scholarly journals Reducing the risk of misdiagnosis of indirect ELISA by normalizing serum-specific background noise: The example of detecting anti-FGFR3 autoantibodies

2019 ◽  
Author(s):  
Christian Peter Moritz
Keyword(s):  
False Positive ◽  
Background Noise ◽  
Indirect Elisa ◽  
Solid Phase ◽  
False Negative ◽  
Rabbit Antiserum ◽  
Growth Factor Receptor ◽  
Normalization Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
False Positive Diagnosis

Indirect enzyme-linked immunosorbent assay (ELISA) is an important diagnostic method as it enables the quantification of the presence of autoantibodies in human blood sera. However, unspecific binding of antibodies to the solid phase causes considerable serum-specific background noise (SSBN), involving the risk of false positive diagnosis. Therefore, we present a simple and concise, yet obvious proof-of-principle of a recently suggested normalization method. The method is based on subtracting SSBN by using non-coated ELISA wells as a control for each serum-of-interest. We performed ELISA to quantify anti-fibroblast growth factor receptor 3 (FGFR3) antibody levels in three positive controls (two anti-FGFR3-positive patients and a rabbit antiserum against FGFR3) and 58 negative controls (healthy blood donors). In all subjects, we found considerable unspecific reactivity which strongly varied among subjects. The conventional normalization method was not able to balance this strong SSBN, as demonstrated by 2/58 false positive healthy controls and one FGFR3-positive patient that was hidden in the noise (false negative). SSBN normalization reduced the frequency of false-positives to 0/58. Further, all three anti-FGFR3-positive sera were successfully detected and even doubled their z-score used to determine positivity. Albeit occupying more space on the ELISA plate, we strongly recommend considering this normalization method when working with blood sera. To better put the idea across to the community, we depict the SSBN issue and its solution in a graphic scheme. We conclude that SSBN normalization increases the sensitivity and specificity of indirect ELISA and thereby reduces the risk of false positive and false negative diagnosis.

An Enzyme-Linked Immunosorbent Assay for the Detection of Aspergillus parasiticus and Aspergillus flavus

1997 ◽  
Vol 60 (8) ◽  
pp. 978-984 ◽  
Author(s):  
GUO-JANE TSAI ◽  
SHOU-CHIN YU
Keyword(s):  
Aspergillus Flavus ◽  
Immunosorbent Assay ◽  
False Positive ◽  
Aspergillus Parasiticus ◽  
False Negative ◽  
Zealand White Rabbit ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Molecular Weights ◽  
Sds Page

An enzyme-linked immunosorbent assay (ELISA) was established for the specific detection of Aspergillus parasiticus and Aspergillus flavus. A New Zealand white rabbit was immunized intravenously with 100 μg of A. parasiticus CCRC 30117 mycelial protein extracts. The antibodies were separated and purified. The optimal concentration of the antibody and antibody-peroxidase conjugate used in the established ELISA was 10 μg/ml with a detection limit of 1 μg/ml. Among the 126 strains tested (including 21 strains of A. parasiticus, 11 strains of A. flavus, 34 isolates of A. parasiticus/A. flavus from cereals, and 60 strains of non-A. parasiticus/A. flavus fungi), the false-negative and false-positive rates were 1.5 and 3.3%, respectively. Strains of Aspergillus flavofrucatis and Aspergillus sojae produced false-positive reactions. However, their antigens had much lower cross-reactivity with the antibodies raised against A. parasiticus, as shown from I50 values. The molecular weights of the main antigens of A. parasiticus were 94, 82, and 40 kDa. The two heavier antigens had higher sugar contents, as demonstrated by SDS-PAGE and immunoblotting. A good correlation (r = 0.97) was found between mycelium measurement by weighing and by ELISA for A. parasiticus grown in yeast extract sucrose broth (YESB) at 25°C.

scholarly journals Combined Use of Indirect ELISA and Western Blotting with Recombinant Hepatocellular Carcinoma-Associated Antigen 59 Is a Potential Immunodiagnostic Tool for the Detection of Prepatent Haemonchus contortus Infection in Goat

