In this work, a novel conjugate of ractopamine and bovine serum albumin (RAC-BSA) has been developed via the Mannich reaction, with a mole coupling ratio for RAC-BSA of 9:1. The proposed conjugation method provides a simple and one-step method with the use of fewer reagents compared with other conjugation methods for competitive immunoassays. RAC-BSA conjugation was used to fabricate a competitive lateral flow strip test for RAC detection in animal feed. For sample preparation, RAC was spiked in swine feed purchased from the local markets in Thailand, and methanol and running buffer at a volume ratio of 10:90 was used as extraction buffer. The procedures for sample preparation were completed within 25 min. Under optimal conditions, the limit of detection (LOD), assessed by the naked eye within 5 min, was found to be 1 ng/g. A semi-quantitative analysis was also conducted using a smart phone and computer software, with a linearity of 0.075-0.750 ng/g, calculated LOD of 0.10 ng/g, calculated limit of quantitation of 0.33 ng/g, and good correlation of 0.992. The recoveries were found in the range of 96.4%-103.7% with a relative standard deviation of 2.5%-3.6% for intra- and inter-assays. Comparison of the results obtained by the strip test with those obtained by enzyme-linked immunosorbent assay had a good agreement in terms of accuracy. Furthermore, this strip test exhibited highly specific RAC detection without cross reactivity with related compounds. Therefore, the RAC-BSA conjugation via the Mannich reaction can be accepted as a one-step and easy conjugation method and applied to the competitive lateral flow strip test.
Animal Feed/analysis* ; Animals ; Cross Reactions ; Enzyme-Linked Immunosorbent Assay/methods* ; Limit of Detection ; Phenethylamines/chemistry* ; Reagent Strips ; Serum Albumin, Bovine/chemistry* ; Swine

OBJECTIVETo develop a rapid, highly sensitive, and quantitative method for the detection of NT-proBNP levels based on a near-infrared point-of-care diagnostic (POCT) device with wide scope.

METHODSThe lateral flow assay (LFA) strip of NT-proBNP was first prepared to achieve rapid detection. Then, the antibody pairs for NT-proBNP were screened and labeled with the near-infrared fluorescent dye Dylight-800. The capture antibody was fixed on a nitrocellulose membrane by a scribing device. Serial dilutions of serum samples were prepared using NT-proBNP-free serum series. The prepared test strips, combined with a near-infrared POCT device, were validated by known concentrations of clinical samples. The POCT device gave the output of the ratio of the intensity of the fluorescence signal of the detection line to that of the quality control line. The relationship between the ratio value and the concentration of the specimen was plotted as a work curve. The results of 62 clinical specimens obtained from our method were compared in parallel with those obtained from the Roche E411 kit.

RESULTSBased on the log-log plot, the new method demonstrated that there was a good linear relationship between the ratio value and NT-proBNP concentrations ranging from 20 pg/mL to 10 ng/mL. The results of the 62 clinical specimens measured by our method showed a good linear correlation with those measured by the Roche E411 kit.

CONCLUSIONThe new LFA detection method of NT-proBNP levels based on the near-infrared POCT device was rapid and highly sensitive with wide scope and was thus suitable for rapid and early clinical diagnosis of cardiac impairment.

Antibodies ; Biomarkers ; Heart Diseases ; diagnosis ; Humans ; Immunoassay ; methods ; Infrared Rays ; Natriuretic Peptide, Brain ; blood ; Peptide Fragments ; blood ; Point-of-Care Testing ; Reagent Strips ; Sensitivity and Specificity

BACKGROUND: Albuminuria is generally known as a sensitive marker of renal and cardiovascular dysfunction. It can be used to help predict the occurrence of nephropathy and cardiovascular disorders in diabetes. Individuals with prediabetes have a tendency to develop macrovascular and microvascular pathology, resulting in an increased risk of retinopathy, cardiovascular diseases, and chronic renal diseases. We evaluated the clinical value of a strip test for measuring the urinary albumin-to-creatinine ratio (ACR) in prediabetes and diabetes. METHODS: Spot urine samples were obtained from 226 prediabetic and 275 diabetic subjects during regular health checkups. Urinary ACR was measured by using strip and laboratory quantitative tests. RESULTS: The positive rates of albuminuria measured by using the ACR strip test were 15.5% (microalbuminuria, 14.6%; macroalbuminuria, 0.9%) and 30.5% (microalbuminuria, 25.1%; macroalbuminuria, 5.5%) in prediabetes and diabetes, respectively. In the prediabetic population, the sensitivity, specificity, positive predictive value, negative predictive value, and overall accuracy of the ACR strip method were 92.0%, 94.0%, 65.7%, 99.0%, and 93.8%, respectively; the corresponding values in the diabetic population were 80.0%, 91.6%, 81.0%, 91.1%, and 88.0%, respectively. The median [interquartile range] ACR values in the strip tests for measurement ranges of <30, 30-300, and >300 mg/g were 9.4 [6.3-15.4], 46.9 [26.5-87.7], and 368.8 [296.2-575.2] mg/g, respectively, using the laboratory method. CONCLUSIONS: The ACR strip test showed high sensitivity, specificity, and negative predictive value, suggesting that the test can be used to screen for albuminuria in cases of prediabetes and diabetes.
Adult ; Aged ; Aged, 80 and over ; Albumins/*analysis ; Creatinine/*urine ; Diabetes Mellitus, Type 2/pathology/urine ; Female ; Humans ; *Immunoassay ; Male ; Middle Aged ; Prediabetic State/pathology/urine ; Reagent Strips/chemistry

