Simultaneous Detection of Prolactin and Growth Hormone Using a Dual-label Time-resolved Fluorescence Immunoassay.

Prolactin (PRL) and growth hormone (GH) are two important hormones secreted by the pituitary gland, and their abnormal levels are often related to disease status. This study aimed to establish a new dual-label time-resolved fluorescence immunoassay (TRFIA) to quantitatively measure PRL and GH levels in serum. A sandwich TRFIA was optimized and established: anti-PRL/GH antibodies immobilized on 96-well plates captured PRL/GH and then banded together with anti-PRL/GH paired antibodies labeled with europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. Finally, a time-resolved analyzer measured the Eu3+/Sm3+ fluorescence values. Clinical serum samples were used to evaluate the detection performance of this method. The sensitivities of this dual-label TRFIA were 0.35 ng/mL and 0.45 ng/mL, respectively, and the detection range was between 0.1 and 1000 ng/mL. All the cross-reactivities were lower than 1.07%. The intra-assay and interassay coefficients of variation were 2.18-7.85% and 2.25-7.30%, respectively. Compared with the registered TRFIA kits, a high Pearson coefficient (r = 0.9626 and 0.9675) was observed. This dual-label TRFIA has high sensitivity, accuracy and specificity with good clinical detection performance, representing a suitable alternative to existing methods for determining PRL and GH levels, and is expected to be used in the clinic in the future.

Determination of serum CA724 levels using fluorescence immunochromatography.

BACKGROUND: Carbohydrate antigen 724 (CA724) is a sensitive and specific indicator for multiple malignant tumors. The aim of this study was to establish a Eu-time resolved fluorescence immunochromatography (Eu-TRFICO) method for quantitative detection of CA724 in serum. METHODS: Eu-TRFICO strips were optimized and assembled. The sensitivity, specificity and precision were evaluated using CA724 standard dilutions and matrix serum. Meanwhile, the reference interval, comparison, and sensitivity/specificity were performed using clinical negative/positive gastric cancer serum samples. RESULTS: The standard curve equation was y = 9.869 x - 154.12 (R2 = 0.993), and the sensitivity was 0.42 U/mL. The common interferents in serum could not affect the quantitative results with low cross-reactivities (all no more than 1.09%). All average recoveries of the intra- and interbatch ranged from 102.38 to 106.40%, and all CVs were below 10%. The reference interval of the healthy subjects was < 4.68 U/mL and the reference interval of the subjects with grade I/II gastric cancer was > 9.54 U/mL. Additionally, a high Pearson r (0.9503) and sensitivity/specificity (92.86%/94.20%) were obtained. CONCLUSION: This study prepared Eu-TRFICO strips with high sensitivity, specificity, precision and satisfactory clinical testing performance, which provides more options for clinical quantitative and convenient testing of CA724.

Determination of echinococcosis IgG antibodies using magnetic bead-based chemiluminescence immunoassay.

Echinococcosis is a kind of parasitic disease shared by humans and animals. The aim of this study was to establish a new detection method for echinococcosis screening using magnetic bead-based chemiluminescence immunoassay (CLIA). A magnetic bead-based CLIA to determine anti-echinococcosis IgG antibodies was optimized and established. The sensitivity, accuracy, precision and recovery rate were evaluated using the national reference serum, and the reference interval, specificity and comparison assays were performed using the clinical negative/positive echinococcosis serum samples. This study established a new CLIA to determine anti-echinococcosis IgG antibodies. The sensitivity of this CLIA method was higher than that of the registered ELISA kit and the national standard, the conformance rate of the negative/positive references was 100% (8/8), the CVs of the sensitivity reference were all below 5%, and the CVs of the precision reference were 5.7%. There was no obvious cross-reactivity with the common parasitic disease-positive serum and serum interferents. Clinical sample testing found that the cutoff value of this CLIA was 5537.15 (RLU), and there was no significant difference between the CLIA method and the registered ELISA kit. This study established a fully automated CLIA method with high sensitivity, specificity, accuracy, precision, recovery rate, and satisfactory clinical testing performance, which may provide a new choice for echinococcosis screening.

A New Method for Early Screening of Gastric Cancer (G17 and CA724 Dual-Labeled Time-Resolved Fluorescence Immunoassay).

Gastric carcinoma (GC) is one of the most common malignancies in the world with the great early screening challenges. The study is aimed at establishing a new detection method for early screening GC using time-resolved fluorescence immunoassay (TRFIA) via quantitative detection of gastrin-17 (G-17) and carbohydrate antigen 724 (CA724) in serum. Time-resolved analyzer measured the fluorescence intensity. The standards of G-17/CA724 were used for drawing the standard curve, which is used to calculate the concentration of G-17 and CA724 in serum sample. The sensitivity for G-17 was 0.54 pg/mL and for CA724 was 0.28 U/mL with a wide-range analyze concentration (0.1-1000) pg/mL or U/mL. The average recoveries ranged from 100.52% to 110.30% for G-17 and 103.02% to 116.00% for CA724. All CVs of the intra- and interassay were below 10% with high specificity. There was a high Pearson coefficient between this TRFIA method and the commercially available kits (Pearson r 0.9117 for G-17 and 0.9449 for CA724). Additionally, the cutoff value was 88.41 pg/mL and 5.47 U/mL for CA724 in health subjects. This study established a TRFIA method for simultaneous detection of the concentrations of G-17 and CA724 in serum, which provide a new method for sensitive, accurate, and specific early screening of gastric cancer.

Determination of parvovirus antigen in the vaccine using time-resolved fluorescence immunoassay.

