A functional assay for serum detection of antibodies against SARS‐CoV‐2 nucleoprotein

SummaryThe activated partial thromboplastin time test (aPTT) represents one of the most commonly used diagnostic tools in order to monitor patients undergoing heparin therapy. Expression of aPTT coagulation time in seconds represents common practice in order to evaluate the integrity of the coagulation cascade. The prolongation of the aPTT thus can indicate whether or not the heparin level is likely to be within therapeutic range. Unfortunately aPTT results are highly variable depending on patient properties, manufacturer, different reagents and instruments among others but most importantly aPTT’s dose response curve to heparin often lacks linearity. Furthermore, aPTT assays are insensitive to drugs such as, for example, low molecular weight heparin (LMWH) and direct factor Xa (FXa) inhibitors among others. On the other hand, the protrombinase-induced clotting time assay (PiCT®) has been show to be a reliable functional assay sensitive to all heparinoids as well as direct thrombin inhibitors (DTIs). So far, the commercially available PiCT assay (Pefakit®-PiCT®, DSM Nutritional Products Ltd. Branch Pentapharm, Basel, Switzerland) is designed to express results in terms of units with the help of specific calibrators, while aPTT results are most commonly expressed as coagulation time in seconds. In this report, we describe the results of a pilot study indicating that the Pefakit PiCT UC assay is superior to the aPTT for the efficient monitoring of patients undergoing UFH therapy; it is also suitable to determine and quantitate the effect of LMWH therapy. This indicates a distinct benefit when using this new approach over the use of aPPT for heparin monitoring.

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Clinical evaluation of a new thyroglobulin immunoradiometric assay in the follow-up of differentiated thyroid carcinoma

Nuklearmedizin ◽

10.1055/s-0038-1625299 ◽

2003 ◽

Vol 42 (02) ◽

pp. 71-77 ◽

Cited By ~ 4

Author(s):

I. Schreivogel ◽

C. Angerstein ◽

U. Siefker ◽

K. Lehmann ◽

G. Altenvoerde ◽

...

Keyword(s):

Thyroid Carcinoma ◽

Residual Disease ◽

False Negative ◽

Differentiated Thyroid Carcinoma ◽

Threshold Concentration ◽

High Rate ◽

Functional Assay ◽

Hypothyroid Patient ◽

Assay Sensitivity ◽

Significant Difference

SummaryAim: Formal and clinical comparison of a new 3rd-gene-ration-Tg-IRMA (3-G-IRMA; Dynotest®Tg-plus) with a conventional Tg-IRMA (3-G-IRMA; SELco®Tg-assay) for patients with differentiated thyroid carcinoma. In addition we evaluated, if thyroglobulin (Tg) levels above a specific threshold concentration indicate the need for further investigations for residual disease. Patients, methods: Tg concentration of 105 sera of 93 consecutive patients with a differentiated thyroid cancer was determined with both assays and compared at different cut-off values (Dynotest®Tg-plus: 0.2, 1, 2 ng/ml; SELco®Tg-assay: 0.5, 1, 2 ng/ml) with the clinical results in respect to the corresponding TSH concentration. Results: Tg concentration did not show any significant difference (SELco®Tg-assay 0.5 ng/ml, Dynotest® Tg-plus 0.2 ng/ml). The Tg-values of both assays correlated with 97%. However, correlation of recovery in both assays was small (40%). The sensitivities and specificities of both assays at different cut-offs and TSH values did not reveal significant differences. In patients with TSH concentration >30 µU/ml the functional assay sensitivity was superior to arbitrary cut-offs in the decision to start further evaluations. Conclusions: In our study neither formal nor clinical significant differences between two Tg-assays were found. In a hypothyroid patient (TSH >30 µU/ml, Tg concentration exceeding the functional assay sensitivity) further investigations for residual disease are warranted. Higher thresholds are of limited value, due to a inacceptable high rate of false negative results.

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A New Functional Assay for Human Protein S Activity Using Activated Factor Vas Substrate

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647133 ◽

1989 ◽

Vol 62 (04) ◽

pp. 1144-1145 ◽

Cited By ~ 49

Author(s):

Martine Wolf ◽

Catherine Boyer-Neumann ◽

Jean-Luc Martinoli ◽

Catherine Leroy-Matheron ◽

Amiral Jean ◽

...

