scholarly journals Analysis of recombinant proteins for Q fever diagnostics

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Halie K. Miller ◽  
Gilbert J. Kersh
Keyword(s):  
Q Fever ◽  
Characteristic Curve ◽  
Bacterial Pathogen ◽  
Immunofluorescence Assay ◽  
Standard Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Assay Sensitivity ◽  
Healthy Donors ◽  
Infected Goat ◽  
The Individual

AbstractSerology is essential for Q fever diagnostics, a disease caused by the bacterial pathogen Coxiella burnetii. The gold standard test is an immunofluorescence assay utilizing whole cell antigens, which are both dangerous and laborious to produce. Complexities of the antigen coupled with the subjective nature of the assay lead to decreased uniformity of test results and underscore the need for improved methodologies. Thirty-three C. burnetii proteins, previously identified as immunoreactive, were screened for reactivity to naturally infected goat serum. Based on reactivity, 10 proteins were analyzed in a secondary screen against human serum from healthy donors. Assay sensitivity and specificity ranged from 21 to 71% and 90 to 100%, respectively. Three promising antigens were identified based on receiver operating characteristic curve analysis (CBU_1718, CBU_0307, and CBU_1398). Five multiplex assays failed to outperform the individual proteins, with sensitivities and specificities ranging from 29 to 57% and 90 to 100%, respectively. Truncating the top antigen, CBU_1718, had no effect on specificity (90%); yet sensitivity decreased dramatically (71% to 21%). Through this study, we have expanded the subset of C. burnetii immunoreactive proteins validated by enzyme-linked immunosorbent assay and demonstrate the effect of novel antigen combinations and protein truncations on assay performance.

scholarly journals Q fever: baseline monitoring of a sheep and a goat flock associated with human infections

2012 ◽  
Vol 140 (11) ◽  
pp. 1939-1949 ◽  
Author(s):  
R. EIBACH ◽  
F. BOTHE ◽  
M. RUNGE ◽  
S. F. FISCHER ◽  
W. PHILIPP ◽  
...  
Keyword(s):  
Q Fever ◽  
Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sheep Flock ◽  
Blood Samples ◽  
Experimental Station ◽  
Animal Infection ◽  
Human Infections ◽  
Goat Flock ◽  
Swabian Alb

SUMMARYAnimal losses due to abortion and weak offspring during a lambing period amounted up to 25% in a goat flock and up to 18% in a sheep flock kept at an experimental station on the Swabian Alb, Germany. Fifteen out of 23 employees and residents on the farm tested positive forCoxiella burnetiiantibodies by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay. Ninety-four per cent of the goats and 47% of the sheep were seropositive forC. burnetiiby ELISA. Blood samples of 8% of goats and 3% of sheep were PCR positive.C. burnetiiwas shed by all tested animals through vaginal mucus, by 97% of the goats and 78% of the sheep through milk, and by all investigated sheep through faeces (PCR testing). In this outbreak human and animal infection were temporally related suggesting that one was caused by the other.

scholarly journals Coxiella burnetii Shedding Routes and Antibody Response after Outbreaks of Q Fever-Induced Abortion in Dairy Goat Herds

2008 ◽  
Vol 75 (2) ◽  
pp. 428-433 ◽  
Author(s):  
Elodie Rousset ◽  
Mustapha Berri ◽  
Benoit Durand ◽  
Philippe Dufour ◽  
Myriam Prigent ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
Complement Fixation ◽  
Q Fever ◽  
Immunofluorescence Assay ◽  
Dairy Goat ◽  
Pcr Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potential Hazard ◽  
Fecal Shedding ◽  
The Relationship

