First direct assay for intact human proinsulin

Abstract We describe a sensitive two-site sandwich enzyme-linked immunosorbent assay for the measurement of intact human proinsulin in 100 μL of serum or plasma. The assay is based on the use of two monoclonal antibodies specific for epitopes at the C-peptide/insulin A chain junction and at the insulin B chain/C-peptide junction, respectively. Cross-reactivities with insulin, C-peptide, and the four proinsulin conversion intermediates were negligible. The detection limit in buffer was 0.2 pmol/L (3 standard deviations from zero). The working range was 0.2–100 pmol/L. The mean intra- and interassay coefficients of variation were 2.4% and 8.9%, respectively. The mean recovery of added proinsulin was 103%. Dilution curves of 40 serum samples are parallel to the proinsulin calibration curve. Proinsulin concentrations in 20 fasting healthy subjects were all above the limit of detection: median (range), 2.7 pmol/L (1.1–6.9 pmol/L). Six fasting non-insulin-dependent diabetes mellitus and five insulinoma patients had proinsulin concentrations significantly higher than healthy subjects: median (range), 7.7 pmol/L (3.2–18 pmol/L) and 153 pmol/L (98–320 pmol/L), respectively.

Download Full-text

Viral Protein VP4 Is a Target of Human Antibodies Enhancing Coxsackievirus B4- and B3-Induced Synthesis of Alpha Interferon

Journal of Virology ◽

10.1128/jvi.79.22.13882-13891.2005 ◽

2005 ◽

Vol 79 (22) ◽

pp. 13882-13891 ◽

Cited By ~ 32

Author(s):

Wassim Chehadeh ◽

Pierre-Emmanuel Lobert ◽

Pierre Sauter ◽

Anne Goffard ◽

Bernadette Lucas ◽

...

Keyword(s):

Healthy Subjects ◽

Neutralizing Antibodies ◽

Mononuclear Cells ◽

Insulin Dependent Diabetes Mellitus ◽

Alpha Interferon ◽

Dependent Diabetes Mellitus ◽

Enzyme Linked Immunosorbent Assay ◽

Peripheral Blood Mononuclear ◽

Coxsackievirus B4

ABSTRACT Coxsackievirus B4 (CVB4)-induced production of alpha interferon (IFN-α) by peripheral blood mononuclear cells (PBMC) is enhanced in vitro by nonneutralizing anti-CVB4 antibodies from healthy subjects and, to a higher extent, from patients with insulin-dependent diabetes mellitus. In this study, we focused on identification of the viral target of these antibodies in CVB systems. High levels of IFN-α were obtained in supernatants of PBMC incubated with CVB4E2 or CVB3 and plasma from healthy subjects and, to a higher extent, from patients. The VP4 capsid proteins dissociated by heating at 56°C from CVB4E2 (VP4CVB4) and CVB3 (VP4CVB3) but not H antigen preincubated with plasma from healthy subjects or patients inhibited the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-α synthesis. There was no cross-reaction between VP4CVB4 and VP4CVB3 in the inhibiting effect. IFN-α levels in culture supernatants showed dose-dependent correlation with anti-VP4 antibodies eluted from plasma specimens using VP4-coated plates. There were higher index values for anti-VP4 antibodies detected by enzyme-linked immunosorbent assay (ELISA) and higher proportions of positive detection in 40 patients than in 40 healthy subjects (80% versus 15% for anti-VP4CVB4). There was no relationship between the levels of anti-CVB neutralizing antibodies and the detection of anti-VP4 antibodies by ELISA. The CVB plasma-induced IFN-α levels obtained in PBMC cultures in the anti-VP4 antibody-positive groups were significantly higher than those obtained in the anti-VP4 antibody-negative groups regardless of the titers of anti-CVB neutralizing antibodies. These results show that VP4 is the target of antibodies involved in the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-α synthesis by PBMC.

