Vyhodnocení ELISA, imunofluorescence a stanovení


Vyhodnocení ELISA, imunofluorescence a stanovení laterálního průtoku pro SARS-CoV-2 sérologické testy 

Background: The SARS-CoV-2 outbreak has emerged at the end of 2019. Aside from the detection of viral genome with specific RT-PCR, there is a growing need for reliable determination of the serological status. We aimed at evaluating five SARS-CoV-2 serology assays.

Methods: An in-house immunofluorescence assay (IFA), two ELISA kits (EUROIMMUN® ELISA SARS-CoV-2 IgG and NovaLisa® SARS-CoV-2 IgG and IgM) and two lateral flow assays (T-Tek® SARS-CoV-2 IgG/IgM Antibody Test Kit and Sure Bio-tech® SARS-CoV-2 IgM/IgG Antibody Rapid Test) were compared on 40 serums from RT-PCR-confirmed SARS-CoV-2 infected patients and 10 SARS-CoV-2 RT-PCR negative subjects as controls.

Results: Control subjects tested negative for SARS-CoV-2 antibodies with all five systems. Estimated sensitivities varied from 35.5 to 71.0% for IgG detection and from 19.4 to 64.5% for IgM detection. For IgG, in-house IFA, EuroImmun, T-Tek and NovaLisa displayed 50-72.5% agreement with other systems except IFA vs EuroImmun and T-Tek vs NovaLisa. Intermethod agreement for IgM determination was between 30 and 72.5%.

Discussion: The overall intermethod agreement was moderate. This inconsistency could be explained by the diversity of assay methods, antigens used and immunoglobulin isotype tested. Estimated sensitivities were low, highlighting the limited value of antibody detection in CoVID-19.

Conclusion: Comparison of five systems for SARS-CoV-2 IgG and IgM antibodies showed limited sensitivity and overall concordance. The place and indications of serological status assessment with currently available tools in the CoVID-19 pandemic need further evaluations.


Analýza zralého krevního BDNF a proBDNF u poruch nálady se specifickými testy ELISA

Previous studies showed that blood BDNF levels in mood disorders were reduced. However, little is known about the changes of BDNF and its precursor proBDNF in lymphocytes. In addition, earlier studies using commercial ELISA kits cannot distinguish mature BDNF from proBDNF. We aimed to investigate the change of mBDNF and proBDNF levels in the peripheral blood and their diagnostic value in the mood disorders using a specific Enzyme-Linked Immunosorbent Assay (ELISA). Serum mBDNF levels were significantly decreased in major depressive disorder (MDD) (n = 90) and bipolar disorder (BD) (n = 15) groups (P < 0.0001), whereas there was no significant change in suicidal group (n = 14) compared to the control group (n = 96).

In the subgroups of MDD, the serum mBDNF level in MDD patients with severe symptoms was significantly lower than that with moderate symptoms (P < 0.05). The serum mBDNF levels in antidepressant-free patients were significantly lower than in antidepressant-treated patients (P < 0.01). Serum mBDNF yielded good diagnostic effectiveness for MDD and BD with sensitivity and specificity around 80-83%. The levels of mBDNF, proBDNF and its receptor sortilin were upregulated in lymphocytes of MDD patients relative to control subjects. Specific ELISA assays for mature BDNF confirmed the reduction of serum mBDNF level in MDD and BD. The measurement of mBDNF level could be a potential diagnostic marker with a cut-off point at 12.4 ng/ml. Upregulations of proBDNF and mBDNF in lymphocytes of MDD patients might be considered as novel pathological biomarkers for MDD.

Psí dirofilárii a tepelné ošetření: Hodnocení pomocí dobře založeného enzymového imunosorbentního testu (ELISA) a psích sér s potvrzeným stavem infekce dirofilárií

Heat treatment of serum has demonstrated improved detection of Dirofilaria immitis antigen in sera of sheltered dogs without knowing the true infection status of the animals and in dogs confirmed experimentally to be infected with heartworm. Utilizing archived sera with necropsy confirmed heartworm infection status (n = 665) and a micro-titer well based ELISA antigen assay, this study evaluated how the composition of heartworm infections affects antigen test results pre- and post-heat treatment, determined subsequent changes to the antigen test sensitivity and specificity, and application of optical density values. The composition of heartworm infections present in dogs with sera initially testing antigen negative consisted of infections by dead 1/34 (2.9 %), immature 10/34 (29.4 %), male only 7/34 (20.6 %), female only 5/34 (14.7 %), and mixed sex infections 11/34 (32.4 %) with 2-62 heartworms of which 6 were microfilaremic.

The composition of heartworm infections remaining antigen negative post-heat treatment consisted of infections by dead 1/14 (7.1 %), immature 9/14 (64.3 %), male only 2/14 (14.3 %), and mixed sex infections 2/14 (14.3 %) with 6 and 62 heartworms of which 1 was microfilaremic. The overall sensitivity for all infections, mature heartworms, and mature females before heat treatment were 86.9 %, 90.7 %, and 93.3 % and after heat treatment sensitivity increased to 94.6 %, 98.4 %, and 99.2 % respectively.

A decrease in specificity from 97.8%-96.1% was observed following heat treatment of heartworm negative sera. Optical density values for the varying infection intensities present in this study clearly indicate that result intensity is not reflective of the number of heartworms present. This study provides additional context for interpreting post-heat antigen results for dogs originating from animal shelters, demonstrates diagnostic utility of optical density, and highlights the need for improved heartworm diagnostics.

Vývoj obřího seskupení Luteinizační hormon (LH) s enzymovým imunoanalýzou (ELISA) a jeho použití k pochopení sexuálního vývoje v seskupení

A recombinant giant grouper Luteinizing Hormone (LH) consisting of tethered beta and alpha subunits was produced in a yeast expression system. The giant grouper LH β-subunit was also produced and administered to rabbits for antibody development. The recombinant LH and its antibody were used to develop an Enzyme Linked Immunosorbent Assay (ELISA). This ELISA enabled detection of plasma LH levels in groupers at a sensitivity between 391 pg/ml and 200 ng/ml. Different species of grouper were assayed with this ELISA in conjunction with gonadal histology and body condition data to identify links between circulating LH levels and sexual development.

We found that circulating levels of LH decreased when oocytes began to degenerate, and sex-transition gonadal characteristics were apparent when LH levels decreased further. When circulating LH levels were related to body condition (body weight/ body length), transitioning-stage fish had relatively high body condition but low plasma LH levels. This observation was similar across multiple grouper species and indicates that plasma LH levels combined with body condition may be a marker for early male identification in the protogynous hermaphrodite groupers.

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