QIA10 c-ErbB2/c-Neu Rapid Format ELISA Kit

QIA10
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      Species Reactivity
      H

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      QIA10-1EA
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          Detects both c-ErbB2/c-Neu p185 and the extracellular domain p105 cleavage fragment. Positive cell lines: A549, HT29, SK-BR-3, and SKOV-3, or breast, prostate, colon, or ovarian cancer.

          Catalogue NumberQIA10
          Brand Family Calbiochem®
          Synonymsc-Neu ELISA Kit, ErbB2 ELISA Kit, Erythroblastosis Virus ELISA Kit, HER-2 ELISA Kit, HER2 ELISA Kit
          Application Data

          In a limited study (30 cancer and 30 normal sera samples), the assay recognized 17% of cancer samples as positive and 95% of normal samples as negative.
          Materials Required but Not Delivered 2-20 µl, 20-200 µl and 200-1000 µl precision pipetters with disposable tips.
          Wash bottle or multichannel dispenser for washing.
          500 ml graduated cylinder.
          Deionized or distilled H2O.
          Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450/595 nm or 450/540nm.
          References
          ReferencesGauchez, A.S., et al. 2008. Anticancer Res. 28, 3067.
          Hung, M.-C. and Y-K Lau, 1999. Seminars in Oncology 26(Suppl 12), 51.
          Ouyang, X., et al. 1999. Lancet 353, 1591.
          Ross, J.S. and J.A. Fletcher, 1999. Am. J. Clin. Pathol. 112, S53.
          Ross, J.S. and J.A. Fletcher, 1998. Stem Cells 16, 413.
          Disis, M.L., et al. 1997. J. Clin. Oncol. 15, 3363.
          Ross, J.S., et al. 1997. Human Pathology 28, 827.
          Ross, J.S., et al. 1997. Cancer 79, 2162.
          Xia, W., et al. 1997. Clin. Cancer Res. 3, 3.
          Cirisano, F.D. and B.Y. Karlan, 1996. J. Soc. Gynecol. Invest. 3, 99.
          Menden, H., et al. 1994. Cancer Res. Clin. Oncol. 120, 378.
          Kynast, B., et al. 1993. J. Cancer Res. Clin. Oncol. 119, 249.
          Gusterson, B. and R. Gelber, 1992. J. Clin. Oncol. 10, 1049.
          Hetzel, D.J., et al. 1992. Gynecol. Oncol. 47, 179.
          Hou, L., et al. 1992. Cancer Lett. 65, 215.
          Leitzel, K., et al. 1992. J. Clin. Oncol. 10, 1436.
          Carney, W.P., et al. 1991. J. Tumor Marker Oncol. 6, p. 53.
          Clark, G.M. and W.L. McGuire, 1991. Cancer Res. 51, 944.
          Langton, B.C., et al. 1991. Cancer Res. 51, 2593.
          Paterson, M.C., et al. 1991. Cancer Res. 51, 556.
          Zabrecky, J.R., et al .1991. J. Biol. Chem. 266, 1716.
          Berchuck, A., et al. 1990. Cancer Res. 50, 4087.
          Borg, A., et. al. 1990. Cancer Res. 50, 4332.
          Inglehart, J.D., et al. 1990. Cancer Res. 50, 6701.
          Kern, J.A., et al. 1990. Cancer Res. 50, 5189.
          Mori, S., et al. 1990. Jpn. J. Cancer Res. 81, 489.
          Paik, S., et al. 1990. J. Clin. Oncol. 8, 103.
          Maguire, H.C. and M.I. Greene, 1989. Seminars in Oncology 16, 148.
          Ro, J., et al. 1989. Cancer Res. 49, 6941.
          Slamon, D.J., et al. 1989. Science 244, 707.
          Tandon, A.K., et al. 1989. J. Clin. Oncol. 7, 1120.
          Wright, C., et al. 1989. Cancer Res. 49, 2087.
          Yoshida, K., et al. 1989. Virchows Arch. B Cell Pathol. Mol. Pathol. 57, 285.
          Fontaine, J., et al. 1988. Oncology 45, 360.
          Kraus, M.H., et al., 1987. EMBO J. 6, 605.
          Slamon, D., et al. 1987. Science 235, 177.
          King, C.R., et al. 1985. Science 229, 974.
          Shin, C., et al. 1981. Nature 290, 261.
          Henry, R.J., et al. 1974. Clinical Chemistry, Harper and Row, New York.
          Product Information
          Form96 Tests
          Format96-well plate
          Kit containsPre-Coated 96-Well Plate, c-ErbB2/c-Neu Lyophilized Standard, Cell Resuspension Buffer, Antigen Extraction Agent, Sample Diluent, Biotinylated Detector Antibody, Detector Diluent, Streptavidin Peroxidase Conjugate, Conjugate Diluent, Wash Buffer, Substrate, Stop Solution, Adhesive Plate Sealers, and a user protocol.
          Positive controlc-erbB2/c-neu
          Applications
          Biological Information
          Assay range0-3 ng/ml
          Assay time4 h
          Sample TypeCell lysates, biological fluids
          Species Reactivity
          • Human
          Physicochemical Information
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          R PhraseR: 23/24/25-35-36/37/38-40-41-43

