Sensitivity and Specificity of CD19.CAR-T Cell Detection by Flow Cytometry and PCR

Chimeric-antigen-receptor-T (CAR-T) cells are currently revolutionizing the field of cancer immunotherapy. Therefore, there is an urgent need for CAR-T cell monitoring by clinicians to assess cell expansion and persistence in patients. CAR-T cell manufacturers and researchers need to evaluate transduction efficiency and vector copy number for quality control. Here, CAR expression was analyzed in peripheral blood samples from patients and healthy donors by flow cytometry with four commercially available detection reagents and on the gene level by quantitative polymerase chain reaction (qPCR). Flow cytometric analysis of CAR expression showed higher mean CAR expression values for CD19 CAR detection reagent and the F(ab’)2 antibody than Protein L and CD19 Protein. In addition, the CD19 CAR detection reagent showed a significantly lower median background staining of 0.02% (range 0.007–0.06%) when compared to the F(ab’)2 antibody, CD19 protein and Protein L with 0.80% (range 0.47–1.58%), 0.65% (range 0.25–1.35%) and 0.73% (range 0.44–1.23%). Furthermore, flow cytometry-based CAR-T cell frequencies by CD19 CAR detection reagent showed a good correlation with qPCR results. In conclusion, quality control of CAR-T cell products can be performed by FACS and qPCR. For the monitoring of CAR-T cell frequencies by FACS in patients, CAR detection reagents with a low background staining are preferable.

Quantitation of reduced IL-33 levels in human serum: mitigating interference from endogenous binding partners

Aim: IL-33 is a potential therapeutic target but commercially available assays for the quantitation of systemic IL-33 have poor reliability. Results: In commercial IL-33 kits, interference from endogenous binding partners (e.g., soluble ST2) causes under-quantitation. Mitigating this required acid dissociation and addition of the detection reagent simultaneously with the capture step. This enabled detection of total, reduced (active) levels of IL-33 in human serum (LLOQ 6.25 pg/ml). Conclusion: Acid treatment of serum samples dissociates IL-33 from endogenous binding partners, increasing soluble ST2 tolerance to >1000 ng/ml. The modified method was specific for reduced endogenous IL-33. Analysis of over 300 samples from individuals with and without asthma and with different smoking status revealed no difference in serum IL-33.

CD19.CAR-T Cell Analysis Using Flow Cytometry and PCR

Introduction: After approval of CD19.CAR-T cell therapy by both FDA and EMA, chimeric antigen receptor T (CAR-T) cell therapy has been established as a new and innovative therapy method for patients with relapsed/refractory acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). Because of this, there is great need for reliable methods for CAR-T cell monitoring by clinicians to further assess cell expansion, distribution and persistence in patients. This is both necessary for CAR-T cell manufacturing sites and researchers to assess transduction efficiency and vector copy numbers per cell. Therefore, we analyzed CAR expression of CD19.CAR-T cells using flow cytometry as well as quantitative polymerase chain reaction (qPCR). Methods: In this study, four different commercially available CD19.CAR-T cell staining reagents (Protein L, CD19 protein, F(ab`)2 antibody, and CD19.CAR-T cell detection reagent) were used to evaluate CAR-T cell products and peripheral blood samples of both patients and healthy donors by flow cytometry. Furthermore, qPCR was performed with data comparison using flow cytometry results. Results: Flow cytometric analysis of CAR expression showed higher mean CAR expression values for CD19 CAR detection reagent and the F(ab')2 antibody than Protein L and CD19 Protein. Moreove, the CD19 CAR detection reagent showed a significantly lower median background staining of 0.02% (range 0.007-0.06%) when compared to F(ab')2 antibody, CD19 protein and Protein L with 0.80% (range 0.47-1.58%.), 0.68% (range 0.30-1.38%) and 0.73% (range 0.44-1.23%). Furthermore, flow cytometry-based CAR-T cell frequencies by CD19 CAR detection reagent showed a good correlation with qPCR results. Conclusion: CAR-T staining was successfully performed with all tested commercially available CAR detection reagents. Evaluation of manufactured CAR-T cells as well as quality control was comparably done using FACS and qPCR. Because of lower frequencies of CAR-T cells in the patient samples, CAR-T cell staining reagents with a low background staining should be preferably used. Disclosures Sauer: Abbvie: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Matterhorn Biosciences AG: Consultancy, Other: DSMB/SAB Member; Takeda: Consultancy, Other: DSMB/SAB Member. Schubert: Gilead: Consultancy. Müller-Tidow: Janssen: Consultancy, Research Funding; Bioline: Research Funding; Pfizer: Research Funding. Schmitt: TolerogenixX: Current holder of individual stocks in a privately-held company; Novartis: Other: Travel grants, Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees; Kite Gilead: Other: Travel grants; Apogenix: Research Funding; Hexal: Other: Travel grants, Research Funding; Bluebird Bio: Other: Travel grants. Schmitt: Hexal: Other: Travel grant; Therakos/Mallinckrodt: Research Funding; Jazz Pharmaceuticals: Other: Travel grant; TolerogenixX Ltd: Current Employment.

