Time-resolved Fluorescence Immunoassay Research Articles

Epidemiological surveillance of Neospora caninum in sheep (Ovis aries) and European mouflon (Ovis aries musimon) in southern Spain.

Neospora caninum is a protozoan with a facultative heteroxenous life cycle, with canids as the definitive hosts and other mammals, mainly ruminants, acting as intermediate hosts. This parasite is recognized as one of the major abortifacient pathogens in cattle. Although reproductive disorders have also been reported in other domestic and wild ruminants, epidemiological data on N. caninum in ruminant species other than cattle is still limited. Here, we evaluate the seroprevalence and risk factors associated with N. caninum exposure in sheep (Ovis aries) and European mouflon (Ovis aries musimon) in southern Spain. Serum samples from 390 sheep and 387 free-ranging mouflons were tested for antibodies against N. caninum using in-house time-resolved fluorescence immunoassay based on NcGRA7 as a recombinant antigen. The individual seroprevalence was 26.2% (95%CI: 22.0-30.7) in sheep and 5.7% (95%CI: 3.8-8.5) in mouflons. At least one seropositive animal was detected in all the 26 sheep farms (100%) and in eight of the 18 (44.4%) hunting estates sampled. The presence of dogs on the farm (≥3) and the sheep breed (purebred) were potential risk factors associated with exposure to N. caninum in sheep. Our results indicate a high circulation of N. caninum in sheep farms in southern Spain. Control measures should be implemented to limit the exposure to this protozoan in sheep flocks. Although we have confirmed for the first time the presence of anti- N. caninum antibodies in European mouflon in Spain, and, despite the relatively low seroprevalence found, the impact of this parasite on mouflon populations should be further studied.

Highly sensitive time-resolved fluorescent microspheres lateral flow immunoassay for the quantitative detection of triadimefon and its metabolite residues in fruits and vegetables.

Ageneral one-step lateral flow immunochromatographic assay (LFIA) for the quantitative detection of triadimefon (TDF) and triadimenol(TDN) in fruit and vegetable samples was developedusing time-resolved fluorescence microspheres (TRFM) as labels. A specific anti-triadimefon monoclonal antibody (mAb) was conjugated with TRFM to fabricate LFIA test strips. A time-resolved fluorometer as an LFIA reader was applied to obtain quantitative results and assess risk ranges for the LFIA test strips. Under the optimized experimental conditions, the limits of detection (LODs) in buffer/cucumbers/tomatoes/oranges were 0.046ng/mL, 0.135µg/kg, 1.047µg/kg, and 5.811µg/kg, respectively, which are ca. 1000 times lower than that of colloidal gold-labeled strips. The recovery in cucumber/tomato/orange samples was 109.4-116.7%, 87.7-110.9%, and 88.0-111.9%, respectively, indicating that the test strips had good reliability. Coupled with the easily customizable pretreatment procedures for various samples, the LFIA results were obtained within 18min without the need for professional personnel or complicated equipment. TRFM-LFIA for TDF and TDN also shows remarkable specificity and precision. The test strips were also low-cost, portable, and convenientto use. These results indicate the test strips could be utilized as a novel strategy for on-site detection of TDF and TDN, which has the potential to expand and detect other pesticide or insecticide residues in food.

Photonic Microbead Array Digital Time-Resolved Fluorescence Ultrasensitive Platform for Simultaneous Detection of Multiple Mycotoxins.

Limitations in the sensitivity, linear detection range, and cross-reaction of lateral flow immunoassays mainly hamper their application in rapid screening for multiple targets. In this work, we designed a new time-resolved fluorescence immunoassay (TRFIA) platform to overcome these limitations. This platform uses europium chelate polystyrene (PS@Eu) nanoparticles conjugated with monoclonal antibodies to sense multiple mycotoxins. We employed a competitive TRFIA protocol in which the conjugated PS@Eu was used on the surfaces of photonic microbead arrays (PMAs). The TRFIA signal of PMAs on the pad was recorded with the digital time-resolved fluorescence reader. The developed TRFIA shows wide detection linear ranges (0.01-1000 ng/mL for DON, 0.1-100 ng/mL for OTA, and 0.01-100 ng/mL for AFB1), low limits of detection (LODs) (7.9 pg/mL for DON, 18 pg/mL for OTA, and 7.7 pg/mL for AFB1), good specificity, good recovery ratios (76.68-117.26%), and good reproducibility in grain samples. The simulated fluorescence enhancement effect of PMA indicated that the electric field distribution on the surface of PS@Eu on PMA is twice higher than that on the surface of PS@Eu. The new TRFIA for three kinds of mycotoxins was 1000-fold more sensitive than the classical TRFIA, and it has great potential application in rapid screening for multiple targets.

