scholarly journals Establishment of a negative-positive threshold optical density value for the enzyme-linked immunosorbent assay (ELISA) to detect soluble antigen of Renibacterium salmoninarum in Alaskan Pacific salmon

1993 ◽  
Vol 16 ◽  
pp. 191-197 ◽  
Author(s):  
TR Meyers ◽  
S Short ◽  
C Farrington ◽  
K Lipson ◽  
HJ Geiger ◽  
...  
Keyword(s):  
Optical Density ◽  
Immunosorbent Assay ◽  
Pacific Salmon ◽  
Enzyme Linked Immunosorbent Assay ◽  
Soluble Antigen ◽  
Renibacterium Salmoninarum ◽  
Positive Threshold

Enzyme-linked Immunosorbent Assay for a Soluble Antigen of Renibacterium salmoninarum, the Causative Agent of Salmonid Bacterial Kidney Disease

1987 ◽  
Vol 44 (1) ◽  
pp. 183-191 ◽  
Author(s):  
R. J. Pascho ◽  
D. Mulcahy
Keyword(s):  
Immunosorbent Assay ◽  
Incubation Temperature ◽  
Tween 80 ◽  
Broth Culture ◽  
Tween 20 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Soluble Antigen ◽  
Renibacterium Salmoninarum ◽  
Bacterial Kidney Disease ◽  
Wash Buffer

A double-antibody enzyme-linked immunosorbent assay (ELISA) for detection of a soluble fraction of Renibacterium salmoninarum was developed from components extracted from the supernatant of an R. salmoninarum broth culture. The Costar® Serocluster™ EIA microplate gave the highest absorbance and signal-to-noise ratios among seven types tested. Including Tween 80 in the wash buffer resulted in higher absorbances than Tween 20 when antigen was present. Background absorbance did not increase when Tween 80 was added to the wash buffer, but did when Tween 80 replaced Tween 20 in antigen and conjugate diluents. Adsorption of coating antibody peaked within 4 h at 37 °C and 16 h at 4 °C. Antigen attachment to antibody-coated microplate wells depended more on incubation temperature than duration; we adopted a 3-h incubation at 25 °C. Conjugate incubation for longer than 1 h at 37 °C or 3 h at 25 °C resulted in unacceptable background levels. No cross-reactions resulted from heat-extracted antigens of 10 other species of bacteria. The optimized ELISA is a 6-h test that enables detection of levels of soluble antigen as low as 2–20 ng.

scholarly journals Comparison of an IgG-Specific Enzyme-Linked Immunosorbent Assay Cutoff of 0.4 Versus 0.8 and 1.0 Optical Density Units for Heparin-Induced Thrombocytopenia

2016 ◽  
Vol 23 (3) ◽  
pp. 282-286 ◽  
Author(s):  
Brianne M. Ritchie ◽  
Jean M. Connors ◽  
Katelyn W. Sylvester
Keyword(s):  
Optical Density ◽  
Immunosorbent Assay ◽  
Predictive Value ◽  
Retrospective Chart Review ◽  
Serotonin Release ◽  
Enzyme Linked Immunosorbent Assay ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Institutional Review ◽  
Sensitivity Specificity