          Toxic by inhalation, in contact with skin and if swallowed.
          Causes severe burns.
          Irritating to eyes, respiratory system and skin.
          Limited evidence of a carcinogenic effect.
          Risk of serious damage to eyes.
          May cause sensitization by skin contact.
          S PhraseS: 23-26-36/37/39-45

          Do not breathe fumes.
          In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
          Wear suitable protective clothing, gloves and eye/face protection.
          In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
          Product Usage Statements
          Intended useThe Calbiochem® c-erbB2/c-neu Rapid Format ELISA is a non-isotopic immunoassay for the in vitro quantitation of human c-erbB2/HER-2/neu oncoprotein in cell lysates, cell culture medium, sera and plasma.
          Storage and Shipping Information
          Ship Code Dry Ice Only
          Toxicity Multiple Toxicity Values, refer to MSDS
          Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
          Storage -20°C
          Storage ConditionsUpon receipt, store the entire kit at -20°C until use. Do not refreeze after thawing. Lyophilized standards must remain at -20°C. All other components may be stored at 4°C. Do not expose reagents to excessive light. Allow kit reagents to warm to room temperature before use.
          Avoid freeze/thaw Avoid freeze/thaw
          Do not freeze Ok to freeze
          Packaging Information
          Transport Information
          Supplemental Information
          Kit containsPre-Coated 96-Well Plate, c-ErbB2/c-Neu Lyophilized Standard, Cell Resuspension Buffer, Antigen Extraction Agent, Sample Diluent, Biotinylated Detector Antibody, Detector Diluent, Streptavidin Peroxidase Conjugate, Conjugate Diluent, Wash Buffer, Substrate, Stop Solution, Adhesive Plate Sealers, and a user protocol.
          Specifications

          Documentation

          c-ErbB2/c-Neu Rapid Format ELISA Kit SDS

          Title

          Safety Data Sheet (SDS) 

