High-affinity antibodies in a new immunoassay for plasma tissue factor: reduction in apparent intra-individual variation

2005 ◽  
Vol 43 (12) ◽  
Author(s):  
Roy F. M. van der Putten ◽  
Henk te Velthuis ◽  
Lucien A. Aarden ◽  
Hugo ten Cate ◽  
Jan F. C. Glatz ◽  
...  
Keyword(s):  
Detection Limit ◽  
Tissue Factor ◽  
Individual Variation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
High Affinity ◽  
Normal Ranges ◽  
Healthy Donors ◽  
Highly Sensitive

AbstractTissue factor, the main initiator of blood coagulation, is shed into plasma by blood cells and endothelium. While studying such circulating plasma tissue factor with a commercially available immunoassay, we found unsatisfactory results and therefore developed a new and highly sensitive enzyme-linked immunosorbent assay (ELISA). High-affinity monoclonal antibodies raised against recombinant soluble tissue factor were used and the new assay had a detection limit of 40fmol/L, approximately six-fold lower than existing assays. Normal ranges in 20 healthy donors were established in serum and in citrated EDTA and heparinized plasma. Tissue factor was also measured in three successive plasma samples from 43 patients with type 2 diabetes mellitus. In citrated plasma from healthy donors, tissue factor concentrations were 2.5 (1.0–9.3) pmol/L (median with range) and were not significantly different in diabetics. With a commercially available immunoassay, seven plasma samples were below the detection limit. Use of the new assay reduced intra-individual variation in diabetics from 49% to 14% and we conclude that high-affinity antibodies may markedly improve immunoassay performance.

scholarly journals Development of new immunoanalytical test systems for diagnostics of potato blackleg caused by Dickeya spp. bacteria

2021 ◽  
Vol 16 (3) ◽  
pp. 198-214
Author(s):  
Shyatesa Razo ◽  
Pavel A. Galushka ◽  
Yuri A. Varitsev ◽  
Anatoly V. Zherdev ◽  
Irina V. Safenkova ◽  
...  
Keyword(s):  
Detection Limit ◽  
Polyclonal Antibodies ◽  
Test System ◽  
Diagnostic Tools ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potato Seed ◽  
Potato Blackleg ◽  
Bacterial Diseases ◽  
High Affinity ◽  
Test Systems

Potato blackleg caused by Dickeya spp. bacteria is one of the most important bacterial diseases of potatoes. The rapid spread of this disease in the territory of Russia requires new effective diagnostic tools for the timely detection of infection. To solve this problem, antisera specific to Dickeya spp. were obtained. Polyclonal antibodies isolated from antisera have shown high affinity for the main species of Dickeya spp. ( D. solani, D. dianthicola, D. chrysanthemi, D. dadantii, D. paradisiaca ). Enzyme linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA) test systems have been developed based on specific and high affinity antibodies that were obtained. For ELISA, the detection limit was 0.8 105 cells/mL for D. solani and 2 104 cells/mL for D. dianthicola . For LFIA, suitable for use in non-laboratory conditions, the detection limit of D. solani was 2 105 cells/mL and the analysis time was 15 minutes. When testing potato seed material, LFIA test system confirmed positive results of ELISA determination in 75 % of samples, and negative - in 100 % of samples.

Development of a highly sensitive ELISA for determination of darunavir in plasma samples using a polyclonal antibody with high affinity and specificity

Bioanalysis ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 355-366 ◽  
Author(s):  
Abdulrahman A Almehizia ◽  
Rashed N Herqash ◽  
Ibrahim A Darwish
Keyword(s):  
High Throughput ◽  
Polyclonal Antibody ◽  
Routine Analysis ◽  
Therapeutic Monitoring ◽  
Pharmacokinetic Studies ◽  
Plasma Samples ◽  
High Affinity ◽  
Spiked Plasma ◽  
Highly Sensitive

Aim: To support pharmacokinetic studies and therapeutic monitoring of darunavir (DRV), a highly sensitive ELISA was developed for the determination of DRV in plasma samples at picogram levels. Results: The assay LOD and LOQ were 15 and 30 pg ml-1, respectively. The working range of the assay was 20–2000 pg ml-1. Analytical recoveries of DRV from spiked plasma were in the ranges of 98.4–113.0 and 86.0–99.1% for intra-assay and inter-assay runs, respectively. The precision of the assay was satisfactory. Conclusion: The ELISA is characterized by high throughput and it is expected to significantly contribute to routine analysis of DRV in its pharmacokinetic studies and therapeutic monitoring.

scholarly journals Molecularly Imprinted Nanoparticles Assay (MINA) in Pseudo ELISA: An Alternative to Detect and Quantify Octopamine in Water and Human Urine Samples

