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Quantitative Determination of Human Stanniocalcin 1 And QUANTITATIVE DETERMINATION NEW PRODUCT OF HUMAN STANNIOCALCIN 1 AND STANNIOCALCIN 2 Human Stanniocalcin 1 ELISA Human Stanniocalcin 2 ELISA Sensitivity (0,7 pg/ml) Very good analytical characteristics Validated for human serum, plasma (EDTA, citrate, heparin), breast milk and urine Preliminary population data BONE AND CARTILAGE METABOLISM ONCOLOGY REPRODUCTION ∙ CARCINOMAS HUMAN STANNIOCALCIN 1 ELISA Human Stanniocalcin 2 ELISA Introduction Stanniocalcin (STC) is present in humans in two isoforms, Evidence suggests that the functional roles of STCs in the STC1 and STC2, which are encoded by separate genes angiogenic process and regulation of local angiogenesis occur localized on chromosomes 8p21.2 and 5q35.2 respectively via the VEGF/VEGFR2 pathway for STC1 and via the VEGF/ [1]. Both STC1 and STC2 are expressed in a wide variety of VEGFR2 and Ang-2 signaling pathways for STC2 [6]. tissues such as heart, kidney, liver or prostate and they seem to act in a paracrine/autocrine manner. A high proportion of A series of recent studies have also shown that STCs are STC1 in liver and kidney is localized to mitochondria whereas associated with cancer. Higher expression levels of STC1 and STC2 is mostly localized to ER and Golgi [2, 3]. STC2 were observer in many human cancer tissues [7-9], and the higher levels are generally correlated with poor prognostic The recent studies demonstrate that Stanniocalcins might outcome. Contrary to STC1, STC2 is known to be involved in play an important function in several physiological and/or the anti-apoptotic regulatory mechanisms in some cancers by pathological processes including calcium regulation, oxidative inhibiting plasma membrane store-operated Ca2+ entry [3]. stress, anti-inflammation, angiogenesis, ischemia reperfusion, nerve diseases and STC might be involved in the development STCs have been reported to be involved in an extensively and progression of multiple malignancies [4]. growing number of physiological and/or pathological functions including tumorgenesis or apoptosis. However, the current Observations suggest a role for STC1 in the regulation of knowledge about the Stanniocalcin family is still limited and Ca2+/Pi transport. Information about the involvement of STC2 needs to be further elucidated. in Ca2+/Pi transport has been lacking until recently when STC2 was identified as a possible negative modulator of the store-operated calcium channel [5]. BioVendor Human Stanniocalcin 1 ELISA (RD191095200R) BioVendor Human Stanniocalcin 2 ELISA (RD191096200R) Intended use Clinical application The RD191095200R Human Stanniocalcin1 ELISA and Bone and cartilage metabolism RD191096200R Human Stanniocalcin 2 ELISA are the Oncology sandwich enzyme immunoassays for the quantitative Reproduction measurement of human STC1 and STC2. The total assay time is less than 3 hours The kits measure STC1 in serum, plasma (EDTA, citrate, heparin), breast milk, urine and STC2 in serum and plasma (EDTA, citrate, heparin) Assay format is 96 wells Standard is recombinant protein based Components of the kit are provided ready to use, concentrated or lyophilized HUMAN STANNIOCALCIN 1 ELISA QUANTITATIVE DETERMINATION OF HUMAN Human Stanniocalcin 2 ELISA STANNIOCALCIN 1 AND STANNIOCALCIN 2 BioVendor Human Stanniocalcin 1 BioVendor Human Stanniocalcin 2 ELISA (RD191095200R) ELISA (RD191096200R) Test principle Test principle In the BioVendor Human Stanniocalcin1 ELISA, standards In the BioVendor Human Stanniocalcin 2 ELISA, standards and samples are incubated in microplate wells pre-coated and samples are incubated in microplate wells pre-coated with polyclonal anti-human STC1 antibody. After 60 minutes with polyclonal anti-human STC2 antibody. After 60 minutes incubation and washing, biotin-labelled polyclonal anti- incubation and washing, biotin-labelled polyclonal anti- human STC1 antibody is added and incubated with captured human STC2 antibody is added and incubated with captured STC1 for 60 minutes. After another washing, streptavidin-HRP STC2 for 60 minutes. After another washing, streptavidin-HRP conjugate is added. After 30 minutes incubation and the last conjugate is added. After 30 minutes incubation and the last washing step, the remaining conjugate is allowed to react washing step, the remaining conjugate is allowed to react with the substrate solution (TMB). The reaction is stopped by with the substrate solution (TMB). The reaction is stopped by addition of acidic solution and absorbance of the resulting addition of acidic solution and absorbance of the resulting yellow product is measured. The absorbance is proportional yellow product is measured. The absorbance is proportional to the concentration of STC1. A standard curve is constructed to the concentration of STC2. A standard curve is