          c-ErbB2/c-Neu Rapid Format ELISA Kit Certificates of Analysis

          TitleLot Number
          QIA10

          References

          Reference overview
          Gauchez, A.S., et al. 2008. Anticancer Res. 28, 3067.
          Hung, M.-C. and Y-K Lau, 1999. Seminars in Oncology 26(Suppl 12), 51.
          Ouyang, X., et al. 1999. Lancet 353, 1591.
          Ross, J.S. and J.A. Fletcher, 1999. Am. J. Clin. Pathol. 112, S53.
          Ross, J.S. and J.A. Fletcher, 1998. Stem Cells 16, 413.
          Disis, M.L., et al. 1997. J. Clin. Oncol. 15, 3363.
          Ross, J.S., et al. 1997. Human Pathology 28, 827.
          Ross, J.S., et al. 1997. Cancer 79, 2162.
          Xia, W., et al. 1997. Clin. Cancer Res. 3, 3.
          Cirisano, F.D. and B.Y. Karlan, 1996. J. Soc. Gynecol. Invest. 3, 99.
          Menden, H., et al. 1994. Cancer Res. Clin. Oncol. 120, 378.
          Kynast, B., et al. 1993. J. Cancer Res. Clin. Oncol. 119, 249.
          Gusterson, B. and R. Gelber, 1992. J. Clin. Oncol. 10, 1049.
          Hetzel, D.J., et al. 1992. Gynecol. Oncol. 47, 179.
          Hou, L., et al. 1992. Cancer Lett. 65, 215.
          Leitzel, K., et al. 1992. J. Clin. Oncol. 10, 1436.
          Carney, W.P., et al. 1991. J. Tumor Marker Oncol. 6, p. 53.
          Clark, G.M. and W.L. McGuire, 1991. Cancer Res. 51, 944.
          Langton, B.C., et al. 1991. Cancer Res. 51, 2593.
          Paterson, M.C., et al. 1991. Cancer Res. 51, 556.
          Zabrecky, J.R., et al .1991. J. Biol. Chem. 266, 1716.
          Berchuck, A., et al. 1990. Cancer Res. 50, 4087.
          Borg, A., et. al. 1990. Cancer Res. 50, 4332.
          Inglehart, J.D., et al. 1990. Cancer Res. 50, 6701.
          Kern, J.A., et al. 1990. Cancer Res. 50, 5189.
          Mori, S., et al. 1990. Jpn. J. Cancer Res. 81, 489.
          Paik, S., et al. 1990. J. Clin. Oncol. 8, 103.
          Maguire, H.C. and M.I. Greene, 1989. Seminars in Oncology 16, 148.
          Ro, J., et al. 1989. Cancer Res. 49, 6941.
          Slamon, D.J., et al. 1989. Science 244, 707.
          Tandon, A.K., et al. 1989. J. Clin. Oncol. 7, 1120.
          Wright, C., et al. 1989. Cancer Res. 49, 2087.
          Yoshida, K., et al. 1989. Virchows Arch. B Cell Pathol. Mol. Pathol. 57, 285.
          Fontaine, J., et al. 1988. Oncology 45, 360.
          Kraus, M.H., et al., 1987. EMBO J. 6, 605.
          Slamon, D., et al. 1987. Science 235, 177.
          King, C.R., et al. 1985. Science 229, 974.
          Shin, C., et al. 1981. Nature 290, 261.
          Henry, R.J., et al. 1974. Clinical Chemistry, Harper and Row, New York.
          User Protocol

          Revision14-March-2016 JSW
          Synonymsc-Neu ELISA Kit, ErbB2 ELISA Kit, Erythroblastosis Virus ELISA Kit, HER-2 ELISA Kit, HER2 ELISA Kit
          Form96 Tests
          Format96-well plate
          Specieshuman
          StorageUpon receipt, store the entire kit at -20°C until use. Do not refreeze after thawing. Lyophilized standards must remain at -20°C. All other components may be stored at 4°C. Do not expose reagents to excessive light. Allow kit reagents to warm to room temperature before use.
          Intended useThe Calbiochem® c-erbB2/c-neu Rapid Format ELISA is a non-isotopic immunoassay for the in vitro quantitation of human c-erbB2/HER-2/neu oncoprotein in cell lysates, cell culture medium, sera and plasma.
          BackgroundThe c-erbB2/c-neu oncogene (also known as HER2/neu) encodes a 185 kDa glycoprotein (p185) with tyrosine kinase activity. It shares close sequence homolgy with the epidermal growth factor receptor (EGF-R). Signal transduction studies indicate extensive complexity in c-erbB2/c-neu signaling pathways although direct binding ligands have yet to be identified. Amplification and/or over-expression of c-erbB2/c-neu has been observed in 25-30% of human breast cancers as well as in ovarian, lung, endometrial, gastric, oral, and prostate carcinomas. Many studies indicate that c-erbB2/c-neu over-expression is the cause of the malignant phenotype rather than the results of such. In addition, over-expression of c-erbB2/c-neu results in a more aggressive, less responsive carcinoma leading to the belief that quantitation of c-erbB2/c-neu could have important prognostic value. In cells that over-express the c-erbB2/c-neu oncogene the extracellular domain is released from the cell surface resulting in a protein approximately 105 kDa (p105). p105 can be detected in cell culture supernatants and the serum, effusions, and saliva of many cancer patients. The Calbiochem® brand has developed a sensitive, specific ELISA for the quantitation of both p185 and p105 in cell lysates, cell culture supernatants, sera, plasma and other biological fluids. The assay has been calibrated in mass units (ng/ml) and takes 4 h to execute.
          Principles of the assayThe c-erbB2/c-neu Rapid Format ELISA is a "sandwich" enzyme immunoassay employing two mouse monoclonal antibodies. An antibody, specific for the human c-erbB2/c-neu protein, has been immobilized onto the surface of wells provided in the kit. The sample to be assayed is pipetted into the wells and allowed to incubate for 2 h, during which time any c-erbB2/c-neu present binds to the capture antibody. Unbound material is washed away and biotinylated detector monoclonal antibody is added. The detector antibody also recognizes human c-erbB2/c-neu protein, and will bind to any c-erbB2/c-neu which has been retained by the capture antibody. The detector antibody, in turn, is bound by horseradish peroxidase-conjugated streptavidin. The horseradish peroxidase catalyzes the conversion of the chromogenic substrate tetra-methylbenzidine (TMB) from a colorless solution to a blue solution (or yellow after the addition of stopping reagent), the intensity of which is proportional to the amount of c-erbB2/c-neu protein in the sample. The colored reaction product is quantified using a spectrophotometer. Quantitation is achieved by the construction of a standard curve using known concentrations of c-erbB2/c-neu (provided lyophilized). By comparing the absorbance obtained from a sample containing an unknown amount of c-erbB2/c-neu with that obtained from the standards, the concentration of c-erbB2/c-neu in the sample can be determined.
          Materials providedStandards should be assayed in duplicate. A standard curve must be performed on the same plate and at the same time as the samples. The c-erbB2/c-neu ELISA provides sufficient reagents to run two sets of standard curves, and 42 samples (if assayed in duplicate all at once using one standard curve), or 36 samples (if assayed on two separate occasions using two standard curves).

