Evaluation of Analytical Performances of Magnetic Force-Assisted Electrochemical Sandwich Immunoassay for the Quantification of Carcinoembryonic Antigen

Carcinoembryonic antigen (CEA) is a biomarker indicated in different cancers, targeted for quantitative analysis via immunoassay. Here we introduce a new technique called magnetic force-assisted electrochemical sandwich immunoassay (MESIA) for determination of CEA level in a drop of human serum using a fully automated point-of-care testing (POCT) device. The analytical performances of the assay are assessed based on precision, accuracy, limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ), linearity, Hook effect, interference, cross-reactivity, and method comparison following the guidelines of the Clinical Laboratory Standards Institute (CLSI). The LoD is 0.50 ng/ml. A linear relationship is shown in the range of 0.5–200 ng/ml. A high dose effect is not seen up to approximately 500,000 ng/ml. The recovery range is from 94.7 to 108.9%. The %CV of run-to-run and within-lab variations are less than 2.04 and 4.41% across the CEA concentrations, respectively, whereas reproducibility is 4.45–6.24%. Method comparison shows that the assay correlates well with the reference device (R2 = 0.9884). The assay demonstrates acceptable precision, accuracy, LoB, LoD and LoQ, hook effect, linearity, interference, cross-reactivity, and high correlation with its reference device. Thus, the system is suitable for the quantification of CEA in clinical practices with a POCT manner.

A competitive ligand-binding assay for the detection of neutralizing antibodies against dostarlimab (TSR-042)

Dostarlimab is a humanized anti–PD-1 monoclonal antibody. Dostarlimab (JEMPERLI; TSR-042) was recently approved in the USA and in the EU. The presence of neutralizing antibodies (NAbs) is a cause for concern because they block the therapeutic function of the antibody and reduce drug efficacy. Therefore, programs developing therapeutic biologics need to develop and validate assays that adequately assess the presence of NAbs in the serum of patients treated with biologic therapies. Presented here is the development and validation of a competitive ligand-binding assay that specifically detects anti-dostarlimab NAbs in human serum. Precision, sensitivity, hook effect, selectivity, assay robustness, stabilities, and system suitability were evaluated. In addition, drug tolerance of the assay with the implementation of a drug removal process was investigated. The cut point factor for the detection of NAbs in human serum at a 1% false-positive rate was determined. The assay’s precision, sensitivity, hook effect, selectivity, robustness, and drug interference were tested and found to be acceptable. With system suitability and stability established, this assay has been used to evaluate NAbs to guide the development of dostarlimab.Trial registration: Clinicaltrials.gov, NCT02715284. Registered 9 March 2016

Comparison of a point-of-care serum amyloid A analyzer frequently used in equine practice with 2 turbidimetric immunoassays used in human and veterinary medicine

Rapid, accurate detection of serum amyloid A (SAA) is needed in equine practice. We validated a patient-side point-of-care (POC) assay (Stablelab; Zoetis) compared to the turbidimetric immunoassays LZ-SAA (TIA-Hum) and VET-SAA (TIA-Vet; both Eiken Chemical). Analytical performance was assessed at 3 different concentration ranges and with interferences. Inter-method comparison using 49 equine serum samples revealed a significant difference between median SAA results ( p < 0.0001), with the strongest bias between the POC and TIA-Vet (median 1,093 vs. 578 mg/L). The median SAA value obtained with the TIA-Hum method was 752 mg/L. Correlation between POC/TIA-Hum and between POC/TIA-Vet was fair (rs = 0.77 and 0.69) and excellent between both TIAs (rs = 0.93). Bias between POC/TIA-Hum, POC/TIA-Vet, and TIA-Hum/TIA-Vet was −56.7%, –80.9%, and −28.2%, respectively. POC intra- and inter-assay CVs (16.1–30% and 19.8–35.5%) were higher than TIA CVs (generally <12%). Bilirubin and hemoglobin had a negative bias on POC and TIA-Vet results (−16.6 to −45.6%); addition of intralipid yielded a positive bias (35.9–77.4%). The POC had good linearity of SAA concentrations up to 10,312 mg/L ( R2 = 0.92). A hook effect was present at SAA >3,000 mg/L for the POC assay. Equine serum SAA was stable over a median period of 2.5 y when stored at −80°C. Overall, there was excellent-to-moderate correlation between tests, but imprecision and hook effect of the POC, as well as bias between the methods, must be considered.