constructed by plotting absorbance values against concentrations of by plotting absorbance values against concentrations of STC1 standards, and concentrations of unknown samples are STC2 standards, and concentrations of unknown samples are determined using this standard curve. determined using this standard curve. HUMAN Stanniocalcin 1 ELISA HUMAN Stanniocalcin 2 ELISA CAT. NO.: RD191309200R CAT. NO.: RD191096200R Assay format Sandwich ELISA, Biotin-labelled Assay format Sandwich ELISA, Biotin-labelled antibody, 96 wells/kit antibody, 96 wells/kit Samples Serum, Plasma Samples Serum and Plasma (EDTA, citrate, heparin) and Urine (EDTA, citrate, heparin) Standards 15.6 – 1000 pg/ml Standards 62.5 to 4000 pg/ml Limit of detection 0.7 pg/ml Limit of detection 5 pg/ml 3.5 3.5 3.0 3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 Absorbance at 450 nm Absorbance at 450 nm 0.0 0.0 10 100 1000 50 500 5000 Hu STC1 (pg/ml) Hu STC2 (pg/ml) HUMAN STANNIOCALCIN 1 ELISA Human Stanniocalcin 2 ELISA BioVendor Human Stanniocalcin 1 BioVendor Human Stanniocalcin 2 ELISA (RD191095200R) ELISA (RD191096200R) Precision Precision Intra-assay (Within-Run) (n=8) Intra-assay (Within-Run) (n=8) Sample Mean SD CV Sample Mean SD CV (pg/ml) (pg/ml) (%) (ng/ml) (ng/ml) (%) 1 468.1 11.0 2.4 1 15.9 0.6 3.9 2 287.7 9.1 3.2 2 16.9 0.8 4.7 Inter-assay (Run-to-Run) (n=7) Inter-assay (Run-to-Run) (n=7) Sample Mean SD CV Sample Mean SD CV (pg/ml) (pg/ml) (%) (ng/ml) (ng/ml) (%) 1 287.8 14.1 4.9 1 13.8 0.5 3.8 2 484.0 28.5 5.9 2 24.4 0.8 3.2 Spiking recovery Spiking recovery Serum samples were spiked with different amounts of human Serum samples were spiked with different amounts of human STC1 and assayed. STC2 and assayed. Sample Observed Expected Recovery O/E Sample Observed Expected Recovery O/E (pg/ml) (pg/ml) (%) (ng/ml) (ng/ml) (%) 389.6 - - 11.2 - - 567.7 577.1 98 15.6 15.0 104 1 1 761.1 764.6 100 20.6 19.2 107 1130.1 1139.6 99 29.4 28.8 102 288.5 - - 9.6 - - 473.2 476.0 99 14.0 13.5 104 2 2 664.2 663.5 100 18.6 17.6 106 1085.1 1038.5 104 27.1 27.2 99 Linearity Linearity Serum samples were serially diluted with Dilution Buffer and Serum samples were serially diluted with Dilution Buffer and assayed. assayed. Sample Dilution Observed Expected Recovery O/E Sample Dilution Observed Expected Recovery O/E (pg/ml) (pg/ml) (%) (ng/ml) (ng/ml) (%) - 637.5 - - - 16.6 - - 2x 313.2 318.8 98 2x 8.2 8.3 99 1 1 4x 161.5 159.4 101 4x 4.1 4.1 100 8x 86.5 79.7 109 8x 1.9 2.1 94 - 866.3 - - - 17.5 - - 2x 427.2 433.2 99 2x 9.0 8.8 103 2 2 4x 192.5 216.6 89 4x 4.5 4.4 104 8x 107.7 108.3 99 8x 2.2 2.2 101 HUMAN STANNIOCALCIN 1 ELISA QUANTITATIVE DETERMINATION OF HUMAN Human Stanniocalcin 2 ELISA STANNIOCALCIN 1 AND STANNIOCALCIN 2 BioVendor Human Stanniocalcin 1 BioVendor Human Stanniocalcin 2 ELISA (RD191095200R) ELISA (RD191096200R) Effect of sample matrix Effect of sample matrix EDTA, citrate and heparin plasma samples were compared EDTA, citrate and heparin plasma samples were compared to respective serum samples from the same 10 individuals. to respective serum samples from the same 10 individuals. Results are shown below: Results are shown below: 800 35 ¢ Serum ¢ EDTA Plasma ¢ Citrate Plasma ¢ Heparin Plasma ¢ Serum ¢ EDTA Plasma ¢ Citrate Plasma ¢ Heparin Plasma 700 30 600 25 500 20 400 15 300 Concentration of STC2 (ng/ml) Concentration of STC1 (pg/ml) 10 200 100 5 0 0 132 45678910 132 45678910 Volunteers Volunteers Summary of protocol Summary of protocol • Reconstitute Master Standard and prepare set of Standards • Reconstitute Master Standard and prepare set of Standards • Dilute samples (serum/plasma/urine 3x, breast milk 50x) • Dilute samples (serum/plasma 20x) • Add 100 µl Standards and samples • Add 100 µl Standards and samples • Incubate at RT for 1 hour/300 rpm • Incubate at RT for 1 hour/300 rpm • Wash plate 3 times • Wash plate 3 times • Add 100 μl Biotin Labelled Antibody solution • Add 100 μl Biotin Labelled Antibody solution • Incubate at RT for 1 hour/300 rpm • Incubate at RT for 1 hour/300 rpm • Wash plate 3 times • Wash plate 3 times • Add 100 μl Streptavidin – HRP Conjugate • Add 100 μl Streptavidin – HRP Conjugate • Incubate at RT for 30 min/300 rpm • Incubate at RT for 30 min/300 rpm • Wash plate 3 times • Wash plate 3 times • Add 100 μl Substrate Solution • Add 100 μl Substrate Solution • Incubate at RT for 10 min • Incubate at RT for 10 min • Add 100 μl Stop Solution • Add 100 μl Stop Solution • Read absorbance and calculate results • Read absorbance and calculate results HUMAN STANNIOCALCIN 1 ELISA Human Stanniocalcin 2 ELISA BioVendor Human Stanniocalcin 1 ELISA (RD191095200R) Preliminary Population Data The following results were obtained when serum samples from 153 unselected donors (89 men + 66 women) 20–65 years old were assayed with the BioVendor Human Stanniocalcin 1 ELISA in our laboratory.
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