Development of a double-antibody sandwich ELISA targeting the receptor binding domain of TcdB toxin of ST11 type Clostridium difficile of porcine origin.

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  1. Clostridium difficile is an important zoonotic intestinal pathogen, which is widely present in humans and a variety of animals. The ST11 type C. difficile is one of the most widespread and harmful subtypes in the world. As a large country in pig farming, China lacks efficient methods for detecting C. difficile of porcine origin, leaving hidden dangers for the prevention and control of C. difficile.
  2. The aim of this study Bio Med Frontiers was to develop a specific and sensitive double-antibody sandwich ELISA for the epidemiological investigation of ST11 type C. difficile of porcine origin. Firstly, a 97 kDa receptor binding domain (RBD) was expressed in a prokaryotic host and purified.
  3. A hybridoma cell line AE2D3 capable of stably secreting monoclonal antibody targeting the RBD was screened, and the antibody subtype was determined to be IgG2b (κ).
  4. Secondly, a double antibody sandwich ELISA method was developed, where the monoclonal antibody targeting the RBD was used as a detection antibody, and the rabbit polyclonal antibody was used as a capture antibody.
  5. The chessboard method was used to determine the matching concentration of the capture antibody and the detection antibody, the antigen coating conditions, the blocking conditions, the incubation conditions for detection antibody and samples to be tested, as well as the reaction conditions of HRP-conjugated and reaction conditions of TMB chromogenic solution.
  6. The negative cutoff OD450 was 0.152, and no cross-reaction with 13 strains of non-ST11 type C. difficile was found. The minimum detection concentration of RBD was 8.83 ng/mL. This specific and sensitive double-antibody sandwich ELISA provides a reliable serological detection method for epidemiological investigation of the ST11 type C. difficile in pig industry.

High concordance of ELISA and neutralization assays allows for the detection of antibodies to individual AAV serotypes.

  • Prescreening of participants in clinical trials that use adeno-associated virus (AAV) vectors is required to identify naive participants, as preexisting neutralizing antibodies can limit the efficacy of AAV gene therapies. About The presence of antibodies to individual AAV serotypes is typically detected by neutralization assay.
  • To streamline the screening process, we compared an ELISA-based screening method with a neutralization assay for the detection of antibodies against AAV1, AAV8, and AAV9 in a collection of 50 rhesus macaque sera and 20 human sera.
  • We observed a high level of concordance between the two assays (Pearson r > 0.8) for all three serotypes in both sample sets. We thus investigated pre- vs post-vector inoculation sera samples from rhesus macaques that received AAV1 or AAV8 vector inoculations for cross-reactive anti-AAV antibodies.
  • All 12 macaques seroconverted to the vector they received, but many also reacted to the other serotypes.
  • Our results validate an easy-to-use ELISA for reliable detection of antibodies to individual serotypes of AAV.
  • Our results also demonstrate that an antibody response post-AAV inoculation may partially cross-react with other AAV serotypes. Overall, these results suggest that either assay can be used by academic labs for prescreening samples for preexisting anti-AAV antibodies.

A High-throughput Automated ELISA Assay for Detection of IgG Antibodies to the SARS-CoV-2 Spike Protein

The SARS-CoV-2 pandemic and vaccination campaign has illustrated the need for high throughput serological assays to quantitatively measure antibody levels. Here, we present a protocol for a high-throughput colorimetric ELISA assay to detect IgG antibodies against the SARS-CoV-2 spike protein.
The assay robustly distinguishes positive from negative samples, while controlling for potential non-specific binding from serum samples.
To further eliminate background contributions, we demonstrate a computational pipeline for fitting ELISA titration curves, that produces an extremely sensitive antibody signal metric for quantitative comparisons across samples and time.

Testicular Localization and Potential Function of Vimentin Positive Cells during Spermatogonial Differentiation Stages.

