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Glycoprotein methods protocols - biotechnology
ELISA for Detection of Serum MUC1 Antibodies 49549541Detection of Humoral Immune Responses to MucinsSilvia von Mensdorff-Pouilly, Claus Vennegoor,and Joseph Hilgers1. IntroductionMUC1 is displaced and altered in malignancies originating in epithelial tissues. Asa consequence, a molecule with short carbohydrate side chains and exposed repetitiveepitopes on its peptide core is shed into the interstitial space surrounding a tumor andgains access to the circulation (1–3), coming in contact with the immune system.Humoral immune responses to MUC1 have been described in healthy subjects and inpatients with benign and malignant diseases (4–6).Breast and ovarian carcinoma patients have antibodies reactive with the MUC1molecule, bound into circulating immune complexes (7). Preliminary results suggestthat the presence of such immune complexes in breast cancer patients at the time ofdiagnosis is related to a favorable disease outcome (8).Unbound MUC1 IgG and IgM antibodies are present in the circulation of bothhealthy subjects and breast cancer patients (9). Screening of a population of healthywomen with the bovine serum albumin (BSA)-conjugated peptide assay (describedunder Subheading 2.1.) gave median IgG and IgM MUC1 antibody levels of opticaldensity units (OD) 0.580 (range 0.304–1.539) and OD 0.823 (range 0.227–1.589),respectively. In pretreatment serum samples from breast cancer patients, the resultsobtained were OD 0.651 (range 0.328–1.537) for IgG and OD 0.779 (range 0.178–1.655) for IgM MUC1 antibodies. A benefit in survival was found in early stage breastcancer patients with circulating MUC1 antibodies at first diagnosis (von Mensdorff-Pouilly, et al., J. Clin. Oncol., in press), suggesting that active specific immunotherapyof breast cancer patients with MUC1-derived (glyco)peptides after primary surgery, inan adjuvant setting, might favorably influence the outcome of disease.The enzyme-linked immunosorbent assays (ELISAs) described here are useful forpretreatment screening of carcinoma patients for the presence of humoral immuneresponses to MUC1 as well as for monitoring MUC1 antibody levels during MUC1vaccine therapy. Other uses and applications include screening for suitable donors ofFrom:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 496 von Mensdorff-Pouilly et al.MUC1-primed B-lymphocytes to obtain B-cell clones producing MUC1 antibodies(10), creating human phage (anti)body libraries that may hold antibodies that recog-nize MUC1 (11), monitoring antibody production in culture supernatant of MUC1-primed B-lymphocytes (10), and epitope mapping of MUC1 or other antibodies (12).2. Materials2.1. BSA-Conjugated Peptide Assay (PEM.CIg)1. Serum or plasma samples, fresh or frozen. Repeated freezing and thawing of the samples(more than four times) should be avoided because it may alter results.2. ELISA microtiter 96-well plates (Costar, Cambridge MA).3. A synthetic 60mer peptide corresponding to three tandem repeats (TRs) of the MUC1peptide core, i.e., NH2-(HGVTSAPDTRPAPGSTAPPA)3-COOH. The 60 mer peptide isconjugated to BSA (Sigma, St. Louis, MO, RIA grade, cat. no. A-7888) using a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride conjugation kit (Imject™ ImmunogenEDC Conjugation Kit for KLH and BSA, Pierce, Rockford, IL). After conjugation, deter-mine the total protein content of the solution and prepare the BSA-conjugated peptide solu-tion for coating the plates on the basis of the measured value. Store stock solution at –20°C.4. 