Animals ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 548 ◽  
Author(s):  
Muhammad Ali-ul-Husnain Naqvi ◽  
Sana Zahra Naqvi ◽  
Muhammad Ali Memon ◽  
Kalibixiati Aimulajiang ◽  
Muhammad Haseeb ◽  
...  
Keyword(s):  
Western Blot ◽  
Western Blotting ◽  
Haemonchus Contortus ◽  
Indirect Elisa ◽  
Fasciola Hepatica ◽  
False Negative ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Combined Use

Haemonchus contortus is recognized as one of the important health problems in small ruminants, leading to reduced production and economic loss for farmers worldwide. Prepatent diagnosis of H. contortus infection is crucial to improve control strategies as this helminth may remove up to one-fifth of total erythrocytes and may cause anemia, edema, diarrhea, and ultimately death in young animals. In this study, one of the excretory and secretory products, rHc-HCA59, was purified and used as antigen to detect specific antibodies in H. contortus infected goats during prepatent stage of infection using indirect enzyme linked immunosorbent assay (ELISA) as screening test. All goats (n = 38) were housed indoor, experimentally infected with 8000 infective larvae (L3) of H. contortus, and serum samples were collected prior to infection and at 14th day of infection. Immunoblotting was performed to confirm the results of indirect ELISA, evaluate the cross reactivity against rHc-HCA59 in sera of most common co-infecting parasites and rectify the false negative samples. Furthermore, three different batches of rHc-HCA59 were produced to evaluate the repeatability of ELISA. No eggs were detected in feces of all goats collected at 7th and 14th day of infection but, H. contortus eggs were detected at 21 days post infection in the feces. Indirect ELISA performed in this study showed 87% sensitivity and 100% specificity. The western blot analysis confirmed immunoreactivity in serum samples which scored positive in indirect ELISA and recognized the samples as negative which had OD450 lower than negative cut-off value in indirect ELISA. Furthermore, all false negative sera (n = 5) that had OD450 value between positive and negative cut-off value in rHc-HCA59 based ELISA were clearly positive in western blot. Moreover, no cross-reactivity was detected in ELISA and western blotting against rHc-HCA59 in positive sera of Toxoplasma gondii, Fasciola hepatica, and Trichinella spiralis. The results of this study concluded that combined use of indirect ELISA and western blotting with rHc-HCA59 is a potential immunodiagnostic tool for the detection of H. contortus infection during prepatent period in goats.

Indirect Enzyme-Linked Immunosorbent Assay for Saxitoxin in Shellfish

1985 ◽  
Vol 68 (1) ◽  
pp. 13-16 ◽  
Author(s):  
Fun S Chu ◽  
Titan S L Fan
Keyword(s):  
Detection Limit ◽  
Immunosorbent Assay ◽  
Indirect Elisa ◽  
Solid Phase ◽  
Microtiter Plate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chromogenic Substrate ◽  
Rabbit Igg ◽  
Lower Detection ◽  
Peroxidase Conjugate

Abstract An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of saxitoxin (STX). Antibodies against STX were demonstrated in rabbits 5 weeks after immunizing with STX-bovine serum albumin (STX-HCHO-BSA). In the ELISA, STX-HCHO-BSA or polylysine-STX was coated onto the microtiter plate, followed by incubation with standard toxin and anti-STX antibody. The amount of antibody bound to the solid phase was determined by incubation with goat anti-rabbit IgG peroxidase conjugate and a reaction with chromogenic substrate. Competitive indirect ELISA revealed that the antiserum did not cross-react with either carbamoyl-neo-STX-suIfate or tetrodotoxin. The antibodies for STX cross-reacted with decarbamoyl- STX and neo-STX about 56% and 16% as much as they did with STX, respectively. The lower detection limits for STX, decarbamoyl-STX, and neo-STX in this sytem were about 25, 45, and 156 pg per assay, respectively. When STX added to clams or mussels was assayed, the detection limit for STX was about 50-100 ppb, and recoveries were in the range of 86.8-107%.