Download Full-text

Insulin release and pancreatic insulin is reduced in young prediabetic BB rats

Acta Endocrinologica ◽

10.1530/acta.0.1120367 ◽

1986 ◽

Vol 112 (3) ◽

pp. 367-371 ◽

Cited By ~ 16

Author(s):

Annette Svenningsen ◽

Thomas Dyrberg ◽

Helle Markholst ◽

Christian Binder ◽

Åke Lernmark

Keyword(s):

Insulin Release ◽

Insulin Dependent Diabetes Mellitus ◽

Insulin Content ◽

Dependent Diabetes Mellitus ◽

Clinical Onset ◽

Pancreatic Insulin ◽

Pancreatic Insulin Content ◽

Bb Rats ◽

Insulin Dependent ◽

The Mean

Abstract. The pancreases of approximately 50 days old diabetes-prone BB/Hagedorn (BB/H) and of the genetically closely related, but non-diabetic BB w-subline (control BB) rats were perfused to determine the capacity of D-glucose to release insulin before the expected development of diabetes. The BB/H rats were from a colony with 82–84% incidence of insulin-dependent diabetes mellitus (IDDM) by 140 days of age. The total amount of insulin released from the BB/H rat pancreas during stimulation with 20 mmol/l D-glucose was reduced by nearly 50% (P <0.01). The initial peak of insulin release was similar between the two groups of animals, whereas the amount of insulin released during the second peak accounted for the diminished release (P < 0.01). The extractable pancreatic insulin was 30% (P < 0.05) less in the BB/H rats. Total insulin release expressed relative to the pancreatic insulin content, was therefore not different between the two groups. It is concluded that about 20–40 days before the mean age of clinical onset of IDDM in BB/H rats, the capacity to release insulin in response to D-glucose is reduced along with a diminished pancreatic insulin content. This abnormality seems to be preceded only by islet cell surface antibodies but not by insulitis.

Download Full-text

A Simple and Rapid Light-Initiated Chemiluminescence Assay for Quantitation of Artemisia-Specific Immunoglobulin E

International Archives of Allergy and Immunology ◽

10.1159/000520511 ◽

2021 ◽

pp. 1-8

Author(s):

Dandan Liu ◽

Bei Zhang ◽

Lina Zhu ◽

Lisheng Zheng ◽

Shaoshen Li ◽

...

Keyword(s):

Food Allergen ◽

Clinical Guideline ◽

Immunoglobulin E ◽

Limit Of Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Chemiluminescence Assay ◽

Limit Of Quantitation ◽

Specific Immunoglobulin E ◽

Specific Immunoglobulin

Background: Light-initiated chemiluminescence assay (LICA) is a homogeneous assay that has been successfully used for the quantitation of food allergen-specific immunoglobulin E (sIgE), but not inhaled allergen-sIgE. Simultaneously, current assays used to detect allergen-sIgE are serum consuming and/or time consuming. Hence, we established a method for the quantitation of Artemisia-sIgE based on LICA and verified its performance according to the clinical guideline documents, laying a foundation for the quantitation of inhaled and food allergen-sIgE in parallel on LICA. Methods: The assay was established after optimizing the first incubation time and the dilutions of Artemisia-coated chemibeads, biotinylated goat anti-human IgE, and serum. In order to quantitate Artemisia-sIgE, the calibration curve was established with a high positive serum of known concentration. The assay performance was confirmed per the clinical guideline documents. In addition, the correlation between the results of LICA and capture enzyme-linked immunosorbent assay was evaluated. Results: The developed LICA’s coefficients of variation of repeatability and intermediate precision were 3.20%, 2.14%, and 3.85% and 4.30%, 4.00%, and 4.40%, respectively. The limit of detection was 0.10 kUA/L, and the limit of quantitation was 0.11 kUA/L. The range of linearity was from 0.27 kUA/L to 97.53 kUA/L (r = 0.9968). The correlation coefficient (r) for the correlation analysis between results of LICA and capture ELISA was 0.9087. This assay was successfully applied in 64 human serum samples, showing good sensitivity (82.20%) and specificity (100%). Conclusion: An Artemisia-sIgE quantitation assay based on LICA was successfully established. Its performance satisfied the clinical requirements and could be widely used in clinical laboratories.