Associations of the AVPR1A RS1 Microsatellite Locus with the Level of Hormones of the Anterior Pituitary Gland and Personality Traits.

The arginine vasopressin receptor gene (AVPR1A) is one of the genes affecting mental processes. The aim of this study was to search for associations of microsatellite locus RS1, which is related to the AVPR1A expression level, with the level of hormones of the anterior pituitary gland and personality traits. The study sample included Yakut men aged 18-26years (n = 121). The analysis of RS1 locus was carried out using the PCR method and sequencing of the primary nucleotide sequence. Serum hormonal levels of thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and prolactin were determined by the time-resolved fluorescence immunoassay (DELFIA), plasma adrenocorticotropic hormone (ACTH) levels were determined by enzyme-linked immunosorbent assay (ELISA). In the Yakut population "short" (S) alleles of the AVPR1A RS1 locus containing ≤ 10 repeats (63%) and the corresponding SS genotypes (44.6%) were more frequent, while individuals with "long" (LL) and "heterozygous" (SL) genotypes accounted for 18.2 and 37.2%, respectively. The range of concentrations of ACTH and TSH in the group of SS genotype carriers was significantly lower than that observed in the group of LL genotype carriers (p = 0.042 and p = 0.048, respectively); the LH level was significantly higher (p = 0.029). The trend towards higher neuroticism in SS genotype carriers compared to the individuals with LL genotypes (p = 0.05) is revealed. The results obtained indicate the modulating effect of genetic variants of the AVPR1A gene on the level of anterior pituitary hormones, which could slightly affect the level of neuroticism in humans.

Establishment and Clinical Application in Stroke of a Serum Copeptin Time-Resolved Fluorescence Immunoassay.

The serum biomarker copeptin, an innovative and stable substitute biomarker of vasopressin, is associated with stroke. Therefore, establishing a highly sensitive time-resolved fluorescence immunoassay for copeptin (copeptin-TRFIA) is helpful to measure stroke and evaluate its value in clinical applications. Double antibody sandwich was used to establish copeptin-TRFIA. The established method was then assessed. Two coated and Eu3+-labeled copeptin monoclonal specific antibodies targeting different antigen epitopes were employed. The serum fluorescence counts of patients with stroke and healthy volunteers were detected by using thewell-established copeptin-TRFIA. Serum copeptin levels were measured and analyzed statistically. The actual measurement linearity range of the proposed method was 0.13-44.66 ng/mL. Copeptin-TRFIA had the inter-assay coefficient of variation (CV) of 6.49%-9.08% and the intra-assay CV of 4.75%-7.77%. Patients with cerebral infarction (CI) and intracerebral hemorrhage (ICH) had significantly higher serum copeptin levels than healthy subjects. Copeptin concentrations in the serum of patients with stroke were significantly correlated with the scores of the National Institute for Healthy Stroke Scale (NIHSS) and modified Rankin Scale (mRS). A highly sensitive copeptin-TRFIA was successfully established. Serum copeptin has a certain value in the clinical diagnosis and prognosis of stroke.

Controlled labelling of tracer antibodies for time-resolved fluorescence-based immunoassays