Polymers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1497 ◽  
Author(s):  
Ewa Moczko ◽  
Richard Díaz ◽  
Bernabé Rivas ◽  
Camilo García ◽  
Eduardo Pereira ◽  
...  
Keyword(s):  
Detection Limit ◽  
Human Urine ◽  
Solid Phase ◽  
Cross Reactivity ◽  
Urine Samples ◽  
Cold Chain ◽  
Enzyme Linked Immunosorbent Assay ◽  
Molecularly Imprinted ◽  
Human Urine Samples ◽  
High Affinity

In 2004, octopamine was added to the list of drugs banned by the world anti-doping agency (WADA) and prohibited in any sport competition. This work aims to develop a new analytical method to detect octopamine in water and human urine samples. We proposed a pseudo-enzyme-linked immunosorbent assay (pseudo-ELISA) by replacing traditional monoclonal antibodies with molecularly imprinted polymer nanoparticles (nanoMIPs). NanoMIPs were synthesised by a solid-phase approach using a persulfate initiated polymerisation in water. Their performance was analysed in pseudo competitive ELISA based on the competition between free octopamine and octopamine-HRP conjugated. The final assay was able to detect octopamine in water within the range 1 nmol·L−1–0.1 mol·L−1 with a detection limit of 0.047 ± 0.00231 µg·mL−1 and in human urine samples within the range 1 nmol·L−1–0.0001 mol·L−1 with a detection limit of 0.059 ± 0.00281 µg·mL−1. In all experiments, nanoMIPs presented high affinity to the target molecules and almost no cross-reactivity with analogues of octopamine such as pseudophedrine or l-Tyrosine. Only slight interference was observed from the human urine matrix. The high affinity and specificity of nanoMIPs and no need to maintain a cold chain logistics makes the nanoMIPs a competitive alternative to antibodies. Furthermore, this work is the first attempt to use nanoMIPs in pseudo-ELISA assays to detect octopamine.

Tissue factor is the receptor for plasminogen type 1 on 1-LN human prostate cancer cells

Blood ◽  
2002 ◽  
Vol 99 (12) ◽  
pp. 4562-4567 ◽  
Author(s):  
Mario Gonzalez-Gronow ◽  
Govind Gawdi ◽  
Salvatore V. Pizzo
Keyword(s):  
Tissue Factor ◽  
Carbohydrate Chain ◽  
Human Prostate ◽  
Extrinsic Pathway ◽  
High Affinity ◽  
Oligosaccharide Chain ◽  
Prostate Tumor Cells ◽  
Human Prostate Tumor

Tissue factor (TF), the initiator of the extrinsic pathway of coagulation, binds plasminogen (Pg) with high affinity through an interaction between kringles 1-3 of Pg and the extracellular domain of TF. We investigated the binding of Pg type 1 (Pg 1) and Pg type 2 (Pg 2) to highly invasive, TF-expressing, 1-LN human prostate tumor cells and to TF isolated from 1-LN cell membranes. Pg 1, containing both N-linked and O-linked oligosaccharide chains, bound to isolated TF with high affinity, whereas Pg 2, containing only one O-linked oligosaccharide chain, did not bind to TF. Although Pg 1 and Pg 2 bind to 1-LN cells, only anti-TF antibodies inhibited the binding of Pg 1, suggesting that TF functions as the receptor for Pg 1 on 1-LN cells. Binding of Pg 1 to isolated TF was inhibited by 6-aminohexanoic acid and α-methylmannoside, suggesting that Pg 1 l-lysine binding sites and the biantennary, mannose-containing N-linked oligosaccharide chain are involved in this interaction. Binding of Pg 1 to 1-LN cells promoted activation by receptor-bound urinary-type Pg activator (u-PA) and initiated a Ca++ signaling cascade. In previous studies we demonstrated that the Pg 2 O-linked carbohydrate chain is essential for its binding to CD26 on 1-LN cells. The current studies suggest that Pg oligosaccharide chains regulate the binding of Pg 1 and Pg 2 to separate receptors on the cell surface.

Immunoreactivity of Tissue Plasminogen Activator and of Its Inhibitor Complexes

1989 ◽  
Vol 61 (03) ◽  
pp. 409-414 ◽  
Author(s):  
M Rånby ◽  
G Nguyen ◽  
P Y Scarabin ◽  
M Samama
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Degradation Products ◽  
Gel Filtration ◽  
Free Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Significant Difference ◽  
Tissue Plasminogen ◽  
Pai 1