ABSTRACT Q fever is a zoonosis caused by Coxiella burnetii, a bacterium largely carried by ruminants and shed into milk, vaginal mucus, and feces. The main potential hazard to humans and animals is due to shedding of bacteria that can then persist in the environment and be aerosolized. The purpose of this study was to evaluate shedding after an outbreak of Q fever abortion in goat herds and to assess the relationship with the occurrence of abortions and antibody responses. Aborting and nonaborting goats were monitored by PCR for C. burnetii shedding 15 and 30 days after the abortion episodes. PCR analysis of all samples showed that 70% (n = 50) of the aborting and 53% (n = 70) of the nonaborting goats were positive. C. burnetii was shed into vaginal mucus, feces, and milk of 44%, 21%, and 38%, respectively, of goats that aborted and 27%, 20%, and 31%, respectively, of goats that delivered normally. Statistical comparison of these shedding results did not reveal any difference between these two groups. PCR results obtained for the vaginal and fecal routes were concordant in 81% of cases, whereas those for milk correlated with only 49% of cases with either vaginal or fecal shedding status. Serological analysis, using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and complement fixation tests, showed that at least 24% of the seronegative goats shed bacteria. Positive vaginal and fecal shedding, unlike positive milk shedding, was observed more often in animals that were weakly positive or negative by ELISA or IFA. Two opposite shedding trends were thus apparent for the milk and vaginal-fecal routes. Moreover, this study showed that a nonnegligible proportion of seronegative animals that delivered normally could excrete C. burnetii.

Solitary IgM phase II response has a limited predictive value in the diagnosis of acute Q fever

2012 ◽  
Vol 140 (11) ◽  
pp. 1950-1954 ◽  
Author(s):  
C. F. H. RAVEN ◽  
J. L. A. HAUTVAST ◽  
T. HERREMANS ◽  
A. C. A. P. LEENDERS ◽  
P. M. SCHNEEBERGER
Keyword(s):  
Phase Ii ◽  
Predictive Value ◽  
Q Fever ◽  
Immunofluorescence Assay ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Initial Sample ◽  
Acute Q Fever ◽  
Diagnostic Criterion

SUMMARYWe investigated the positive predictive value (PPV) of a solitary positive immunoglobulin M (IgM) phase II response for the serodiagnosis of acute Q fever detected with either an indirect immunofluorescence assay (IFA) or an enzyme-linked immunosorbent assay (ELISA). Initial and follow-up sera from patients suspected of acute Q fever were included if initially only IgM phase II tested positive with IFA in 2008 (n=92), or ELISA in 2009 (n=85). A seroconversion for Q fever was defined as an initial sample being IgG phase II negative but positive in the follow-up sample. The PPV of an initial isolated IgM phase II result detected by IFA or ELISA was 65% and 51%, respectively, and therefore appeared not to adequately predict acute Q fever. For this reason it cannot be used as a diagnostic criterion nor should it be included in public health notification without confirmation with other markers or a follow-up serum sample.

scholarly journals Development of an Enzyme-Linked Immunosorbent Assay-Based Test with a Cocktail of Nucleocapsid and Spike Proteins for Detection of Severe Acute Respiratory Syndrome-Associated Coronavirus-Specific Antibody

2008 ◽  
Vol 16 (2) ◽  
pp. 241-245 ◽  
Author(s):  
Luis G. Giménez ◽  
Jose Rojas ◽  
Almudena Rojas ◽  
Joaquín Mendoza ◽  
Ana G. Camacho
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Immunosorbent Assay ◽  
Immunofluorescence Assay ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Recombinant Polypeptide ◽  
Igm Antibody ◽  
Set Up ◽  
The Individual ◽  
Recombinant Polypeptides

