Detection of infliximab, adalimumab, and anti-drug antibodies: Development and validation of new monotest, automated assays on multiparametric instrument.

Objective: To convert manual ELISA kits to fully automated immunoassays that quantify serum drug levels and anti-drug antibodies levels of infliximab and adalimumab (CHORUS Promonitor kits). Desing and methods: CHORUS Promonitor INFLIXIMAB, CHORUS Promonitor ADALIMUMAB, CHORUS Promonitor ANTI-INFLIXIMAB, and CHORUS Promonitor ANTI-ADALIMUMAB kits were compared with the corresponding Promonitor kits to determine sensitivity and specificity of the assays. For the automated assays, the entire procedure -from samples dilution to final readouts-was performed by CHORUS TRIO instrument (DIESSE, Italy). Residual human serum samples from clinical laboratory investigations and samples resulting from the addition of specific drugs (IFX or ADL) or anti-drug antibodies (anti-IFX or anti-ADL) were used for the characterization and validation of the tests. Results: CHORUS Promonitor kits showed an excellent agreement (Cohen's coefficient = 1) with the Promonitor kits and were linear within predefined ranges. All assays were accurate and repeatable, as an acceptable variability were observed within runs, between runs, lots, and instruments. No difference in detecting the reference drug or biosimilars emerged. Conclusion: During preclinical development, these kits resulted as sensitive, specific, accurate, and able to quantify either the reference drug or the corresponding biosimilars. All these features support their use in clinical practice for therapeutic drug monitoring of patients with inflammatory diseases under treatment with IFX or ADL.

Validation, performance, and reliability of two automated tests for vitamin B12 and folate assay.

Background: Deficiency of Vitamin B12 and folate may determine hematological, neurological, and metabolic alterations; therefore, an accurate quantification of their serum levels is required, especially in the presence of symptoms that might suggest a deficiency. CHORUS VIT B12 and CHORUS FOLATE are two automated immunoassays, developed to quantify vitamin B12 and folate, respectively, in human serum. Design and methods: This single-center, non-pharmacological, diagnostic study described the validation and characterization of CHORUS VIT B12 and CHORUS FOLATE, with a specific focus on performance, precision, and reliability. For each assay, 500 serum samples were analyzed. A comparison between CHORUS assays and commercially available kit was also performed. Results: For CHORUS VIT B12 the lower limit of quantification (LLoQ) was 165.0 pg/mL and the upper LoQ (ULoQ) was 1846.8 pg/mL. The assay was linear within the calibration range (150-2000 pg/mL) and the accuracy was described with the International Standard Vitamin B12, Serum Folate, HOLO TC (NIBSC code: 03/178), with a mean recovery on two lots of 111%. For CHORUS FOLATE (calibration range of 2.0-20.0 ng/mL), LLoQ was 2.0 ng/mL and ULoQ 19.6 ng/mL. The linearity was demonstrated from 2.4 to 20.0 ng/mL; the accuracy was described with the International Standard mentioned above, achieving a mean recovery on three lots of 92%. The lowest and highest values of both CHORUS and COBAS kits were similar and the median values did not significantly vary. Conclusion: CHORUS VIT B12 and CHORUS FOLATE performed well, accurately, and reliably in quantifying vitamin B12 and folate in human serum.

Development of an Anti-Zika and Anti-Dengue IgM ELISA Assay: Evaluation of Cross Reactivity and Validation.

Zika and dengue viruses (ZIKV and DENV) have been considered major global threats to humans in the past decade. The two infections display similar epidemiological and clinical manifestations. They are transmitted by the same primary vector, accounting for the co-circulation of the two viruses in regions where they are endemic. Highly specific and sensitive serological assays that are able to detect ZIKV and DENV antibodies (Abs) during the acute and convalescent phases of infections would help to improve clinical management and disease control. We report the development and characterisation of two monoclonal Abs, the ZIKV 8-8-11 and the DENV 8G2-12-21, which recognise the Zika non-structural protein 1 (NS1) and the dengue virus type 2 envelope protein, respectively. Both mAbs were used to set up enzyme-linked immunosorbent assays (ELISAs) specific for the detection of anti-Zika immunoglobulin M (IgM) and anti-dengue IgM and whose performance was similar to commercially available kits. These kits, intended to be used with the CHORUS Instruments, are rapid and require ≤ 50 µL of human serum. These tests could represent an affordable and reliable option for the rapid diagnosis of both ZIKV and DENV infections in developing countries, where these flaviviruses are endemic.