Design, Synthesis, and Evaluation of Trivalent PROTACs Having a Functionalization Site with Controlled Orientation

Trivalent PROTACs having a functionalization site with controlled orientation were designed, synthesized, and evaluated. Based on the X-ray structure of BRD protein degrader MZ1 (1) in complex with human VHL and BRD4BD2, we expected that the 1,2-disubstituted ethyl group near the JQ-1 moiety in MZ1 (1) could be replaced by a planar benzene tether as a platform for further functionalization. To test this hypothesis, we first designed six divalent MZ1 derivatives, 2a-c and 3a-c, by combining three variations of substitution patterns on the benzene ring (1,2-, 1,3-, and 1,4-substitution) and two variations in the number of ethylene glycol units (1 or 2). We then tested the synthesized compounds for the BRD4 degradation activity of each. As expected, we found that 1,2D-EG2-MZ1 (2a), an MZ1 derivative with 1,2-disubstituted benzene possessing two ethylene glycol units, had an activity profile similar to that of MZ1 (1). Based on the structure of 2a, we then synthesized and evaluated four isomeric trivalent MZ1 derivatives, 15a-15d, having a tert-butyl ester unit on the benzene ring as a handle for further functionalization. Among the four isomers, 1,2,5T-EG2-MZ1 (15c) retained a level of BRD4 depletion activity similar to that of 2a without inducing a measurable Hook effect, and its BRD4 depletion kinetics was the same as that of MZ1 (1). Other isomers were also shown to retain BRD4 depletion activity. Thus, the trivalent PROTACs we synthesized here may serve as efficient platforms for further applications.

Design, Synthesis, and Evaluation of Trivalent PROTACs Having a Functionalization Site with Controlled Orientation

Trivalent PROTACs having a functionalization site with controlled orientation were designed, synthesized, and evaluated. Based on the X-ray structure of BRD protein degrader MZ1 (1) in complex with human VHL and BRD4BD2, we expected that the 1,2-disubstituted ethyl group near the JQ-1 moiety in MZ1 (1) could be replaced by a planar benzene tether as a platform for further functionalization. To test this hypothesis, we first designed six divalent MZ1 derivatives, 2a-c and 3a-c, by combining three variations of substitution patterns on the benzene ring (1,2-, 1,3-, and 1,4-substitution) and two variations in the number of ethylene glycol units (1 or 2). We then tested the synthesized compounds for the BRD4 degradation activity of each. As expected, we found that 1,2D-EG2-MZ1 (2a), an MZ1 derivative with 1,2-disubstituted benzene possessing two ethylene glycol units, had an activity profile similar to that of MZ1 (1). Based on the structure of 2a, we then synthesized and evaluated four isomeric trivalent MZ1 derivatives, 15a-15d, having a tert-butyl ester unit on the benzene ring as a handle for further functionalization. Among the four isomers, 1,2,5T-EG2-MZ1 (15c) retained a level of BRD4 depletion activity similar to that of 2a without inducing a measurable Hook effect, and its BRD4 depletion kinetics was the same as that of MZ1 (1). Other isomers were also shown to retain BRD4 depletion activity. Thus, the trivalent PROTACs we synthesized here may serve as efficient platforms for further applications.

Weak Precipitin Ring from A Fecal Specimen with Markedly Elevated Alpha-1 Antitrypsin Level Measured by Radial Immunodiffusion

Radial immunodiffusion (RID) is a classic methodology for antigen quantification that relies on the development of a distinct precipitin ring from precipitated antigen-antibody complex. As the precipitin ring grows, the precipitate at the inner edge of the ring constantly dissolves due to excess antigen flooding from the point of application, while new precipitate forms at the leading edge of the ring. RID plates with anti-human alpha-1-antitrypsin are prepared in our lab to measure fecal alpha-1 antitrypsin (A1AT) to aid in the diagnosis and monitoring of Protein Losing Enteropathy (PLE). The procedure has routinely produced precipitin rings with small radii and distinct edges after incubating at room temperature for 48 hours. Unexpectedly, a fecal specimen from a patient produced an extremely weak and large precipitin ring that could have been easily overlooked. Dilution studies confirmed a highly elevated A1AT level of 67 mg/g dry stool. The very weak and large precipitin ring was reproduced with a spiked specimen with similar A1AT concentration and kept expanding for several days until a distinct ring was formed eventually. Our data highlights a rare example of high-dose hook effect in RID and calls for meticulous attention and caution when reading and interpreting gel-based immunoassays with unexpected markedly elevated results to avoid additional confirmatory testing. In these cases, we recommend repeat testing with diluted specimens.

Common Pitfalls in the Interpretation of Endocrine Tests

Endocrine tests are the cornerstone of diagnosing multiple diseases that primary care physicians are frequently faced with. Some of these tests can be affected by situations that affect the proper interpretation, leading to incorrect diagnoses and unnecessary treatment, such as the interference of biotin with thyroid function test, falsely elevated prolactin values in presence of macroprolactinemia or falsely normal due to the “hook effect” in macroprolactinomas. Recognizing these situations is essential for the clinician to make an adequate interpretation of these tests as well as an accurate diagnosis that guarantees the best outcomes for the patient.