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- Johansson, Malin, 1972-, et al.
(författare)
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Quantitative analysis of tyrosinase transcripts in blood
- 2000
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Ingår i: Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 46:7, s. 921-927
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Tidskriftsartikel (refereegranskat)abstract
- Background: Tyrosinase is an enzyme unique to pigment-forming cells. Methods using this transcript for detection of melanoma cells in blood have given divergent results. Quantitative analytical procedures are therefore needed to study the analytical performance of the methods.Methods: Mononucleated cells were isolated by Percoll centrifugation. RNA was isolated by each of three methods: UltraspecTM-II RNA isolation system, FastRNATM GREEN Kit, and QIAamp RNA Blood Mini Kit. cDNA was synthesized using random hexamer primers. A tyrosinase-specific product of 207 bp was amplified by PCR. As an internal standard (and competitor) we used a 207-bp cDNA with a base sequence identical to the tyrosinase target except for a 20-bp probe-binding region. The PCR products were identified by 2,4-dinitrophenol (DNP)-labeled probes specific for tyrosinase (5′DNP-GGGGAGCCTTGGGGTTCTGG-3′) and internal standard (5′DNP-CGGAGCCCCGAAACCACATC-3′) and quantified by ELISA.Results: The calibration curves were linear and had a broad dynamic measuring range. A detection limit (2 SD above zero) of 48 transcripts/mL of blood was obtained from a low control. The analytical imprecision was 50% and 48% at concentrations of 1775 and 17 929 transcripts/mL (n = 12 and 14, respectively). With the cell line SK-Mel 28 added to blood and RNA extracted with the Ultraspec, Fast RNA, and QIAamp RNA methods, we found (mean ± SD) 1716 ± 1341, 2670 ± 3174, and 24 320 ± 5332 transcripts/mL of blood. Corresponding values were 527 ± 497, 2497 ± 1033, 14 930 ± 1927 transcripts/mL of blood when the cell line JKM86-4 was added. One high-risk patient was followed by repeated analysis of tyrosinase transcripts in blood. The melanoma marker 5-S-cysteinyldopa in serum and urine was within reference values, but tyrosinase mRNA was slightly increased (120–168 transcripts/mL of blood). The tyrosinase mRNA increased to 1860 transcripts/mL concomitant with the increase in 5-S-cysteinyldopa; later a spleen metastasis was found.Conclusions: The results obtained with different RNA extraction methods illustrate the importance of quantitative methods for validation of methods. The use of QIAamp RNA improved the extraction efficiency considerably. Data from a case study suggest the assay is suitable in the follow-up of patients with high risk of developing metastases.
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- Nurmikko, P., et al.
(författare)
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Production and characterization of novel anti-prostate-specific antigen (PSA) monoclonal antibodies that do not detect internally cleaved Lys145-Lys146 inactive PSA
- 2000
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 46:10, s. 1610-1618
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Tidskriftsartikel (refereegranskat)abstract
- Background: The nature of free, uncomplexed prostate-specific antigen (s) in the circulation is still unknown. In this study, we developed novel anti-PSA antibodies using PSA produced by a metastasized cancer cell line, LNCaP, as an immunogen. Methods: Hybridoma cell lines were screened with different methods that aimed at finding antibodies specific for the forms of free PSA produced by LNCaP cell line. Obtained antibodies were further studied for their characteristics related to previously characterized monoclonal antibodies. Results: Numerous anti-PSA antibodies were obtained, of which four represented unique epitopes previously unrecognized by us. One free-PSA-specific antibody was bound to PSA on two distinct epitopes, and one antibody was bound to the carboxyl-terminal peptide of PSA. Two antibodies were found to bind to the peptide sequence adjacent to the internal cleavage site Lys145-Lys146. These antibodies failed to recognize internally cleaved PSA at Lys145-Lys146. We could not find anti-proPSA antibodies despite the fact that LNCaP PSA contained more than one-half of the zymogen form of PSA. Conclusions: We report, for the first time, novel anti-PSA antibodies that do not recognize internally cleaved PSA at Lys145-Lys146 and thus are specific for intact, unclipped PSA. (C) 2000 American Association for Clinical Chemistry.
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- Scorilas, A., et al.
