Measurement of a more stable region of osteocalcin in serum by ELISA with two monoclonal antibodies

1995 ◽  
Vol 41 (10) ◽  
pp. 1439-1445 ◽  
Author(s):  
C Rosenquist ◽  
P Qvist ◽  
N Bjarnason ◽  
C Christiansen
Keyword(s):  
Monoclonal Antibodies ◽  
Hormone Replacement ◽  
Premenopausal Women ◽  
Stable Region ◽  
Serum Samples ◽  
Early Postmenopausal Women ◽  
Intact Molecule ◽  
Capture Antibody ◽  
The Mean ◽  
Human Osteocalcin

Abstract Intact human osteocalcin purified from femoral bones as well as tryptic fragments of the intact molecule [amino acids (aa) 1-19, 20-43, and 45-49] were used to raise and screen monoclonal antibodies (MAbs). A two-site ELISA for measurement of human osteocalcin in serum was developed with use of these MAbs. A MAb recognizing midregion human osteocalcin (aa20-43) was used as capture antibody, and an NH2 terminus (aa7-19)-specific peroxidase-conjugated MAb was used for detection. Human osteocalcin obtained from bone was used for calibration, and parallelism was observed for osteocalcin from serum samples, NH2-terminal midfragments (aa1-43), and synthetic human osteocalcin. Both inter- and intraassay variations were < 7%. Serum osteocalcin in healthy premenopausal women (n = 49) was 18.3 +/- 4.2 micrograms/L (mean +/- SD) and 28.6 +/- 9.7 micrograms/L in early postmenopausal women (n = 114). The mean serum concentration (n = 10) decreased by 10% after 7 days of storage at 4 degrees C, whereas the concentration of intact human osteocalcin was reduced 63%. The N-MID ELISA and an IRMA measuring intact human osteocalcin were used to monitor the effect of hormone replacement therapy in a retrospective study. A significant decrease to the premenopausal concentration was detected only in the N-MID ELISA.

scholarly journals Serum CrossLaps One Step ELISA. First application of monoclonal antibodies for measurement in serum of bone-related degradation products from C-terminal telopeptides of type I collagen

1998 ◽  
Vol 44 (11) ◽  
pp. 2281-2289 ◽  
Author(s):  
Christian Rosenquist ◽  
Christian Fledelius ◽  
Stephan Christgau ◽  
Brian J Pedersen ◽  
Martin Bonde ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Postmenopausal Women ◽  
Type I Collagen ◽  
Degradation Products ◽  
Hormone Replacement ◽  
Premenopausal Women ◽  
Type I ◽  
Serum Samples ◽  
Aspartic Acid Residue ◽  
One Step

Abstract We have developed a two-site ELISA for measurement in serum of bone-related degradation products derived from C-terminal telopeptides of type I collagen. The assay is based on the application of two highly specific monoclonal antibodies against the amino acid sequence of AHD-β-GGR, where the aspartic acid residue (D) is β-isomerized. In a one-step incubation procedure, the degradation products containing cross-linked diisomerized EKAHD-β-GGR peptides are captured by a biotinylated antibody and a peroxidase-conjugated antibody. The generated complex is then bound to the streptavidin surface via the biotin conjugate. Desalted urinary antigens are used for standardization, and parallelism is observed with serum samples. Results are obtained in <2.5 h, and both inter- and intraassay imprecision are <8%. The serum CrossLaps™ concentration was 1748 ± 740 pmol/L (mean ± SD) in premenopausal women (n = 65) and 2952 ± 1325 pmol/L in a group of healthy postmenopausal women (n = 169). The Serum CrossLaps One Step ELISA was capable of detecting a highly significant (P <0.001) effect of hormone replacement therapy in a retrospective study involving 22 postmenopausal women.

scholarly journals Oestrogen levels in serum and urine of premenopausal women eating low and high amounts of meat