ABSTRACT A new enzyme-linked immunosorbent assay (ELISA)-based immunoglobulin G (IgG)-plus-IgM antibody detection test for severe acute respiratory syndrome (SARS) has been developed by using a cocktail of four recombinant polypeptides as the antigen. These recombinant fragments were designed as parts of two different structural proteins from SARS-associated coronavirus (SARS-CoV). One recombinant polypeptide, S251-683, was designed as part of the spike glycoprotein, and the other three polypeptides comprised almost the whole nucleocapsid protein, avoiding the last 25 C-terminal amino acids. Immunization with a cocktail of these four polypeptides yielded a specific polyclonal antibody that is able to recognize SARS-CoV-infected cells by an immunofluorescence assay. This polypeptide cocktail was also used to set up an ELISA-based IgG-plus-IgM antibody detection test, which showed 99% specificity and 90% sensitivity upon evaluation using sera from 100 healthy negative controls and 20 SARS patients. Separate immunoreactivity assays with each recombinant polypeptide demonstrated that a combination of N and S protein fragments was more suitable than the individual peptides for developing a serological assay for SARS-CoV.

scholarly journals Validation of a Novel Commercial ELISA Test for the Detection of Antibodies against Coxiella burnetii

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1075
Author(s):  
Salvatore Ledda ◽  
Cinzia Santucciu ◽  
Valentina Chisu ◽  
Giovanna Masala
Keyword(s):  
Diagnostic Accuracy ◽  
Phase Ii ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Animal Health ◽  
Elisa Test ◽  
Clinical Diagnostics ◽  
Complex Life Cycle ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specificity And Sensitivity

Q fever is a zoonosis caused by Coxiella burnetii, a Gram-negative pathogen with a complex life cycle and a high impact on public and animal health all over the world. The symptoms are indistinguishable from those belonging to other diseases, and the disease could be symptomless. For these reasons, reliable laboratory tests are essential for an accurate diagnosis. The aim of this study was to validate a novel enzyme-linked immunosorbent assay (ELISA) test, named the Chorus Q Fever Phase II IgG and IgM Kit (DIESSE, Diagnostica Senese S.p.A), which is performed by an instrument named Chorus, a new device in medical diagnostics. This diagnostic test is employed for the detection of antibodies against C. burnetii Phase II antigens in acute disease. Our validation protocol was performed according to the Italian Accreditation Body (ACCREDIA) (Regulation UNI CEI EN ISO/IEC 17025:2018 and 17043:2010), OIE (World Organization for Animal Health), and Statement for Reporting Studies of Diagnostic Accuracy (STARD). Operator performance was evaluated along with the analytical specificity and sensitivity (ASp and ASe) and diagnostic accuracy of the kit, with parameters such as diagnostic specificity and sensitivity (DSp and DSe) and positive and negative predictive values (PPV and NPV), in addition to the repeatability. According to the evaluated parameters, the diagnostic ELISA test was shown to be suitable for validation and commercialization as a screening method in human sera and a valid support for clinical diagnostics.

scholarly journals Etiology, clinical characteristics and coinfection status of bronchiolitis in Suzhou

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jiahong Tan ◽  
Jinfeng Wu ◽  
Wujun Jiang ◽  
Li Huang ◽  
Wei Ji ◽  
...  
Keyword(s):  
Virus Infection ◽  
Clinical Characteristics ◽  
Immunofluorescence Assay ◽  
Clinical Syndrome ◽  
Enzyme Linked Immunosorbent Assay ◽  
Direct Immunofluorescence ◽  
Increased Risk ◽  
Syncytial Virus ◽  
Direct Immunofluorescence Assay ◽  
Mixed Virus Infection