          • c-erbB2/c-neu Antibody COATED PLATE (Kit Component No. JA1980-1EA): 96 removable wells coated with c-erbB2/c-neu antibody.
          • c-erbB2/c-neu STANDARD (Kit Component No. JA1986): two vials containing lyophilized c-erbB2/c-neu protein. Standards have been calibrated against recombinant sp185HER2/neu. Reconstituted standards should be discarded after one use.
          NOTE: Standards have been calibrated in nanograms per ml (ng/ml). To convert to arbitrary Human Neu Units (HNU) multiply the number of ng/ml by the conversion factor 400. For example there are 1200 HNU in the 3 ng/ml c-erbB2/c-neu standard. This is equivalent to 16 femtomole per ml (fm/ml).
          • c-erbB2/c-neu DETECTOR ANTIBODY (Kit Component No. JA1983): Biotinylated monoclonal anti-human c-erbB2/c-neu antibody.
          • c-erbB2/c-neu 500X CONJUGATE (Kit Component No. JA1985): Streptavidin-Peroxidase Conjugate: 500-fold concentrated solution.
          • CONJUGATE DILUENT (Kit Component No. JA1984): The buffer for dilution of 500X Conjugate.
          • RESUSPENSION BUFFER (Kit Component No. JA1978): A buffer for the preparation of cell and tissue lysates. It is recommended that this buffer be adjusted to contain 5 mM EDTA, 0.2 mM PMSF, 1 µg/ml pepstatin, and 0.5 µg/ml leupeptin before use.
          • AEA (Kit Component No. JA1977): Antigen Extraction Agent. Use as directed in Sample Preparation for the extraction of c-erbB2/c-neu from cell and tissue preparations.
          • SUBSTRATE (Kit Component No. JA1608): The chromogenic substrate, tetra-methylbenzidine (TMB).
          • c-erbB2/c-neu SAMPLE DILUENT (Kit Component No. JA1979): A buffer used to dilute standards and samples.
          • 20X PLATE WASH CONCENTRATE (Kit Component No. JA1617): 20-fold concentrated solution of PBS and surfactant. Contains 2% chloroacetamide.
          • STOP SOLUTION (Kit Component No. JA1616): 2.5N sulfuric acid.
          • PLATE SEALERS (Kit Component No. JB155): To cover plates during incubations.
          Materials Required but not provided 2-20 µl, 20-200 µl and 200-1000 µl precision pipetters with disposable tips.
          Wash bottle or multichannel dispenser for washing.
          500 ml graduated cylinder.
          Deionized or distilled H2O.
          Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450/595 nm or 450/540nm.
          Precautions and recommendationsUpon receipt, store the entire kit at -20°C until use. Do not refreeze after thawing. Lyophilized standards must remain at -20°C. All other components may be stored at 4°C. Allow kit reagents to warm to room temperature before use. Do not expose reagents to excessive light.
          Wear disposable gloves and eye protection.
          Use only the wells provided with the kit.
          Do not mix reagents from different kits.
          Do not mouth pipette or ingest any of the reagents.
          The buffers and reagents used in this kit contain anti-microbial and anti-fungal reagents. Care should be taken to prevent direct contact with these products.
          Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are handled.
          Human samples may be contaminated with infectious agents. Do not ingest, expose to open wounds, or breathe aerosols. Dispose of samples properly.
          Preparation• Cell Lysates: Numerous extraction protocols can be used. The following protocol has been shown to work with a number of cell lines. It is provided as an example of a suitable extraction procedure, but should not be construed as necessarily being the method of choice. Users may wish to experiment with extraction procedures that work best in their laboratory. 1. For suspension cells, pellet by centrifugation, remove supernatant, resuspend with PBS and pellet by centrifugation. For attached cells, remove supernatant from cells (you may save the supernatant for testing in the ELISA). Wash cells once with PBS, harvest cells by trypsinization followed by gentle centrifugation. 2. Aspirate any remaining liquid leaving an intact cell pellet. At this point the cell pellet can be frozen at -80°C and lysed at a later date. We recommend for every 5-7 x 106 cells, resuspend the pellet in 1 ml of Resuspension Buffer (provided) containing 5 mM EDTA, 0.2 mM PMSF, 1 µg/ml pepstatin, and 0.5 µg/ml leupeptin. 3. Add 20 µl of Antigen Extraction Agent (AEA, provided) for every 100 µl of cell suspension. 4. Incubate 30 min on ice with occasional vortexing. 