Download Full-text

Urine CA-2 as a biomarker for diagnosis urinary stone and prediction of its complications

10.21203/rs.3.rs-59394/v1 ◽

2020 ◽

Author(s):

Yuan-jing Leng ◽

Hai-bin Zhou ◽

Jiang-ling Fu ◽

Wen-juan Wang

Keyword(s):

Healthy Subjects ◽

Characteristic Curve ◽

Urinary Stone ◽

Urinary Stones ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Healthy Control ◽

The Mean ◽

The Difference ◽

The Relationship

Abstract PURPOSECarbonic anhydrase-2 (CA-2) plays a role in mineralization and calcification in organism. Strong evidence suggests that CA-2 is associated with urolithiasis. However, the relationship between CA-2 and urinary stone remains unclear. The study aimed to assess the association of urine CA-2 (uCA-2) level and the potential risk of urinary stone.METHODSFrom March 2017 to November 2019, a prospective cohort study was conducted on patients with urinary stones and healthy subjects to determine the pretreatment uCA-2 level detection by Enzyme linked immunosorbent assay (ELISA). The difference of uCA-2 level between patients with urinary stones and healthy subjects was compared. Then comparison between stone patients with complications and without complications was carried out as well as correlation analysis to detect factors associated with biomarker expression.RESULTS118 patients with urinary stones were into urinary stones group and 42 healthy subjects were into healthy control group. The mean pretreatment uCA-2 level was significantly higher in patients with urinary stones group than healthy controls group (P=0.028). Furthermore, The uCA-2 level was positive correlation with urinary stones complications (R=0.379, P=0.000), especially pain complications (R=0.524, P=0.000) and hematuria complications (R=0.374, P=0.000). Receiver operating characteristic curve (ROC) analysis that a uCA-2 level threshold of 10.94 ng/mL had 83.67% sensitivity and 68.12% specificity for predicting urinary stones complications. CONCLUSIONSExcessive uCA-2 excretion is a major risk factor for urinary stone. Our findings suggested that uCA-2 may be used as an unappreciated biomarker for the diagnosis urinary stone in patients and to predict its complications.

Download Full-text

Detection of antibodies to recombinant truncated flagellin and sonicated whole cell antigen of Burkholderia pseudomallei in acute melioidosis and in healthy Bangladeshi individuals

IMC Journal of Medical Science ◽

10.3329/imcjms.v14i1.47455 ◽

2020 ◽

Vol 14 (1) ◽

pp. 47-52

Author(s):

Md Shariful Alam Jilani ◽

Tang Thean Hock ◽

Sraboni Mazumder ◽

Fahmida Rahman ◽

Md Mohiuddin ◽

...

Keyword(s):