2013 ◽  
Vol 17 (9) ◽  
pp. 2087-2093 ◽  
Author(s):  
Brook E Harmon ◽  
Yukiko Morimoto ◽  
Fanchon Beckford ◽  
Adrian A Franke ◽  
Frank Z Stanczyk ◽  
...  
Keyword(s):  
Mixed Models ◽  
Repeated Measures ◽  
Premenopausal Women ◽  
Least Square ◽  
Serum Samples ◽  
Meat Eating ◽  
Three Samples ◽  
Blood And Urine Samples ◽  
The Mean ◽  
Serum And Urine

AbstractObjectiveBased on the hypothesis that high-meat diets may increase breast cancer risk through hormonal pathways, the present analysis compared oestrogens in serum and urine by meat-eating status.DesignIntervention with repeated measures.SettingTwo randomized soya trials (BEAN1 and BEAN2) among premenopausal healthy women.SubjectsBEAN1 participants completed seven unannounced 24 h dietary recalls and donated five blood and urine samples over 2 years. BEAN2 women provided seven recalls and three samples over 13 months. Serum samples were analysed for oestrone (E1) and oestradiol (E2) using RIA. Nine oestrogen metabolites were measured in urine by LC–MS. Semi-vegetarians included women who reported consuming <30 g of red meat, poultry and fish daily, and pescatarians those who reported consuming <20 g of meat/poultry but >10 g of fish daily. All other women were classified as non-vegetarians. We applied mixed models to compute least-square means by vegetarian status adjusted for potential confounders.ResultsThe mean age of the 272 participants was 41·9 (sd 4·5) years. Serum E1 (85 v. 100 pg/ml, P = 0·04) and E2 (140 v. 154 pg/ml, P = 0·04) levels were lower in the thirty-seven semi-vegetarians than in the 235 non-vegetarians. The sum of the nine urinary oestrogen metabolites (183 v. 200 pmol/mg creatinine, P = 0·27) and the proportions of individual oestrogens and pathways did not differ by meat-eating status. Restricting the models to the samples collected during the luteal phase strengthened the associations.ConclusionsGiven the limitations of the study, the lower levels of serum oestrogens in semi-vegetarians than non-vegetarians need confirmation in larger populations.

scholarly journals Development and Evaluation of Three Immunofluorometric Assays That Measure Different Forms of Osteocalcin in Serum

2000 ◽  
Vol 46 (3) ◽  
pp. 332-337 ◽  
Author(s):  
Sanna-Maria Käkönen ◽  
Jukka Hellman ◽  
Matti Karp ◽  
Pirjo Laaksonen ◽  
Karl J Obrant ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Hormone Replacement Therapy ◽  
Bone Formation ◽  
Diurnal Variation ◽  
Replacement Therapy ◽  
Hormone Replacement ◽  
Control Group ◽  
Plasma Samples ◽  
Edta Plasma ◽  
Human Osteocalcin

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 NH2-terminal mid-fragment and the intact hOC. In addition, assay 9 required hOC to be γ-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 °C, followed by assay 4 and assay 2. Results from the last assay were &lt;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.

Generation and characterization of monoclonal antibodies to human type-5 tartrate-resistant acid phosphatase: development of a specific immunoassay of the isoenzyme in serum

1995 ◽  
Vol 41 (10) ◽  
pp. 1495-1499 ◽  
Author(s):  
P Chamberlain ◽  
J Compston ◽  
T M Cox ◽  
A R Hayman ◽  
R C Imrie ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Acid Phosphatase ◽  
Premenopausal Women ◽  
Tartrate Resistant Acid Phosphatase ◽  
Time Resolved ◽  
Specific Serum ◽  
Serum Trap ◽  
Human Osteoclast ◽  
The Mean

