Calibration–free concentration analysis for quantification of anti-drug specific antibodies in polyclonal positive control antibodies and in clinical samples

2021 ◽  
pp. 113002
Author(s):  
Christina Aniol-Nielsen ◽  
Henrik Toft-Hansen ◽  
Madeleine Dahlbäck ◽  
Claus Henrik Nielsen ◽  
Helene Solberg
Keyword(s):  
Positive Control ◽  
Clinical Samples ◽  
Free Concentration ◽  
Specific Antibodies ◽  
Calibration Free

scholarly journals ANTIBACTERIAL RESISTANCE PATTERN OF PSEUDOMONAS AERUGINOSA ISOLATED FROM CLINICAL SAMPLES AT A GENERAL HOSPITAL IN PADANG, WEST SUMATRA, INDONESIA

2017 ◽  
Vol 10 (8) ◽  
pp. 158
Author(s):  
Rustini Rustini ◽  
Jamsari Jamsari ◽  
Marlina Marlina ◽  
Nasrul Zubir ◽  
Yori Yuliandra
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Resistance ◽  
Clinical Sample ◽  
Opportunistic Pathogen ◽  
Positive Control ◽  
Diffusion Method ◽  
Clinical Samples ◽  
Resistance Pattern ◽  
Tract Infections ◽  
Almost All

Objectives: Pseudomonas aeruginosa is an opportunistic pathogen that has an innate resistance to some antibiotics. This bacterium is one of the mostcommon causes of nosocomial infections that include surgical wound infections, burns, and urinary tract infections. The bacteria have been reportedlyresistant to many antibiotics and have developed multidrug resistance (MDR). The objective of the study was to determine the resistance pattern ofP. aeruginosa isolated from clinical samples of patients against some major antibiotics.Methods: Isolates of P. aeruginosa were obtained from clinical sample of urine, sputum, swabs, pus, feces, and blood and cultured in cetrimide agar. P.aeruginosa ATCC 27853 was used as a positive control. The antibacterial susceptibility testing was conducted against 13 antibiotics: Ceftazidime, cefotaxime,ceftriaxone, cefoperazone, ciprofloxacin, levofloxacin, ofloxacin, gentamicin, amikacin, piperacillin, ticarcillin, meropenem, and imipenem. The examinationwas carried out using agar diffusion method of Kirby-Bauer and following the standards from Clinical and Laboratory Standards Institute (CLSI).Results: The results showed that bacterial resistance was established against all tested antibiotics. The highest number of resistance was shownagainst ceftriaxone (44.21%), whereas the most susceptibility was exhibited against amikacin (only 9.47% of resistance). MDR P. aeruginosa (MDRPA)was detected on almost all clinical samples tested, except the feces. The sample with the highest percentage of MDRPA was the pus.Conclusion: The study concludes that the most effective antibiotic against P. aeruginosa is amikacin (91.51%), whereas the most resistance is exhibited to ceftriaxone (43.16%).

scholarly journals Clinical Assessment of a Nocardia PCR-Based Assay for Diagnosis of Nocardiosis

2018 ◽  
Vol 56 (6) ◽  
Author(s):  
Claire Rouzaud ◽  
Véronica Rodriguez-Nava ◽  
Emilie Catherinot ◽  
Frédéric Méchaï ◽  
Emmanuelle Bergeron ◽  
...  
Keyword(s):  
Clinical Sample ◽  
Positive Control ◽  
Negative Control ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
Underlying Condition ◽  
Alternative Diagnosis ◽  
Specificity And Sensitivity ◽  
Assay Result

