Evriklia S. Lianidou

Comparative study of fluorescent ternary terbium complexes. Application in enzyme amplified fluorimetric immunoassay for α-fetoprotein

Abstract A systematic spectrofluorimetric study of the formation of ternary complexes of terbium ions (Tb3+) with ethylenediamine tetraacetic acid (EDTA) and a variety of bidentate organic ligands has been performed. Fourteen ligands (salicylic acid, quinolones and hydroxybenzene sulfonic acid derivatives) were examined as efficient energy donating chelators for Tb3+. The fluorescence properties of the ligands, alone as well as of the ternary complexes with Tb3+ and EDTA have been examined. Optimization studies, including the effect of pH, buffer systems and the use of EDTA analogs, were performed. A possible relationship between the structure and the ability of the ligand to act as an efficient light absorbing and energy donating chelator for Tb3+ is discussed. The phosphate ester of diflunisal, one of the most sensitive ligands for Tb3+, was used as an alkaline-phosphatase substrate for the determination of α-fetoprotein (AFP) in serum by a highly sensitive enzyme amplified lanthanide luminescence immunoassay by means of second derivative synchronous fluorescence spectroscopy as a detection technique. By using this substrate, AFP was determined in serum with a limit of detection (3 × s.d. blank) of 5 pg ml−1, coefficients of variation in the 2.4–5.3% range and mean recovery from four pooled serum samples at four concentration levels (5, 40, 100 and 250 ng ml−1) equal to (99 ± 3)%.

BRCA1 Mutation Analysis in Breast/Ovarian Cancer Families from Greece

Germline mutations in BRCA1 gene account for varying proportions of breast/ovarian cancer families, and demonstrate considerable variation in mutational spectra coincident with ethnic and geographical diversity. We have screened for mutations the entire coding sequence of BRCA1 in 30 breast/ovarian cancer women with family history of two or more cases of breast cancer under age 50 and/or ovarian cancer at any age. Genomic DNA from patient was initially analyzed for truncating mutations in exon 11 with PTT followed by DNA sequencing. In the cases where no frameshift mutation was observed in exon 11, all other exons were screened with direct sequencing. Two novel (3099delT, 3277insG) and three already described (3741insA, 1623del5‐TTAAA, 5382insC‐twice) truncating mutations were identified. In addition, 6 point mutations (L771L, P871L, E1038G, K1183R, S1436S, S1613G) which are already classified as polymorphisms were identified. Three unclassified intronic variants (IVS16‐68 G>A, IVS16‐92 G>A, IVS18+65G>A) were also detected. These results show that BRCA1 deleterious mutations are present in a fraction (20%) of Greek breast/ovarian cancer families similar to other European countries. Mutations were detected in high‐ (≥3 members) as well as in moderate‐risk (2 members) families. This is the first report of BRCA1 mutation analysis in Greece. Hum Mutat 16:272–273, 2000.

Determination of tumor necrosis factor-α (TNF-α) in serum by a highly sensitive enzyme amplified lanthanide luminescence immunoassay

Abstract Objectives: To develop a highly sensitive enzyme amplified lanthanide luminescence (EALL) immunoassay for tumor necrosis factor-α (TNF-α). Methods: The method is based on the use of two monoclonal antibodies against TNF-α, one “capture” antibody and one labeled with biotin, in a “sandwich type” assay format. Alkaline phosphatase (ALP) conjugated to an antibiotin-polyclonal antibody is used as the enzyme label. ALP cleaves phosphate from diflunisal phosphate (DIFP) to produce diflunisal (DIF). The detection system is based on the combination of enzymatic amplification introduced by ALP and the formation of a highly fluorescent terbium complex that can be monitored by time resolved or conventional fluorimetry. Results: By using 50 μL of sample, the dynamic range of the assay extends up to 2000 ng/L of TNF-α, with a detection limit of 1 ng/L, within-run CVs ranging from 3 to 15% and recoveries of 97 ± 2%. By using 100 μL of sample the dynamic range of the assay extends up to 1000 ng/L of TNF-α with a detection limit of 0.2 ng/L, recoveries of 94 ± 13%, within-run CVs ranging from 2 to 6.5% and between-run CVs ranging from 5 to 15%, in a total incubation time of 3h. No interference by the presence of other cytokines (IL-1β, IL-2, IL-4, IL-6, IFN-γ) or by rheumatoid factors has been observed. The results obtained by the proposed method and by a commercially available kit (Medgenix TNF-α EASIA) correlated well (n = 26, r = 0.934). Conclusion: The proposed method is highly sensitive, simple and rapid and can reliably measure TNF-α in the ng range in biological specimens.

Synchronous scanning second derivative spectrofluorimetry for the simultaneous determination of diflunisal and salicylic acid added to serum and urine as ternary complexes with terbium and EDTA

Abstract By using second derivative synchronous scanning fluorescence spectrometry the simple resolution of two nonsteroidal, antiinflammatory drugs diflunisal and salicylic acid, as ternary complexes with terbium and EDTA, is accomplished. The method developed is simple, sensitive and rapid, and has been successfully applied for the determination of both compounds in 10 μl of untreated human serum and urine samples. The detection limits for diflunisal were 0.9 and 1.8 mg l −1 , and for salicylic acid 1.2 and 1.7 mg l −1 , in serum and urine, respectively. The mean analytical recoveries from serum and urine samples spiked with diflunisal (25–100 mg l −1 ) and salicylic acid (28–138 mg l −1 ) were 99 ± 8% (serum) and 102 ± 8% (urine) for diflunisal and 102 ± 9% (serum) and 95 ± 8% (urine) for salicylic acid. The precision of all determinations varied from 2.5 to 10%.