Tracer antibodies, which are labelled with fluorescent or other type of reporter molecules, are widely employed in diagnostic immunoassays. Time-resolved fluorescence immunoassay (TRFIA), recognized as one of the most sensitive immunoassay techniques, utilizes tracers labelled with lanthanide ion (Ln) chelates. The conventional approach for conjugating isothiocyanate (ITC) Ln-chelates to antibodies involves random chemical targeting of the primary amino group of Lys residues, requiring typically overnight exposure to an elevated pH of 9–9.3 and leading to heterogeneity. Moreover, efforts to enhance the sensitivity of the assays by introducing a higher number of Ln-chelates per tracer antibody are associated with an elevated risk of targeting critical amino acid residues in the binding site, compromising the binding properties of the antibody. Herein, we report a method to precisely label recombinant antibodies with a defined number of Ln-chelates in a well-controlled manner by employing the SpyTag/SpyCatcher protein ligation technology. We demonstrate the functionality of the method with a full-length recombinant antibody (IgG) as well as an antibody fragment by producing site-specifically labelled antibodies for TRFIA for cardiac troponin I (cTnI) detection with a significant improvement in assay sensitivity compared to that with conventionally labelled tracer antibodies. Overall, our data clearly illustrates the benefits of the site-specific labelling strategy for generating high-performing tracer antibodies for TRF immunoassays.

A rapid and sensitive fluorescent chromatography with cloud system for MPXV point-of-care diagnosis

Monkeypox (mpox) is spreading around the world, and its rapid diagnosis is of great significance. In the present study, a rapid and sensitive fluorescent chromatography assisted with cloud system was developed for point-of-care diagnosis of mpox. To screen high affinity antibodies, nanoparticle antigen AaLS-A29 was generated by conjugating A29 onto scaffold AaLS. Immunization with AaLS-A29 induced significantly higher antibody titers and monoclonal antibodies were generated with the immunized mice. A pair of monoclonal antibodies, MXV 14 and MXV 15, were selected for fluorescence chromatography development. The Time-Resolved Fluorescence Immunoassay (TRFIA) was used to develop the chromatography assay. After optimization of the label and concentration of antibodies, a sensitive TRFIA assay with detection limit of 20 pg/mL and good repeatability was developed. The detection of the surrogate Vaccinia virus (VACA) strain Tian Tan showed that the TRFIA assay was more sensitive than the SYBR green I based quantitative PCR. In real samples, the detection result of this assay were highly consistent with the judgement of Quantitative Real-Time PCR (Concordance Rate = 90.48%) as well as the clinical diagnosis (Kappa Value = 0.844, P < 0.001). By combining the portable detection and online cloud system, the detection results could be uploaded and shared, making this detection system an ideal system for point-of-care diagnosis of mpox both in field laboratory and outbreak investigation.

Development of a time-resolved fluorescence immunoassay kit for detecting canine coronavirus and parvovirus through double labeling

ObjectiveCanine enteric coronavirus (CCV) and canine parvovirus type 2 (CPV-2) are the main pathogens responsible for acute gastroenteritis in dogs, and both single and mixed infections are common. This study aimed to establish a double-labeling time-resolved fluorescence immunoassay (TRFIA) to test and distinguish CCV and CPV-2 diseases.MethodsA sandwich double-labeling TRFIA method was established and optimized using europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. CCV/CPV-2 antigens were first captured by the immobilized antibodies. Then, combined with Eu3+/Sm3+-labeled paired antibodies, the Eu3+/Sm3+ fluorescence values were detected after dissociation to calculate the CCV/CPV-2 ratios. The performance, clinical performance and methodology used for laboratory (sensitivity, specificity, accuracy and stability) testing were evaluated.ResultsA double-label TRFIA for CCV and CPV-2 detection was optimized and established. The sensitivity of this TRFIA kit was 0.51 ng/mL for CCV and 0.80 ng/mL for CPV-2, with high specificity for CCV and CPV-2. All the accuracy data were less than 10%, and the recovery ranged from 101.21 to 110.28%. The kits can be temporarily stored for 20 days at 4 °C and can be stored for 12 months at temperatures less than − 20 °C. Based on a methodology comparison of 137 clinically suspected patients, there was no statistically significant difference between the TRFIA kit and the PCR method. Additionally, for CCV detection, the clinical sensitivity was 95.74%, and the clinical specificity was 93.33%. For CPV-2 detection, the clinical sensitivity was 92.86%, and the clinical specificity was 96.97%.ConclusionIn this study, a double-label TRFIA kit was prepared for CCV and CPV-2 detection with high laboratory sensitivity, specificity, accuracy, stability, clinical sensitivity and specificity. This kit provides a new option for screening/distinguishing between CCV and CPV-2 and may help improve strategies to prevent and control animal infectious diseases in the future.