A quantitative assay for detection of SARS-CoV-2 neutralizing antibodies.

OBJECTIVES: Serological assays for SARS-CoV-2 have a critical role not only in diagnosis of COVID-19, but also in assessing the degree and duration of response of specific antibodies against the virus obtained through infection or vaccination. We present the results obtained with a competitive immunoenzymatic method (Chorus SARS-CoV-2 "Neutralizing" Ab) for quantitative determination of total neutralizing anti-S1 SARS-CoV-2 antibodies (IgG, IgM, and IgA) in human serum obtained on a disposable device with the Chorus TRIO instrument using a recombinant strong neutralizing antibody as tracer. METHODS: A total of 694 sera were evaluated for SARS-CoV-2 neutralizing antibodies: 407 uninfected, 201 symptomatic subjects, 37 post-infection patients, and 49 vaccinated. Sixty-eight of the previous sera were used to compare the Chorus SARS-CoV-2 "Neutralizing" Ab results with those obtained with micro-neutralization of the Alpha and original variants. A set of 74 positive sera for other respiratory infections were analyzed to evaluate the possible cross reaction to SARS-CoV-2 virus. RESULTS: Of the 694 samples, only 3 had discordant results between micro-neutralization and values measured by Chorus SARS-CoV-2 "Neutralizing" Ab: 1 false negative and 2 false positives. Values of sensitivity and specificity were very high: percent positive agreement (sensitivity) 99.6% (95% CI: 97.7 - 99.9) and percent negative agreement (specificity) 99.6% (95% CI: 98.0 -99.9). Concordance was high with a Gwet's Ac1 of 0.992. No significant differences were observed between the alpha and original variants. CONCLUSIONS: The Chorus SARS-CoV-2 "Neutralizing" Ab test was highly sensitive and specific, and varies from most other currently available tests since it analyzes only antibodies with viral-neutralizing capacity.

Large scale production and characterization of SARS-CoV-2 whole antigen for serological test development.

BACKGROUND: The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated a pandemic with alarming rates of fatality worldwide. This situation has had a major impact on clinical laboratories that have attempted to answer the urgent need for diagnostic tools, since the identification of coronavirus disease 2019 (COVID-19). Development of a reliable serological diagnostic immunoassay, with high levels of sensitivity and specificity to detect SARS-CoV-2 antibodies with improved differential diagnosis from other circulating viruses, is mandatory. METHODS: An enzyme-linked immunosorbent assay (ELISA) using whole inactivated virus cultured in vitro, was developed to detect viral antigens. WB and ELISA investigations were carried out with sera of convalescent patients and negative sera samples. Both analyses were concurrently performed with recombinant MABs to verify the findings. RESULTS: Preliminary data from 10 sera (5 patients with COVID-19, and 5 healthy controls) using this immunoassay are very promising, successfully identifying all of the confirmed SARS-CoV-2-positive individuals. CONCLUSION: This ELISA appears to be a specific and reliable method for detecting COVID-19 antibodies (IgG, IgM, and IgA), and a useful tool for identifying individuals which have developed immunity to the virus.

Development and characterization of a monoclonal antibody directed against human telomerase reverse transcriptase (hTERT).

Telomerase activity plays an important role in the two complementary processes of cellular immortalization and senescence. This enzyme is active in almost all tumors, but also in inflammatory and many normal proliferating cells. Therefore, the main limits of molecular determinations, such as telomeric repeat amplification protocol assay is that they are not able to discriminate between the enzymatic activity of tumor and normal cells. The most appropriate technique for this would be immunohistochemical determination using monoclonal antibodies. Very few monoclonal antibodies (Mabs) directed against the human telomerase reverse transcriptase (hTERT) are commercially available and in the present study, we developed a new Mab directed against this protein (TERT-3 36-10) to investigate the possibility of detecting immunoreactivity to this Mab by immunohistochemical and flow cytometric approaches. Immunohistochemical determination showed a lack of reactivity to the Mab in highly differentiated striated muscle tissue, a variable reactivity in dysplastic cervical epithelial tissue and similar and widespread immunoreactivity in cell lines and clinical tumors. Furthermore, we demonstrated the ability of this Mab to inhibit enzyme activity in cell extract from MCR bladder tumor cell line.