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.

Multiplexed High-Throughput Electrokinetically-Controlled Immunoassay on a Chip for the Detection of Specific Bacterial Antibodies in Human Serum

2007 ◽  
Author(s):  
Yali Gao ◽  
Philip M. Sherman ◽  
Yu Sun ◽  
Dongqing Li
Keyword(s):  
Human Serum ◽  
Specific Binding ◽  
Transport Processes ◽  
Escherichia Coli O157 ◽  
Serum Samples ◽  
E Coli ◽  
Human Serum Samples ◽  
Clinical Environments ◽  
H Pylori ◽  
Serum Components

This work presents a multiplexed electrokinetically-controlled heterogeneous immunoassay that can process ten samples in parallel. The immunoassay microchip was soft-lithographically fabricated using poly(dimethylsiloxane) and glass. Controlling parameters of the electrokinetically-driven flow in the microfluidic network was determined by numerically simulating transport processes. Multiple passively adsorbed antigens captured antibodies present in samples, which then bound with TRITC-labeled detection antibodies to generate fluorescent signals. Antibodies against Escherichia coli O157:H7 and Helicobacter pylori were studied as model analytes. After conditions for antigen-coating were optimized, a 24-minute assay detected E. coli O157:H7 antibody in the concentration range of 0.02–10 μg/mL, and H. pylori antibody in the range of 0.1–50 μg/mL. In testing human serum samples, non-specific binding of serum components was effectively suppressed by using 10% (w/v) bovine serum albumin. An accuracy of 100% was achieved in detecting either E. coli O157:H7 antibody or H. pylori antibody from human serum samples. Simultaneous screening of both antibodies was also successfully demonstrated. The immunoassay chip shows an excellent potential for efficiently detecting multiple pathogenic infections in clinical environments.

The Determination of Thiamine in Small Amounts of Whole Blood and Serum by a Simplified Thiochrome Method

1965 ◽  
Vol 11 (6) ◽  
pp. 617-623 ◽  
Author(s):  
Than Myint ◽  
Harold B Houser
Keyword(s):  
Whole Blood ◽  
Serum Concentrations ◽  
Serum Samples ◽  
Adsorption Column ◽  
The Mean ◽  
Mean Differences ◽  
Hydrolysis Of ◽  
Sample Dilution ◽  
Low Serum

Abstract This simplified thiochrome method for the determination of thiamine eliminates deproteinization, sample dilution (other than reagent), and purification by adsorption column; pH need not be adjusted as it is automatically controlled. The method depends upon the hydrolysis of whole blood and serum with N HCI, N/10 HCI, and diastase enzyme. Reproducibility was good; the mean differences (± S.D.) between duplicate blood and serum samples were 2.36 ± 2.87 and 1.5 ± 1.70 mµg./ml., respectively. Recovery of added thiamine ranged from 94 to 104% with a mean of 99.5 ± 3.41%. Storage of hydrolysates for 30 days did not change the results, and low serum concentrations could be measured in serum. Whole blood and serum values of thiamine in 44 healthy adults ranged from 11.3 to 47.8 mµg./ml. (mean, 29.3) and from trace amounts to 20.5 mµg./ml. (mean, 10.2), respectively.

RADIOIMMUNOASSAY FOR TRIIODOTHYRONINE IN HUMAN SERUM

1973 ◽  
Vol 72 (3) ◽  
pp. 464-474 ◽  
Author(s):  
M. Hüfner ◽  
R.-D. Hesch
Keyword(s):  
Statistical Data ◽  
Specific Binding ◽  
Plasma Samples ◽  
Serum Samples ◽  
Exchange Method ◽  
Binding Reaction ◽  
Direct Measurements ◽  
The Mean ◽  
Ion Exchange Method ◽  
Ph Buffer

ABSTRACT A radioimmunoassay for triiodothyronine (T3) is demonstrated. Details of the T3 antibody binding reaction are discussed and the statistical data of the assay system are presented. Plasma samples were first extracted by a simple ion exchange method and the extracts then tested by radioimmunoassay. The mean normal concentration was 1.35 ± 0.29 ng/ml. Later, direct measurements in serum samples were performed after partial inhibition of T3 binding to TBG by merthiolate and high pH buffer. After correction for the residual non-specific binding a mean normal plasma level of 1.2 ± 0.3 ng/ml was obtained.