Second derivative synchronous scanning fluorescence spectrometry as a sensitive detection technique in immunoassays. Application to the determination of α-fetoprotein

Abstract Second derivative synchronous (scanning) fluorescence spectrometry (SDSFS) has been used for the first time as an alternative to time-resolved fluorescence for the detection of Tb 3+ chelates. This approach minimizes the background signal by taking advantage of the large Stokes shift properties of Tb 3+ chelates and offers high sensitivity by narrowing the spectral bands. The analytical performance of this detection technique has been evaluated by choosing the well-defined enzyme-amplified lanthanide luminescence (EALL) immunoassay of α-fetoprotein (AFP) as a model. Monoclonal “capture” antibodies and monoclonal biotin-labelled antibodies in a “sandwich-type” assay configuration in a microwell format have been used. Alkaline phosphatase (ALP) conjugated to an antibiotin antibody was used as an enzyme label. ALP cleaves phosphate from salicylphosphate to produce salicylic acid which forms a highly fluorescent ternary complex with Tb 3+ and EDTA, which is monitored by SDSFS. The method allows the measurement of AFP with a limit of detection of 2 pg ml −1 , coefficients of variation in the range 4.5–9.9% and mean recovery from four pooled serum samples at two concentration levels equal to 94 ± 11%.

Simple, rapid and sensitive spectrofluorimetric determination of diflunisal in serum and urine based on its ternary complex with terbium and EDTA

Abstract A very simple, rapid and highly sensitive fluorimetric method for the determination of diflunisal in serum and urine is described. The method is based on the formation of a ternary complex between diflunisal, Tb3+ and EDTA in alkaline aqueous solutions. This complex exhibits very intense terbium ion luminescence with a main emission maximum at 546 nm when excited at 284 nm. Optimum conditions for the complex formation have been investigated. The detection limit for diflunisal is 2.4 μg 1−1, while the range of application is 0.01–6.00 mg 1−1. The method has been successfully applied for the determination of diflunisal in untreated human serum and urine samples. Analytical recoveries from serum and urine samples spiked with diflunisal were in the ranges of 96.8–101.2% and 98.0–102.0%, respectively.

Characterization of the BRCA1-like immunoreactivity of human seminal plasma.

OBJECTIVES The subcellular localization of the breast cancer susceptibility gene product BRCA1 has been controversial. Discrepant results have been reported during the past 3 years, partially because of the unavailability of highly specific reagents for BRCA1 protein. Our objective was to characterize the BRCA1-like immunoreactivity that is detected in human seminal plasma by using monoclonal and polyclonal antibodies that are supposedly specific for BRCA1 protein. METHODS We used immunologic, chromatographic, and protein sequencing techniques to detect the immunoreactivity of BRCA1 in seminal plasma and to purify and partially identify the immunoreactive species. RESULTS We present data indicating that two BRCA1 antibodies, SG-11 and D-20, which were thought to be free of cross-reactivities, strongly interact with proteins present in human seminal plasma. This cross-reactivity is detectable even at seminal plasma dilutions as high as 10(6)-fold, and it is effectively blocked by peptides that capture the binding site of either SG-11 or D-20 antibodies. Purification and characterization of the immunoreactive compound revealed that this consists of a macromolecular complex that contains semenogelins. The D-20 polyclonal antibody was found to cross-react with purified semenogelins I and II; the SG-11 monoclonal antibody appeared to recognize a component of the macromolecular complex that was not semenogelin. CONCLUSIONS Our data demonstrate that the BRCA1 antibodies SG-11 and D-20 strongly interact with seminal plasma proteins and are not highly specific for BRCA1 protein. It is thus suggested that BRCA1 antibodies should be used with caution until reagents free of interference are developed and evaluated. In light of the very high cross-reactivity of the two antibodies with seminal plasma proteins, we recommend that new BRCA1 antibodies should be examined for cross-reactivity with seminal plasma proteins to verify specificity.

Fragment analysis of the p53 gene in ovarian tumors

he role of the p53 tumor suppressor gene inhuman carcinogenesis has been extensivelystudied. One central function of the p53 protein iscontrol of cellular growth after DNA damagethrough mechanisms involving growth arrest andapoptosis (1–3). These functions are believed to be atleast partially mediated by the ability of p53 to actas a transcription factor. Mutations in the p53 geneare found in most human malignancies and currentresearch is focusing on their role in cancer initiationand progression. p53 gene mutations can lead todefective cellular responses after DNA damage, dys-regulated cell growth, and tumor formation. Theidentification of p53 gene mutations in tumor cells isof diagnostic and therapeutic importance because:(a) tumors with a mutant p53 gene are usuallyresistant to certain chemotherapeutic agents or ra-diation; (b) a variety of tumors bearing a mutant p53gene have a less favorable prognosis than tumors ofthe same type with a wild-type p53 gene (4).The frequency of p53 gene mutations is high incancers of the colon (5), breast (6), lung (7), ovary (8),and brain (9). About 10% of the mutations aredeletions or insertions (10). Insertions range from 1to 14 nucleotides in length and in most cases, theinserted nucleotides duplicate the sequences of theneighboring region. Deletions range from 1 to 37nucleotides. Presence of deletions/insertions in 6 outof 11 newly established ovarian carcinoma cell lineshave been reported (11). In this report, we studiedthe presence of deletions and insertions in the p53gene in 89 primary ovarian tumors.