5. Transfer extracts to microcentrifuge tubes and centrifuge for 5 min at 1000 x g at 4°C. 6. Aliquot cleared lysate to clean microfuge tubes. The sample should be aliquotted to avoid multiple freeze/thaws. These samples are now ready for analysis according to the instructions provided in the Detailed Protocol. Samples may be stored at -20°C for future testing in the c-erbB2/c-neu ELISA. Samples found to contain greater than 3 ng/ml c-erbB2/c-neu (i.e., outside the range of the standard curve) must be diluted with Sample Diluent (provided), so that the c-erbB2/c-neu concentration falls within the range spanned by the standard curve, and assayed again. Using this protocol dilutions of cell lysates range from 1:10 to 1:1000 thus must be determined emperically. The following cell lysates contain c-erbB2/c-neu: HT29, SK-BR3, A549, MD-MBA-361, MCF7, HeLa, SK-OV3 and A-431. • Culture Medium: For suspension cells: Pellet by centrifugation (1000 x g for 5 min, 4°C) and remove supernatant for testing. Samples may be stored at -20°C. For adherent cells: Remove tissue culture media, centrifuge tissue culture media (1000 x g for 5 min, 4°C), and remove supernatant for testing. Samples may be stored at -20°C. • Sera and Plasma: A final dilution of 1:20 in Sample Diluent is suggested. Normal mouse IgG has been added to this solution as a precaution against anti-mouse antibodies in the samples. Such antibodies could potentially bind to the mouse capture antibody causing false results. • Tissue: Steps 1-3 are normally performed on tissue specimens to prepare a cytosol for the measurement of estrogen and progesterone receptors using dextran-coated charcoal binding assays. A sample of the homogenate can be withdrawn just before the ultracentrifugation step and used for the measurement of c-erbB2/neu protein after further processing as described beginning with Step 4. 1. Weigh the frozen specimen and slice the frozen tissue into small pieces. 2. Add cold (4°C) Resuspension Buffer to the pieces of tissue. Use a buffer:tissue ratio 10:1 (v/w); for example, add 10 ml of buffer to 1 gm of tissue. Resuspension Buffer contains 10 mM Tris-HCl (pH 7.4), 1.5 mM EDTA, 10% glycerol and anti-microbial agents. An effective cocktail of protease inhibitors consists of 0.5 µg/ml leupeptin, 1 µg/ml pepstatin and 0.2 mM PMSF. 3. Just before use, 0.1% monothioglycerol (MTG) is added to the Resuspension Buffer for ligand binding assays. MTG is not necessary for the c-erbB2/c-neu ELISA but in a limited study, decreased the assay slightly. Molybdate (10 mM) is added to the Resuspension Buffer in some laboratories. It has no effect on the measurement of c-erbB2/c-neu protein using the c-erbB2/c-neu ELISA up to 10 mM. 4. Homogenize on ice using a Polytron homogenizer or equivalent with two 5-s bursts at 1/2 speed (setting 6). Allow 15-20 s cooling time on ice between bursts. 5. Mix 10 µl of Antigen Extraction Agent (AEA) with 50 µl of homogenate in a microcentrifuge tube (a 1:6 dilution). Incubate 5 min at room temperature with mixing. These specimens may be stored frozen at -80°C for at least 1 month. 6. Centrifuge at 15,000 rpm (about 14,000-16,000 x g) for 10 min in a microcentrifuge. Recover the supernatant. 7. Measure the protein concentration using an established method such as the Micro-BCA assay (Pierce, Rockford, IL), the Bio-Rad Protein Microassay (Bio-Rad Laboratories, Richmond, CA) or the Lowry method. Since the supernatant will usually contain >2 mg protein/ml when prepared as described above, use of any of the recommended microassays allows measurement of the protein content in a 1:100 or greater dilution of the sample. At least a 1:100 dilution of the sample must be made to prevent interference by AEA in the Bio-Rad Protein Microassay. DO NOT use the Pierce Micro-BCA protein assay if resuspension buffer contains MTG. At a 1:20 or greater dilution, the sample processing methods described here cause no apparent interference with protein measurement by Lowry. The same solution used to dilute the sample for protein measurement should be used to prepare the protein standards and the assay blank. PBS works well. (Sample Diluent cannot be used because it contains BSA; Resuspension Buffer is inappropriate since it contains 10% glycerol). 7. Prior to measurement of the c-erbB2/neu protein levels in the ELISA, the sample must be diluted to at an appropriate protein concentration. A starting lysate protein concentration of 5 µg/ml in Sample Diluent is recommended. Some samples may require additional dilutions.