Rapid Detection of Chloramphenicol Residues in Fermented Foods Based on UPLC-DAD

in the detection of chloramphenicol residues in fermented food, there are often problems of slow detection speed. Using UPLC-DAD method, a rapid detection method of chloramphenicol residues in fermented food based on UPLC-DAD method is designed. According to the characteristics of chloramphenicol, set up the detection reagent, select the detection equipment, and form the detection laboratory. It is usingUPLC-DAD method to design the test paper, using the set test reagent to deal with the sample to be tested, according to the design results of the test process, combining the reagent with the sample, to determine its specificity. Chloramphenicol residue was detected by test paper. So far, the rapid detection method of chloramphenicol residues in fermented food based on UPLC-DAD method has been designed. Compared with the original detection method, the detection speed of the detection method designed in this paper is significantly higher than the original method. In conclusion, the rapid detection method of chloramphenicol residues in fermented food based on UPLC-DAD method is effective.

Method for detecting acetylated PD-L1 in cell lysates v1

Here we present a method that allows detection of acetylated PD-L1 and is applicable to a wide range of cell lines. The method captures >90% of acetylated PD-L1 species, is semi-quantitative and simple to perform in any lab equipped with tissue culture and western blot equipment. The method involves processing cells in a lysis buffer that has been optimized for efficient immunoprecipitation (IP) of acetylated species, an IP enrichment step utilizing an acetyl-lysine affinity matrix and western blot detection of both total and acetylated PD-L1 on the same blot. This technique compliments the alternative IP approach utilizing a PD-L1 antibody as the IP reagent and an anti-acetyl lysine antibody as the detection reagent. However, because the protocol described here enables the detection of both total and acetylated PD-L1 on the same blot, this method has the advantage of allowing quantitation of the percent of PD-L1 that is acetylated, an important parameter for mechanistic interpretation. The method described here utilizes beads that are covalently linked to the affinity antibody, resulting in extremely clean IP results. Western blots can be re-probed with a pan anti-acetyl lysine antibody to visualize the total protein acetylation profile in any given lysate, a property that is useful when examining PD-L1 acetylation in the presence of HDAC inhibitors or other treatments affecting global acetylation.

Sensitive assay design for detection of anti-drug antibodies to biotherapeutics that lack an immunoglobulin Fc domain

Today the evaluation of unwanted immunogenicity is a key component in the clinical safety evaluation of new biotherapeutic drugs and macromolecular delivery strategies. However, the evolving structural complexity in contemporary biotherapeutics creates a need for on-going innovation in assay designs for reliable detection of anti-drug antibodies, especially for biotherapeutics that may not be well-suited for testing by a bridging assay. We, therefore, initiated systematic optimization of the direct binding assay to adapt it for routine use in regulatory-compliant assays of serum anti-drug antibodies. Accordingly, we first prepared a SULFO-TAG labeled conjugate of recombinant Protein-A/G to create a sensitive electrochemiluminescent secondary detection reagent with broad reactivity to antibodies across many species. Secondly, we evaluated candidate blocker-diluents to identify ones producing the highest signal-to-noise response ratios. Lastly, we introduced use of the ratio of signal responses in biotherapeutic-coated and uncoated wells as a data transformation strategy to identify biological outliers. This alternative data normalization approach improved normality, reduced skewness, and facilitated application of a parametric screening cut point. We believe the optimized direct binding assay design employing SULFO-TAG labeled Protein-A/G represents a useful analytical design for detecting serum ADA to biotherapeutics that lack an immunoglobulin Fc domain.