SummaryAn enzyme linked immunosorbent assay (ELISA) based on goat polyclonal antibodies against human tissue plasminogen activator (tPA) was evaluated. The relative immunoreactivity of tPA in free form and tPA in complex with inhibitors was estimated by ELISA and found to be 100, 74, 94, 92 and 8l% for free tPA and tPA in complex with PAI-1, PAI-2, α2-antiplasmin and C1-inhibitor, respectively. Addition of tPA to PAI-1 rich plasma resulted in rapid and total loss of tPA activity without detectable loss of ELISA response, indicating an immunoreactivity of tPA in tPA/PAI-1 complex of about l00%. Three different treatments of citrated plasma samples (acidification/reneutralization, addition of 5 mM EDTA or of 0.5 M lysine) prior to determination by ELISA all resulted in increased tPA levels. The fact that the increase was equally large in all three cases along with good analytical recovery of tPA added to plasffi, supported the notion that all tPA antigen present in plasma samples is measured by the ELISA. Analysis by ELISA of fractions obtained by gel filtration of plasma from a patient undergoing tPA treatment identified tPA/inhibitor complexes and free tPA but no low molecular weight degradation products of tPA. Determinations of tPA antigen were made at seven French clinical laboratories on coded and randomized plasma samples with known tPA antigen content. For undiluted samples there was no significant difference between the tPA levels found and those known to be present. The between-assay coefficient of variation was 7 to 10%. In conclusion, the ELISA appeared suited for determination of total tPA antigen in human plasma samples.

Predictive Value of Osteoprotegerin for Detecting Coronary Artery Calcification in Type 2 Diabetes Mellitus Patients in Correlation with Extent of Calcification Detected by Multidetector Computed Tomography

2019 ◽  
Vol 19 (6) ◽  
pp. 845-851 ◽  
Author(s):  
Sahar Ahmed ◽  
Rasha Sobh
Keyword(s):  
Coronary Artery ◽  
Coronary Artery Calcification ◽  
Enzyme Linked Immunosorbent Assay ◽  
Diabetic Patients ◽  
Coronary Artery Calcification Score ◽  
Type 2 Diabetic Patients ◽  
Duration Of Diabetes ◽  
Calcification Score ◽  
Type 2 Diabetic

Background:Osteoprotegerin (OPG) is a tumor necrosis factor receptor super-family member. It specifically acts on bone by increasing bone mineral density and bone volume. Recent studies have evidenced its close relation to the development of atherosclerosis and plaque destabilization. Elevated OPG level has also been associated with the degree of coronary calcification in the general population and it has been considered to be a marker of coronary atherosclerosis.Objective:The aim of this study was to determine the relation between OPG levels and Coronary Artery Calcification score (CACs) in Type 2 diabetic patients in comparison to healthy controls.Methods:Our study included 45 type 2 diabetic patients (mean age 51.7 years; 51.1% male) without evidence of previous CVD and 45 healthy age and sex matched subjects as control. All participants were subjected to full history, full examination and lab investigations. Serum OPG concentration was measured by an enzyme-linked immunosorbent assay (ELISA) and CAC imaging was performed using non contrast Multi detector CT of the heart.Results:Significant CAC (<10 Agatston units) was seen in 23 patients (51.11 %).:OPG was significantly high in diabetic patients in comparison to controls with mean 12.9±5.7 pmol/l in cases, and 8.6±0.5 pmol/l in controls (P value < 0.001).:The Coronary Artery Calcification Score (CACS) was positively correlated with age and duration of diabetes. The OPG was positively correlated with age, fasting blood sugar and duration of diabetes. The CACS showed a significantly positive correlation with OPG.Conclusion:Findings suggested that increasing in serum OPG was consistent with CAC and could be used for the early diagnosis of subclinical atherosclerosis.

African Origin of Human T-Lymphotropic Virus Type 2 (HTLV-2) Supported by a Potential New HTLV-2d Subtype in Congolese Bambuti Efe Pygmies

1998 ◽  
Vol 72 (5) ◽  
pp. 4327-4340 ◽  
Author(s):  
Anne-Mieke Vandamme ◽  
Marco Salemi ◽  
Marianne Van Brussel ◽  
Hsin-Fu Liu ◽  
Kristel Van Laethem ◽  
...  
Keyword(s):  
T Cell ◽  
Virus Type ◽  
Phylogenetic Analyses ◽  
Enzyme Linked Immunosorbent Assay ◽  
African Origin ◽  
Entire Genome ◽  
T Lymphotropic Virus ◽  
Tax Protein ◽  
Human T Cell