Vimentin is a type of intermediate filament (IF) and one of the first filaments expressed in spermatogenesis.
Vimentin plays numerous roles, consisting of the determination of cell shape, differentiation, cell motility, the maintenance of cell junctions, intracellular trafficking, and assisting in keeping normal differentiating germ cell morphology.
This study investigated the vimentin expression in two populations of undifferentiated and differentiated spermatogonia.
We examined vimentin expression in vivo and in vitro by immunocytochemistry (ICC), immunohistochemistry (IMH), and Fluidigm real-time polymerase chain reaction. IMH data showed that the high vimentin expression was localized in the middle of seminiferous tubules, and low expression was in the basal membrane. ICC analysis of the colonies by isolated differentiated spermatogonia indicated the positive expression for the vimentin antibody, but vimentin’s expression level in the undifferentiated population was negative under in vitro conditions.
Fluidigm real-time PCR analysis showed significant vimentin expression in differentiated spermatogonia compared to undifferentiated spermatogonia (p < 0.05). Our results showed that vimentin is upregulated in the differentiation stages of spermatogenesis, proving that vimentin is an intermediate filament with crucial roles in the differentiation stages of testicular germ cells. These results support the advanced investigations of the spermatogenic process, both in vitro and in vivo.

PD-L1 promotes myofibroblastic activation of hepatic stellate cells by distinct mechanisms selective for TGF-β receptor I versus II

  • Intrahepatic cholangiocarcinoma (ICC) contains abundant myofibroblasts derived from hepatic stellate cells (HSCs) through an activation process mediated by TGF-β. To determine the role of programmed death-ligand 1 (PD-L1) in myofibroblastic activation of HSCs, we disrupted PD-L1 of HSCs by shRNA or anti-PD-L1 antibody.
  • We find that PD-L1, produced by HSCs, is required for HSC activation by stabilizing TGF-β receptors I (TβRI) and II (TβRII). While the extracellular domain of PD-L1 (amino acids 19-238) targets TβRII protein to the plasma membrane and protects it from lysosomal degradation, a C-terminal 260-RLRKGR-265 motif on PD-L1 protects TβRI mRNA from degradation by the RNA exosome complex.
  • PD-L1 is required for HSC expression of tumor-promoting factors, and targeting HSC PD-L1 by shRNA or Cre/loxP recombination suppresses HSC activation and ICC growth in mice. Thus, myofibroblast PD-L1 can modulate the tumor microenvironment and tumor growth by a mechanism independent of immune suppression.

High concordance of programmed death-ligand 1 expression with immunohistochemistry detection between antibody clones 22C3 and E1L3N in non-small cell lung cancer biopsy samples.

The detection of programmed death-ligand 1 (PD-L1) expression can enrich for patients who respond to anti-programmed cell death 1 (PD-1)/PD-L1 therapies. Though, for most laboratories, the cost of PD-L1 22C3 pharmDx is prohibitive for widespread use, whereas the laboratory-developed test (LDT) PD-L1 E1L3N antibody clone is widely available and inexpensive.
This study aims to explore the analytical performance of E1L3N on the Dako Autostainer Link-48 platform and further evaluate the concordance of E1L3N and 22C3 expression in non-small cell lung cancer (NSCLC) biopsy samples.
A total of 171 NSCLC biopsy samples were utilized in this study. Cases with less than 100 tumor cells were excluded.
Serial sections of representative blocks were used for immunohistochemistry (IHC) staining. The staining protocol was performed according to the standard PD-L1 IHC 22C3 pharmDx package. PD-L1 staining on the tumor cell membrane was detected by immunofluorescence.
 At a 1% cutoff value, PD-L1 was positive in 46.2% of patients using clone 22C3 and 42.1% of patients using E1L3N assays. At a 50% cutoff value, PD-L1 was positive in 16.4% of patients using clone 22C3 and 15.2% of the patients using E1L3N assays.
Cohen’s kappa was used to evaluate the concordance of the PD-L1 expression between clone 22C3 and E1L3N.
The kappa values were 0.893 [95% confidence interval (CI): 0.826-1] at the 1% cutoff and 0.868 (95% CI: 0.764-1) at the 50% cutoff. An evaluation of the intraclass correlation coefficients (ICCs) between the antibodies was used to quantify the interassay variability for PD-L1 expression in tumor cells.
ICCs showed high concordance between the two antibodies (0.955, 95% CI: 0.939-0.967).

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Cohen’s kappa was also used to assess the consistency of the PD-L1 evaluation between two pathologists.
The kappa values were 0.941 and 0.912 at the 1% cutoff, and 0.904 and 0.909 at the 50% cutoff for clone 22C3 and E1L3N expression, respectively.
 The results indicated that the clone E1L3N assay has a high concordance with 22C3. The PD-L1 clone E1L3N assay is reliable and cost-effective and could be used as a primary screening agent for PD-L1 IHC staining in pathological laboratories, especially in a research setting.

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