Application of gold immunochromatographic assay strip combined with digital evaluation for early detection of Toxoplasma gondii infection in multiple species.

BACKGROUND: Timely diagnosis of Toxoplasma gondii infection is necessary to prevent and control toxoplasmosis transmission. The gold immunochromatographic assay (GICA) is a means of rapidly detecting pathogen in samples. GICA-based diagnostic methods have been developed to accurately detect pathogens with high sensitivity and specificity, and their application in T. gondii diagnosis is expected to yield good results. METHODS: Colloidal gold test strips were produced using T. gondii C-terminal truncated apical membrane antigen 1 (AMA1C). Colloidal gold-AMA1C and colloidal gold-murine protein conjugate were synthesized under optimal conditions. A nitrocellulose membrane was treated with AMA1C and goat anti-mouse antibody as the test line and control line, respectively. In total, 90 cat serum samples were tested using AMA1C-GICA and a commercial enzyme linked immunosorbent assay (ELISA) kit. The GICA results were digitally displayed using a portable colloidal gold immunochromatographic test strip analyzer (HMREADER). The sensitivity, specificity, and stability of AMA1C-GICA were assessed, and this was then used to examine clinical samples, including 203 human sera, 266 cat sera, and 81 dog sera. RESULTS: AMA1C-GICA had a detection threshold of 1:32 for T. gondii-positive serum. The GICA strips specifically detected T. gondii antibodies and exhibited no reactivity with Plasmodium vivax, Paragonimus kellicotti, Schistosoma japonicum, Clonorchis sinensis, and Schistosoma mansoni. Consequently, 15 (16.7%) positive samples were detected using the AMA1C-GICA and commercial ELISA kits for each of the assays. The receiver-operating characteristic curve showed that GICA had a relative sensitivity of 85.3% and specificity of 92%, with an area under the curve of 98%. After analyzing clinical samples using HMREADER, 1.2%-23.4% of these samples were found to be positive for T. gondii. CONCLUSIONS: This study presents a novel assay that enables timely and efficient detection of serum antibodies against T. gondii, thereby allowing for its early clinical diagnosis. Furthermore, the integration of digital detection using HMREADER can enhance the implementation of GICA.

Ultrasensitive detection of imatinib in human serum using a gold-based paper sensor.

Imatinib is the tyrosine kinase inhibitor of choice for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. However, imatinib has drawbacks such as drug resistance and significant differences in pharmacokinetics within patients. Therefore, a colloidal gold-based immunochromatographic assay (CG-IA) was developed for measuring and monitoring imatinib in human serum. An imatinib derivative containing carboxyl groups was used for the synthesis of the immunogen, and 4-(4-methyl-1-piperazinylmethyl) benzoic acid was selected as the hapten for the heterologous coating antigen. Next, a highly sensitive and specific monoclonal antibody (mAb), 2F7 was screened for the construction of a CG-IA, with an IC50 value of 0.091 ng/mL. For the qualification of imatinib in human serum, the visual limit of detection (vLOD) and cut-off values of the CG-IA were 2 and 20 ng/mL, respectively. For quantitative detection, the calculated LOD value of the CG-IA was 0.068 ng/mL, with a linearity range of 1.004 and 23.087 ng/mL. The recovery rate of spiked serum samples was between 88.24 % and 104.75 %. In addition, the concentration of imatinib in the serum samples from 10 patients was detected by CG-IA and revealed a good correlation with those from LC-MS/MS. These results indicated that the developed gold-based paper sensor could become an effective tool for the rapid monitoring of imatinib in human serum samples.

Rapid Colloidal Gold Immunoassay for Pharmacokinetic Evaluation of Vancomycin in the Cerebrospinal Fluid and Plasma of Beagle Dogs.

Vancomycin (VAN), a glycopeptide antibiotic, is the preferred therapeutic agent for treating Gram-positive bacteria. Rapid and precise quantification of VAN levels in cerebrospinal fluid (CSF) and plasma is crucial for optimized drug administration, particularly among elderly patients. Herein, we introduce a novel clinical test strip utilizing colloidal gold competitive immunoassay technology for the expedient detection of VAN. This test strip enables the detection of VAN concentrations in clinical samples such as plasma within 10 min and has a limit of detection of 10.3 ng/mL, with an inhibitory concentration 50% (IC50) value of 44.5 ng/mL. Furthermore, we used the test strip for pharmacokinetic analysis of VAN in the CSF and plasma of beagle dogs. Our results provide valuable insights into the fluctuations of the drug concentration in the CSF and plasma over a 24 h period after a single intravenous dose of 12 mg/kg. The test strip results were compared with the results obtained via liquid chromatography-mass spectrometry methods, and the measured VAN concentrations in the CSF and plasma via both of the methods showed excellent agreement.