ABSTRACT The diagnosis of nocardiosis, a severe opportunistic infection, is challenging. We assessed the specificity and sensitivity of a 16S rRNA Nocardia PCR-based assay performed on clinical samples. In this multicenter study (January 2014 to April 2015), patients who were admitted to three hospitals and had an underlying condition favoring nocardiosis, clinical and radiological signs consistent with nocardiosis, and a Nocardia PCR assay result for a clinical sample were included. Patients were classified as negative control (NC) (negative Nocardia culture results and proven alternative diagnosis or improvement at 6 months without anti- Nocardia treatment), positive control (PC) (positive Nocardia culture results), or probable nocardiosis (positive Nocardia PCR results, negative Nocardia culture results, and no alternative diagnosis). Sixty-eight patients were included; 47 were classified as NC, 8 as PC, and 13 as probable nocardiosis. PCR results were negative for 35/47 NC patients (74%). For the 12 NC patients with positive PCR results, the PCR assay had been performed with respiratory samples. These NC patients had chronic bronchopulmonary disease more frequently than did the NC patients with negative PCR results (8/12 patients [67%] versus 11/35 patients [31%]; P = 0.044). PCR results were positive for 7/8 PC patients (88%). There were 13 cases of probable nocardiosis, diagnosed solely using the PCR results; 9 of those patients (69%) had lung involvement (consolidation or nodule). Nocardia PCR testing had a specificity of 74% and a sensitivity of 88% for the diagnosis of nocardiosis. Nocardia PCR testing may be helpful for the diagnosis of nocardiosis in immunocompromised patients but interpretation of PCR results from respiratory samples is difficult, because the PCR assay may also detect colonization.

scholarly journals Design and Evaluation of a Multiplexed Assay to Assess Human Immunogenicity Against Humira®

2020 ◽  
Vol 22 (5) ◽  
Author(s):  
Matthew Alleyn ◽  
Kristin Closson ◽  
Adam Gentile ◽  
Nathan Gulbis ◽  
Christopher Taylor ◽  
...  
Keyword(s):  
Immune Responses ◽  
Drug Efficacy ◽  
Clinical Samples ◽  
Screening Assay ◽  
Specific Antibodies ◽  
Tiered Approach ◽  
Clinical Events ◽  
Industry Standards ◽  
Recommended Guidelines ◽  
Immunogenicity Assays

Abstract The use of biologic-based therapeutics has revolutionized our ability to treat complex diseases such as cancer- and autoimmune-related disorders. Biologic-based therapeutics are known to generate anti-drug immune responses or immunogenicity in clinical patients which can lead to altered pharmacokinetics, decreased drug efficacy, and unwanted adverse clinical events. Assays designed to detect and assess anti-drug immune responses are used to help monitor patients and improve drug safety. Utilizing a tiered approach, screening assays are developed first to identify patients that are potentially positive for anti-drug-specific antibodies. Patients that screen positive are subjected to additional tiers of testing that include a confirmation assay to confirm the presence of expected anti-drug-specific antibodies, a titer assay to assess relative levels of anti-drug-specific antibodies, and, depending on the drug’s mechanism of action or concerns of adverse clinical reactions, further characterization such as drug neutralization and anti-drug antibody isotyping. This tiered approach can prove to be detrimental to clinical samples from exposure to multiple cycles of testing, freeze thaws, and repeated handling by lab personnel. Multiplexing some of these assays together may streamline the characterization of anti-drug immune responses and help reduce the repeated usage of clinical samples. In this study, we combined a screening assay and anti-drug isotyping assays into one multiplexed assay using the Luminex® xMAP® Technology. The multiplexed assay was developed and validated to meet the FDA recommended guidelines for immunogenicity assessments. These results show that multiplexed assays perform comparably to industry standards. This study should encourage labs to explore the use of multiplexing immunogenicity assays to characterize anti-drug antibody responses quickly, with less repeat testing and reduced sample handling.

