Comparison between immunofluorescence and immunohistochemistry for Listeria monocytogenes detection in formalin-fixed paraffin-embedded tissues

ABSTRACT: Listeria monocytogenes is a bacterium that infect humans and animals and causes a zoonotic disease characterized by encephalitis, septicemia or abortion. In addition, listeriosis leads to significant economic losses due to animal death and sacrifice. This research compared the technique of immunofluorescence (IF) and immunohistochemistry (IHC) for the diagnosis of L. monocytogenes in formalin-fixed and paraffin-embedded (FFPE) tissues. A total of 30 tissue blocks from 15 animals with history and/or lesions compatible with listeriosis were selected. For both IHC and IF, the same diluted (1:200) polyclonal primary antibody was used against L. monocytogenes serotypes 1 and 4. For IHC, a polymer secondary antibody conjugated to peroxidase (HRP) was used. For IF, samples were incubated with a fluorescein-labeled anti-rabbit IgG secondary antibody. Each sample was classified according to the presence and percentage of immunolabeling area. From 30 samples, 10 were positive at least for one technique, whereas eight samples were positive for both IHC and IF with similar score. There was strong immunolabeling in tissue samples from bovines experimentally infected with L. monocytogenes ATCC 7644, as well as in nervous tissues from naturally infected ruminants. Additionally, IF did not show any difference in sensitivity when compared to IHC. Using processed biological materials for IF, instead of fresh tissues, is a quite unique technique, since there are few protocols described. Therefore, this study demonstrated that both techniques are efficient to detect L. monocytogenes in FFPE tissues.

Upconverting phosphor technology-based lateral flow assay for the rapid and sensitive detection of anti-Trichinella spiralis IgG antibodies in pig serum

Background Trichinella spiralis is a zoonotic food-borne parasite. A disease caused by infection with T. spiralis is called trichinellosis in humans. It is important to investigate the epidemic situation and the surveillance of herds and then prevent infection in humans. Therefore, this study is to develop a rapid and sensitive diagnostic method for on-site test in domestic and wild animals. Methods Upconverting phosphor nanoparticles (UCNPs), an excellent optical label, were conjugated with the excretory-secretory (ES) antigens from T. spiralis muscle larvae (ML) or goat anti-rabbit IgG, and a lateral flow (LF) assay based on these probes (UCNPs-ES/goat anti-rabbit IgG) was developed for the rapid and sensitive detection of anti-T. spiralis IgG antibodies in pig serum. The assay is named the UPT-LF-ES assay. In addition, the probes were characterized, and the assay was optimized. A cut-off threshold of the assay was also identified by using 169 known negative pig samples. Performance of the assay to T. spiralis with different infective numbers, cross-reactivity with other parasitic infections, the single-blinded experiment, and coincidence were evaluated with the assay. Results The UPT-LF-ES assay was successfully constructed and optimized based on the probes of UCNPs-ES/goat anti-rabbit IgG. In the pigs infected with 100, 1000, and 10,000 ML, positive results were first presented at 35 days post-infection (dpi), 30 dpi, and 25 dpi, respectively. The assay had no cross-reaction with other parasitic infections. A single-blinded experiment indicated that the sensitivity and specificity of the UPT-LF-ES assay were 100% and 100%, respectively, the area under the receiver operating characteristic (ROC) curve was 1.000. In addition, the value detected by the UPT-LF-ES assay was significantly different between positive and negative samples. Moreover, compared with the “gold standard” magnetic stirrer method, the coincidence rate of the UPT-LF-ES assay was 87.27%, and the kappa (K) coefficient was 0.7454, showing a substantial agreement. Conclusions The UPT-LF-ES assay is a useful point-of-care test (POCT) with T. spiralis in the detection of pig, which contributes to preventing human trichinellosis. Graphical Abstract

Combination of Aptamer Amplifier and Antigen-Binding Fragment Probe as a Novel Strategy to Improve Detection Limit of Silicon Nanowire Field-Effect Transistor Immunosensors

