Kit for the quantitative determination of total prostatespecific antigen in the serum of human blood by the method of one-step enzyme-linked immunosorbent assay

 

(57) Abstract:

The invention relates to the field of biology and medicine and can be used for the diagnosis of prostate cancer, monitoring the effectiveness of the treatment of prostate tumors, in screening programs to identify risk, forecast of development of tumor and detection of recurrence. The essence of the invention is a reagent kit for the quantitative determination prostatespecific antigen in serum containing media with immobilized first mouse monoclonal antibodies produced by the hybrid strain of cultured animal cells Mus.musculus L NO. RCCC(P)D, the second conjugate of mouse monoclonal antibodies produced by the hybrid strain of cultured animal cells Mus.musculus L NO. RCCC(P)D with peroxidase, the calibration sample PSA (0; 2,5; 5,0; 10,0 and about 30.0 ng/ml PSA) in Tris buffer with 1% bovine serum albumin. The technical result is the quantitative determination of total prostatespecific antigen in the serum at any vertical scanning photometer. 5 table.

The kit is intended for the quantitative determination of the concentration of a specific antigen PDF clinical practice for screening and monitoring treatment of prostate diseases, for prognosis of prostate cancer.

The set contains the tablet with immobilized monoclonal antibodies, six calibration samples ( containing 0; 1; 2,5; 5; 10 and 30 ng/ml (PSA) conjugate is a mouse monoclonal antibody with horseradish peroxidase reagents for enzymatic reactions (dye tetramethylbenzidine, citrate buffer with hydrogen peroxide and stop reagent sulfuric acid). Media immobilized mouse monoclonal antibodies produced by the hybrid strain of cultured animal cells Mus.musculus L. No. RCCC(P)D. The conjugate contains mouse monoclonal antibodies produced by the hybrid strain of cultured animal cells Mus. Musculus L NO. RCCC(P)D. As the calibration samples used DOG with 1% serum albumin.

The set allows to detect 100% of patients with malignant prostate tumors among patients with cancer (5 tables).

The invention relates to biology and medicine and can be used to diagnose prostate cancer, monitoring the effectiveness of the treatment of prostate tumors, in screening programs to identify risk groups among the male population, forecast developed logit set company Hoffman La Roche, designed for the quantitative measurement of the concentration of PSA in serum and plasma of human blood by the method of enzyme immunoassay analyzer COBAS CORE ( Cancer Res., v.51, 222-226 (1991).

The set consists of polystyrene beads coated with mouse MAB to the DOG man, the conjugate of Fab fragment of the second anti-PSA MAB with horseradish peroxidase in buffer with stabilizer and preservative, calibrators 0 and 25 ng/ml PSA, control serum containing a known amount of PSA in Tris buffer with protein stabilizer and preservative, a dilution buffer with protein stabilizer and preservative. For carrying out the enzymatic reaction set is assembled with the substrate solution TMB (tetramethylbenzidine). The definition can only be performed on a fully automated analyzer COBAS CORE. On the other equipment, this set may not be used. So the set is not universal and may not be used in laboratories where there is no automated analyzer COBAS CORE.

The purpose of the invention is to remedy these disadvantages.

This goal is achieved using a pair of highly specific mouse MAB produced by strains hybridnet polystyrene, which allow the detection of any photometer horizontal scan.

The set contains the tablet with immobilized monoclonal antibodies, six calibration samples (containing 0; 1; 2,5; 5; 10 and 30 ng/ml PSA) conjugate is a mouse monoclonal antibody with horseradish peroxidase, control serum with known PSA, reagents for enzymatic reactions (dye tetramethylbenzidine and citrate buffer with hydrogen peroxide, the stop reagent sulfuric acid). Media immobilized mouse monoclonal antibodies produced by the hybrid strain of cultured animal cells Mus.musculus L. No. RCCC/ P/ D. The conjugate contains mouse monoclonal antibodies produced by the hybrid strain of cultured animal cells Mus. Musculus L NO. RCCC(P)661 D. as the calibration samples used PSA in 1% serum albumin. For carrying out the enzymatic reaction is used substrate buffer with hydrogen peroxide, TMB (tetramethylbenzidine) and stop registrera acid.

Analysis.

To release the tablet from the package. In wells add 50 ál of each calibration sample containing 0; 1; 2,5; 5; 10; 30 ng/ ml PSA, in other wells add in the t to all wells 100 ál of working strength conjugate and incubate at 37°C for 1 h After incubation wash the wells 4 times with a working solution for washing tablet. Add to all wells, 100 μl of developing solution, incubate for 15 min at 37°C. stop the reaction by adding into each well, 100 μl of 4.8% sulfuric acid. Measure the optical density in the wells on the vertical scanning photometer at a wavelength of 450 nm.

The main characteristics of the set are shown in table 1.

Example 1. Serum 10 female donors were investigated for the presence of PSA, defined with the help of this set. In none of the cases did not observe a positive result. Serum 40 donors-men investigated for the presence of antigen PSA, defined with the help of this set. The average value of PSA in the serum of the group amounted to (0,46±0,06) ng/ml.

Example 2. Samples of 14 sera from patients with tumors other than prostate cancer, localizations (colorectal cancer, gastric cancer, liver cancer, colon cancer, colon cancer, lung cancer, pancreatic cancer). The mean value for this group was (1,1±0,3) ng/ml PSA.

Example 3. Sera of 11 patients with adenocarcinoma of the prostate histologically confirmed, was investigated using Dunn is with BPH, confirmed histologically, researched with the help of this set. The mean PSA value for this group amounted to (17,4±6,5) ng /ml. the results of the experiments are presented in tables 2-5. From the tables it is seen that with this set you can identify patients with tumors of the prostate.

Reagent kit for the quantitative determination prostatespecific antigen in serum containing media with immobilized first monoclonal antibody, conjugate second mouse monoclonal antibodies with peroxidase, the calibration sample PSA (0; 2,5; 5,0; 10,0 and about 30.0 ng/ml PSA) in Tris buffer with 1% bovine serum albumin, wherein as the first antibodies using mouse monoclonal antibodies produced by the hybrid strain of cultured animal cells Mus.musculus L NO. RCCC(P)D, and as secondary antibodies using mouse monoclonal antibodies produced by the hybrid strain of cultured animal cells Mus.musculus L NO. RCCC(P)D, the reaction proceeds in one stage, with 1 h incubation and detection can be performed on any vertical scanning photometer.



 

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