Calibration free concentration analysis by surface plasmon resonance in a capture mode

Talanta ◽  
2016 ◽  
Vol 148 ◽  
pp. 478-485 ◽  
Author(s):  
Jonathan Visentin ◽  
Laetitia Minder ◽  
Jar-How Lee ◽  
Jean-Luc Taupin ◽  
Carmelo Di Primo
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Free Concentration ◽  
Calibration Free

scholarly journals High Number of RNA Copies in Asymptomatic Individuals Infected with SARS-CoV-2 in an Area of the Colombian Caribbean

2020 ◽  
Author(s):  
Salim Mattar ◽  
Caty Martinez-Bravo ◽  
Ricardo Rivero ◽  
Hector Contreras ◽  
Alvaro Faccini-Martinez ◽  
...  
Keyword(s):  
Public Health ◽  
Real Time ◽  
Target Genes ◽  
Simultaneous Detection ◽  
Positive Control ◽  
Clinical Samples ◽  
Viral Kinetics ◽  
Standard Curve ◽  
Polymerase Chain ◽  
Asymptomatic Individuals

Abstract Background. Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) is an emerging pandemical virus that has caused millions of reported cases and hundreds of thousands of deaths in less than six months. South America has suffered the pandemic because it lacks the hospital and economic capacities needed to contain the pandemic's advance. Public health implications of transmission, while asymptomatic is a critical concern at the current pandemic.Objective: Describe the socio-demographic, clinical, and viral kinetics features of a cohort of SARS-CoV-2 infected individuals from the Colombian Caribbean. Methods: Six hundred eighty-six clinical samples from several hospital centers in the province were received between April 9th and May 16th, 2020. RNA was extracted using lysis buffers and spin columns. The samples were tested for SARS-CoV-2 by RT-qPCR (Reverse transcription real-time polymerase chain reaction) using commercially available multiplex real-time PCR assay for simultaneous detection of 3 target genes of SARS-CoV-2 (AllplexTM, 2019-nCoV assay, Korea). Viral copies quantification was done using a standard curve constructed from seriated dilutions of a SARS-CoV-2 positive control. Statics descriptive methods were used. Results: Thirty-five nasopharyngeal samples were positive for SARS-CoV-2 infection; the average age was 43 (range, 1-95 years). Seventeen of 35 (49%) of the patients showed symptoms. Most of them had cough, fever, and odynophagia, 3 of the patients reported having arthralgia. Only two patients required hospitalization. None of the patients had known co-morbidities. RT-qPCR results show that two of the symptomatic patients had significantly higher RNA copies than the rest of the patients. Eighteen of 35 (51%) individuals were asymptomatic, the average age was 30 (range, 6-61 years. Four individuals showed a higher copy than some symptomatic patients. Nonetheless, the average of RNA copies 8.26 x10+10 was lower than the symptomatic.Conclusions: the population studied was young with an average of 43 years in symptomatic and 30 years of asymptomatic; this is important because of the high impact in the economy, and probably it is the cause of the reduced lethality observed in the department. Because a large proportion of infections probably result from transmission from asymptomatic or pre-symptomatic persons, the usefulness of public health interventions in Colombian provinces should be based in the molecular screening in a vast conglomerate's population and to quantify the viral load.

scholarly journals Evaluation of bio-conjugation methods for obtaining of synthetic positive control for IgM-capture ELISA

2014 ◽  
Vol 5 (2) ◽  
pp. 85-89
Author(s):  
O. Y. Galkin ◽  
Y. V. Gorshunov
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Horseradish Peroxidase ◽  
Blood Serum ◽  
Positive Control ◽  
Specific Antibodies ◽  
Diagnostic Kits ◽  
Capture Elisa ◽  
Normal Human ◽  
Torch Infections