Quantitative detection and prognostic value of antibodies against M-type phospholipase A2 receptor and its cysteine-rich ricin domain and C-type lectin domains 1 and 6-7-8 in patients with idiopathic membranous nephropathy.

M-type phospholipase A2 receptor (PLA2R) is the major autoantigen in adult idiopathic membranous nephropathy (IMN). Although reactive epitopes in the PLA2R domains have been identified, the clinical value of these domains recognized by anti-PLA2R antibodies remains controversial. Accordingly, this study aimed to quantitatively detect changes in the concentrations of different antibodies against epitopes of PLA2R in patients with IMN before and after treatment to evaluate the clinical value of epitope spreading. Highly sensitive time-resolved fluorescence immunoassay was used to quantitatively analyze the concentrations of specific IgG and IgG4 antibodies against PLA2R and its epitopes (CysR, CTLD1, CTLD6-7-8) in a cohort of 25 patients with PLA2R-associated membranous nephropathy (13 and 12 in the remission and non-remission groups, respectively) before and after treatment, and the results were analyzed in conjunction with clinical biochemical indicators. The concentration of specific IgG (IgG4) antibodies against PLA2R and its epitopes (CysR, CTLD1 and CTLD6-7-8) in non-remission group was higher than that in remission group. The multipliers of elevation of IgG (IgG4) antibody were 5.6(6.2) fold, 3.0(24.3) fold, 1.6(9.0) fold, and 4.2(2.6) fold in the non-remission/remission group, respectively. However, the difference in antibody concentrations between the two groups at the end of follow-up was 5.6 (85.2), 1.7 (13.1), 1.0 (5.1), and 1.5 (22.3) times higher, respectively. When detecting concentrations of specific IgG antibodies against PLA2R and its different epitopes, the remission rate was 66.67% for only one epitope at M0 and 36.36% for three epitopes at M0. When detecting concentrations of specific IgG4 antibodies against PLA2R and its different epitopes, the remission rate was 100.00% for only one epitope at M0 and 50.00% for three epitopes at M0. A trivariate logistic regression model for the combined detection of eGFR, anti-CTLD678 IgG4, and urinary protein had an AUC of 100.00%. Low concentrations of anti-CysR-IgG4, anti-CTLD1-IgG4, and anti-CTLD6-7-8-IgG4 at initial diagnosis predict rapid remission after treatment. The use of specific IgG4 against PLA2R and its different epitopes combined with eGFR and urinary protein provides a better assessment of the prognostic outcome of IMN.

INSL3 Variation in Dogs Following Suppression and Recovery of the HPG Axis.

Insulin-like peptide 3 (INSL3) is a constitutive product of mature, adult-type Leydig cells of the testes and consequently in most mammals is an ideal biomarker with which to monitor pubertal development. A new heterologous time-resolved fluorescence immunoassay was developed and validated to measure circulating INSL3 in the blood of adult male dogs. Compared to other species, INSL3 concentration is low with marked variation between individuals, which appears to be independent of breed, age, or weight. A model system was then used in which a cohort of beagle dogs was subject to a GnRH-agonist implant to suppress the HPG axis and spermatogenesis, followed by implant removal and recovery. Unlike testosterone, INSL3 levels were not fully suppressed in all animals by the GnRH agonist, nor was the recovery of Leydig cell function following implant removal uniform or complete, even after several weeks. In dogs, and dissimilar from other species (including humans), Leydig-cell INSL3 appears to be quite variable between individual dogs and only weakly connected to the physiology of the HPG axis after its suppression by a GnRH-agonist implant and recovery. Consequently, INSL3 may be less useful in this species for the assessment of testis function.

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.

Quantitative Determination of Aflatoxin B1 in Maize and Feed by ELISA and Time-Resolved Fluorescent Immunoassay Based on Monoclonal Antibodies.