Establishment of a Colloidal Gold Immunochromatographic Method for the Detection of Cypermethrin in Tobacco.

In this study, the establishment of a colloidal gold immunochromatographic method for the detection of cypermethrin in tobacco was achieved by using colloidal gold immunochromatography: strong specificity and high sensitivity of cypermethrin semi-antigens and encapsulants were prepared during the study. The best colloidal gold solution was prepared by spectrophotometer and transmission electron microscope screening; the preparation process of gold-labeled antibodies was optimized, and finally the product of colloidal gold rapid detection test strips for cypermethrin was developed. The results of technical parameters and detection indexes showed that the detection limit of cypermethrin in tobacco was 1 mg/kg, and there was no cross-reaction with bifenthrin, cypermethrin, cyfluthrin and phenothrin, and the detection results of 30 tobacco samples were consistent with those of gas chromatography.

Development of Colloidal Gold Test Strips for the Detection of Oxamyl Residues in Tobacco.

In this study, monoclonal antibodies against oxamyl were prepared, and colloidal gold immunochromatography was used to design a rapid test strip product for the detection of oxamyl in tobacco with high specificity, accuracy and stability without cross-reactivity to commonly used tobacco fungicides based on the optimization of conditions such as pH value of diluent, diluent dosage, concentration of antibody marker, type of confining solution and complex solution. 5 The results of five samples of post-harvest ready-to-bake tobacco and first-harvest tobacco were consistent with the gas chromatographic method, which proved the reliability of the test strips. The limits of detection for the post-harvest and first-harvest tobacco samples were 0.1 mg/kg, and the test strips developed in this study are suitable for mass testing in tobacco laboratories with good application prospects because of their short detection time, simple pre-treatment and detection methods.

Development of Colloidal Gold Test Strips for the Detection of Triadimenol Residues in Tobacco.

The rapid and accurate determination of triadimenol residues is of great significance. In this study, based on the advantages of high efficiency, rapidity, reliability, simplicity and low cost of immunology, a test strip product for the rapid detection of triadimenol residues in tobacco was designed based on the optimization of conditions such as pH and dosage of diluent, concentration of antibody stock solution, type of confining solution and complex solution, with high specificity, accuracy and The results of 20 samples of fresh and first roasted tobacco were all consistent with the method of gas chromatography, which proved the reliability of the test strips. The detection limit for fresh and roasted tobacco was 5 mg/kg, and the test strips developed in this study are suitable for mass testing of tobacco samples in tobacco-related laboratories because of their short detection time, simple pre-treatment and detection methods, and good application prospects.

Design and development of a lanthanide-labeled immunochromatographic strip for simultaneous detection of morphine, methamphetamine and ketamine in hair.

Colloidal gold immunoassay is the most widely used method in the field of drug detection. However, this method often has poor quantitative identification ability and low analytical sensitivity, which is not suitable for the analysis of hair poisoning ingredients. In order to solve these limitations, we developed an immunochromatographic test strip for simultaneously screening multiple drugs in this study. This hand-held test strip used fluorescent nanoparticles loaded with lanthanide chelates as the signal carrier of fluorescence reading, and conducted quantitative testing of various drugs based on the competitive immune reaction between the analyte and antigen. Under the optimal conditions, the competition curves of morphine (MOP), methamphetamine (MET) and ketamine (KET) were obtained on a single band. The detection limit (LOD) of this analytical method was 100-1000 times lower than that of colloidal gold test strips. The detection limits of MOP, MET and KET were 0.06 ng mL-1, 0.1 ng mL-1 and 1.0 ng mL-1, respectively. No cross-reaction was observed when morphine, methamphetamine and ketamine were tested simultaneously with this method. And 184 hair samples were tested simultaneously, and the detected amount was very close to the results of LC-MS. The immunochromatographic strip showed good stability in repeated tests, and the coefficient of variation was less than 15%. Fluorescence immunochromatography strips and handheld strip readers have the characteristics of portability, speed, ease of operation and high sensitivity, and may become powerful tools for screening drug abuse in hair in forensic medicine.

