NAP (Bull. Cane 1986, 73, 1) there should be at least four CEA-specific epitopes. Cross-reactive epitopes exist in a number of at least eight. From these data it can be seen that the proof of specificity for anti-CEA antibodies, especially by excluding a reaction with nonspecific CEA-related A.itigen, requires careful analysis. When used in bilateral binding assays, at least the so-called solid-phase antibodies ("catchers") or the second antibodies ("tracers") or both antibodies must be CEA-specific in order to rule out the detection of the clinically non-relevant, CEA-related antigens in the test. In general, the antibody specificity is tested during the test establishment by extensive investigations with highly purified antigen preparations (CEA, NCA, etc.).

-2- 3C1844

Difficulties in the development of a monoclonal assay are evidently that with individual antibodies, the relatively high molecular weight and heterogeneous as well as serum proteins possibly masked glycoprotein is not bound with sufficient reliability and thus made available for the detection. Therefore, test systems based on conventional antisera or a combination of such an antiserum with a monoclonal antibody are still commercially available.

Object of the invention

The invention has the goal to build an EIA, which is easy to handle in routine operation and satisfies high demands in its reliability and sensitivity. Furthermore, a selection method is to be specified, under the use of which antibodies are tested for their suitability as components of such an EIA and selected.

Explanation of the essence of the invention

It is an object of the invention to provide a CEA EIA which is based on the use of monoclonal antibodies, each of which is present alone or in a defined mixture. An EIA is to be presented, which works on the solid-phase principle and can be carried out in microtiter plates. It is a further object of the invention to divide monoclonal antibodies into "CEA-specific" and "CEA-responsible, but not CEA-specific" by setting up a selection regime, to then a further division into preferably as a solid-phase antibody ("catcher") usable and peroxidase-marked Make secondary antibodies ("tracers").

The object is achieved according to the invention by providing an EIA for the quantitative determination of CEA which contains the following monoclonal antibodies as components (abbreviations, see Table 1).

A. 1. Solid Phase Antibody ("Catcher")

FC 3, DG 2, CC 11 (each alone or in any combination) 2. Second antibody ("Tracer")

AH11, AB 10, AD11 (each alone or in any combination) or

B. Solid Phase Antibody

FC 3, DG 2, CC 11 (each alone or in any combination) 2. Second antibody HD11.

In both cases, the antibodies bind up to three CEA-specific epitopes on the solid phase. That is a favorable condition for the test. The epitopes for the "catcher" and "tracer" antibodies are each different. The EIA variant A shows a sensitivity of 55% for a specificity of 95%, the EIA variant B with the same specificity a sensitivity of 48% (comparison of 100 patients with colorectal carcinomas and 100 blood donors). A comparison with a test kit of a leading company gives a sensitivity of 42% for the same serum samples with a specificity of 95%. Over 20 investigated antibody combinations are ruled out for a practicable test for insufficient antibody binding to plastic or for insufficiently high extinctions in the overall test system. After testing several combinations for missing false-positive reactions with so-called problem genes (rheumatoid factor, bilirubin, lipids, hemoglobin) and due to the specificity-sensitivity comparisons with a reference method, the variants CEA-EIAA. and B. proved to be the cheapest. It is safe and proven by experiments that the effect of the combination of the specified monoclonal antibodies based on a synergistic effect, which is apparently due to the individual properties of the monoclonal antibodies used.

With the CEA-EIA A. and B. the CEA determination is preferably carried out in microtiter plates in a conventional manner.

The method according to the invention for the selection of antibodies suitable for the quantitative determination of CEA is carried out in the following individual steps:

1. The immunohistological reaction of antibodies is extensively tested on paraffin sections of CEA-containing (colon carcinoma), NCA-1-containing (granulocytes, pulmonocytes, breast carcinomas) and BGP-1-containing tissue (bile ducts in the liver). This results in a subdivision possibility of the antibodies in "CEA-specific" and "CEA-responsive, but not specific".