In this study, a highly sensitive monoclonal antibody (mAb) was developed for the detection of aflatoxin B1 (AFB1) in maize and feed. Additionally, indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and time-resolved fluorescence immunoassay assay (TRFICA) were established. Firstly, the hapten AFB1-CMO was synthesized and conjugated with carrier proteins to prepare the immunogen for mouse immunization. Subsequently, mAb was generated using the classical hybridoma technique. The lowest half-maximal inhibitory concentration (IC50) of ic-ELISA was 38.6 ng/kg with a linear range of 6.25-100 ng/kg. The limits of detections (LODs) were 6.58 ng/kg and 5.54 ng/kg in maize and feed, respectively, with the recoveries ranging from 72% to 94%. The TRFICA was developed with a significantly reduced detection time of only 21 min, from sample processing to reading. Additionally, the limits of detection (LODs) for maize and feed were determined to be 62.7 ng/kg and 121 ng/kg, respectively. The linear ranges were 100-4000 ng/kg, with the recoveries ranging from 90% to 98%. In conclusion, the development of AFB1 mAb and the establishment of ic-ELISA for high-throughput sample detection, as well as TRFICA for rapid detection presented robust tools for versatile AFB1 detection in different scenarios.

Clinical value of serum MMP-3 in chronic kidney disease

BackgroundChronic kidney disease (CKD) is defined as the progressive deterioration of renal parenchyma and decline in renal unit function. In the early stages of CKD(G1 + G2), symptoms are usually not obvious and cannot be effectively recognized on the basis of available clinical markers. Progression to the middle and late stages of CKD results in severe kidney damage with multiple complications causing adverse outcomes, including death. Therefore, the early diagnosis and monitoring of CKD is critical. Matrix metalloproteinase-3 (MMP-3), an extracellular matrix–degrading enzyme, plays an important role in kidney diseases. However, the clinical significance of serum MMP-3 levels in CKD has rarely been reported. MethodsWe quantified the serum MMP-3 levels of 237 patients with CKD and 96 healthy individuals by using a highly sensitive time-resolved fluorescence immunoassay and analyzed differences in MMP-3 levels among the stages of CKD and the correlations of these changes with clinical indicators. ResultsThe serum MMP-3 concentrations of patients with CKD (171.76 ± 165.22 ng/mL) were significantly higher than those of healthy controls (34.05 ± 22.93 ng/mL; P < 0.0001). In CKD, serum MMP-3 levels were significantly correlated with estimated glomerular filtration rate (eGFR) (r = − 0.5804, P < 0.0001), serum creatinine (CREA) (r = 0.5823, P < 0.0001), blood urea nitrogen (BUN) (r = 0.6106, P < 0.0001), and protein-to-creatinine ratio (r = 0.4992, P < 0.0001). Randomized forest analysis finds CREA, BUN, and MMP-3 most significant influences on CKD disease severity. The critical value of MMP-3 concentration of 40.39 ng/mL combined with eGFR was effective in diagnosing positive patients in the early (G1 + G2) stage of CKD and showed a positivity rate of 73.45 %. Moreover, in the early stages of CKD, patients with CKD who had serum MMP-3 concentration > 100 ng/mL had more severe renal impairment and inflammation than those with CKD who have lower serum MMP-3 concentrations. ConclusionElevated serum MMP-3 levels are correlated with decreased kidney function in CKD progression, and patients with concomitant inflammation may express high levels of serum MMP-3. Serum MMP-3 may assist eGFR in improving the diagnosis of patients with early CKD.

Time-resolved fluorescence immunoassay based on glucose oxidase-encapsulated metal-organic framework for amplified detection of foodborne pathogen

BackgroundFluorescence immunoassays are commonly employed for the detection of pathogenic bacteria as a means of ensuring food safety and preserving public health. However, the challenges such as poor photostability and background interference have limited their sensitivity and accuracy. The emergence of metal-organic frameworks (MOFs) as a label probe offers a promising solution for advancing fluorescence immunoassays owing to their tunable nature. Nonetheless, the low fluorescence efficiency of MOFs and the potential risk of dye leakage pose obstacles to achieving high detection sensitivity. Therefore, there exists a pressing need to fully utilize the potential of MOF composites in fluorescence immunoassays. ResultsWe explored the potential of glucose oxidase-encapsulated zeolitic imidazole framework-90 (GOx@ZIF-90) as a label probe to construct a time-resolved fluorescence immunoassay with amplified detection signal. This immunoassay involved functionalizing Fe3O4 nanoparticle with porcine antibody to specifically capture and separate the target bacteria, Staphylococcus aureus (S. aureus). The captured S. aureus was then bound by GOx@ZIF-90 modified with vancomycin, resulting in a fluorescence response in the europium tetracycline (EuTc). The encapsulation of GOx in ZIF-90 provided a confinement effect that significantly enhanced the catalytic activity and stability of GOx. This led to a highly efficient conversion of glucose to H2O2, amplifying the fluorescence signal of EuTc. The immunoassay demonstrated a high sensitivity in detecting S. aureus, with a detection limit of 2 CFU/mL. We also obtained satisfactory results in milk samples. Attractively, the time-resolved detection mode of EuTc allowed the immunoassay to eliminate background fluorescence and enhance accuracy. SignificanceThis study not only presented a new method for detecting foodborne pathogens but also highlighted the potential of enzyme-encapsulated MOF composites as label probes in immunoassays, providing valuable insights for the design and fabrication of MOF composites for various applications.