BACKGROUND: Microscopic examinations are usually performed to confirm urine sediments in samples flagged in automated urinalysis. The aim of this study was to analyze the review rates and the difference in urinalysis results according to review rules. METHODS: A total of 1,408 urine samples submitted for health screening were collected. The urine chemistry test and urine sediment test were performed using EikenUS 3100 (Eiken Chemical Co. Ltd., Japan) and Sysmex UF-1000i (Sysmex Co., Japan), respectively. We assessed the rate of agreement between the 2 analyses and the kappa values for white blood cells (WBCs) and red blood cells (RBCs). Microscopic examinations were performed for all cases of discordant results between the urine strip and automated sediment analysis, some cases of concordant results, and cases of albuminuria. RESULTS: The review rate was 14.3%. Microscopic examinations were additionally performed on 77 samples (77/1,207, 6.4%) including 29 and 56 samples flagged for WBCs and RBCs, respectively. Based on the results of microscopic examination, the false-positive and the false-negative results of the urine chemistry test and automatic sediment analysis were corrected. Among concordant results between two methods, a clinically significant number of false-negatives were identified (6 results of WBC detection [6/125, 4.8%] and 4 of RBC detection [4/145, 2.8%]). Among the 22 unflagged cases of albuminuria, pathologic casts were detected in 21 cases (21/22, 95.5%). CONCLUSIONS: Microscopic examination based on the combined results of the two analyses improved the quality of the test.
Albuminuria ; Chemistry ; Erythrocytes ; Flow Cytometry ; Leukocytes ; Mass Screening ; Microscopy ; Quality Improvement* ; Reagent Strips ; Urinalysis*

A strip reader based lateral flow immunoassay (LFIA) was established for the rapid and quantitative detection of ractopamine (RAC) in swine urine. The ratio of the optical densities (ODs) of the test line (AT) to that of the control line (AC) was used to effectively minimize interference among strips and sample variations. The linear range for the quantitative detection of RAC was 0.2 ng/mL to 3.5 ng/mL with a median inhibitory concentration (IC50) of 0.59 ± 0.06 ng/mL. The limit of detection (LOD) of the LFIA was 0.13 ng/mL. The intra-assay recovery rates were 92.97%, 97.25%, and 107.41%, whereas the inter-assay rates were 80.07%, 108.17%, and 93.7%, respectively.
Animals ; Immunoassay ; Phenethylamines ; urine ; Reagent Strips ; Swine ; urine

BACKGROUND: The urine dipstick is widely used as an initial screening tool for the evaluation of proteinuria; however, its diagnostic accuracy has not yet been sufficiently evaluated. Therefore, we evaluated its diagnostic accuracy using spot urine albumin/creatinine ratio (ACR) and total protein/creatinine ratio (PCR) in proteinuria. METHODS: Using PCR > or = 0.2g/g or > or = 0.5g/g and ACR > or = 300mg/g or > or = 30mg/g as the reference standard, we calculated the diagnostic accuracy profile: sensitivity, specificity, positive and negative predictive value, and the area under the curve (AUC) of the receiver operating characteristic curve. RESULTS: PCR and ACR were available for 10,348 and 3,873 instances of dipstick testing. The proportions with PCR > or = 0.2g/g, > or = 0.5g/g and ACR > or = 300mg/g, > or = 30mg/g were 38.2%, 24.6% and 8.9%, 31.7%, respectively. The AUCs for PCR > or = 0.2g/g, > or = 0.5g/g, and ACR > or = 300mg/g were 0.935 (trace: closest to ideal point), 0.968 (1+), and 0.983 (1+), respectively. Both sensitivity and specificity were > 80% except for PCR > or = 0.5g/g with trace cutoff. For the reference standard of ACR > or = 30mg/g, the AUC was 0.797 (trace) and the sensitivity was 63.5%. CONCLUSION: Urine dipstick test can be used for screening in older outpatients with ACR > or = 300mg/g or PCR as the reference standard for proteinuria. However, we cannot recommend the test as a screening tool with ACR > or = 30mg/g as the reference owing to its low sensitivity.
Albuminuria ; Area Under Curve ; Humans ; Mass Screening ; Outpatients* ; Polymerase Chain Reaction ; Proteinuria* ; Reagent Strips ; ROC Curve ; Sensitivity and Specificity