10 mM Sodium phosphate/0.15 M sodium chloride buffer, pH 7.0. To prepare 2 L of 5Xconcentrated phosphate/salt buffer, dissolve 9.56 g of Na2HPO4, 4.5 g of Na2H2PO4·H2Oand 87.66 g of NaCl in demineralized water. Add demineralized water to 2 L. Experienceshows that the pH of the concentrated buffer is 6.6–6.7. No pH adjustments are needed:after 5X dilution with demineralized water the pH of the phosphate/salt buffer shouldmeasure 7.0.5. Phosphate/salt buffer containing 0.5% BSA, to dilute the conjugate.6. Blocking buffer (also for serum/plasma dilutions): phosphate/salt buffer containing 1%BSA and 0.1% sodium azide.7. Washing buffer-phosphate/salt buffer with 0.05% Tween-20. Approximately 30 mL ofwashing buffer are necessary per washing turn (480 mL/plate).8. Horseradish peroxidase-labeled rabbit antihuman IgG and IgM, both from Dako A/S, Glostrup,Denmark. Dilute the conjugate 1:10,000 in phosphate/salt buffer containing 0.5% BSA.9. Substrate solution containing 0.06 mg/mL of tetramethylbenzidine (TMB) diluted in 0.1 Msodium acetate/citric acid buffer solution, pH 4.0, and 0.03% hydrogen peroxide. Sub-strate solution should be prepared immediately before use as required.a. Stock solution TMB: 6 mg 3,3',5,5'-tetramethylbenzidine (Sigma, cat. no. T-2885) in1 mL of dimethyl sulfoxide. Store in the dark at room temperature.b. 0.1 M sodium-acetate/citric acid buffer, pH 4.0. This pH is reached with a solutioncontaining 0.0627 M sodium acetate and 0.0374 M citric acid.c. 30% Hydrogen peroxide.10. 1.6 N sulfuric acid.2.2. Biotinylated Peptide Assay1. Streptavidin (Sigma-Aldrich, Zwijndrecht, Netherlands). Store stock solution (4 mg/mL)in 50-µL portions at –80°C. Streptavidin solution for coating the plates: 4 µg/mL ofstreptavidin in 10 mM sodium phosphate/0.15 M sodium chloride, pH 7.0, containing0.02% sodium azide.2. A 60mer synthetic peptide corresponding to three TR of the MUC1 peptide corebiotinylated at the amino terminal end. Three alanines should be incorporated at the amino ELISA for Detection of Serum MUC1 Antibodies 497terminal end to account for the streptavidin pocket. The final sequence is biotin-AAA-(HGVTSAPDTRPAPGSTAPPA)3-COOH. Stock solution peptide: 1mg/ml in phosphate-buffered saline (PBS). Keep at –70°C.3. All other materials as in Subheading 2.1.3. Methods3.1. BSA-Conjugated Peptide Assay (PEM.CIg)1. Apply 250 ng of BSA-conjugated peptide (see Note 1) diluted in 75 µL of phosphate/saltbuffer per well and 75 µL of 1% BSA solution per well to plates in duplicate alternaterows. Seal the wells with plastic plate sealers and incubate overnight at room tempera-ture. Plates can be stored, well sealed, for several months at –70°C.2. Rinse the plates twice with demineralized water, shake the plates empty, and fill the wellswith demineralized water. Repeat once.3. Fill the wells with 300 µL of blocking buffer, seal the plates, and incubate overnight at4°C (or 3 h at 37°C).4. Wash twice with washing buffer, shake the plates empty, and fill the wells with 300 µL ofwashing buffer per well. After the last wash leave the washing buffer in the wells untilimmediately before applying serum dilutions, because the wells should be left dry foronly the shortest time possible.5. Shake the plates empty, one by one, immediately before applying serum. Apply 75 µL ofpreviously diluted human serum per well (see Note 2). Dilute human serum samples 1:100(for IgG determinations) or 1:500 (for IgM determinations) in blocking buffer. Applyeach serum sample in duplicate, i.e., fill two wells coated with peptide and two wellscoated with BSA (see Notes 3–6). In each plate, fill one duplicate set of wells with block-ing buffer only. Seal the plates and incubate overnight at 4°C.6. Wash seven times with washing buffer: after the last wash leave the washing buffer inwells until ready to fill with conjugate. Shake the plates empty, one by one, and dry upperand underside of plates with clean tissue carefully before applying conjugate.7. Drop into each well 75 µL of horseradish peroxidase-conjugated antihuman IgG (or anti-human IgM) diluted 1:10,000 in 0.5% BSA solution. Avoid touching the bottoms andsides of wells. Seal the plates. Incubate for 60 min at room temperature. Keep a smallamount of conjugate to test the substrate solution before you apply it. At equal amounts,the mixture should immediately become an intense blue colour.8. Wash seven times with washing buffer and rinse the