Background: Previous studies have demonstrated optimized diagnostic accuracy in utilizing higher antiheparin–platelet factor 4 (PF4) enzyme-linked immunosorbent assay (ELISA) optical density (OD) thresholds for diagnosing heparin-induced thrombocytopenia (HIT). We describe the incidence of positive serotonin release assay (SRA) results, as well as performance characteristics, for antiheparin–PF4 ELISA thresholds ≥0.4, ≥0.8, and ≥1.0 OD units in the diagnosis of HIT at our institution. Methods: Following institutional review board approval, we conducted a single-center retrospective chart review on adult inpatients with a differential diagnosis of HIT evaluated by both antiheparin–PF4 ELISA and SRA from 2012 to 2014. The major endpoints were to assess incidence of positive SRA results, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy at antiheparin–PF4 ELISA values ≥0.4 OD units when compared to values ≥0.8 and ≥1.0 OD units. Clinical characteristics, including demographics, laboratory values, clinical and safety outcomes, length of stay, and mortality, were collected. Results: A total of 140 patients with 140 antiheparin–PF4 ELISA and SRA values were evaluated, of which 23 patients were SRA positive (16.4%) and 117 patients were SRA negative (83.6%). We identified a sensitivity of 91.3% versus 82.6% and 73.9%, specificity of 61.5% versus 87.2% and 91.5%, PPV of 31.8% versus 55.9% and 63.0%, NPV of 97.3% versus 96.2% and 94.7%, and accuracy of 66.4% versus 86.4% and 88.6% at antiheparin–PF4 ELISA thresholds ≥0.4, ≥0.8, and ≥1.0 OD units, respectively. Conclusion: Our study suggests an increased antiheparin–PF4 ELISA threshold of 0.8 or 1.0 OD units enhances specificity, PPV, and accuracy while maintaining NPV with decreased sensitivity.

scholarly journals Detection of Pseudorabies Virus Infection in Subunit-Vaccinated and Nonvaccinated Pigs using a Nucleocapsid-Based Enzyme-Linked Immunosorbent Assay

1992 ◽  
Vol 4 (2) ◽  
pp. 164-169 ◽  
Author(s):  
M. J. McGinley ◽  
D. L. Todd ◽  
H. T. Hill ◽  
K. B. Platt
Keyword(s):  
Immunosorbent Assay ◽  
Specific Antibody ◽  
Nucleocapsid Protein ◽  
Pseudorabies Virus ◽  
Antibody Responses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Screening Assay ◽  
Virus Specific Antibody ◽  
Positive Threshold ◽  
Virus Exposure

The potential of a pseudorabies virus (PRV) nucleocapsid protein (NC)-based enzyme-linked immunosorbent assay (ELISA) as a screening assay for PRV infection in subunit-vaccinated and nonvaccinated pigs was studied. The NC-ELISA compared favorably to a commercial ELISA for detecting PRV infection in nonvaccinated pigs. Virus-specific antibody was first detected by the NC-ELISA between days 14 and 21 in 5 pigs challenged intranasally with 104PFU of virus. Antibody continued to be detected in these pigs through day 42, when the experiment was terminated. The NC-ELISA also detected antibody in 23 of 24 pigs from PRV-infected herds. In contrast, the commercial ELISA detected antibody 1 week earlier than the NC-ELISA in experimentally infected pigs but failed to detect antibody in 3 naturally exposed pigs that were identified by the NC-ELISA. Infection in these animals was confirmed by radioimmunoprecipitation analysis. The potential usefulness of the NC-ELISA for detecting infection in vaccinated pigs was also evaluated. The nucleocapsid-specific antibody responses of 10 PRV envelope glycoprotein subunit-vaccinated pigs were monitored prior to and following nasal exposure to a low dose (1023PFU) of PRV. Sera were collected periodically for 113 days after infection. Nucleocapsid-specific antibody responses measured by the NC-ELISA remained below the positive threshold before challenge but increased dramatically following virus exposure. Maximum ELISA responses were obtained on day 32 postchallenge (p.c.). Mean ELISA responses decreased thereafter but remained well above the positive threshold on day 113 p.c. PRV nucleocapsid protein can be used effectively as antigen in the ELISA for detecting PRV infection in both nonvaccinated and subunit-vaccinated pigs.

scholarly journals Rubella-specific IgG subclass avidity ELISA and its role in the differentiation between primary rubella and rubella reinfection

1988 ◽  
Vol 101 (3) ◽  
pp. 591-598 ◽  
Author(s):  
H. I. J. Thomas ◽  
P. Morgan-Capner
Keyword(s):  
Optical Density ◽  
Immunosorbent Assay ◽  
Primary Infection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Subclass ◽  
Antibody Avidity ◽  
Specific Antibodies ◽  
Specific Igg ◽  
Protein Denaturant