Rural Areas ◽

Burkholderia Pseudomallei ◽

Enzyme Linked Immunosorbent Assay ◽

Igg Antibodies ◽

Healthy Individuals ◽

Serum Samples ◽

Igg Antibody ◽

Whole Cell ◽

Cell Antigen ◽

The Mean

Background and objectives: Several types of Burkholderia pseudomallei antigens have been used to determine the antibody response in acute and asymptomatic cases. In the present study, we have detected immunoglobulin G (IgG) antibody to recombinant truncated flagellin antigen (RTFA) of B. pseudomallei in the sera of acute melioidosis cases and healthy individuals from melioidosis endemic areas of Bangladesh by indirect enzyme-linked immunosorbent assay (ELISA). In parallel, IgG antibody to sonicated whole cell antigen (SWCA) of B. pseudomallei was determined to compare with anti-RTFA antibody. Methodology: Serum samples from culture confirmed melioidosis cases and from healthy individuals aged 21 years and above residing in melioidosis endemic rural areas were included in the study. Serum IgG antibody to RTFA and SWCA of B. pseudomallei was determined by indirect ELISA. Results: Out of 8 culture confirmed acute melioidosis cases, 7 (87.5%) and 8 (100%) were positive for anti-B. pseudomallei IgG antibodies by RTFA and SWCA methods respectively. Among 361 healthy individuals, the rate of seropositivity by RTFA-ELISA was significantly less than that of SWCA-ELISA (16.1% versus 26.8%; p = 0.001). The mean optical density (OD) of RTFA-ELISA of positive cases was significantly less than that of SWCA-ELISA in both melioidosis and healthy individuals (0.79±0.11 versus 2.4±0.08, p = 0.0001; 0.67±0.01 versus 1.27±0.02, p = 0.0001). The sensitivity and specificity of RTFA-ELISA were 88.9% and 100% respectively. Conclusion: Findings of the study suggest that multiple or combination of antigens should be used to study the seroprevalence of B. pseudomallei infection in a community. Also, prospective study is necessary to find out the duration of persistence of antibodies to different antigenic components of B. pseudomallei after exposure. Ibrahim Med. Coll. J. 2020; 14(1): 47-52

Download Full-text

High 24-Hour Urinary C-Peptide Excretion in Non-Insulin Dependent Diabetes Mellitus

The Korean Journal of Internal Medicine ◽

10.3904/kjim.1986.1.2.172 ◽

1986 ◽

Vol 1 (2) ◽

pp. 172-178

Author(s):

Young Hwan Chung ◽

Kyong Soo Park ◽

Ki Up Lee ◽

Seong Yeon Kim ◽

Hong Kyu Lee ◽

...

Keyword(s):

Diabetes Mellitus ◽

Insulin Dependent Diabetes Mellitus ◽

Insulin Dependent Diabetes ◽

Dependent Diabetes Mellitus ◽

C Peptide ◽

Insulin Dependent

Download Full-text

Foodborne Snow Mountain Agent Gastroenteritis in a School Cafeteria

PEDIATRICS ◽

10.1542/peds.79.4.559 ◽

1987 ◽

Vol 79 (4) ◽

pp. 559-563

Author(s):

Charles Guest ◽

Kenneth C. Spitalny ◽

H. Paul Madore ◽

Katherine Pray ◽

Raphael Dolin ◽

...

Keyword(s):

Relative Risk ◽

Strong Association ◽

Case Definition ◽

Enzyme Linked Immunosorbent Assay ◽

Fisher Exact Test ◽

Confidence Limits ◽

Serum Samples ◽

Exact Test ◽

Relative Risks ◽

The Mean

In 1984, an outbreak of gastroenteritis occurred at a school with 1,860 students in Brooklyn, NY. In a single-stage cluster sample of 375 students, 129 (34%) had illnesses that met our case definition of vomiting or diarrhea. The mean incubation period was 26 hours, and the mean illness duration was 24 hours. All case students had eaten in the cafeteria on at least one day between Nov 13 and 16, compared with 174/214 (81%) noncase students (P = 10-8, Fisher exact test). Foods implicated were french fries (relative risk 1.7, 95% confidence limits 1.4, 2.0) and hamburgers (relative risk 1.6, 95%, confidence limits 1.2, 2.1). Two cafeteria employees had served those foods while affected by diarrhea. By a recently developed blocking enzyme-linked immunosorbent assay, six of 11 (55%) case students showed fourfold antibody increases between acute-and convalescent-phase serum samples for Snow Mountain agent, a Norwalk-like virus, compared with one of ten (10%) noncase students (P = .04, Fisher exact test). We strongly suspect, but cannot document conclusively, that the Snow Mountain agent was spread to students on a vector of hot foods contaminated by ill food handlers. Implicated foods conferred low relative risks and could only have accounted for 74% of cases of illness. The strong association between cafeteria exposure and illness, therefore, suggests that additional modes of spread occurred.