Abstract We have characterized four monoclonal antibodies (mAbs) to the purple ("tartrate-resistant," band 5) acid phosphatase of the human osteoclast (TRAP) and used these to develop a specific serum immunoassay. All four mAbs are of high affinity (Ka = 1-5 x 10(8) L/mol) with a very fast Kassoc (0.2-2.0 x 10(5) L mol-1 s-1) and a moderate Kdissoc (1-3 x 10(-3) s). Two of the mAbs were selected to develop a time-resolved fluorescence immunoassay to measure serum concentrations of TRAP. The mean serum immunoreactive TRAP in a group of healthy premenopausal women and men was 3.7 +/- 1.8 micrograms/L (mean +/- SD) and 3.5 +/- 1.6 micrograms/L, respectively. Significantly higher concentrations of TRAP were found in postmenopausal women (6.3 +/- 2.3 micrograms/L) and in eight patients with Gaucher disease (19.3 +/- 4.7 micrograms/L). Further studies are required to investigate the value of serum TRAP as a marker of bone resorption.

Immunologic Assessment of Patients Treated with Bovine Fibrin as a Hemostatic Agent

1996 ◽  
Vol 76 (06) ◽  
pp. 0925-0931 ◽  
Author(s):  
John F Carroll ◽  
Keith A Moskowitz ◽  
Niloo M Edwards ◽  
Thomas J Hickey ◽  
Eric A Rose ◽  
...  
Keyword(s):  
Coagulation Factors ◽  
Allergic Reactions ◽  
Factor V ◽  
Normal Range ◽  
Serum Samples ◽  
Human Fibrinogen ◽  
Bovine Thrombin ◽  
Thrombotic Complications ◽  
The Mean ◽  
Clinically Significant

SummaryTwenty-one cardiothoracic surgical patients have been treated with fibrin as a topical hemostatic/sealing agent, prepared from bovine fibrinogen clotted with bovine thrombin. Serum samples have been collected before treatment with fibrin and postoperatively between 1 and 9 days, 3 and 12 weeks, and 6 and 8 months. The titers of anti-bovine fibrinogen antibodies, measured by ELISA specific for immunoglobulins IgG or IgM, increased to maximal values after about 8 or 6 weeks, respectively. After 8 months, IgG titers were on average 20-fold lower than the mean maximal value, while IgM titers returned to the normal range. IgG was the predominant anti-bovine fibrinogen immunoglobulin as documented by ELISA, affinity chromatography and electrophoresis. Anti-bovine fibrinogen antibodies present in patients reacted readily with bovine fibrinogen, but did not cross-react with human fibrinogen as measured by ELISA or by immunoelectrophoresis. A significant amount of antibodies against bovine thrombin and factor V has been found, many cross-reacting with the human counterparts. No hemorrhagic or thrombotic complications, or clinically significant allergic reactions, occurred in any patient, in spite of antibody presence against some bovine and human coagulation factors. The treatment of patients with bovine fibrin, without induction of immunologic response against human fibrinogen, appeared to be an effective topical hemostatic/sealing measure.

Autoantibodies to Thrombomodulin: Development of an Enzyme Immunoassay and a Survey of their Frequency in Patients with the Lupus Anticoagulant

1992 ◽  
Vol 67 (05) ◽  
pp. 507-509 ◽  
Author(s):  
John Gibson ◽  
Margaret Nelson ◽  
Ross Brown ◽  
Hatem Salem ◽  
Harry Kronenberg
Keyword(s):  
Enzyme Immunoassay ◽  
Lupus Anticoagulant ◽  
Specific Binding ◽  
Technical Problem ◽  
Serum Samples ◽  
Citrate Buffer ◽  
The Mean ◽  
Sample Dilution ◽  
Negative Population ◽  
Serum Components