SUMMARYAn antiglobulin enzyme-linked immunosorbent assay for rubella-specific IgG1and IgG3was adapted to measure antibody avidity by incorporating a mild protein denaturant, diethylamine (DEA), into the serum diluent. Sera were tested at varying dilutions, both with and without DEA, if they contained sufficient specific IgG1or IgG3. The optical density (OD) was measured and curves were plotted. The highest OD (V) was noted and halved (V/2). The distance between the OD curves at V/2 was measured as the DEA shift value.Sera were examined from people whose sera contained rubella-specific antibodies as a consequence of infection or vaccination in the distant past (24 sera), recent primary rubella (66 sera), symptomatic reinfection (11 sera) or asymptomatic reinfection (64 sera). For specific IgG1the DEA shift value was <O·6 for cases of rubella in the distant past, compared with >0·8 for the first month after primary infection. The maximum DEA shift value for the sera from cases of reinfection was 0·65.No serum from cases of rubella in the distant past contained sufficient specific IgG3to estimate avidity. The sera collected within 1 month of onset of primary rubella gave DEA shift values >O·7 compared with sera from reinfections, which gave DEA shift values <O·6, except for two sera from a case of symptomatic reinfection.Thus the assessment of specific IgG subclass avidity is of value in differentiating serologically primary rubella from reinfection.

scholarly journals Identifikasi Penambahan Daging Babi pada Pangan Berbahan Dasar Daging Sapi Menggunakan ELISA dan qPCR

2019 ◽  
Vol 7 (2) ◽  
pp. 17-25
Author(s):  
Diyan Cahyaningsari ◽  
Hadri Latif ◽  
Etih Sudarnika
Keyword(s):  
Real Time ◽  
Optical Density ◽  
Immunosorbent Assay ◽  
Real Time Pcr ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cycle Threshold

Penelitian ini bertujuan untuk mengidentifikasi dan menganalisis penambahan daging babi ternak dan babi hutan baik yang mentah (raw) maupun yang diolah (cooked) di dalam pangan asal hewan berbahan dasar daging sapi menggunakan metode uji enzyme-linked immunosorbent assay (ELISA)  dan real-time PCR (qPCR). Sebanyak 40 sampel yang terdiri dari 20 daging babi ternak dan 20 daging babi hutan dihomogenisasi dengan daging sapi dalam bentuk mentah maupun olahan (bakso). Konsentrasi setiap daging babi ternak dan babi hutan dalam bentuk mentah dan olahan bakso pada campuran daging sapi antara lain 2%, 1%, 0.5%, 0.25%, dan 0.125%. Hasil uji ELISA pada penelitian ini menunjukkan bahwa uji ini mampu mengidentifikasi adanya penambahan daging babi ternak dan babi hutan dalam pangan asal hewan baik dalam bentuk mentah maupun olahan bakso hingga konsentrasi 0.25%. Hasil uji t terhadap nilai optical density (OD) uji ELISA menunjukkan bahwa tidak terdapat perbedaan dalam mendeteksi spesies babi ternak dengan babi hutan (p≥0.05), tetapi terdapat perbedaan pada sampel bentuk mentah dengan bakso (p<0.05). Hasil uji qPCR mampu mengidentifikasi adanya penambahan daging babi ternak dan babi hutan dalam pangan asal hewan baik dalam bentuk mentah maupun olahan bakso hingga konsentrasi 0.125%. Hasil uji t  terhadap nilai cycle threshold (Ct) uji qPCR menunjukkan bahwa tidak terdapat perbedaan dalam mendeteksi spesies babi ternak dengan babi hutan (p≥0.05), tetapi terdapat perbedaan pada sampel bentuk mentah dengan bakso (p<0.05). Metode ELISA dan qPCR dapat dijadikan sebagai metode pengujian untuk mengidentifikasi pencampuran spesies yang tidak dikehendaki, khususnya daging babi pada pangan asal hewan berbahan dasar daging sapi yang beredar di masyarakat.

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