A sensitive antibody-based lateral flow dipstick was developed for ginsenoside Re (GRe) detection. The stick consisted of a sample pad, a conjugate pad, membrane and an absorbent pad. The membrane was coated with two capture reagents, GRe-BSA conjugate and goat anti-mouse antibodies, forming a test line and a control line, respectively. The conjugate pad was saturated with colloidal gold particles coated with affinity purified monoclonal anti-GRe antibody. The visual detection limit was 200 microg x L(-1) of GRe and the reaction time was 10 min. The Panax ginseng roots were identified after these samples (10 mg) were extracted with 5 mL tap water for 30 min at room temperature, and the extracts were tested by the dipsticks and ELISA kit. The true and false P. ginseng could be distinguished with dipsticks. The dipstick could be used to detect the quality of the P. ginseng samples when the extract was diluted 100-folds. The results were compared with those obtained using an indirect competitive enzyme-linked immunosorbent assay (icELISA). The dipstick assay proved to be a sensitive and rapid tool for quality control of P. ginseng.
Animals ; Antibodies, Monoclonal ; immunology ; Counterfeit Drugs ; analysis ; Ginsenosides ; analysis ; Immunoassay ; instrumentation ; methods ; Mice ; Panax ; chemistry ; Reagent Strips ; Time Factors

This paper presents a vision-based method for the width of NC membrane online inspection. In the production of bio-test strip, the number of antigen or antibody which is coated on the membrane depends on the width and the uniformity of test line T and control line C. People should control the width and the uniformity strictly to ensure the accuracy of lines in order to achieve quantitative inspection with high sensitivity. And online inspection must be done, it cannot be processed when the solution has been dry up. This paper gives a design of online inspection system based on linear charge-coupled device (linear CCD), it makes such advantages in terms of speed, accuracy, and the operation to achieve real-time, online inspection.
Biosensing Techniques ; instrumentation ; Diagnostic Techniques, Ophthalmological ; Enzyme-Linked Immunosorbent Assay ; Gold Colloid ; chemistry ; Humans ; Immunosorbent Techniques ; instrumentation ; Online Systems ; Photometry ; instrumentation ; Reagent Strips ; Vision Tests ; instrumentation ; methods ; Vision, Ocular ; Visual Acuity

To develop a specific, rapid, and convenient immunochromatography assay (ICA) to detect melamine residues in dairy products and feed sample. Colloidal gold particles labeled with purified monoclonal antibody against anti-melamine were used as the detector reagent. The MEL-OVA (the conjugate of melamine and ovalbumin) and goat anti-mouse melamine IgG were blotted on the test and control regions of nitrocellulose membrane. The strip was then assembled with sample pad, absorbing pad, and dorsal shield. The limit of detection (LOD) is 50 microg/L. The test trip was applied to detect melamine in milk, milk powder, and animal feeds, with detection limits of 100 microg/L for milk, 100 ng/g for milk powder, 200 ng/g for feeds. Compared to LC-MS/MS, the ICA could be used to screen a large number of dairy products and feed samples for melamine residue.
Animals ; Antibodies, Monoclonal ; chemistry ; Cattle ; Dairy Products ; analysis ; Food Contamination ; analysis ; Gold Colloid ; chemistry ; Immunochromatography ; methods ; Milk ; chemistry ; Reagent Strips ; chemical synthesis ; chemistry ; Sensitivity and Specificity ; Triazines ; analysis

BACKGROUND: Ascorbic acid can cause false negative results in the detection of urinary hemoglobin (Hb) with reagent test strips. The fully automated urine analyzer URiSCAN SUPER (YD Diagnostics, Korea), which can measure ascorbic acid, was recently developed. We compared the URiSCAN SUPER to the semi-automated urine analyzer URiSCAN PRO II (YD Diagnostics) and evaluated the usefulness of a new reagent strip to detect ascorbic acid. METHODS: A total of 641 urine samples were used to evaluate the agreement between the two analyzers. In addition we performed urine microscopic examinations to investigate the sensitivity and specificity of urinary Hb and white blood cells. We determined the detection limit of urine ascorbic acid. We also determined the positive rate of ascorbic acid and the false negative rate for urinary Hb. Additionally, the interference effect of ascorbic acid for urinary Hb was investigated. RESULTS: The agreement rate between the two analyzers was greater than 98% for all tests except for urinary specific gravity. The detection limit of urine ascorbic acid was 10 mg/dL. The positive rate of ascorbic acid was 49.8%. The false-negative rate for urinary Hb was 6.0% and 2.8% (P>0.05) in the presence and absence of urine ascorbic acid, respectively. CONCLUSIONS: The performances of the two urine analyzers were comparable. URiSCAN SUPER for the detection of urinary Hb was resistant to interference by ascorbic acid. Since the URiSCAN SUPER performs a fully automated analysis, it would be useful in urine screening.
Ascorbic Acid ; Automation ; False Negative Reactions ; Hemoglobins ; Leukocytes ; Limit of Detection ; Mass Screening ; Reagent Strips ; Sensitivity and Specificity ; Specific Gravity ; Urinalysis