2. In view of the need to use antibodies as "tracers", the peroxidase taggeability of each individual antibody is determined by a modification of the conventional FDNB method (TR BURKOT et al., J. Immunol., Meth. 1985, 84, 25). The modification consists in that the examination is carried out in previously unknown manner on paraffin sections of tissue containing CEA, as a result of which it is possible to decide which antibodies can be used as a "catcher" and which as a second antibody ("tracer") are.

3. All candidate "catcher" and "tracer" antibodies are selected in single, double or triple combinations to achieve sufficiently high reaction with the antigen (measured as absorbance after substrate reaction with the "tracer" enzyme In this case, up to three antibodies are used both on the "catcher" side and on the "tracer" side, as shown in the exemplary embodiment, and the reaction with the protein Antigen can be significantly increased if two or three antibodies instead of only one antibody on the respective side of the test system can be applied .. Optimal reactions with the antigen carried out in the described procedure, if in each test step ("catcher" and "tracer") three antibodies be applied.

Due to the omission of individual antibodies in the two test steps, the minimum number of antibodies is determined in a suitable combination, depending on the extent of the reduction in the reaction caused. In principle, at least in one test step ("catcher" and / or "tracer" antibody), pure CEA-specific antibodies are used.

4. Examination of a larger number of serum samples is crucial for the selection of antibody combinations according to the criteria of "specificity" and "sensitivity".

5. Other important quality features of such a test for detection of a marker in serum or plasma are the recovery rate of CEA in serum samples, the linearity of the determined CEA values at serum dilution and the precision in multiple determination. This applies in particular to the test procedure with undiluted serum, which in many cases has a CEA-masking effect. This effect can be largely eliminated by the antibody multiple combinations.

The monoclonal antibodies used are accessible, according to the patent DD-PS 252 200th

embodiment

1. Examination of existing, listed in Table 1 monoclonal antibody whose reaction with highly purified CEA is detected (methods after B.Micheeletal., J. Immunol., Meth. 1981,45,41) and whose reaction with various epitopes is already known, by means of indirect Immunoperoxidase technique on paraffin sections of human tissues to determine their reaction with NCA-1 (in granulocytosis, pulmonocytes, breast carcinoma tissue) and BGP-1 (in the bile duct epithelium in the liver tissue) and confirmation of reaction with CEA-containing colon carcinomas.

2. Testing of Peroxidase Markability by FDNB Method on Paraffin Sections of CEA-Containing Tissue (high sensitivity to pretreatment with FDNB is synonymous with low or absent peroxidase labelability).

Table 2: Grouping of antibodies after testing for specificity and peroxidase (POD) taggeability (the antibody short names are given, see Table 1).

CEA specific cross-reactive

goodPOD-markable AB10, AD11

less good POD-markable FC3, DG2, CC11 AH11, HD11 not POD-markable BB 9

The result from 1 and 2 is shown in Table 2.

Thereafter, of eight antibodies tested, three are CEA specific but less well suited for peroxidase labeling.

Of five antibodies that react with CEA-related antigens, ie are not CEA-specific, two antibodies prove to be well-labeled, two less well-labeled and one antibody unsuitable. This leads to the conclusion that the specific antibodies are most suitable as "catchers" and that two to a maximum of four non-specific antibodies are suitable as "tracers".

3. According to the results of 1 and 2, the specific antibodies are used individually or in various combinations as "catchers" and the four antibodies which may also be considered as "tracers" are also tested individually or in combination in the test system.