Detecting proteins at low concentrations in high-ionic-strength conditions by silicon nanowire field-effect transistors (SiNWFETs) is severely hindered due to the weakened signal, primarily caused by screening effects. In this study, aptamer as a signal amplifier, which has already been reported by our group, is integrated into SiNWFET immunosensors employing antigen-binding fragments (Fab) as the receptors to improve its detection limit for the first time. The Fab-SiNWFET immunosensors were developed by immobilizing Fab onto Si surfaces modified with either 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) (Fab/APTES-SiNWFETs), or mixed self-assembled monolayers (mSAMs) of polyethylene glycol (PEG) and GA (Fab/PEG-SiNWFETs), to detect the rabbit IgG at different concentrations in a high-ionic-strength environment (150 mM Bis-Tris Propane) followed by incubation with R18, an aptamer which can specifically target rabbit IgG, for signal enhancement. Empirical results revealed that the signal produced by the sensors with Fab probes was greatly enhanced compared to the ones with whole antibody (Wab) after detecting similar concentrations of rabbit IgG. The Fab/PEG-SiNWFET immunosensors exhibited an especially improved limit of detection to determine the IgG level down to 1 pg/mL, which has not been achieved by the Wab/PEG-SiNWFET immunosensors.

Establishment and Application of a Method for the Determination of Ganoderic Acid A

A method for the quantitative determination of ganoderic acid A was constructed using the principle of indirect competitive enzyme-linked immunosorbent assay (ELISA), and this method was used to determine the ganoderic A contents of Ganoderma lucidum samples in the market. The conjugate of ganoderic acid A and bovine serum albumin was used for four rounds of immunization on test rabbits to obtain rabbit antiganoderic acid A antibody IgG. The enzyme-labeled plate was coated with the conjugate of ganoderic acid A and ovalbumin. The first stage reaction in the indirect competitive ELISA was that the conjugate of ganoderic acid A in the sample competed with the conjugate coated on the enzyme-labeled plate to bind rabbit antibodies. The second stage reaction was the combination of goat anti-rabbit IgG–horseradish peroxidase and rabbit antiganoderic acid A antibody IgG. The results of the determination of ganoderic acid A standard by this method showed that the coefficient of variation of repeated wells in the group was <5%, the detection limit of ganoderic acid A was 0.6 μg/L, and ganoderic acid A had a substantial dose-response relationship in the content range of 0.9–72.9 μg/L (R2 = 0.994). This method was used to measure the ganoderic A content of 12 varieties of G. lucidum in the market and showed the obvious differences in the ganoderic acid A contents of the different varieties. This method is simple, fast, and of great importance to the quality control of Ganoderma products.

Preparasi Strip Imunokromatografi Koloid Emas untuk Deteksi Cepat Aeromonas hydrophila

Aeromonas hydrophila merupakan agen penyebab Motile Aeromonas Septicemia (MAS) yang menyebabkan kerugian ekonomi pada industri akuakultur air tawar. Saat ini, metode diagnostik yang tersedia untuk determinasi A. hydrophila membutuhkan waktu lama dan tidak sesuai diaplikasikan di lapangan. Oleh karena itu, dibutuhkan adanya metode lain yang dapat digunakan sebagai diagnostik yang cepat dan aplikatif di lapangan. Tujuan dari penelitian ini adalah membuat alat diagnostik yang mampu digunakan sebagai pendeteksi agen penyebab MAS. Secara singkat, partikel koloid emas berukuran 31,88 nm dibuat melalui reduksi kimia asam kloroaurat dengan natrium sitrat. Koloid emas sebagai detektor dikonjugasikan dengan antibodi poliklonal anti-A. hydrophila 50 μg/ml pada pH 7. Membran nitroselulosa sebagai membran reaksi, ditetesi dengan antibodi poliklonal anti-A. hydrophila 2 mg/ml di garis T dan antibodi goat anti-rabbit IgG 1 mg/ml di garis C. Hasil dari penelitian ini, strip imunokromatografi yang dikembangkan mampu mendeteksi A. hydrophila dengan deteksi minimum 1,2x105 CFU/ml. Strip bersifat spesifik terhadap A. hydrophila, tidak ada reaksi silang yang ditemukan ketika direaksikan dengan bakteri lain. Analisis sampel menggunakan strip hanya membutuhkan waktu 10 menit. Strip imunokromatografi yang dikembangkan dapat mendeteksi A. hydrophila dengan cepat dan memiliki potensi untuk digunakan di lapangan.