          Detailed protocolThe c-erbB2/c-neu ELISA is provided with removable strips of wells so the assay can be carried out on two separate occasions. Since conditions may vary, a standard curve MUST be determined each time the assay is performed. Standards should be assayed in duplicate. Disposable pipette tips and reagent troughs should be used for all transfers to avoid cross-contamination of reagents or samples.
          1. Remove the appropriate number of wells from the foil pouch keeping them in the well holder. Return any unused wells to the foil pouch, reseal and store at 4°C.
          2. Prepare a working solution (1X) of Wash Buffer by adding 25 ml of the 20X concentrated solution (provided), to 475 ml of deionized water. Mix well.
          3. Each time an assay is performed, reconstitute a lyophilized Standard as described on the Lyophilized c-erbB2/c-neu Standard vial label to give a concentration of 3 ng/ml by carefully and accurately pipetting the stated amount of dH2O and if required, sample diluent. Let the reconstituted standard sit for 15 min at room temperature, with occasional swirling. Avoid excessive agitation of the standard. After reconstituting the c-erbB2/c-neu Standard it should be diluted with Sample Diluent. Obtain six tubes and label them 3, 1.5, 0.75, 0.375, 0.188, and 0 ng/ml. Add 220 µl of Sample Diluent into each tube except the 3 ng/ml tube (first tube) which gets "undiluted" reconstituted standard. Remove 440 µl from the original vial of lyophilized material and add it to the first tube. Remove 220 µl from the first tube (3 ng/ml) and add it to the second tube (1.5 ng/ml) and mix gently. Repeat this procedure until you reach the fifth tube (0.188 ng/ml). The last tube (0 ng/ml) should just be Sample Diluent. Reconstituted standards should be discarded after one use.
          4. Prepare all samples. Samples should be diluted with Sample Diluent (provided).
          5. Add samples and each of the c-erbB2/c-neu standards (in duplicate) by pipetting 100 µl into appropriate wells using clean pipette tips for each sample.
          6. Cover wells with a plate sealer and incubate at room temperature for 2 h.
          7. Wash wells 3 times with 1X Wash Buffer making sure each well is filled completely.
          8. Pipette 100 µl of the Detector Antibody into each well, cover with a plate sealer and incubate at room temperature for 1 h.
          9. Wash wells 3 times with 1X Wash Buffer making sure each well is filled completely.
          10. Dilute a sufficient amount of the 500X Conjugate 1:500 in Conjugate Diluent to provide 100 µl of 1X solution for each sample and standard well (For example: add 24 µl to 12 ml of Conjugate Diluent), mix gently. Filter with a 0.2 mm syringe filter prior to use.
          11. Pipette 100 µl of the 1X Conjugate into each well, cover with a plate sealer and incubate at room temperature for 30 min. Discard any unused 1X Conjugate.
          12. Wash wells 3 times with 1X Wash Buffer making sure each well is filled completely.
          13. FLOOD ENTIRE PLATE WITH dH2O. Remove contents of wells by inverting over sink and tapping on paper towels.
          14. Add 100 µl of Substrate Solution to each well and incubate IN THE DARK at room temperature for 30 min.
          15. Add 100 µl of Stop Solution to each well in the same order as the previously added Substrate Solution.
          16. Measure absorbance in each well using a spectrophotometric plate reader at dual wavelengths of 450/595 nm (or 450/540nm). Wells must be read within 30 min of adding the Stop Solution.
          CalculationsEvaluation of Results Average the duplicate absorbance values for each standard, including the zero and all sample values. 1. On graph paper, plot the mean absorbance values for each of the standards on the Y axis, versus the concentration of each standard (ng/ml) on the X axis. 2. Determine the concentration of unknowns by interpolation from the standard curve. There are a variety of plate reader software packages available (Softmax, Molecular Devices Corporation, Menlo Park, CA; KinetiCalc, BioTek Instruments, Inc. Winooski, VT) for analysis of plate data, which simplifies this process. All concentrations of c-erbB2/c-neu reported in this booklet used point to point curve fitting.
          Assay characteristics and examples