plates once with demineralized water,proceeding as in step 6.9. Drop 75 µL of substrate solution into each well, avoid touching the bottoms and sides of wells.Incubate for 60 min in the dark at room temperature. Positive wells will color blue. BSA-coated wellsshould color pale blue, very faintly; to see the color place the plate against a white background.10. Add into each well 100 µL of 1.6 N sulfuric acid to stop the reaction. Blue will turn to yellow.11. Measure the reaction at 450 nm in a microwell plate reader.3.2. Biotinylated Peptide Assay1. Apply 75 µL of streptavidin solution to each well, seal the plates, and incubate overnightat 4°C. Plates can be stored, well sealed, for several months at -70 C.2. Wash the wells three times with washing buffer, shake the plates empty, and fill the wellswith 300 µL of washing buffer per well. After the last wash leave the buffer in the wells.3. Shake the wells until empty and fill with blocking buffer, 300 µL/well. Seal the plates andincubate at room temperature for 60 min (or overnight at 4°C). 498 von Mensdorff-Pouilly et al.Table 1Examples of IgG and IgM Readings of Individual WellsIgG DeterminationsSample 60mer 60mer BSA BSA ODBlocking buffer 0.150 0.150 0.044 0.044 0.106Standard 1:250 1.091 1.111 0.099 0.095 1.004Standard 1:500 0.801 0.808 0.077 0.080 0.726Standard 1:1000 0.539 0.534 0.065 0.070 0.469Standard 1:2000 0.384 0.385 0.056 0.057 0.328Low sample 0.322 0.333 0.062 0.059 0.267Middle sample 0.913 0.949 0.060 0.087 0.858High sample 1.735 1.783 0.098 0.110 1.655IgM DeterminationsSample 60mer 60mer BSA BSA ODBlocking buffer 0.216 0.215 0.046 0.047 0.169Standard 1:250 1.417 1.422 0.073 0.073 1.347Standard 1:500 1.244 1.244 0.064 0.062 1.181Standard 1:1000 1.024 1.051 0.055 0.056 0.982Standard 1:2000 0.785 0.811 0.053 0.053 0.745Low sample 0.238 0.237 0.073 0.068 0.167Middle sample 0.917 0.926 0.073 0.078 0.846High sample 1.523 1.524 0.079 0.085 1.442Figure 1. ELISA for Detection of Serum MUC1 Antibodies 4994. Wash the wells twice with washing buffer. After the last wash, leave the buffer in wells.5. Aspirate the buffer from the wells and fill with 75 µL of biotinylated peptide solution,diluted to a concentration of 1 µg/mL peptide in 1% BSA solution. Seal the plates andincubate for 60 min at room temperature.6. Wash the wells three times as described in step 2. Proceed further as in Subheading 2.2.,steps 5–11.4. Notes1. A comparison of the two assay methods showed the BSA-conjugated peptide assay to bemore sensitive for the detection of human MUC1 antibodies in serum and plasma samplesthan the one with biotin-conjugated peptide. The reason could be the more random pre-sentation of the peptide when conjugated to BSA, in comparison to its more orderly pre-sentation, which could lead to steric hinderance, in the streptavidin-biotin system. Thebiotinylated peptide assay has been used with excellent results to detect and monitor theproduction of MUC1 antibodies by MUC1-primed B-lymphocytes in culture.2. In our experience, prediluted serum and plasma samples (1:20 in phosphate/salt buffer/0.1%BSA/0.1% sodium azide) give reproducible results even after 2 to 3 mo of storage at 4°C.3. Individual results are calculated as the mean difference between the readings in opticaldensity units in the two peptide-coated and the two BSA-coated wells. Table 1 illustratessome results.4. To standardize the assay, construct a four-point standard curve for each individual plateby testing a positive serum sample in four consecutive dilutions. Ascribe an arbitraryvalue of 1 to the highest serum dilution, and calculate the value of the samples testedwithin each plate in relation to the standard curve by least-square regression analysis(Fig. 1). Measurements that fall outside the standard curve should be retested at higherdilutions.5. The choice of standards should be a matter of careful consideration. Humanized mono-clonal antibodies provide suitable standards for IgG determinations (Fig. 2) whereasFigure 2. 