Abstract Background Bronchiolitis is a clinical syndrome commonly encountered in practice, particularly among infants and young children. To investigate the prevalence of pathogens in hospitalized children with bronchiolitis and study the clinical characteristics of bronchiolitis with or without coinfections. Methods We investigated the respiratory specimens and clinical data of 1012 children with bronchiolitis who were treated at the Children’s Hospital of Soochow University between November 2011 and December 2018. The nasopharyngeal aspirates were examined to detect viruses by direct immunofluorescence assay or polymerase chain reaction (PCR). Mycoplasma pneumoniae (MP) was tested by PCR and enzyme-linked immunosorbent assay. Results Of the 1134 children less than 2 years with bronchiolitis, 122 were excluded by exclusion criteria. Causative pathogen was detected in 83.2% (842 of 1012). The majority of these (614 [72.9%] of 842) were single virus infection. The most common pathogens detected were respiratory syncytial virus (RSV) (44.4%), MP (15.6%), and human rhinovirus (HRV) (14.4%). Coinfection was identified in 13.5% (137 of 1012) of the patients. Coinfection included mixed virus infection and virus infection with MP infection. Children with single virus infection had a higher rate of oxygen therapy compared with single MP infection. Conclusions The most common pathogen detected in children with bronchiolitis is RSV, followed by MP and HRV. Coinfection leads to a longer period of illness, increased severity of the symptoms and increased risk of hypoxemia.

scholarly journals Plasma interleukin-17 and alpha-fetoprotein combination effectively predicts imminent hepatocellular carcinoma occurrence in liver cirrhotic patients

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Kung-Hao Liang ◽  
Ming-Wei Lai ◽  
Yang-Hsiang Lin ◽  
Yu-De Chu ◽  
Chih-Lang Lin ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Characteristic Curve ◽  
Alpha Fetoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Interleukin 17 ◽  
Medical Need ◽  
Tumor Tissues ◽  
Resected Tumor ◽  
Cirrhotic Patients ◽  
A Prospective Study

Abstract Background Predicting imminent hepatocellular carcinoma (HCC) in liver cirrhotic patients is an unmet medical need. We aimed to investigate circulatory biomarkers and their optimum combinations in a prospective study. Methods We investigated plasma interleukin 17 (IL-17) concentrations, quantified using enzyme-linked immunosorbent assay (ELISA), for the prediction of HCC in a large cohort of 404 HCC-naïve liver cirrhotic patients regularly followed after recruitment. Additionally, IL-17 in surgically resected tumor tissues were evaluated using immunohistochemistry staining. Results IL-17 was detected in HCC tissues. The IL-17 concentrations in the peripheral blood do not have correlation with an extensive list of 31 common demographic, metabolic and liver function variables in the cohort of liver cirrhotic patients. Furthermore, patients stratified by IL-17 and alpha-fetoprotein (AFP) showed distinctive cumulative incidence of HCC. Imminent HCC, defined here as HCC occurrence within 1 year, can be predicted by IL-17 alone with an area under the receiver operating characteristic curve [AUC] of 0.762 (P = 0.002). An multivariate analysis showed that age, hepatitis C viral infection, AFP and IL-17 were four independent factors associated with imminent HCC (adjusted P = 0.03, 0.041, 0.024 and 0.008 respectively). An explicit risk score (R) combining the concentrations of two plasma biomarkers, AFP and IL-17, achieved a high AUC of 0.933 (95% confidence interval 0.893–0.972, P < 0.001) in predicting imminent HCC, with 100% sensitivity and 79.9% specificity at the optimum cutoff. The score is defined as: $${\text{R}} = (2.6914)*{\text{IL-17}} + (0.3909)*{\text{AFP}} - (0.80812875)*{\text{IL-17}}^{2} + (0.10288876884)*{\text{IL-17}}^{2} *{\text{AFP}}.$$ R = ( 2.6914 ) ∗ IL-17 + ( 0.3909 ) ∗ AFP - ( 0.80812875 ) ∗ IL-17 2 + ( 0.10288876884 ) ∗ IL-17 2 ∗ AFP . Conclusions The circulatory IL-17 concentration is a predictor of subsequent HCC occurrence in liver cirrhotic patients. The combination of AFP and IL-17 is highly effective in predicting imminent HCC within 1 year.