(författare)
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Streptavidin-polyvinylamine conjugates labeled with a europium chelate : Applications in immunoassay, immunohistochemistry, and microarrays
- 2000
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 46:9, s. 1450-1455
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Tidskriftsartikel (refereegranskat)abstract
- Background: The favorable properties of lanthanide chelates compared with conventional fluorescent probes have attracted considerable interest. A Eu3+ chelator, 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA), has been synthesized previously. Methods: We here describe immunoassay, immunohistochemistry, and microarray applications of a new streptavidin-based universal polyvinylamine (PVA) detection reagent that is multiply labeled with the europium chelate of BCPDA. Solid-phase time-resolved immunofluorometric assays for biotinylated mouse IgG and prostate-specific antigen (PSA) were developed using the new conjugate as a detection reagent. The new conjugate was also used for the immunohistochemical localization of PSA expression in paraffin-embedded prostatic tissues. A model microarray with spotted biotinylated antibody as target was also performed. Results: Approximately 50-100 BCPDA moieties were covalently bound to PVA, which was then linked to streptavidin via biotin interaction. The macromolecular complex successfully recognized and bound biotinylated detection reagents, e.g., antibodies. The new reagent enabled measurement of solid phase-immobilized biotinylated mouse IgG with a detection limit of ~1 pg/assay and demonstrated excellent linearity. In an ELISA-type sandwich PSA assay that included two PSA monoclonal antibodies using the new conjugate as detection reagent, we detected 0.001 μg/L PSA (~100 fg or ~3 amol/assay). Serum samples analyzed for PSA by this method and a commercial assay gave highly correlated results. The new reagent enabled excellent immunohistochemical localization of PSA expression in prostate tissues. Using the new reagent in a model microarray experiment with biotinylated mouse IgG as target, we demonstrated excellent spatial resolution of 5-to 10-nL microspots. Conclusions: The new detection reagent may find important applications in biotechnology. (C) 2000 American Association for Clinical Chemistry.
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- Becker, Charlotte, et al.
(författare)
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Sensitive and specific immunodetection of human glandular kallikrein 2 in serum
- 2000
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Ingår i: Clinical Chemistry. - 0009-9147. ; 46:2, s. 198-206
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Human glandular kallikrein 2 (hK2) is expressed in the prostate and is present in serum from men with prostate cancer. Specific detection in serum is difficult mainly because of low concentrations and immunological cross-reactivity with prostate-specific antigen (PSA). Our objectives were to design an assay with improved analytical detection and functional sensitivity and nonsignificant cross-reactivity with PSA, and to characterize different immunoreactive forms of hK2. METHODS: In the assay, critical PSA epitopes were blocked with four monoclonal antibodies (MAbs) specific for PSA. Subsequently, hK2 was captured using a MAb against hK2 (5% cross-reactivity with PSA), and after washing, hK2 was detected by a europium-labeled MAb with identical affinity for hK2 and PSA. RESULTS: The analytical detection limit was <10 ng/L, and functional sensitivity was 30 ng/L. Cross-reaction with PSA was <0.01%. Between-assay imprecision was 3.1% for 1600 ng/L hK2 and 4. 8% for 160 ng/L hK2; corresponding values for within-assay precision were 1.9% and 4.5%, respectively. Complexes of hK2-alpha(1)-antichymotrypsin (ACT) were detected in vitro with -6% bias compared with the free form of hK2. Gel filtration of patient samples showed that hK2 correlated in size mainly with free hK2; only 4-19% corresponded to hK2 possibly complexed with ACT or protein C inhibitor. CONCLUSIONS: Our assay had extremely low cross-reactivity with PSA, provided a very low detection limit, and allowed close to equimolar detection of the free and complexed forms of hK2. Moreover, we found that free hK2 is the predominant immunoreactive form of hK2 in serum.
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- Kakonen, S M, et al.
(författare)
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Development and evaluation of three immunofluorometric assays that measure different forms of osteocalcin in serum
- 2000
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Ingår i: Clinical Chemistry. - 0009-9147. ; 46:3, s. 332-337
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Circulating human osteocalcin (hOC) has been used as a marker of bone formation. Our aim was to validate three immunofluorometric assays (IFMAs), measuring different forms of hOC. METHODS: The two-site IFMAs were based on previously characterized monoclonal antibodies. Assay 2 recognized intact hOC, assays 4 and 9 measured the NH(2)-terminal mid-fragment and the intact hOC. In addition, assay 9 required hOC to be gamma-carboxylated. RESULTS: A 76-79% increase of serum immunoreactive hOC was found in the postmenopausal group compared with the premenopausal group with all IFMAs. With EDTA-plasma samples, the observed increases were lower (49-65%). The hOC concentration in the postmenopausal group receiving hormone replacement therapy was 42-44% lower than that in the postmenopausal control group in both serum and EDTA-plasma samples. The depressed carboxylation in warfarin-treated patients was accompanied by lower results in assay 9. The ratio of assay 9 to assay 4 totally discriminated the warfarin-treated patients from the controls. Assay 9 showed the smallest decreases in measured hOC after storage of serum or plasma for 4 weeks at 4 degrees C, followed by assay 4 and assay 2. Results from the last assay were <17% of their initial values after 4 weeks of storage. No diurnal variation was observed with assay 9 as opposed to the two other IFMAs. CONCLUSION: The three assays with their distinct specificity profiles (intact vs fragmented and carboxylated vs decarboxylated hOC) may provide valuable tools for investigating the significance of different hOC forms in various bone-related diseases.
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