Establishment of ultrasensitive immunoassay strip based on colloidal gold-McAb probe for detecting cyproheptadine hydrochloride and six phenothiazines in feed.

An ultrasensitive and broad-specific monoclonal antibody recognising cyproheptadine hydrochloride and six phenothiazines was produced. The 50% inhibition concentration against cyproheptadine hydrochloride was 0.036 ng/mL, and the cross-reactivities for six phenothiazines were from 6.33% to 63.16%. Based on the developed monoclonal antibody, an immunochromatographic strip was established, with the visual detection limits (cut-off values) of seven drugs ranging from 5 to 100 ng/g in feedstuffs. With the strip reader, the 50% inhibition concentration of the developed immunochromatographic strip for seven drugs ranged from 0.570 to 7.750 ng/g. The intra-assay recoveries were from 79.8% to 103.4% with the highest coefficient of variation of 11.3%. The inter-assay recoveries were from 79.0% to 96.6% with the highest coefficient of variation of 12.7%. In summary, the proposed immunochromatographic strip was considered suitable for simultaneously monitoring cyproheptadine hydrochloride and phenothiazines in feedstuffs.

Colloidal gold-based immunochromatographic biosensor for quantitative detection of S100B in serum samples.

Traumatic brain injury has become a serious public health problem. Timely detection, diagnosis and treatment of brain injury are closely related to the prognosis of patients, so identification of highly sensitive and specific biochemical markers of brain injury has important clinical value. Currently, the most studied and most promising marker is the protein S100B. In this study, a rapid quantitative biosensor for S100B was established using colloidal gold labeling and double antibody (8C10-6B8) sandwich immunochromatography. The biosensor was capable of quantifying S100B within 15 min, and showed no cross-reactivity with S100A, NSE, GFAP, or PGP9.5. The detection limit was determined to be 4.6 pg mL-1 with a linear range of 0.01-2 ng mL-1. Recovery experiments also indicated that the method had an acceptable accuracy. Moreover, the quantitative colloidal gold assay correlated well with the results of a chemiluminescence immunoassay when testing 40 clinical serum samples. Our developed colloidal gold quantitative immunochromatographic biosensor is a rapid, sensitive, specific and accurate method for the detection of S100B protein in serum, which is useful in the clinic for early diagnosis, as well as assessment of disease progression and prognosis of traumatic brain injury.

A colloidal gold immunoassay strip assay for cadmium detection in oilfield chemicals.

Cadmium ions (Cd2+) are some of the major pollutants in oilfield chemicals. To reduce the pollution of oilfield chemicals, it is necessary to detect and control the content of Cd2+. In this study, we synthesized a highly sensitive and specific monoclonal antibody against Cd2+ with an IC50 of 1.97 ng mL-1 and no cross-reactivity. Based on this antibody, a colloidal gold immunoassay strip detection assay with an IC50 of 1 mg kg-1 and a detection range of 1.0-20 mg kg-1 in oilfield chemicals was developed. This assay could be completed in 20 min and can be used for Cd2+ on-site testing in oilfield chemicals and improve supervision efficiency in oil exploration and development.

Immunochromatographic test strip for quantitative and rapid detection of tolfenpyrad in food samples.

In the study, a hapten was designed to preserve the molecular structure of tolfenpyrad while introducing a carboxyl group and was coupled with a carrier protein to synthesize an immunogen and coating antigen. A monoclonal antibody was fabricated against tolfenpyrad and its performance was assessed by indirect competitive enzyme-linked immunosorbent assay. Finally, we developed a colloidal gold nanoparticle immunochromatographic test strip (CGN-ICTS) for the detection of tolfenpyrad in kale, Chinese cabbage, and eggplant samples. The results shows that CGN-ICTS was sensitive, with calculated detection limits of 0.49 ng/g for kale and Chinese cabbage and 0.99 ng/g for eggplant. Subsequently, CGN-ICTS and LC-MS were used to analyze the tolfenpyrad-spiked samples. The recovery rate of the CGN-ICTS for kale samples was 97.1-103.0%, for Chinese cabbage samples was 93.7-103.4%, and for eggplant samples was 92.7-105.7%. Recovery rates were similar between CGN-ICTS and LC-MS. Therefore, CGN-ICTS can be used to quickly screen tolfenpyrad residues in foods.

Development of a Lateral Flow Immunoassay Based on a Highly Specific Monoclonal Antibody To Detect 4-Methylaminoantipyrine.