500 von Mensdorff-Pouilly et al.screening of a number of serum samples will provide suitable candidates for an IgMstandard. Perform a dilution curve and choose four-point dilutions from the linear portionof the curve (OD 0.3–OD 1.3). Choose a sample from which a suitable amount is avail-able (1 mL of serum will be enough for ~400 plates).6. The intra- and interassay coefficients of variation of the PEM.CIg assay are, respec-tively, 2 and 12% for the IgG determinations and 1.2 and 3% for the IgM determinations.References1. Hilkens, J., Kroezen, V., Bonfrer, J. M. G., De Jong-Bakker, M., and Bruning, P. F. (1986)MAM-6, a new serum marker for breast cancer monitoring. Cancer Res. 46, 2582–2587.2. Sikut, R., Zhang, K., Baeckström, D., and Hansson, G. C. (1996) Distinct sub-populationsof carcinoma-associated MUC1 mucins as detected by the monoclonal antibody 9H8 andantibodies against the sialyl-Lewis a and sialyl-Lewis x epitopes in the circulation of breastcancer patients. Int. J. Cancer 66, 617–623.3. Bon, G. G., Kenemans, P., Van Kamp, G. J., Yedema, C. A., and Hilgers, J. (1990) Reviewon the clinical value of polymorphic epithelial mucin tumor markers for the managementof carcinoma patients. J. Nucl. Med. Allied. Sci. 34, 151–62.4. Hinoda, Y., Nakagawa, N., Nakamura, H., Makiguchi, Y., Itoh, F., Adachi, M., Yabana,T., Imai, K., and Yachi, A. (1993) Detection of a circulating antibody against a peptideepitope on a mucin core protein, MUC1, in ulcerative colitis. Immunol. Lett. 35, 163–168.5. Kotera, Y., Fontenot, D. J., Pecher, G., Metzgar, R. S., and Finn, O. J. (1994) Humoralimmunity against a tandem repeat epitope of human mucin MUC-1 in sera from breast,pancreatic, and colon cancer patients. Cancer Res. 54, 2856–2860.6. Rughetti, A., Turchi, V., Ghetti, C. A., Scambia, G., Panici, P. B., Roncucci, G., Mancuso,S., Frati, L., and Nuti, M. (1993) Human B-cell immune response to the polymorphicepithelial mucin. Cancer Res. 53, 2457–2459.7. Gourevitch, M. M., von Mensdorff-Pouilly, S., Litvinov, S. V., Kenemans, P., Van Kamp,G. J., Verstraeten, A. A., and Hilgers, J. (1995) Polymorphic epithelial mucin (MUC-1)-containing circulating immune complexes in carcinoma patients. Br. J. Cancer 72, 934–938.8. von Mensdorff-Pouilly, . S, Gourevitch, M. M., Kenemans, P., Verstraeten, A. A.,Litvinov, S. V., Van Kamp, G. J., Meijer, S., Vermorken, J., and Hilgers, J. (1996) Humoralimmune response to polymorphic epithelial mucin (MUC-1) in patients with benign andmalignant breast tumours. Eur. J. Cancer 32A, 1325–1331.9. von Mensdorff-Pouilly, S., Gourevitch, M. M., Kenemans, P., Verstraeten, A. A., VanKamp, G. J., Kok, A., Van Uffelen. K., Snijdewint, F. G. M., Paul, M. A., Meijer, S. andHilgers, J. (1998) An enzyme-linked immunosorbent assay for the measurement of circu-lating antibodies to polymorphic epithelial mucin (MUC1). Tumor Biol. 19, 186–195.10. Petrarca, C., Casalino, B., von Mensdorff-Pouilly, S., Rughetti, A., Rahimi, H., Scambia,G., Hilgers, J., Frati, L., and Nuti, M. Isolation of MUC1-primed B lymphocytes fromtumor draining lymph nodes using immunomagnetic beads. Cancer Immunol. Immunother.47, 272–277.11. Tureci, O., Sahin, U., and Pfreundschuh, M. (1997) Serological analysis of human tumorantigens: molecular definition and implications. Mol. Med. Today 3, 342–349.12. Vennegoor, C. J. G. M., Nijman, H. W., Drijfhout, J. W., Vernie, L., Verstraeten, A. A.,von Mensdorff-Pouilly, S., Hilgers, J., Verheijen, R. H. M., Kast, W. M., Melief, C. J. M.,and Kenemans, P. (1997) Autoantibodies to p53 in ovarian cancer patients and healthywomen: a comparison between whole p53 protein and 18-mer peptides for screening pur-poses. Cancer Lett. 116, 93–101. . NH 2-( HGVTSAPDTRPAPGSTAPPA)3-COOH. The 60 mer peptide isconjugated to BSA (Sigma, St. Louis, MO, RIA grade, cat. no. A-7888) using a 1-ethyl- 3-( 3-dimethylaminopropyl). © Humana Press Inc., Totowa, NJ 496 von Mensdorff-Pouilly et al.MUC1-primed B-lymphocytes to obtain B-cell clones producing MUC1 antibodies(10), creating