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. This interference results in measurement of artifactual levels of analyte, e.g. , carcinoembryonic antigen (CEA) .
• CEA carcinoembryonic antigen
• HAMA human anti-mouse antibodies
• the administered doses of antibody increase (>20mg) or multiple injections are given, the incidence of HAMA increases.
• the interference of HAMA on the accurate measurement of plasma levels of CEA has been documented, see Dillman, R.O., et al., J. Biol. Response Mod., 5 :394-410, (1986); Jaffers, G.J., et al..
• Modifications of the i munoassay 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. Surq. , 123:1242-6 (1988) and heat extraction of the plasma (see Hansen, H.J. , et al., Clin. Chem. , 35(1) :146-51 (1989).
• Recently the commercially available CEA-Rocha ⁇ EIA has been modified by the addition of a single MAB to reduce the interference of potential heterophilic antibodies.
• 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.
• Plasma CEA values (ng/ml) obtained using the unmodified EIA + mlgG ( B) and the modified EIA ( %__ ) were expressed as a ratio of the CEA values measured by the unmodified EIA alone ( Q) 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 suppressible CEA titers.
• 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) .
• Assays are conducted in known manner, e.g. , by utilization, in the manner set forth in the accompanying instructions, of a commercially available CEA-Roche EIA kit.
• a publicly available package insert from such a kit has been concurrently filed and is incorporated herein by reference.
• the 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 4,873,313, issued on October 10, 1989.
• 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.
• Partially suppressible CEA Plasma with elevated CEA values which were lowered by the addition of mlgG but still remained above the normal CEA values (>5mg/ml) and thus were classified clinically to be CEA positive.
• 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 37 ⁇ C prior to performing the assay.
• the appropriate dilution corrections were included in the calculations.
• CEA measurements obtained using the modified EIA kit were performed on all plasma samples, according to the concurrently filed package instructions incorporated herein by reference. Each sample was tested in duplicate at two different dilutions.
• 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. Table 1
• Plasma CEA a Plasma HAMA b ng/ml (S.D.) ug/ml (S.D.)
• Plasma samples from 30 patients who had received 20-40mg l ⁇ :L In-labeled anti-CEA MAB for tumor immunoscintigraphy and who developed a rising titer of CEA were used in this analysis.
• 58 were found to have elevated (>5.0ng/ml) CEA values when measured using the unmodified EIA.
• the number of elevated CEA samples dropped to 47 (81%) .
• Addition of 200 ⁇ g mlgG to the plasma prior to running the unmodified EIA resulted in only 37 CEA positive (>5.0ng/ml) samples (64%).
• 21 samples, which were positive for CEA in the unmodified EIA proved to be normal for CEA in the
• 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.
• samples in this group showed suppression of CEA values of >18% with an overall mean of 65.7%.
• 200ug mlgG were added to plasma samples prior to assay using either unmodified or modified Roche EIA.
• 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 titer of HAMA in the samples that continued to give positive CEA (>5.0ng/ml) values with the modified EIA were on the whole higher (18.9 + 24.9) than those that gave negative ( ⁇ 5.0ng/ml) CEA values (6.99 + 8.17). However, this difference was not statistically significant due to the wide spread of values and concomitant high standard deviations. Thus no statistical correlation between HAMA level and ability of the modified EIA to correct the false positive CEA values was obtained.
• no statistically significant relationship was found between the level of HAMA and the difference in suppression of CEA levels obtained with the modified assay and the unmodified assay + mlgG (p>0.03).
• 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 respectively (Table 3) .
• the modified assay gave 10/47 (21.5%) false positive CEA determinations and 12/47 (25.5%) partially suppressible CEA titers (Figure 3b) .
• 10 (48%) continued to show false positive CEA values with the modified assay.
• 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).
• a false elevation of the plasma CEA may lead to extensive unnecessary testing or even unnecessary surgical procedures. Therefore, elimination of false positive CEA values is clinically most desirable.
• the imaging agent administered to the patients was a murine IgGl 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.
• the exemplified combination of the three monoclonals (1:1:1, lOO ⁇ g total) gave CEA values which most consistently reflected those produced with the addition of polyclonal mlgG.
• the addition of lOO ⁇ g of IgGl MAB was not as effective as the mixture in eliminating HAMA interference, even though the immunogen given to the patients was an IgGl 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)

Citations (1)

Family Cites Families (2)

• 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

Patent Citations (1)

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events