Blocking Interleukin-33 Alleviates the Joint Inflammation and Inhibits the Development of Collagen-Induced Arthritis in Mice

Rheumatoid arthritis (RA) is considered a systemic chronic inflammatory joint disease characterized by chronic synovitis and cartilage and bone destruction. Interleukin-33 (IL-33) is a proinflammatory cytokine which is highly expressed in the synovium of RA patients and the joints of mice with collagen-induced arthritis (CIA) and exacerbates CIA in mice. However, the role of the IL-33-neutralizing antibody in the murine model of CIA remains unclear. In the present study, CIA mice were given intraperitoneally with polyclonal rabbit anti-murine IL-33 antibody (anti-IL-33) or normal rabbit IgG control after the first signs of arthritis. Administration of anti-IL-33 after the onset of disease significantly reduced the severity of CIA and joint damage compared with controls treated with normal rabbit IgG. Anti-IL-33 treatment also significantly decreased the serum levels of interferon-γ(IFN-γ),IL-6, IL-12, IL-33, and tumor necrosis factor-α (TNF-α). Moreover, anti-IL-33 treatment significantly downregulated the production of IFN-γ, IL-6, IL-12, IL-33, and TNF-α in ex vivo-stimulated spleen cells. Together, our results indicate that the IL-33-neutralizing antibody may provide a therapeutic strategy for RA by inhibiting the release of proinflammatory cytokines.

Surface Modification of a Stable CdSeZnS/ZnS Alloy Quantum Dot for Immunoassay

Quantum dots (QDs) are powerful materials in various bioapplications based on their excellent optical and electronic properties. For the application of various fields of QDs, surface modification of QDs is necessary. However, surface modification in QDs may result in a reduction in quantum yield (QY). This reduction of QY causes many weaknesses in the biological application of QDs. In this study, CdSeZnS/ZnS alloy QDs were used to prepare antibody-conjugated QDs for a sandwich immunoassay. The alloy QDs displayed a QY of 84.5% that was maintained at 83.0% (98.2% of QY was maintained) after surface modification with the anti-rabbit IgG as a model study. Surface-modified QDs successfully detected their corresponding target through antibody-antigen binding. The limit of detection was 1.1 × 10 2 ng mL-1 for rabbit IgG.

A Sensitive Two-Analyte Immunochromatographic Strip for Simultaneously Detecting Aflatoxin M1 and Chloramphenicol in Milk

A two-analyte immunochromatographic strip (immunostrip) was developed for the simultaneous detection of aflatoxin M1 (AFM1) and chloramphenicol (CAP) in milk. Protein conjugates (AFM1-ovalbumin (OVA) and CAP-OVA) and goat anti-rabbit IgG were respectively drawn on nitrocellulose membrane as two test lines (T1 and T2) and a control line (C). The immunostrip was dipped into a well that contained a 200 μL milk sample, 5 μL AFM1 antibody-gold conjugates, and 8 μL CAP antibody-gold conjugates; the whole assay was completed in 15 min and the results could be interpreted visually or using a reader. This immunostrip has cut-off levels of 0.1 ng/mL and 0.5 ng/mL for AFM1 and CAP, respectively. Analysis of CAP and AFM1 in milk samples revealed that data from the immunostrip test agreed closely with those obtained from ELISA. The two-analyte immunostrip is a rapid way for on-site simultaneous detection of AFM1 and CAP in milk.