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Immunoassays for specific determination of SCCA isoforms

An immunoassay, SCCA2 technology, applied in the direction of anti-animal/human immunoglobulin, immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve problems such as incomplete understanding

Inactive Publication Date: 2005-10-19
CANAG DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The biological roles of SCCA1 and SCCA2 are not fully understood

Method used

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  • Immunoassays for specific determination of SCCA isoforms
  • Immunoassays for specific determination of SCCA isoforms
  • Immunoassays for specific determination of SCCA isoforms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Preparation of recombinant SCCA

[0015] 1.1 Clone SCCA

[0016] The mRNAs from cell line Caski (cervix), C-4I (cervix), A549 (lung) and RPMI2650 (pharynx) were prepared using QuickPrep Micro mRNA Purification Kit (Pharmacia Company), cDNA was prepared using the First-Strand cDNA Synthesis Kit (Pharmacia). In 100μl containing 10mM Tris-HCl pH 8.85, 25mM KCl, 5mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 (Boehringer Company), 0.2mM dNTP (Pharmacia Company), 10μM SCCA 1-7F (the DNA sequences of all primers are listed in Table 1), 10μM SCCA 391-397B, 2μl cDNA and 2.5U Pwo-polymerase (Boehringer Company ) in the reaction solution, the 1218bp DNA fragment covering the SCCA coding sequence was amplified by PCR reaction. After denaturing the samples at 96°C for 5 minutes, a total of 30 cycles were performed, each cycle consisting of denaturation at 96°C for 15 seconds, annealing at 60°C for 15 seconds, and stretching at 72°C for 30 seconds. Finally, stretch at 72°C for 10 minutes to ...

Embodiment 2

[0033] Establishment of hybridomas and monoclonal antibodies

[0034] 2.1 Immunization and initial selection of anti-SCCA hybridomas

[0035] Polyclonal antisera reactive with the SCC antigen were obtained by immunizing rabbits subcutaneously with the recombinant SCC antigen and collecting immune sera according to standard procedures. The titer of the polyclonal antisera was checked by measuring the reactivity of the antisera with biotinylated SCCA2 and SCCA1 immobilized in streptavidin plates (Labsystems Oy, Helsinki, Finland). Recombinant SCCA2 and SCCA1 were biotinylated with biotin-N-succinimidyl hexanoate according to standard procedures.

[0036] Monoclonal antibodies reactive with SCCA1 and SCCA2 were obtained by immunizing Balb / c mice intraperitoneally with 10-50 μg of recombinant SCCA in Ribi adjuvant. After immunization, after receiving 2-4 booster doses within 60-90 days, the splenocytes of the immunized mice were fused with the P3×63Ag 8 myeloma cells.

[0037] ...

Embodiment 3

[0131] Establishment of Immunoassay

[0132] Using established monoclonal antibodies and recombinant proteins, it is likely to be possible to develop immunoassays for the separate determination of total SCCA and "free" SCCA, and for the separate determination of total SCCA1 and "free" SCCA1. Assays and Assays for Individual Determination of Total SCCA2 and "Free" SCCA2.

[0133] 3.1 Immunoassay for the determination of total SCCA

[0134] 3.1.1 Immunoassay for the determination of "Total SCCA"

[0135] SCCA-specific assays were designed using antibodies from A1a (Table 1) in combination with antibodies from group A2 or A3a, i.e. "free" SCCA1, "free" SCCA2, complexed SCCA1 and complexed The sum of SCCA2.

[0136] A preferred combination is antibody SCC113 as capture antibody and SCC107 as detection antibody.

[0137] SCC113 monoclonal antibody was biotinylated with biotin NHRS hexanoate (Sigma Chemical Company, USA) using standard procedures and used as capture antibody. T...

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Abstract

The present invention relates to monoclonal antibodies capable of distinguishing squamous cell cancer antigens, SCCA, in either free or complex bound forms, preferably antigens SCCA1 and SCCA2, as well as hybridomas recognizing such antibodies, method for diagnosing SCC, as well as diagnostic kits for detecting SCCAs.

Description

technical field [0001] The present invention relates to the individual determination of the various isoforms of SCCA, and the use of the serum concentrations of the different isoforms and their ratios as a method of diagnosing cancer. Background technique [0002] Squamous cell carcinoma antigen (SCCA) is a serological marker of squamous cell carcinoma (SCC) of the uterus, cervix, lung, head and neck, vulva and esophagus (1, 2). It was first purified in the late 1970s by Kato and coworkers from squamous cell carcinoma of the human cervix, TA-4 complex with a molecular weight of 42-48 kDa (1,3). Electrophoresis of the TA-4 complex revealed more than 10 fragments, and isoelectric aggregation of the antigen suggested two subfragments, an acidic (pI<6.25) and a neutral (pI≥6.25) isomorphic Construct (4). [0003] Cloning of the cDNA of SCCA revealed that it belongs to the family of serine protease inhibitors (serpins) (6). Further cloning of the gene region on chromosome 1...

Claims

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Application Information

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IPC IPC(8): C07K16/30G01N33/574
CPCC07K16/3007G01N33/57484C07K16/30A61K39/395G01N33/574
Inventor 伊万·罗杰尼尔松·奥利
Owner CANAG DIAGNOSTICS
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