Keyword(s):

Protein S ◽

Human Protein ◽

Functional Assay ◽

Protein S Activity

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A Novel Functional Assay of Protein C in Human Plasma and fts Comparison with Amidolytic and Anticoagulant Assays

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648377 ◽

1992 ◽

Vol 67 (01) ◽

pp. 046-049 ◽

Cited By ~ 10

Author(s):

H A Guglielmone ◽

M A Vides

Keyword(s):

Oral Anticoagulant ◽

Protein C ◽

Activated Protein C ◽

Protein S ◽

Normal Subjects ◽

Test Sample ◽

Functional Assay ◽

Fast Method ◽

Factor Va ◽

Protein C Activity

SummaryA simple and fast method for the quantitative determination of protein C activity in plasma is here described. The first step consists in the conversion of protein C in the test sample into activated protein C by means of an activator isolated from Southern Copperhead venom. Subsequently, the degradation of factor Va, in presence of protein C-deficient plasma, is measured by the prolongation of the prothrombin time which is proportional to the amount of protein C in the sample. The dose-response curve showed a linear relationship from 6 to 150% protein C activity and the inter- and intra-assay reproducibility was 3.5% and 5.6% respectively. In normal subjects, a mean of protein C level of 98 ± 15% of normal pooled plasma was found. Comparison with the anticoagulant assay in samples of patients with oral anticoagulant, liver cirrhosis, disseminated intravascular coagulation and severe preeclampsia revealed an excellent correlation (r = 0.94, p <0.001). Also, a similar correlation (r = 0.93, p <0.001) existed between amidolytic assay and the method here proposed for all the samples studied without including the oral anticoagulant group. These results allowed us to infer that this method evaluates the ability of protein C to interact with protein S, phospholipids, calcium ions and factor Va.

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Behaviour and Quantitation of Extrinsic (Tissue-Type) Plasminogen Activator in Human Blood

Thrombosis and Haemostasis ◽

10.1055/s-0038-1665244 ◽

1983 ◽

Vol 50 (02) ◽

pp. 518-523 ◽

Cited By ~ 16

Author(s):

C Kluft ◽

A F H Jie ◽

R A Allen

Keyword(s):

Plasminogen Activator ◽

Venous Occlusion ◽

Tissue Type ◽

Functional Assay ◽

Uterine Tissue ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Baseline Values

SummaryFunctional assay of extrinsic (tissue-type) plasminogen activator (EPA) in plasma on fibrin plates was evaluated. Using specific quenching antibodies, we demonstrated the method to be specific for EPA under all conditions tested. Contributions of urokinases and intrinsic activators were excluded. The quantity of EPA in blood samples, as compared with purified uterine tissue activator, shows 1 blood activator unit (BAU) to be comparable to 0.93 ng.The median values for EPA activity for healthy volunteers were: baseline, 1.9 BAU/ml (n = 123); diurnal, 5.5 BAU/ml (n = 12); DDAVP administration, 11.7 BAU/ml (n = 39); exhaustive exercise, 25 BAU/ml (n = 24); venous occlusion (15 min), 35 BAU/ml (n = 61). A large inter-individual variation in EPA activity was found, while individual baseline values tended to be constant for periods of weeks.In vitro in blood EPA activity shows a disappearance of 50% in about 90 min at 37° C; EPA activity in euglobulin fractions is stable for ≤2 hr at 37° C.A rapid decrease in EPA activity occurs in vivo, as noted after extracorporal circulation and exercise stimulation (t½ decay, 2-5 min).

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Rubella Antibodies in Human Serum: Detection by the Indirect Fluorescent-Antibody Technique

Science ◽

10.1126/science.145.3635.943 ◽

1964 ◽

Vol 145 (3635) ◽

pp. 943-945 ◽

Cited By ~ 39

Author(s):

G. C. Brown ◽

H. F. Maassab ◽

J. A. Veronelli ◽

T. J. Francis

Keyword(s):

Human Serum ◽

Fluorescent Antibody ◽

Fluorescent Antibody Technique ◽

Indirect Fluorescent Antibody ◽

Antibody Technique ◽

Serum Detection

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Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽

10.1182/blood.v89.11.3919 ◽

1997 ◽

Vol 89 (11) ◽

pp. 3919-3924 ◽

Cited By ~ 328

Author(s):

Jean C.Y. Wang ◽

Monica Doedens ◽

John E. Dick

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Allogeneic Transplantation ◽

Functional Assay ◽

Hematopoietic Stem ◽

Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

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Blood functional assay for rapid clinical interpretation of germline TP53 variants

Journal of Medical Genetics ◽

10.1136/jmedgenet-2020-107059 ◽

2020 ◽

pp. jmedgenet-2020-107059 ◽

Cited By ~ 1

Author(s):

Sabine Raad ◽

Marion Rolain ◽

Sophie Coutant ◽

Céline Derambure ◽

Raphael Lanos ◽

...