The enzyme-linked immunosorbent assay (ELISA) is the most informative and versatile method of serological diagnostics. The possibility of detecting by ELISA specific antibodies of different classes allow to differentiate primary infectious process and its remission, exacerbation and chronic disease (differential diagnosis). This approach is implemented in the methodology for evaluation of patients for the presence of humoral immune response to TORCH-infections pathogens (toxoplasmosis, rubella, cytomegalovirus, herpes simplex viruses’ infections, and some others). Therefore, testing for presence of specific IgG and IgM antibodies against TORCH-infections pathogens in blood serum is an important element of motherhood and childhood protection. The essential problem in the production of IgM-capture ELISA diagnostic kits is obtaining of positive control. The classic version of positive control is human blood serum (plasma) containing specific antibodies. But specific IgM-positive sera are insignificant raw materials. This fact can seriously restrict the production of diagnostic kits, especially in the event of large-scale production. We have suggested the methodological approach to using of synthetic positive controls in IgM-capture ELISA kits based on conjugate of normal human IgM and monoclonal antibodies against horseradish peroxidase. It is found that this task can be fulfilled by means of NHS ester-maleimide-mediated conjugation (by sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), reductive amination-mediated conjugation (by sodium periodate) and glutaraldehyde-mediated conjugation. It was found that conjugates of normal human IgM and monoclonal antibodies against horseradish peroxidase obtained using NHS ester-mediated maleimide conjugation and periodate method were similar by molecular weight, whereas conjugate synthesized by glutaraldehyde method comprised at least three types of biopolymers with close molecular weight. It was found that synthetic positive control obtained by different methods was characterized by higher titer compared to IgM-positive high-titer serum. However, positive control obtained by NHS ester-mediated maleimide conjugation had the best titration profile characteristics. 

scholarly journals Molecular Detection of Bartonella spp. in China and St. Kitts

2019 ◽  
Vol 2019 ◽  
pp. 1-9
Author(s):  
Ke Huang ◽  
Patrick John Kelly ◽  
Jilei Zhang ◽  
Yi Yang ◽  
Weiguo Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Bartonella Henselae ◽  
Variable Region ◽  
Positive Control ◽  
Clinical Samples ◽  
Complete Agreement ◽  
Copy Numbers ◽  
Specificity And Sensitivity ◽  
Or Groups ◽  
Pcr Products

Bartonella are vector-borne hemotropic bacteria that infect a wide variety of hosts, including people. While there are PCR assays that can identify individual or groups of Bartonella, there is no reliable molecular method to simultaneously detect all species while maintaining genus specificity and sensitivity. By comparing highly conserved 16S rRNA sequences of the better-recognized Bartonella spp. on GenBank, we selected primers and probes for a genus-specific pan-Bartonella FRET-qPCR. Then, a gltA-based Bartonella PCR was established by selecting primers for a highly variable region of gltA, of which the sequenced amplicons could identify individual Bartonella spp. The pan-Bartonella FRET-qPCR did not detect negative controls (Brucella spp., Anaplasma spp., Rickettsia spp., Coxiella burnetii, and Wolbachia) but reliably detected as few as two copies of the positive control (Bartonella henselae) per reaction. There was complete agreement between the pan-Bartonella FRET-qPCR and the gltA-based Bartonella PCR in detecting Bartonella in convenience test samples from China and St. Kitts: cats (26%; 81/310), Ctenocephalides felis (20%; 12/60), cattle (24%; 23/98), and donkeys (4%; 1/20). Sequencing of the gltA-based Bartonella PCR products revealed B. henselae (70%; 57/81) and B. clarridgeiae (30%; 24/81) in cats and C. felis (67%; 8/12, and 33%; 4/12, respectively) and B. bovis in cattle (23.5%; 23/98) and donkeys (4.0%; 1/24). The pan-Bartonella FRET-qPCR and gltA-based Bartonella PCR we developed are highly sensitive and specific in detecting recognized Bartonella spp. in a single reaction. The pan-Bartonella FRET-qPCR is convenient requiring no gel electrophoresis and providing copy numbers, while the gltA-based Bartonella PCR reliably differentiates individual Bartonella species. The use of these PCRs should greatly facilitate large-scale surveillance studies and the diagnosis of infections in clinical samples.

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