EE315 The Budget Impact of Including the Automated Time-Resolved Fluorescence Immunoassay As Point-of-Care Testing on Cardiac Markers in an Emergency Department in China

EE315 The Budget Impact of Including the Automated Time-Resolved Fluorescence Immunoassay As Point-of-Care Testing on Cardiac Markers in an Emergency Department in China

Age-related changes in circulating INSL3 concentrations and their associations with ovarian conditions in Japanese Black beef cattle

Information on circulating levels of insulin-like peptide 3 (INSL3) in female domesticated animals is limited, as their concentrations are significantly lower than in males. The objectives of the present study were to 1) develop a sandwich time-resolved fluorescence immunoassay (TRFIA) with higher detectability to measure blood INSL3 concentrations in female cattle, 2) determine INSL3 concentrations in female cattle among age groups and reproductive conditions, and 3) explore associations between INSL3 levels and ultrasonographic ovarian measurements. Blood was collected repeatedly from Japanese Black beef female calves (n = 12; 0－8 mo), heifers (n = 10; 10－26 mo), and cows (n = 20; 27－200 mo). Blood was taken from the cows (n = 13) at follicular, post-ovulatory, and luteal phases, and from cows with follicular cysts (n = 12). Ultrasonography of ovaries was conducted in the calves (n = 12) and the cows without ovarian diseases (n = 9). The ovarian area, as well as the number and diameters of antral follicles ≥ 2 mm, were determined in each ovary. The proposed method detected a difference in plasma INSL3 between calves (0.01 ng/mL) and heifers (0.18 ng/mL). However, the conventional assay showed similar levels for calves and heifers (1.82 vs 2.07 ng/mL). Plasma INSL3 and testosterone concentrations increased from calves to heifers (P < 0.0001), but only INSL3 rose from heifers to cows (P < 0.0001). INSL3 and testosterone concentrations did not change across the estrus cycle in cows, and the levels of both hormones in follicular cystic cows did not differ from those in the follicular phase. Ovarian area, maximal and average follicular diameters, and total volume of all follicles per animal were higher in cows than calves (P < 0.001). Plasma INSL3 concentrations correlated positively with the total volumes of all follicles in calves (P < 0.05) and cows (P < 0.05), whereas testosterone concentrations did not correlate with ovarian follicular measurements. In conclusion, plasma INSL3 concentrations measured by the proposed sandwich TRFIA showed a clear increase from female calves to cows in beef cattle. These results suggest that circulating levels of INSL3, but not of testosterone, are associated with the total volume of all antral follicles in both ovaries per animal in female cattle.

Establishment of growth stimulating gene 2 protein time-resolved fluorescence immunoassay and its application in sepsis

AimThis study aimed to establish a highly sensitive time-resolved fluorescence immunoassay of growth stimulating express gene 2 protein (ST2-TRFIA) and evaluate its application value for sepsis. MethodsTwo types of ST2 monoclonal specific antibodies against different epitopes of antigen molecule were used as coating and Eu3+-labeled antibodies. The double-antibody sandwich method was used in establishing ST2-TRFIA, and the methodology was evaluated. The established ST2-TRFIA was used in detecting ST2 concentration in the plasma samples of healthy controls and sepsis. ResultsThe linear range of ST2-TRFIA was 1.446–500 ng/mL. Plasma ST2 concentrations detected through ST2-TRFIA were consistent with the results of fluorescence quantitative immunochromatography (ρ = 0.946). The plasma ST2 concentrations of patients with sepsis were significantly higher than those of healthy controls (P < 0.01). ConclusionThis study successfully established a highly sensitive ST2-TRFIA, which was highly comparable to commercially available fluorescent quantitative immunochromatographic kits and can facilitate the timely diagnosis of sepsis.