Keyword(s):

Mononuclear Cells ◽

Transcriptional Response ◽

Functional Assay ◽

Mrna Levels ◽

Peripheral Blood Mononuclear ◽

Patients With Cancer ◽

Pathogenic Variants ◽

Functional Variants ◽

Multiplex Ligation Probe Amplification ◽

P53 Mrna

BackgroundThe interpretation of germline TP53 variants is critical to ensure appropriate medical management of patients with cancer and follow-up of variant carriers. This interpretation remains complex and is becoming a growing challenge considering the exponential increase in TP53 tests. We developed a functional assay directly performed on patients’ blood.MethodsPeripheral blood mononuclear cells were cultured, activated, exposed to doxorubicin and the p53-mediated transcriptional response was quantified using reverse transcription–multiplex ligation probe amplification and RT-QMPSF assays, including 10 p53 targets selected from transcriptome analysis, and two amplicons to measure p53 mRNA levels. We applied this blood functional assay to 77 patients addressed for TP53 analysis.ResultsIn 51 wild-type TP53 individuals, the mean p53 functionality score was 12.7 (range 7.5–22.8). Among eight individuals harbouring likely pathogenic or pathogenic variants, the scores were reduced (mean 4.8, range 3.1–7.1), and p53 mRNA levels were reduced in patients harbouring truncating variants. We tested 14 rare unclassified variants (p.(Pro72His), p.(Gly105Asp), p.(Arg110His), p.(Phe134Leu), p.(Arg158Cys), p.(Pro191Arg), p.(Pro278Arg), p.(Arg283Cys), p.(Leu348Ser), p.(Asp352Tyr), p.(Gly108_Phe109delinsVal), p.(Asn131del), p.(Leu265del), c.-117G>T) and 12 yielded functionally abnormal scores. Remarkably, the assay revealed that the c.*1175A>C polymorphic variant within TP53 poly-adenylation site can impact p53 function with the same magnitude as a null variant, when present on both alleles, and may act as a modifying factor in pathogenic variant carriers.ConclusionThis blood p53 assay should therefore be a useful tool for the rapid clinical classification of germline TP53 variants and detection of non-coding functional variants.

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A non-human primate in vitro functional assay for the early evaluation of TB vaccine candidates

npj Vaccines ◽

10.1038/s41541-020-00263-7 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Rachel Tanner ◽

Andrew D. White ◽

Charelle Boot ◽

Claudia C. Sombroek ◽

Matthew K. O’Shea ◽

...

Keyword(s):

Growth Inhibition ◽

Mononuclear Cells ◽

Functional Assay ◽

Intermediate Precision ◽

Peripheral Blood Mononuclear ◽

Growth Inhibition Assay ◽

Early Evaluation ◽

Human Primate

AbstractWe present a non-human primate mycobacterial growth inhibition assay (MGIA) using in vitro blood or cell co-culture with the aim of refining and expediting early tuberculosis vaccine testing. We have taken steps to optimise the assay using cryopreserved peripheral blood mononuclear cells, transfer it to end-user institutes, and assess technical and biological validity. Increasing cell concentration or mycobacterial input and co-culturing in static 48-well plates compared with rotating tubes improved intra-assay repeatability and sensitivity. Standardisation and harmonisation efforts resulted in high consistency agreements, with repeatability and intermediate precision <10% coefficient of variation (CV) and inter-site reproducibility <20% CV; although some systematic differences were observed. As proof-of-concept, we demonstrated ability to detect a BCG vaccine-induced improvement in growth inhibition in macaque samples, and a correlation between MGIA outcome and measures of protection from in vivo disease development following challenge with either intradermal BCG or aerosol/endobronchial Mycobacterium tuberculosis (M.tb) at a group and individual animal level.

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