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.

scholarly journals Immunoglobulin-like Domain of HsFcμR as a Capture Molecule for Detection of Crimean-Congo Hemorrhagic Fever Virus- and Zika Virus-Specific IgM Antibodies

2019 ◽  
Vol 65 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Anne Rackow ◽  
Christa Ehmen ◽  
Ronald von Possel ◽  
Raquel Medialdea-Carrera ◽  
David Brown ◽  
...  
Keyword(s):  
Zika Virus ◽  
Specific Binding ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Size Exclusion ◽  
Animal Origin ◽  
Serum Samples ◽  
Crimean Congo Hemorrhagic Fever ◽  
Hemorrhagic Fever Virus ◽  
Igm Antibodies

Abstract BACKGROUND The cellular surface molecule HsTOSO/FAIM3/HsFcμR has been identified as an IgM-specific Fc receptor expressed on lymphocytes. Here, we show that its extracellular immunoglobulin-like domain (HsFcμR-Igl) specifically binds to IgM/antigen immune complexes (ICs) and exploit this property for the development of novel detection systems for IgM antibodies directed against Crimean-Congo hemorrhagic fever virus (CCHFV) and Zika virus (ZIKV). METHODS His-tagged HsFcμR-Igl was expressed in Escherichia coli and purified by affinity chromatography, oxidative refolding, and size-exclusion chromatography. Specific binding of HsFcμR-Igl to IgM/antigen ICs was confirmed, and 2 prototypic ELISAs for the detection of anti-CCHFV and anti-ZIKV IgM antibodies were developed. Thereby, patient sera and virus-specific recombinant antigens directly labeled with horseradish peroxidase (HRP) were coincubated on HsFcμR-Igl-coated ELISA plates. Bound ICs were quantified by measuring turnover of a chromogenic HRP substrate. RESULTS Assay validation was performed using paired serum samples from 15 Kosovar patients with a PCR-confirmed CCHFV infection and 28 Brazilian patients with a PCR-confirmed ZIKV infection, along with a panel of a priori CCHFV/ZIKV-IgM-negative serum samples. Both ELISAs were highly reproducible. Sensitivity and specificity were comparable with or even exceeded in-house gold standard testing and commercial kits. Furthermore, latex beads coated with HsFcμR-Igl aggregated upon coincubation with an IgM-positive serum and HRP-labeled antigen but not with either component alone, revealing a potential for use of HsFcμR-Igl as a capture molecule in aggregation-based rapid tests. CONCLUSIONS Recombinant HsFcμR-Igl is a versatile capture molecule for IgM/antigen ICs of human and animal origin and can be applied for the development of both plate- and bead-based serological tests.

scholarly journals Quantification of Serum Proteins Using Capillary Electrophoresis

1995 ◽  
Vol 32 (5) ◽  
pp. 493-497 ◽  
Author(s):  
M A Jenkins ◽  
M D Guerin
Keyword(s):  
Capillary Electrophoresis ◽  
Gel Electrophoresis ◽  
Serum Proteins ◽  
Clinical Laboratory ◽  
Charged Particles ◽  
Protein Electrophoresis ◽  
Agarose Gel Electrophoresis ◽  
Serum Samples ◽  
Routine Clinical Laboratory ◽  
Sample Dilution

Capillary electrophoresis is a technique that can be automated for the separation of charged particles. By investigating suitable sample dilution and injection time and adhering to a strict washing procedure we have been able to quantify paraproteins in serum samples. This has enabled us to use the technique of capillary electrophoresis for the provision of serum protein electrophoresis in a routine clinical laboratory. We present our findings of 260 serum samples, which included 76 samples with paraproteins analysed by both capillary electrophoresis (EC) and high resolution agarose gel electrophoresis (HRAGE). CE was able to detect all the monoclonal bands detected by HRAGE, and, in particular, better able to detect IgA monoclonal bands occurring in the beta region. The major advantages of CE over HRAGE relate to the automated nature of CE with the elimination of the need for a densitometer.

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.

scholarly journals Levels of Circulating TIMP-2 and MMP2-TIMP2 Complex Are Decreased in Squamous Cervical Carcinoma

2010 ◽  
Vol 2010 ◽  
pp. 1-3 ◽  
Author(s):  
Talvensaari-Mattila Anne ◽  
Turpeenniemi-Hujanen Taina
Keyword(s):  
Cervical Carcinoma ◽  
Matrix Metalloproteinase 2 ◽  
Healthy Controls ◽  
Matrix Degradation ◽  
Serum Samples ◽  
Study Population ◽  
The Mean ◽  
Natural Tissue ◽  
Squamous Cervical Carcinoma