          Figure 1: Clinical Utility of the c-erbB2/c-neu ELISA

          The rapid format assay (Top) has a 17 percent clinical sensitivity (ability to recognize affected individuals) at a 95 % clinical specificity (ability to recognize unaffected individuals). This compares favorably with the overnight ELISA (Bottom) which has a 10 % clinical sensitivity with the same samples. The normal and cancer ranges are similar in both assays.

          Standard curve

          Figure 2: Standard Curve

          The mean signal of each standard run in replicates of three in eight assays using two different lots of plates and two different lots of detector antibody.

          Sensitivity Notes

          Figure 3: Sensitivity

          The lower limit of detection (LLD), commonly used to define sensitivity, was measured by assaying nine replicates of zero eight times using two different lots of plates and two different lots of detector antibody. The grand mean signal and pooled standard deviation of zero was calculated. The grand mean of each standard (run in replicates of three in the eight assays) was used for the standard curve (Figure 2), and the response, mean signal of zero plus two standard deviations, read in dose from the standard curve is the LLD; that is, the smallest dose that is not zero with 95% confidence.

          Assay Range0-3 ng/ml
          Precision

          Figure 4: Precision

          The pooled coefficients of variation (according to the formula of Henry et. al., 1974) and between assay coefficients of variation are plotted against c-neu concentrations. The pooled data were collected from samples run four times using two different lots of plates and two different lots of detector antibody in replicates of three.

          Parallelism

          Figure 5: Parallelism

          The study tested dilution-recovery of 28 positive samples. Dilutions were run in the ELISA and the "found" doses were plotted against the "expected" doses to determine linearity of dilution. The slope is not significantly different from one and the intercept is not significantly different from zero. These studies are consistent with the absence of cross-reacting and matrix effects such as pH, salts, and endogenous binders that interfere with the reagents used in the assay. Note: NHS = Normal Human Sera; Lysates = cellular lysates of cultured cells, and Supernatants = conditioned media of growing cells.

          Specificity

          Figure 6: Specificity

          Levels of c-erbB2/c-neu in cell culture supernatants, cancer serum and recombinant protein detected by the ELISA after immunoaffinity extraction of c-erbB2/c-neu by six different c-erbB2/c-neu antibodies that are not used in the ELISA.

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