Urine β-2-glycoprotein 1 as a biomarker for diagnosis of systemic lupus erythematosus

Lupus ◽  
2021 ◽  
pp. 096120332110142
Author(s):  
Jung Sun Lee ◽  
Eun-Ju Lee ◽  
Jeonghun Yeom ◽  
Ji Seon Oh ◽  
Seokchan Hong ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Lupus Erythematosus ◽  
Medical Center ◽  
Characteristic Curve ◽  
Area Under The Curve ◽  
Diagnostic Value ◽  
Urine Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Systemic Lupus ◽  
Asan Medical

Objective The need for a biomarker with robust sensitivity and specificity in diagnosing systemic lupus erythematosus (SLE) remains unmet. Compared with blood samples, urine samples are more easily collected; thus, we aimed to identify such a biomarker based on urinary proteomics which could distinguish patients with SLE from healthy controls (HCs). Methods Urine samples were collected from 76 SLE patients who visited rheumatology clinic in 2019 at Asan medical center and from 25 HCs. Urine proteins were analyzed using sequential windowed acquisition of all theoretical fragment ion spectra-mass spectrometry, and the candidate marker was confirmed by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristic curve analysis was used to determine the diagnostic value of the candidate biomarker. Results Of 1157 proteins quantified, 153 were differentially expressed in urine samples from HCs. Among them were previously known markers including α-1-acid glycoprotein 1, α-2-HS-glycoprotein, ceruloplasmin, and prostaglandin-H2 D-isomerase. Moreover, the amount of β-2 glycoprotein (APOH) was increased in the urine of patients with SLE. The ELISA results also showed the level of urine APOH was higher in patients with SLE than in HCs and patients with rheumatoid arthritis. Moreover, the level was not different between SLE patients with and without nephritis. The urine APOH had an area under the curve value of 0.946 at a cut-off value of 228.53 ng/mg (sensitivity 91.5%, specificity 92.0%) for the diagnosis of SLE. Conclusion The results indicate that the urine APOH level can be an appropriate screening tool in a clinical setting when SLE is suspected.

SENSITIVE AND SPECIFIC ENZYME-LINKED IMMUNOSORBENT ASSAY FOR UROKINASE-TYPE PLASMINOGEN ACTIVATOR IN HUMAN PLASMA

1987 ◽  
Author(s):  
J Grøndahl-HANSEN ◽  
N Agerlin ◽  
L S Nielsen ◽  
K Danø
Keyword(s):  
Plasminogen Activator ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mean Values ◽  
Amino Terminal ◽  
Type Plasminogen Activator ◽  
Healthy Donors ◽  
Urokinase Type Plasminogen Activator ◽  
The Mean

An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of human urokinase-type plasminogen activator (u-PA) in plasma and serum. Microtiter plates were coated with a monoclonal antibody and incubated with standard or sample. Bound u-PA was quantitated with polyclonal antibodies conjugated with biotin, followed by avidin-peroxidase. The assay was 10-fold as sensitive as other previously reported ELISAs, the detection limit being approximately 1 pg of u-PA in a volume of 100 μl with a linear dose-response up to 15 pg of u-PA. The assay detected active u-PA and its inactive proenzyme form equally well and the recovery of both forms was higher than 90% in plasma. A variety of structurally related proteins, including t-PA, were tested, but no reaction with proteins other than u-PA and its amino-terminal degradation product were observed. The intra-assay and inter-assay coefficients of variation for determination of u-PA in plasma were 7.6% and 8.4%, respectively. The assay was equally applicable to serum. The values obtained with plasma and serum were similar, and the results were not affected by small variations in the preparation of the samples. The ELISA was used to measure the concentration of u-PA in plasma from 34 healthy donors. The mean values for u-PA in plasma from healthy donors was 1.1 ng/ml ± 0.3 ng/ml (SD) (range 0.6 - 1.5 ng/ml). No significant differences were found between men and women and no correlation between u-PA concentration and age could be demonstrated.The mean u-PA concentration in plasma from healthy donors obtained in this study is substantially lower than that reported by others. This might be due to different methods of determination of the protein content of the standard preparations or to differences in the specificity of the assays.

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