Diagnostic Performance of Various Methodologies for Group B Streptococcus Screening in Pregnant Woman in China

Maternal vaginal/rectal colonization of group B streptococcus (GBS) is a main risk for neonatal invasive infection. Efficient determination of GBS colonization in pregnant women is crucial. This study aimed to investigate the prevalence of GBS carriage and evaluate the diagnostic performance of six methodologies for GBS screening conducted in China, including blood agar plate, liquid chromogenic medium, and loop-mediated isothermal amplification (LAMP) without pre-enrichment, chromogenic agar plate with pre-enrichment, and GBS antigen detection without and with pre-enrichment in comparison with the standard reference method (Lim broth-enriched subculture with plating on 5% sheep blood agar). Vaginal/rectal swabs were collected from 1,281 pregnant women at 35–37 weeks of gestation. Of them, 309 were taken in triplicate, one for Lim broth-enriched subculture, one for blood agar plate, and the third for GBS antigen detection (Reagent W); 177 were acquired in duplicate, one for Lim broth-enriched subculture and the other for GBS antigen detection (Reagent H); 502 were obtained in duplicate, one for Lim broth-enriched subculture and the other for liquid chromogenic medium; 158 were collected in duplicate, one for Lim broth-enriched subculture and the other for LAMP; and 135 were inoculated in Lim broth-enriched for GBS antigen detection (Reagent W) and subculture with chromogenic agar plate and 5% blood agar plate. The overall prevalence of GBS carriage was 10.1% (130/1,281, 95% CI: 8.5–12.1%) according to the standard reference method. Compared with the standard reference method, the LAMP had excellent performance of sensitivity (100%, 95%CI: 83.4–100%), specificity (94%, 95%CI: 88.1–97.1%), and Yoden index (0.940); as well as the blood agar plate with sensitivity (81.5%, 95%CI: 61.3–93.0%), specificity (100%, 95%CI: 98.3–100.0%), and Yoden index (0.815). The other four methods were not sufficient to reach the threshold in terms of sensitivity or specificity compared to the standard reference method. Furthermore, for LAMP, results can be obtained within 0.5–1 h, while for blood agar plate, which needed 24–48 h, and further identification was required. Our data suggested that the performance of LAMP was highly comparable to the standard Lim broth-enriched subculture and LAMP is considered as an alternative for fast and accurate GBS screening.

Phage Display Screening of Bovine Antibodies to Foot-and-Mouth Disease Virus and Their Application in a Competitive ELISA for Serodiagnosis

For serodiagnosis of foot-and-mouth disease virus (FMDV), monoclonal antibody (MAb)-based competitive ELISA (cELISA) is commonly used since it allows simple and reproducible detection of antibody response to FMDV. However, the use of mouse-origin MAb as a detection reagent is questionable, as antibody responses to FMDV in mice may differ in epitope structure and preference from those in natural hosts such as cattle and pigs. To take advantage of natural host-derived antibodies, a phage-displayed scFv library was constructed from FMDV-immune cattle and subjected to two separate pannings against inactivated FMDV type O and A. Subsequent ELISA screening revealed high-affinity scFv antibodies specific to a serotype (O or A) as well as those with pan-serotype specificity. When BvO17, an scFv antibody specific to FMDV type O, was tested as a detection reagent in cELISA, it successfully detected FMDV type O antibodies for both serum samples from vaccinated cattle and virus-challenged pigs with even higher sensitivity than a mouse MAb-based commercial FMDV type O antibody detection kit. These results demonstrate the feasibility of using natural host-derived antibodies such as bovine scFv instead of mouse MAb in cELISA for serological detection of antibody response to FMDV in the susceptible animals.

Generation and characterization of monoclonal antibodies against mature hepcidin and its application to neutralization and quantitative alteration assay

ABSTRACT Hepcidin regulates the quantity of ferroportin (FPN) on cellular membrane. In our cell assay expressing ferroportin labeled with green fluorescence, FPN was internalized and degraded only after treatment with hepcidin-25, not hepcidin-22 or hepcidin-20, leading to accumulation of cellular iron. Thus we generated murine monoclonal antibodies (mAbs) against hepcidin-25, and then characterized and validated their functions. Among them, several mAbs showed a neutralizing activity that may prevent ferroportin internalization induced by hepcidin-25. To measure hepcidin level in various fluids, mAbs specific for human and rat hepcidin-25 were selected. As for rat, a sandwich ELISA developed using clone rHN1 as capture antibody and biotinylated clone mHW1 as a detection reagent had high sensitivity, allowing for the detection of 1-100 ng/mL of hepcidin-25. Rat hepcidin-25 level in plasma was measured at an average concentration of 63.0 ng/mL in healthy condition, and at 218.2 ng/mL after stimulation of lipopolysaccharide.