scholarly journals Rubella Surveillance and Diagnostic Testing among a Low-Prevalence Population, New York City, 2012–2013

2017 ◽  
Vol 24 (9) ◽  
Author(s):  
Beth M. Isaac ◽  
Jane R. Zucker ◽  
Francesca R. Giancotti ◽  
Emily Abernathy ◽  
Joseph Icenogle ◽  
...  
Keyword(s):  
New York ◽  
New York City ◽  
York City ◽  
False Positive ◽  
Diagnostic Tests ◽  
Indirect Elisa ◽  
Diagnostic Testing ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Department Of Health

ABSTRACT The New York City Department of Health and Mental Hygiene (DOHMH) receives clinical and laboratory reports for rubella. Because rubella immunoglobulin M (IgM) assays may produce false-positive results and rubella infections may be asymptomatic, interpretation of positive IgM results can be challenging. Rubella reports received by DOHMH in 2012 to 2013 were reviewed. The rubella IgM testing purpose was determined through case investigation. Results of IgM testing by indirect enzyme-linked immunosorbent assay (ELISA) and capture enzyme immunoassay (EIA) were compared to determine positive predictive value (PPV) and specificity. DOHMH received 199 rubella reports; 2 were true cases. Of all reports, 77.9% were tested for rubella IgM erroneously, 19.6% were tested for diagnostic purposes, 2.0% had unknown test purpose, and 0.5% were not tested. PPV of indirect ELISA was 6% overall, 14% for diagnostic tests, and 0% for tests ordered erroneously. PPV of capture EIA was 29% overall, 50% for diagnostic tests, and 0% for tests ordered erroneously. Overall, specificity was 52% for indirect ELISA and 85% for capture EIA. Limiting rubella IgM testing to patients for whom rubella diagnosis is suspected and using a more specific IgM assay have the potential to reduce false-positive rubella IgM results.

scholarly journals Rapid Mycobacterial Liquid Culture-Screening Method for Mycobacterium avium Complex Based on Secreted Antigen-Capture Enzyme-Linked Immunosorbent Assay

2009 ◽  
Vol 16 (5) ◽  
pp. 613-620 ◽  
Author(s):  
Sung Jae Shin ◽  
Kelly Anklam ◽  
Elizabeth J. B. Manning ◽  
Michael T. Collins
Keyword(s):  
Immunosorbent Assay ◽  
Mycobacterium Avium ◽  
False Positive ◽  
High Performance ◽  
Liquid Culture ◽  
False Negative ◽  
Screening Method ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Test

ABSTRACT Sensors in automated liquid culture systems for mycobacteria, such as MGIT, BacT/Alert 3D, and Trek ESP II, flag growth of any type of bacteria; a positive signal does not mean that the target mycobacteria are present. All signal-positive cultures thus require additional and often laborious testing. An immunoassay was developed to screen liquid mycobacterial cultures for evidence of Mycobacterium avium complex (MAC). The method, called the MAC-enzyme-linked immunosorbent assay (ELISA), relies on detection of MAC-specific secreted antigens in liquid culture. Secreted MAC antigens were captured by the MAC-ELISA with polyclonal anti- Mycobacterium avium subsp. paratuberculosis chicken immunoglobulin Y (IgY), detected using rabbit anti-MAC IgG, and then revealed using horseradish peroxidase-conjugated goat anti-rabbit IgG. When the MAC-ELISA was evaluated using pure cultures of known mycobacterial (n = 75) and nonmycobacterial (n = 17) organisms, no false-positive or false-negative MAC-ELISA results were found. By receiver operator characteristic (ROC) analysis of 1,275 previously identified clinical isolates, at the assay optimal cutoff the diagnostic sensitivity and specificity of the MAC-ELISA were 92.6% (95% confidence interval [95% CI], 90.3 to 94.5) and 99.9% (95% CI, 99.2 to 100), respectively, with an area under the ROC curve of 0.992. Prospective evaluation of the MAC-ELISA with an additional 652 clinical samples inoculated into MGIT ParaTB medium and signaling positive per the manufacturer's instructions found that the MAC-ELISA was effective in determining those cultures that actually contained MAC species and warranting the resources required to identify the organism by PCR. Of these 652 MGIT-positive cultures, the MAC-ELISA correctly identified 96.8% (of 219 MAC-ELISA-positive cultures) as truly containing MAC mycobacteria, based on PCR or high-performance liquid chromatography (HPLC) as reference tests. Only 6 of 433 MGIT signal-positive cultures (1.4%) were MAC-ELISA false negative, and only 7 of 219 MGIT signal-negative cultures (3.2%) were false positive. The MAC-ELISA is a low-cost, rapid, sensitive, and specific test for MAC in liquid cultures. It could be used in conjunction with or independent of automated culture reading instrumentation. For maximal accuracy and subspecies-specific identification, use of a confirmatory multiplex MAC PCR is recommended.

