Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57565—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen [CEA]

Definitions

• This invention relates to removal of interference observed in double antibody assays consequent from the presence of human anti-murine and heterophilic antibodies present in human plasma.
• CEA carcinoembryonic antigen
• HAMA human anti-mouse antibodies
• Modifications of the immunoassay for CEA designed to obviate the effect of HAMA include preincubation of the plasma with polyclonal murine IgG (see Morton, B.A., et al., Arch. Surg., 123:1242-6 (1988) and heat extraction of the plasma (see Hansen, H.J., et al., Clin. Chem., 35(1):146-51 (1989).
• CEA-Roche ® EIA has been modified by the addition of a single MAB to reduce the interference of potential heterophilic
• CEA-Roche ® EIA itself involves the addition of a single monoclonal to the kit. This still results in a 22% false positive CEA for patients who have received a murine monoclonal antibody for diagnosis or therapy, see Price, T., et al., Clin. Chem.,
• Pursuant to this invention falsely elevated analyte levels in double antibody EIA assays are corrected by the addition of a mixture of IgGl, IgG2a and IgG2b to human plasma specimens.
• modified EIA were expressed as a ratio of the CEA values measured by the unmodified EIA alone ( ⁇ ) for each of the four patient groups.
• the patient groups were: group 1, non-imaged controls; group 2, imaged patients with true positive CEA titers; group 3, imaged patients with false positive CEA titers; and group 4, imaged patients with partially
• Heterophilic antibody interference in double antibody immunoassays has been a recognized problem in the quantification of hormones, enzymes, and cell surface antigens for diagnostic purposes. This interference can produce either false positive or false negative results. Boscato and Stuart reported that 40% of normal plasma contain detectable
• non-analyte substances which are capable of broad specificity antibody binding and have the potential of interfering with double antibody immunoassays, see Boscato, L.M., et al., Clin. Chem., 32(8):1491-5 (1986).
• HAMA appears to be the cause of false positive results in the current clinically used double antibody immunoassays for CEA.
• HAMA and heterophilic antibody interference in double EIA assays is reduced or eliminated by incubating human plasma or serum specimens prior to assay with a mixture of IgG
• CEA-Roche EIA kit includes a hybridoma or cell line on deposit with the ATCC, Accession No. HB8747, and described and claimed as T84.66 in U.S. Patent
• the mixture of monoclonal IgG isotypes (about 15 to about 25 ⁇ l), e.g., Ig2a, IgEa and Ig2b, is incubated at about 30°C to 50°C, preferably 37°C, for an appropriate time, e.g., 15 to 90 minutes, preferably from about 30 to 60 minutes, with from about 75 to about 150 ⁇ l of a diluted plasma or serum specimen.
• the monoclonal antibody mixture preferably but not necessarily, contains equal proportions by weight of each MAB.
• CEA-Roche ® EIA Roche Diagnostic System, Nutley, NJ.
• Plasma samples of 13 patients who had never received murine antibodies and had CEA values ranging from normal ( ⁇ 5ng/ml) to 53.9ng/ml were used as controls.
• CEA level of each plasma sample was measured using both the standard (unmodified) CEA-Roche ® EIA kit (see the concurrently filed package insert) and a version of this kit modified by including a single MAB.
• all plasma samples were incubated with either 20 ⁇ l PBS or 20 ⁇ l (200 ⁇ g) polyclonal mlgG for one hour at 37oC prior to performing the assay.
• HAMA human anti-murine antibody
• Plasma samples obtained from a limited population of 13 patients that had a range of CEA values but who had never received a murine monoclonal antibody for diagnosis or therapy were run in the unmodified EIA ⁇ mlgG and the modified EIA to compare CEA values and determine if addition of 200 ⁇ g mlgG affected the measurement of CEA. The results are reported in Table 1.
• the 58 plasma samples determined positive for CEA using the unmodified EIA were grouped into three categories based on the effect of MiGG incubation on the resultant plasma CEA value as described, infra.
• the first category contained fifteen samples (26%) whose CEA values were not significantly (p>0.05) affected by the addition of mlgG, as shown by the data in Table 2.
• the correlation coefficient between the CEA values obtained using the modified EIA and the unmodified EIA + mlgG was 0.73. This correlation was better than that found in the false-positive group
• monoclonal antibodies also decreased the CEA levels measured in each case; however, the effectiveness was more variable.