Background. The role of matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) in matrix degradation and metastasis has been described in various tumors. Their action is inhibited by their natural tissue inhibitor molecules TIMP-1 and -2.Methods. The study population consisted of 12 squamous cervical carcinoma patients and 27 healthy volunteer control patients. MMP-9, MMP-2-TIMP-2 complex, TIMP-1, and TIMP-2 were analyzed from serum samples using enzyme-linked immunoassay (ELISA).Results. The mean levels of serum TIMP-2 and of MMP-2-TIMP-2 complex were higher in healthy controls compared to patients with a malignant tumor. Serum TIMP-2 values decreased significantly from healthy controls (median 323 g/l, range 305–342 g/l) to malignant (median 136 g/l, range 120–151 g/l) squamous cervical carcinoma patients . Also, serum proMMP2-TIMP2 complex values decreased from control patients to squamous cervical carcinoma patients .Conclusion. This paper shows that the levels of circulating TIMP-2 and that of MMP-2-TIMP-2 complex are lower in squamous cervical carcinoma patients than in healthy women.

Immunoreactive IGF-II in serum of healthy subjects and patients with growth hormone disturbances and uraemia

1984 ◽  
Vol 107 (2) ◽  
pp. 164-170 ◽  
Author(s):  
Gösta Enberg ◽  
Kerstin Hall
Keyword(s):  
Confidence Limit ◽  
Binding Protein ◽  
Protein S ◽  
Serum Levels ◽  
Cross Reaction ◽  
Diabetic Patients ◽  
Serum Samples ◽  
Igf I ◽  
Age Dependent ◽  
The Mean

Abstract. A radioimmunoassay has been developed for IGF-II, using Sepharose-coupled antibodies. Porcine insulin, human insulin and human proinsulin showed no cross-reaction, whereas the cross-reaction for IGF-I was 10%. To minimize the influence of the binding protein(s), all serum samples were extracted with acid ethanol before assay. The mean serum level of immunoreactive IGF-II and 95% confidence limit in 46 healthy adults were 587 ng/ml and 354–974 ng/ml, respectively. In contrast to the declining levels of IGF-I with increasing age, no such age-dependent decrease was found for IGF-II levels between 20 to 70 years. No difference in IGF-II levels was found between patients with acromegaly and healthy adult controls. In cord serum and serum from adult patients with GH deficiency the levels were significantly lower (P < 0.001) compared to controls. In diabetic patients with uraemia the mean level and 95% confidence limit were 1222 ng/ml and 532–2808 ng/ml, respectively. Thus, significantly increased serum levels of immunoreactive IGF-II have only been found in serum from patients with uraemia. Whether this is due to an increased production of IGF-II, or secondary to other factors such as the binding protein(s), will require further investigation.

Free thyroxine assessed with three assays in sera of patients with nonthyroidal illness and of subjects with abnormal concentrations of thyroxine-binding proteins

1993 ◽  
Vol 39 (8) ◽  
pp. 1668-1674 ◽  
Author(s):  
R Docter ◽  
H van Toor ◽  
E P Krenning ◽  
M de Jong ◽  
G Hennemann
Keyword(s):  
Blood Donors ◽  
Reference Range ◽  
Equilibrium Dialysis ◽  
Free Thyroxine ◽  
Serum Samples ◽  
Nonthyroidal Illness ◽  
Nonesterified Fatty Acids ◽  
Thyroxine Binding Globulin ◽  
Control Value ◽  
The Mean

Abstract Three methods for estimating free thyroxine (FT4) in serum were studied: equilibrium dialysis, the SPAC-ET FT4 radioimmunoassay kit, and the Amerlite MAB FT4 luminometric assay. Serum samples from 10 subjects with above-normal thyroxine-binding globulin (TBG), 6 with low TBG, 30 with familial dysalbuminemic hyperthyroxinemia (FDH), 13 with nonesterified fatty acids (NEFA) concentrations in serum &gt; 1.0 mmol/L, and 178 patients with various degrees of nonthyroidal illness (NTI) were measured and compared with samples from 42 euthyroid blood donors. The Amerlite MAB FT4 assay compared well with equilibrium dialysis, whereas the SPAC-ET assay averaged 40% lower. All three assays were not influenced by changes in TBG and showed no or only little changes in the presence of NEFA. Mean FT4 values in the FDH samples were somewhat higher than in controls when measured with the SPAC-ET assay, about equal with equilibrium dialysis, and somewhat below the mean control value with the Amerlite MAB FT4 assay, although individual results were within the control reference range. In NTI patients, no FT4 values were below the control reference range by the Amerlite MAB FT4 assay, 4 of 178 were below this range by equilibrium dialysis, and 1 of 178 was below this range by the SPAC-ET assay. In all assays a large proportion of the NTI samples showed FT4 values above the control reference range, a result that will interfere with the efficacy of these assays for assessing thyroid function in NTI patients.

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