The doubtful value of tympanometry in the diagnosis of otosclerosis

1985 ◽  
Vol 99 (6) ◽  
pp. 545-547 ◽  
Author(s):  
G. G. Browning ◽  
I. R. C. Swan ◽  
S. Gatehouse
Keyword(s):  
False Positive ◽  
False Negative ◽  
Positive Diagnosis ◽  
False Positive Diagnosis ◽  
Practical Usefulness ◽  
The Mean ◽  
Mean Compliance ◽  
Range Of Values ◽  
Normal Controls ◽  
Made In

AbstractMany consider that the compliance of the middle ear as measured from the tympanogram can be helpful in diagnosing otosclerosis. To test this assertion, the compliance in 34 individuals with surgically proven otosclerosis was compared with the compliance in 34 age and sex matched, normal controls, randomly selected from the population. Though the mean compliance was different in the two groups, there was considerable overlap in the range of values which severely limits the practical usefulness of tympanometry.If the level of compliance is taken at which a false negative diagnosis would be made in 10 per cent of otosclerotic ears, a false positive diagnosis of otosclerosis would be made in 88 per cent of normal ears. If the level of compliance is taken at which a false positive diagnosis of otosclerosis would be made in 10 per cent of normal ears, 72 per cent of ears with otosclerosis would be considered normal.It is concluded that tympanometry will not help to arrive at a diagnosis of otosclerosis.

scholarly journals Color feature extraction of HER2 Score 2+ overexpression on breast cancer using Image Processing

2018 ◽  
Vol 154 ◽  
pp. 03016
Author(s):  
Izzati Muhimmah ◽  
Dadang Heksaputra ◽  
Indrayanti
Keyword(s):  
Image Segmentation ◽  
False Positive ◽  
Gold Standard ◽  
Image Acquisition ◽  
False Negative ◽  
Growth Factor Receptor ◽  
Her2 Status ◽  
Color Features ◽  
Color Deconvolution ◽  
Expert Validation

One of the major challenges in the development of early diagnosis to assess HER2 status is recognized in the form of Gold Standard. The accuracy, validity and refraction of the Gold Standard HER2 methods are widely used in laboratory (Perez, et al., 2014). Method determining the status of HER2 (human epidermal growth factor receptor 2) is affected by reproductive problems and not reliable in predicting the benefit from anti-HER2 therapy (Nuciforo, et al., 2016). We extracted color features by methods adopting Statistics-based segmentation using a continuous-scale naïve Bayes approach. In this study, there were three parts of the main groups, namely image acquisition, image segmentation, and image testing. The stages of image acquisition consisted of image data collection and color deconvolution. The stages of image segmentation consisted of color features, classifier training, classifier prediction, and skeletonization. The stages of image testing were image testing, expert validation, and expert validation results. Area segmentation of the membrane is false positive and false negative. False positive and false negative from area are called the area of system failure. The failure of the system can be validated by experts that the results of segmentation region is not membrane HER2 (noise) and the segmentation of the cytoplasm region. The average from 40 data of HER2 score 2+ membrane images show that 75.13% of the area is successfully recognized by the system.