The test system is based on the principle of a two-side binding test and consists of the following individual steps:

1. Coating of the microtiter plates (VEB Polyplast Halberstadt) with the catcher antibodies (at neutral or alkaline pH)

2. Saturation of the plastic of the microtiter plates with suitable protein solution

3. Incubation with CEA antigen preparations of various concentrations (reference curve) or serum or plasma samples

4. Wash the microtiter wells with a suitable wash buffer

5. Incubation with peroxidase-labeled "tracer" antibodies

6. Washing

7. Addition of chromogen and H ₂ O ₂ (to measure the peroxidase activity bound by the antigen-antibody reaction)

8. Stopping the enzyme reaction, for example with 1 NH ₂ SO ₄

9. Measurement of the color intensity (absorption) in the individual microtiter plate wells (the color intensity is proportional to the antigen concentration detected in the test).

Result:

When using a relatively high concentration of CEA (50pg / l), for variants with only one "catcher" or "tracer" antibody, absorptions of at most A = 0.5 result. The highest absolute extinctions are achieved with the use of three "catcher" and three "tracer" antibodies (A = 1.4). For routine purposes, an absorption of A = 0.8-1.0 is required.

When using an antigen reference curve (ranging from 0-100pg / l CEA), well evaluable waveforms are only achieved if three to four antibodies are applied in the entire system.

Combined use of multiple antibodies will save absolute antibody levels due to a potentiating effect (eg, combined use of three catcher antibodies compared to only one antibody, saving by a factor of 5-10).

Recovery studies for CEA ^: Sorum samples are between 90 and 115% when six antibodies are used, allowing reliable CEA determination in undiluted sera and good linearity in serial serum dilutions. The precision of the test variant corresponds with coefficients of variation of 3-5% (intraassay) or 7-10% (interassay) high standards.

Table 1: Overview of the immunohistochemical reactions of eight selected anti-CEA antibodies to paraffin sections, including labeling barotest

antibody Short epitope granulofilamentosa designation cytes' ä \ + D11-A / B10 FROM 10 d, D11-D / G2 DG 2 e + B4-A / H11 AH11 e + B4-B7B9 BB 9 f + D11-A / D11 AD11 f + D14-H / D11 HD11 G D11-C / C11 CC11 H B4-F / C3 FC 3

Immunohistochemical reaction with pulmonary bile duct colonic mammary

zyten 'epithelium' carcinoma ² carcinoma ²

FDNB sensitivity ³

9.9

9.9

9.9

9.9

n.d.

9.9

9.9

9.9

5.6

0/6

1.6

2.6

n.d.

6.6

0/6

0/6

1 test of the reaction on at least four tissue samples.

2 Number of CEA-containing colon carcinomas and NCA-containing breast carcinomas (positive / tested).

3 Examination of the labeling bar with peroxidase (by means of FDNB treatment of the antibodies before immunohistological reaction on paraffin sections of CEA-containing colon carcinoma tissue).

Claims (3)

1. Monoclonal enzyme immunoassay (EIA) for the quantitative determination of carcinoembryonic antigen (CEA) in biological fluids using the two-sided binding technique, characterized in that as solid phase antibodies the monoclonal antibodies B4-F / C3, D11-D / G 2, D11-C / C11 each alone or in any combination with each other, as secondary antibodies the monoclonal antibodies B4-A / H11, D11-A / B10, D11-A / D11 alone or in any combination with each other or the monoclonal antibody D14-H / D11 alone next to conventional tools for a CEA EIA. 2. Monoclonal enzyme immunoassay according to claim 1, characterized in that the enzyme immunoassay is designed for the microtiter plate technique. 3. A method for the selection of monoclonal antibodies for use in an EIA for the quantitative determination of CEA, characterized in that by immunohistological reaction to paraffin sections of CEA, NCA-1, BGP-1-containing tissues monoclonal antibodies in CEA-specific and CEA responsible, but not specific, to be grouped; by examining the peroxidase markability by means of an immunohistological procedure carried out on paraffin sections of CEA-containing tissue, the monoclonal antibodies are differentiated into solid-phase and secondary antibodies, the antigen reaction of the antibodies is checked by extinction measurement after substrate reaction of the secondary antibodies and combinations of solid-phase antibodies and secondary antibodies and, respectively, synergism and the cheapest combinations are selected.