SummaryIn order to investigate the possibility that autoantibodies to thrombomodulin (TM) may exist in patients with the lupus anticoagulant (LA) and perhaps be implicated in the pathogenesis of recurrent thrombosis seen in such patients, we developed an enzyme-immunoassay to screen serum samples for anti-human TM activity. The major technical problem encountered in developing this assay was to reduce the non-specific binding of serum components from both the LA positive and the negative population. Considerable reduction of non-specific binding was achieved by use of a phosphate/citrate buffer at pH 8.0 and the use of an optimal sample dilution of 1/40. In addition, samples were always tested in parallel in blank wells and results are expressed as an OD ratio. Samples from 113 patients with the LA were assayed and compared to 78 patients referred for LA testing but found to be negative. The mean OD values for the LA positive patients (± SD) was 1.36 (0.44) with a range of 0.78-2.57. This was virtually identical to the values for the LA negative population (1.38 ± 0.40, range 0.76-2.77). The results of this study indicate that there is no evidence for the presence of a significant autoantibody activity to TM in patients with the LA when compared to LA negative patients. If such autoantibodies do exist their frequency must be quite low.

scholarly journals Exogenous female sex steroid hormones and risk of asthma and asthma-like symptoms: a cross sectional study of the general population

Thorax ◽  
2001 ◽  
Vol 56 (8) ◽  
pp. 613-616
Author(s):  
P Lange ◽  
J Parner ◽  
E Prescott ◽  
C Suppli Ulrik ◽  
J Vestbo
Keyword(s):  
General Population ◽  
Postmenopausal Women ◽  
Hormone Replacement ◽  
Premenopausal Women ◽  
Cross Sectional Study ◽  
Asthma Medication ◽  
Hormonal Contraceptives ◽  
Cross Sectional ◽  
Heart Study ◽  
Prescription Practice

BACKGROUNDRecent evidence suggests a role for hormonal factors in the aetiology of asthma.METHODSData from a large study of women selected from the general population were used to relate treatment with oral hormonal contraceptives (OCP) and postmenopausal hormone replacement therapy (HRT) to the following asthma indicators: self-reported asthma, wheezing, cough at exertion, and use of medication for asthma. The study sample comprised 1536 premenopausal and 3016 postmenopausal women who participated in the third round of the Copenhagen City Heart Study in 1991–4. A total of 377 women were taking OCP (24.5% of premenopausal women) and 458 were on HRT (15.2% of postmenopausal women).RESULTSIn premenopausal women 4.8% reported having asthma. The prevalence of self-reported asthma, wheeze, use of asthma medication, and cough at exertion was not significantly related to use of OCP. In postmenopausal women the prevalence of self-reported asthma was 6.2%. A weak but consistent association was observed between HRT and self-reported asthma (OR 1.42 (95% CI 0.95 to 2.12)), wheeze (OR 1.29 (95% CI 1.02 to 1.64)), cough at exertion (OR 1.34 (95% CI 1.01 to 1.77)), and use of asthma medication (OR 1.45 (95% CI 0.97 to 2.18)).CONCLUSIONSIn this study of the general population no relationship was found between the use of OCP and asthma. Although an association was observed between HRT and asthma and asthma-like symptoms, this was relatively weak and it is concluded that there is no necessity to change present prescription practice.

scholarly journals Development of Sensitive Immunoassays for Free and Total Human Glandular Kallikrein 2

2004 ◽  
Vol 50 (9) ◽  
pp. 1607-1617 ◽  
Author(s):  
Ville Väisänen ◽  
Susann Eriksson ◽  
Kaisa K Ivaska ◽  
Hans Lilja ◽  
Martti Nurmi ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Detection Limit ◽  
Solid Phase ◽  
Specific Antigen ◽  
Control Group ◽  
Serum Samples ◽  
Human Kallikrein 2 ◽  
Kallikrein 2 ◽  
Capture Antibody ◽  
Total Psa