scholarly journals ELISA versus Nested PCR for Detection of Hepatitis B Co-infection in HIV-infected People in Bandung Indonesia

2021 ◽  
pp. 991-1001
Author(s):  
Patricia Gita Naully
Keyword(s):  
Hepatitis B ◽  
Nested Pcr ◽  
False Positive ◽  
Hepatitis B Surface Antigen ◽  
False Negative ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hiv Patients ◽  
Nested Polymerase Chain Reaction ◽  
Pcr Method ◽  
Sensitivity Specificity

The number of Human Immunodeficiency Virus infected patients in Bandung is increasing. HIV patients are often suffering from Hepatitis B Virusco-infection. The co-infection status must be detected using the accurate method. This study aims to compare the accuracy of Enzyme-Linked Immunosorbent Assay and nested Polymerase Chain Reaction in detecting HIV-HBV co-infection in Bandung. The research method used is experimental. The sample used in this study was 50 people. All samples had met the inclusion criteria. The presence of hepatitis B surface antigen in the serum was detected using a qualitative ELISA Wantai kit, while sHBsAg gene in the whole blood was amplified using 2 pairs of the specific primary. The results obtained from both methods were analyzed to determine sensitivity, specificity, and accuracy values. The result of Hepatitis B detection using ELISA method showed that 15 samples (30%) were positive, 1 sample (2%) was a false positive, and 3 samples (6%) were the false negative. Moreover, the nested PCR method showed that 18 samples (36%) were positive without false positive or false negative result. These results showed that ELISA has a sensitivity value of 83.33%, specificity of 96.87%, and the accuracy of 92%, while Nested PCR has a value of 100% for all three parameters. Therefore, it can be concluded that the Nested PCR method was more accurate than ELISA to detect Hepatitis B co-infection in HIV patients in Bandung.The Nested PCR can be recommended for detecting the other co-infection cases.   Keywords: accuracy, HBsAg, co-infection, sensitivity, specificity

Transvaginal duplex ultrasonography appears to be the gold standard investigation for the haemodynamic evaluation of pelvic venous reflux in the ovarian and internal iliac veins in women

2014 ◽  
Vol 30 (10) ◽  
pp. 706-713 ◽  
Author(s):  
MS Whiteley ◽  
SJ Dos Santos ◽  
CC Harrison ◽  
JM Holdstock ◽  
AJ Lopez
Keyword(s):  
False Positive ◽  
Gold Standard ◽  
False Negative ◽  
Duplex Ultrasonography ◽  
Coil Embolisation ◽  
Pelvic Vein ◽  
False Positive Diagnosis ◽  
Internal Iliac ◽  
Pre Treatment ◽  
Vein Reflux

Objectives To assess the suitability of transvaginal duplex ultrasonography to identify pathological reflux in the ovarian and internal iliac veins in women. Methods A retrospective study of patients treated in 2011 and 2012 was performed in a specialised vein clinic. Diagnostic transvaginal duplex ultrasonography in women presenting with symptoms or signs of pelvic vein reflux were compared with the outcomes of treatment from pelvic vein embolisation. A repeat transvaginal duplex ultrasonography was performed 6 weeks later by a blinded observer and any residual reflux was identified. Results Results from 100 sequential patients were analysed. Mean age 44.2 years (32–69) with mode average parity of 3 (0–5 deliveries). Pre-treatment, 289/400 veins were refluxing (ovarian – 29 right, 81 left; internal iliac – 93 right, 86 left). Coil embolisation was successful in 86/100 patients and failed partially in 14/100 – 5 due to failure to cannulate the target vein. One false-positive diagnosis was made. Conclusion Currently there is no accepted gold standard for pelvic vein incompetence. Comparing transvaginal duplex ultrasonography with the outcome from selectively treating the veins identified as having pathological reflux with coil embolisation, there were no false-negative diagnoses and only one false-positive. This study suggests that transvaginal duplex ultrasonography could be the gold standard in assessing pelvic vein reflux.

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