A time-resolved fluorescence immunoassay for rapid and precise automatic quality control of human papillomavirus type 68 VLPs in human papillomavirus vaccine

The effectiveness and necessity of human papillomavirus (HPV) vaccination to prevent HPV infection and cervical cancer are increasingly recognized by people. The 15-valent HPV vaccine, which protects against almost high-risk types of HPV viruses identified by WHO, has attracted much attention. However, as the valence of vaccines increases, quality control in the HPV vaccine production process is facing more challenges. The precise quality control of the HPV type 68 virus-like particles (VLPs), one of the unique components of the 15-valent HPV vaccine that distinguishes it from existing vaccines, is the new requirement for vaccine manufacturers. Here we developed a novel time-resolved fluorescence immunoassay (TRFIA) for rapid and precise automatic quality control of HPV68 VLPs in HPV vaccine. Two murine monoclonal antibodies specifically targeting the HPV68 L1 protein were used to establish a classical sandwich assay. Except for pretreating the vaccine sample, the whole analysis process was performed by a fully automated machine, which saves detection time and gets rid of manual error. Multiple experiments established that the current novel TRFIA can efficiently and reliably analyses HPV68 VLPs. Present novel TRFIA has exhibited merits with speed, robustness, high sensitivity with a minimum detection value of 0.08 ng/mL, considerable accuracy, a wide detection range (up to 1000 ng/mL) and excellent specificity. It is also expected to provide a new detection method for quality control for each HPV type VLPs. To summarize, the novel TRFIA is of great interest for application in HPV vaccine quality control.

Sensitive amplified luminescent proximity homogeneous assay for the quantitative detection of CA242

We here developed a sensitive and stable amplified luminescent proximity homogeneous assay (AlphaLISA) method for fast quantification of CA242 in human serum. Donor and acceptor beads modified with carboxyl groups could be coupled with CA242 antibodies after activation in the AlphaLISA method. CA242 was rapidly detected by the double antibody sandwich immunoassay. The method yielded good linearity (>0.996) and detection range (0.16–400 U/mL). The intra-assay precisions of CA242-AlphaLISA were between 3.43% and 6.81% (< 10%), and the inter-assay precisions were between 4.06% and 9.56% (< 15%). The relative recoveries ranged from 89.61% to 107.29%. Detection time for the CA242-AlphaLISA method was only 20 min. Moreover, results of CA242-AlphaLISA and time-resolved fluorescence immunoassay had satisfactory correlation and consistency (ρ = 0.9852). The method was successfully applied to the analysis of human serum samples. Meanwhile, serum CA242 has a good detection value in the identification and diagnosis of pancreatic cancer and the monitoring of disease degree. Furthermore, the proposed AlphaLISA method is expected to be an alternative to traditional detection methods, laying a good foundation for the further development of kits to detect other biomarkers in future studies.

Immediate, sensitive and specific time-resolved fluorescent immunoassay strips based on immune competition for the detection of procymidone in vegetables

As an organochlorine pesticide, procymidone has become an increasing serious problem in vegetables in recent years. In order to solve a series of problems such as low sensitivity of the immunological methods, a time-resolved fluorescent immunoassay strip (TRFIAS) was developed with low limit of detection (LOD). Based on the principle of antigen-antibody immune competition, TRFIAS could detect procymidone in vegetables. After the targets being added, the samples were incubated at 37 °C for 10 min. TRFIAS was observed under a UV lamp of 365 nm. The results were processed by ImageJ software to complete the qualitative and quantitative analysis of TRFIAS. Under the optimal working conditions, a linear range of TRFIAS was 5–250 ng/mL with a LOD of 0.33 ng/mL and a visual limit of detection (VDL) of 50 ng/mL. And the TRFIAS had good specificity, precision and accuracy. Comparing the results of TRFIAS with those of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), the two assays were in good agreement. Therefore, TRFIAS has the advantages of high sensitivity, high specificity, simple operation and relatively short assay time. Meanwhile, it shows absolutely great advantages in both quantitative and qualitative analysis.