Abstract Background: Free and total human kallikrein 2 (hK2) might improve the discrimination between prostate cancer and benign prostatic hyperplasia. Concentrations of hK2 are 100-fold lower than concentrations of prostate-specific antigen (PSA); therefore, an hK2 assay must have a low detection limit and good specificity. Methods: PSA- and hK2-specific monoclonal antibodies were used in solid-phase, two-site immunofluorometric assays to detect free and total hK2. The total hK2 assay used PSA-specific antibodies to block nonspecific signal. The capture antibody of the free hK2 assay did not cross-react with PSA. To determine the hK2 concentrations in the male bloodstream, total hK2 was measured in a control group consisting of 426 noncharacterized serum samples. Free and total hK2 were measured in plasma from 103 patients with confirmed prostate cancer. Results: All 426 males in the control group had a total hK2 concentration above the detection limit of 0.0008 μg/L. The median total hK2 concentration was 0.022 μg/L (range, 0.0015–0.37 μg/L). hK2 concentrations were 0.1–58% of total PSA (median, 3.6%). hK2 concentrations were similar in men 41–50 and 51–60 years of age. The ratio of hK2 to PSA steadily decreased from 5–30% at PSA &lt;1 μg/L to 1–2% at higher PSA concentrations. In 103 patients with prostate cancer, the median hK2 concentration in plasma was 0.079 μg/L (range, 0.0015–16.2 μg/L). The median free hK2 concentration was 0.070 (range, 0.005–12.2) μg/L. The proportion of free to total hK2 varied from 17% to 131% (mean, 85%). Conclusions: The wide variation in the free-to-total hK2 ratio suggests that hK2 in blood plasma is not consistently in the free, noncomplexed form in patients with prostate cancer. The new assay is sufficiently sensitive to be used to study the diagnostic accuracies of free and total hK2 for prostate cancer.

Automated immunoassays for the autoantibodies to carbamylated or citrullinated telopeptides of type I and II collagens

2015 ◽  
Vol 53 (9) ◽  
Author(s):  
Jere Häyrynen ◽  
Maija Kärkkäinen ◽  
Aulikki Kononoff ◽  
Leena Arstila ◽  
Pia Elfving ◽  
...  
Keyword(s):  
Early Arthritis ◽  
Joint Disease ◽  
Type I ◽  
Type Ii ◽  
Newly Diagnosed ◽  
Seronegative Arthritis ◽  
Inflammatory Joint Disease ◽  
Undifferentiated Arthritis ◽  
Serum Samples ◽  
The Mean

AbstractThe aim of the study was to describe automated immunoassays for autoantibodies to homocitrulline or citrulline containing telopeptides of type I and II collagen in various disease categories in an early arthritis series.Serum samples were collected from 142 patients over 16 years of age with newly diagnosed inflammatory joint disease. All samples were analyzed with an automated inhibition chemiluminescence immunoassay (CLIA) using four different peptide pairs, each consisting of a biotinylated antigen and an inhibiting peptide. Assays were performed with an IDS-iSYS analyzer. Autoantibodies binding to homocitrulline and citrulline containing C-telopeptides of type I (HTELO-I, TELO-I) and type II collagens (HTELO-II, TELO-II) were analyzed.The mean ratio of HTELO-I inhibition in seropositive and seronegative rheumatoid arthritis (RA) was 3.07 (95% CI 1.41–11.60), p=0.003, and in seropositive and seronegative undifferentiated arthritis (UA) 4.90 (1.85–14.49), p<0.001. The respective mean ratios in seropositive and seronegative RA and UA were in TELO-I 8.72 (3.68–58.01), p<0.001 and 3.13 (1.49–6.16), p=0.008, in HTELO-II 7.57 (3.18–56.60), p<0.001 and 2.97 (1.23–6.69), p=0.037, and in TELO-II 3.01 (1.30–9.51), p=0.002 and 3.64 (1.86–7.65), p=0.008. In reactive arthritis, ankylosing spondylitis, psoriatic arthritis and unspecified spondyloarthritis the inhibition levels were similar to those observed in seronegative RA or UA.Autoantibodies binding to homocitrulline or citrulline containing telopeptides of type I and II collagen did not differ significantly. They were highest among patients with seropositive disease and they differentiated seropositive and seronegative arthritis.

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