As a highly contagious and potentially fatal disease of dogs, canine parvovirus type 2 (CPV-2) usually causes severe myocarditis and gastroenteritis, while vaccine injection has greatly reduced the incidence of CPV-2 diseases. However, there is currently a lack of simple and effective method for quantitative detection of CPV-2 in vaccine. Therefore, this study aims to prepare an accurate method to determine the CPV-2 antigen (CPV-2-Ag) in vaccine. Here, a sandwich time-resolved fluorescence immunoassay (TRFIA) was established and optimized. Anti-CPV-2 antibodies were immobilized on 96-well plates to capture CPV-2-Ag, and then bound together with the detection antibodies labeled with Europium(III) (Eu3+ ) chelates; finally, time-resolved fluorometry was employed to measure the fluorescence intensity. Vaccination was performed to evaluate the relationship between CPV-2-Ag concentration and antibody titer. The sensitivity is 1.15 mEU/mL (LogY = 1.524 + 0.8667 × LogX, R2  = 0.9933), and the average recovery is among 91.00% to 106.39% without cross-reactions with the other canine viral antigen. The correlation between ELISA assay and this method is up to 0.9861. And, there is high correlation between the CPV-2-Ag concentration and antibody titers (R2  = 0.9234). This immunoassay established has high sensitivity, accuracy, and specificity, which indicate that this method could be suitable for quantitative detection of CPV-2-Ag in vaccine evaluation.

Astragaloside IV inhibits excessive mesangial cell proliferation and renal fibrosis caused by diabetic nephropathy via modulation of the TGF-ß1/Smad/miR-192 signaling pathway.

Astragaloside IV (ASI) exhibits a wide variety of pharmacological effects in cardiovascular diseases, hepatitis and kidney disease and due to this, ASI has recently become an attractive research target. The present study aimed to determine the effect of ASI on renal fibrosis and the mechanisms underlying its therapeutic effects in diabetic nephropathy (DN). In vitro, ASI was added to rat mesangial cells (RMCs) and cultured with a high level of glucose (HG) to observe the effects exhibited on proliferation and fibrosis-related mRNA and protein expression. In vivo, a DN model was established using streptozotocin administration in rats, and renal injury was evaluated using renal histological examination. The expression levels of related mRNAs and proteins were analyzed using reverse transcription-quantitative PCR, western blot analysis and immunohistochemistry. ASI was demonstrated to downregulate miR-192 expression and inhibit excessive proliferation of RMCs, which was induced by HG, in a dose-dependent manner. Additionally, ASI exhibited a therapeutic effect on DN rats. ASI was also demonstrated to decrease the miR-192 expression and mRNA and protein expression of transforming growth factor-ß1 (TGF-ß1), Smad3, α-smooth muscle actin (α-SMA) and collagen type 1 (col1), and increase the mRNA and protein expression of Smad7 in vitro and in vivo. These results suggested that ASI exhibited a therapeutic effect on DN, possibly due to the inhibition of excessive mesangial proliferation and renal fibrosis via the TGF-ß1/Smad/miR-192 signaling pathway.

Determination of parvovirus antibodies in canine serum using magnetic bead-based chemiluminescence immunoassay.

Canine parvovirus type 2 (CPV-2), as a highly contagious and potentially fatal disease of dogs and many other carnivores, usually causes severe gastroenteritis and myocarditis. Therefore, it is very necessary and urgent to have an accurate method to determine the CPV-2 antibodies (CPV-2-Ab) in canine samples. Here, a magnetic bead-based chemiluminescence immunoassay was established and optimized to detect the concentration of CPV-2-Ab in serum. And a commercial assay was also used to evaluate the consistency with our method. After optimization of the detective system, the CPV-2-Ab was captured by CPV-antigen-magnetic bead (8.3 µg/mL); then combined with the conjugation of anti-canine IgG antibody-acridinium ester (0.36 µg/mL). Finally, collected the signal (read the luminosity) after 1 H reaction time. The linear correlation coefficient (R2 ) is 0.9924. The limit of detection (sensitivity) is 0.36 ng/mL (the linear dynamic range: 1.32-93.75 ng/mL), and the average recovery is 100.89% without cross-reactions with other canine viral antibodies. The results' correlation between commercial assays and this method is 0.9888. This immunoassay establishes that it has high sensitivity, accuracy, and specificity in clinical analysis, indicating that this method could be suitable for quantitative detection of CPV-2-Ab and evaluation of vaccination effect.

Effects of Acupuncture on 1-chloro-2,4-dinitrochlorobenzene-induced Allergic Contact Dermatitis in Mice.

Allergic contact dermatitis (ACD) is a chronic inflammatory skin disease. Topical corticosteroids are the first-line therapy for ACD despite their significant adverse effects. Acupuncture has been widely used in the treatment of various skin diseases, but its underlying mechanism remains unrevealed. In this study, we investigated the characteristics of acupuncture treatment based on effectiveness and mechanism. BALB/c mice received 1-chloro-2,4-dinitrobenzene (DNCB) application to build AD-like model. Results showed that acupuncture was an effective treatment method in inhibiting inflammatory conditions, serum IgE levels, and expression of proinflammatory cytokine Th2 (IL-4, IL-6), and Th2 (IL-1ß, TNF-α) mRNA compared with DNCB treatment. Acupuncture treatment also inhibited nuclear factor-κB p65, phosphorylation of IκBα, and phosphorylation of occludin proteins expression. Furthermore, it could improve the expression of epidermal growth factor in both mRNA and protein levels. These results suggest that acupuncture, as an alternative therapy treatment for its no significant side effects, was effective in alleviating ACD by reducing proinflammatory cytokines and changing proteins' expression.