Download Full-text

P–314 Serum Stem Cell Factor as a predictor of top quality blasotcysts formation in women with endometriosis

Human Reproduction ◽

10.1093/humrep/deab130.313 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

J Liss ◽

M Kuczynska ◽

A Knight ◽

K Lukaszuk

Keyword(s):

Stem Cell ◽

Ovarian Stimulation ◽

Stem Cell Factor ◽

Enzyme Linked Immunosorbent Assay ◽

Tyrosine Kinase Receptor ◽

Target Cells ◽

Serum Samples ◽

The Mean ◽

Ovary Reserve

Abstract Study question To evaluate the correlation between the serum level of stem cell factor (s-SCF) during the stimulation and results of embryo culture. Summary answer The serum SCF concentration at the stimulation stage may be a potential predictor of IVF outcome in endometriosis patients. What is known already Stem cell factor (SCF) is a pleiotropic cytokine that affects the target cells via the c-kit receptor, a tyrosine kinase receptor. Recent evidence indicates that SCF and c-kit may play a role in regulation and growth of ovarian follicular function. It is unclear whether endometriosis primarily affects in vitro fertilization outcomes via oocyte quality. SCF is produced during the human follicular phase, immediately before the ovulatory phase, and may play an important role in folliculogenesis and in the mechanism of ovulation. It may reflect a successful stimulation with ample follicle maturation. Study design, size, duration This was a prospective case-control study and consisted four group of patients: 10 with endometriosis, 24 PCOs, 20 with normal (AMH 1.2–4.0 ng/ml) and 11 with lower (AMH<1.2 ng/ml) ovary reserve who were undergoing IVF treatment with the assessment of serum SCF concentration between August 2019 and March 2020 at INVICTA Fertility Centre, Poland. The age of the patients ranged from 22 to 42 years (median 34 years). Participants/materials, setting, methods s-SCF was measured in duplicate by enzyme-linked immunosorbent assay (ELISA) kit in 195 serum samples collected during ovarian stimulation on days 1 and 8 and on the day of oocyte retrieval. We analysed correlation between s-SCF level and formation of top quality (TQ) blastocysts on day 5 formation in the study groups. Main results and the role of chance We have compared mean level of s-SCF within each group dividing the patients into two subgroups – those with at least one TQ blastocyst (TQ) on day 5 vs. those with no TQ blastocysts (no-TQ). There were no significant differences in mean s-SCF level on day 1 of stimulation between no-TQ and TQ patients in PCOs, normal and lower ovary reserve groups (41.1 pg/ml vs. 40.9 pg/ml; 34.8 pg/ml vs. 38.9 pg/ml and 32.3pg/ml vs. 28.7 pg/ml respectively). The mean level of s-SCF in endometriosis patients was higher in case of no-TQ compared to the TQ subgroup and were 41.1 pg/ml and 29.1 pg/ml respectively. Also no significant differences were also observed in the mean level of s-SCF in the no-TQ and TQ subgroups on the 8 day of stimulation and pick-up in PCOs, normal and lower ovary reserve patients. However, again in the case of endometriosis patients, the mean level of s-SCF was significantly lower on the 8 day of stimulation (28.1 pg/ml vs. 49.1 pg/ml; p < 0.05) and pick-up day (33.4 pg/ml vs. 50.4 pg/ml; p < 0.005) in samples from patients who had at least one TQ blastocysts on day 5 of culture. Limitations, reasons for caution More data are required to confirm the corelation of s-SCF level and presence of top quality blastocysts in patients with endometriosis. Wider implications of the findings: Our study suggests that the level of serum SCF during ovarian stimulation in patients with endometriosis of less 30 pg/ml may potentially be a predictor for the chance of obtaining at least one top quality blastocyst on day 5 and thus a chance to successful treatment. Trial registration number Not applicable

Download Full-text