• HAMA HAMA for the indirect EIA used in this study was determined from a sampling of 44 patients who had never received murine antibodies and who had normal plasma CEA levels.
• the normal range obtained from the mean HAMA value plus two standard deviations, was 0.36 ⁇ g/ml.
• Table 1 all of the non-imaged control patients had HAMA values in the normal range. All samples in the true positive CEA group (Table 2) also showed normal HAMA values.
• HAMA values for the plasma in the false positive CEA group showed a broad range from normal levels ( ⁇ 0.36 ⁇ g/ml to 84.5 ⁇ g/ml. Five samples were ⁇ 0.36 ⁇ g/ml. The degree of false elevation of CEA in the unmodified EIA did not show a significant
• HAMA values In the partially suppressible CEA group (Table 4) the HAMA values also ranged from normal levels (two samples) to high values (65.6 ⁇ g/ml). In this patient population, there was a significant
• the CEA values of the samples obtained with modified EIA and the unmodified EIA + mlgG were expressed as a ratio of the corresponding values obtained in the standard unmodified EIA alone ( Figure 2). This was done for each of the four patient groups. Plasma CEA values were similar in all three assays for patients who had not been given murine antibodies (control group 1), or who had little or no measurable HAMA (true positive group 2). However, for patients with measurable HAMA (groups 3 and 4), there was a substantial difference between the CEA values obtained using the unmodified assay and those obtained with the other two assays. The most marked differences were seen in group 3, the false positive group in which an overall average percent suppression of 95.3% and 98.9% was obtained with the modified Roche EIA and the unmodified Roche EIA + mlgG
• CEA plasma levels of CEA are used in the follow-up management of patients who have undergone a potentially curative operative procedure. Increases in the CEA titer are an early indicator of residual or recurrent colorectal cancer and as such may result in further diagnostic tests or surgical explorations (i.e., second look laparotomy) for localization of the site of the disease, see Shively, J.E., et al., Crit. Rev. One./Hem., 2:355-399 (1985). Thus, a false elevation of the plasma CEA may lead to
• the modified EIA corrected the HAMA interference in approximately half of the samples tested. Sixteen of these patient plasma samples in which there was a discrepancy in value between the unmodified assay + mlgG and the modified assay were reassayed with the addition of polyclonal mlgG to the plasma prior to running the modified kit. In each case, addition of the mlgG to the modified assay returned the CEA to levels comparable to those obtained in the unmodified assay plus mlgG.
• the imaging agent administered to the patients was a murine IgG1 monoclonal antibody
• the effectiveness of adding murine monoclonal antibodies to correct the remaining falsely elevated CEA samples in the modified EIA was examined. It was found that addition of each monoclonal antibody alone had some effect on decreasing the CEA levels. However, the exemplified combination of the three monoclonals (1:1:1, 100 ⁇ g total) gave CEA values which most consistently reflected those produced with the addition of polyclonal mlgG. Interestingly, the addition of 100 ⁇ g of IgG1 MAB was not as effective as the mixture in eliminating HAMA interference, even though the immunogen given to the patients was an IgG1 monoclonal antibody. Why removal of HAMA interference is more effective using a polyclonal mlgG or a mixture of monoclonal IgG subclasses remains unclear.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Immunology (AREA)
• Chemical & Material Sciences (AREA)
• Urology & Nephrology (AREA)
• Hematology (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Medicinal Chemistry (AREA)
• Analytical Chemistry (AREA)
• Cell Biology (AREA)
• Pathology (AREA)
• Food Science & Technology (AREA)
• Biotechnology (AREA)
• Physics & Mathematics (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Peptides Or Proteins (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Applications Claiming Priority (1)

Publications (2)

Family

ID=22220815

Family Applications (1)

Country Status (4)

Families Citing this family (11)

Family Cites Families (3)

• 1990
  • 1990-04-25 WO PCT/US1990/002239 patent/WO1991016627A1/en not_active Ceased
  • 1990-04-25 AU AU59652/90A patent/AU645981B2/en not_active Expired - Fee Related
  • 1990-04-25 JP JP2510079A patent/JPH04506866A/ja active Pending
  • 1990-04-25 EP EP90911145A patent/EP0479901A1/en not_active Withdrawn

Also Published As

Similar Documents

Legal Events