ABSTRACT We identified a potential new subtype within human T-cell lymphotropic virus type 2 (HTLV-2), HTLV-2d, present in members of an isolated Efe Bambuti Pygmy tribe. Two of 23 Efe Pygmies were HTLV-2 seropositive, with HTLV-2 Western blot and enzyme-linked immunosorbent assay reactivities. From one of them the entire genome of the HTLV-2 strain Efe2 could be amplified and sequenced. In all gene regions analyzed, this strain was the most divergent HTLV-2 strain, differing by 2.4% (tax/rex) to 10.7% (long terminal repeat) from both subtypes HTLV-2a and HTLV-2b, yet major functional elements are conserved. The similarity between the HTLV-2 Efe2 Gag and Env proteins and the corresponding HTLV-2a and -2b proteins is consistent with the observed serological reactivity. In the proximal pX region, one of the two alternative splice acceptor sites is abolished in HTLV-2 Efe2. Another interesting feature of this potential new subtype is that it has a Tax protein of 344 amino acids (aa), which is intermediate in length between the HTLV-2a Tax protein (331 aa) and the HTLV-2b and -2c Tax proteins (356 aa) and similar to the simian T-cell lymphotropic virus type 2 (STLV-2) PP1664 Tax protein. Together these two findings suggest a different phenotype for the HTLV-2 Efe2 strain. Phylogenetic analyses confirmed that the Pygmy Efe2 strain potentially belonged to a new and quite divergent subtype, HTLV-2d. When the STLV-2 bonobo viruses PP1664 and PanP were used as an outgroup, it was clear that the Pygmy HTLV-2 Efe2 strain had the longest independent evolution and that HTLV-2 evolution is consistent with an African origin.

scholarly journals Advanced glycation end product levels were correlated with inflammation and carotid atherosclerosis in type 2 diabetes patients

2020 ◽  
Vol 15 (1) ◽  
pp. 364-372 ◽  
Author(s):  
Jie Li ◽  
Haiyan Shangguan ◽  
Xiaoqian Chen ◽  
Xiao Ye ◽  
Bin Zhong ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Type 2 Diabetes ◽  
Color Doppler ◽  
Enzyme Linked Immunosorbent Assay ◽  
Advanced Glycation ◽  
Advanced Glycation End Product ◽  
Inflammatory Status ◽  
Tnf Α ◽  
Ifn Γ

AbstractDiabetes mellitus with atherosclerosis (AS) adds to the social burden. This study aimed to investigate whether advanced glycation end product (AGE) levels were correlated with inflammation and carotid AS (CAS) in type 2 diabetes mellitus (T2DM) patients. A total of 50 elderly T2DM patients and 50 age-matched senior healthy subjects were recruited in this study. T2DM patients were classified into two groups based on the intima–media thickness (IMT) of the carotid artery from color Doppler ultrasonography. Patients with IMT > 1 mm were classified into the T2DM + CAS group (n = 28), and patients with IMT < 1 mm were assigned as the T2DM + non-atherosclerosis (NAS) group (n = 22). The plasma levels of AGEs, receptor for AGE (RAGE), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) of all subjects were measured by enzyme-linked immunosorbent assay. The T-lymphocyte subsets were analyzed by a flow detector. T2DM + CAS patients showed significantly higher concentrations of AGEs, RAGE, TNF-α, and IFN-γ in the peripheral blood. The highest levels of CD4+ T cells were observed in the T2DM + CAS group. The AGE level was positively correlated with the concentrations of RAGE, TNF-α, IFN-γ, and CD4+. In summary, the results showed that the levels of AGEs may be correlated with the inflammatory status in T2DM patients with CAS.

scholarly journals The frequency of Tim-3 on circulating Tfh cells was increased in type 2 diabetes mellitus patients

2020 ◽  
Vol 18 ◽  
pp. 205873922098280
Author(s):  
Shuai Guo ◽  
Xujie Yu ◽  
Limei Wang ◽  
Jing Jing ◽  
Yuanyuan Sun ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Type 2 Diabetes ◽  
Type 2 Diabetes Mellitus ◽  
High Glucose ◽  
C Peptide ◽  
Glucose Levels ◽  
Tfh Cells ◽  
Fasting Plasma ◽  
Healthy Donors

Type 2 diabetes mellitus (T2DM) is a chronic, low-grade inflammation disease. T follicular helper (Tfh) cells and T cell immunoglobulin and mucin domain 3 (Tim-3) are implicated in many immune diseases. This study aims to explore whether Tim-3 expression on Tfh cells is associated with T2DM progression. White blood cells (WBCs) were harvested from 30 patients with T2DM and 20 healthy donors. The abundance of circulating Tfh cells (cTfh) and the frequency of Tim-3 were analyzed by flow cytometry. Levels of fasting plasma glucose (FPG), insulin, hemoglobin A1C (HbA1C), and fasting plasma C-peptide were measured. Body mass index (BMI) and diabetes duration were also recorded. Patients with T2DM had higher numbers of cTfh cells. In addition, cTfh cells showed a negative correlation with HbA1C and diabetes duration, a positive correlation with fasting plasma C-peptide. The frequency of Tim-3 on cTfh cells was higher among T2DM patients compared with healthy donors. The in vitro experiment showed that high glucose levels increased the abundance cTfh cells but had no effect on Tim-3 expression. Our results suggest that cTfh cells and associated Tim-3 frequency may contribute to the progression of T2DM, and high glucose levels may influence cTfh cells directly.

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