To avoid false-positive results in immunoassays due to cross-reactivity of antibodies with structural analogues, especially metabolites of target compounds, the preparation of highly specific antibodies is crucial. Preserving the characteristic structure of a target compound when designing a hapten is important when preparing highly specific antibodies. Here, we designed a novel hapten, 4-(((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4yl)amino)methyl)benzoic acid, named AA-BA, to improve the specificity of antibodies for detection of 4-methylaminoantipyrine (MAA), a residual marker of dipyrone, an important antipyretic-analgesic and anti-inflammatory drug. The structural features of the hapten remained almost the same as those of MAA. After experimental validation, monoclonal antibody 6A4 (mAb 6A4) was prepared with the half maximal inhibitory concentration (IC50) value of 4.03 ng/mL and negligible cross-reactivity with dipyrone metabolites and other antibiotics. In addition, a specific lateral flow immunoassay (LFA) strip based on colloidal gold was developed for screening MAA with a cutoff value of 25 ng/mL in milk. The developed LFA is a useful tool for rapid and accurate detection of MAA.

Colloidal gold and fluorescent immunochromatographic test strips for canine parvovirus detection.

Canine parvovirus (CPV) is an acute and highly infectious virus causing disease in puppies and, thus, affecting the global dog industry. The current CPV detection methods are limited by their sensitivity and specificity. Hence, the current study sought to develop a rapid, sensitive, simple, and accurate immunochromatographic (ICS) test to detect and control the spread and prevalence of CPV infection. More specifically, 6A8, a monoclonal antibody (mAb) with high specificity and sensitivity, was obtained by preliminary screening. The 6A8 antibody was labelled with colloidal gold particles. Subsequently, 6A8 and goat anti-mouse antibodies were coated onto a nitrocellulose membrane (NC) as the test and control lines, respectively. Furthermore, 6A8 and rabbit IgG antibodies were labelled with fluorescent microspheres and evenly sprayed onto a glass fibre membrane. Both strips could be prepared in 15 min with no noticeable cross-reactivity with other common canine intestinal pathogens. The strips were simultaneously used to detect CPV in 60 clinical samples using real-time quantitative PCR, hemagglutination, and hemagglutination inhibition assays. The colloidal gold (fluorescent) ICS test strip was stable for 6 (7) and 4 (5) months at 4 °C and room temperature (18-25 °C). Both test strips were easy to prepare and rapidly detected CPV with high sensitivity and specificity. Moreover, the results were easily interpretable. This study establishes a simple method for two CPV diseases, colloidal gold and fluorescent immunochromatographic (ICS) test strips. KEY POINTS: • CPV test strips do not exhibit cross-reactivity with other canine intestinal pathogens. • The strips are stable for months at 4 °C and at room temperature (18-25 °C). • These strips are a promising approach for the timely diagnosis and treatment of CPV.

Hapten synthesis and a colloidal gold immunochromatographic strip assay to detect nitrofen and bifenox in fruits.

In this study, we synthesized two haptens similar in structure to nitrofen (NIT), and screened out five monoclonal antibodies with the ability to recognize NIT and bifenox (BIF) by competitive ELISA, with the lowest IC50 values of 0.87 ng mL-1 and 0.86 ng mL-1, respectively. The antibody 5G7 was selected to be combined with colloidal gold to establish a lateral flow immunochromatographic assay strip. This method was shown to qualitatively and quantitatively detect the residues of NIT and BIF in fruit samples. The visual limits of detection for qualitative detection were 5 µg kg-1 and 10 µg kg-1 for NIT and BIF, respectively. The calculated limits of detection for quantitative detection were 0.75 µg kg-1, 1.77 µg kg-1 and 2.55 µg kg-1 respectively, for nitrofen in orange, apple and grapes, and 3.54 µg kg-1, 4.96 µg kg-1 and 5.26 µg kg-1, respectively, for bifenox. Thus the strip assay could be used for rapid analysis of fruit samples.

A gold-based immunochromatographic strip for the specific detection of tacrolimus in whole blood.

Tacrolimus is a macrolide immunosuppressant widely used in organ transplantation. Due to the narrow treatment window, therapeutic drug monitoring of the clinical application of tacrolimus is necessary. In this study, a carboxyl group introduced at the hydroxyl or carbon positions of tacrolimus was used to couple with carrier protein to synthesize complete antigens. After screening different immunogens and coating antigens, a highly-sensitive and specific monoclonal antibody (mAb) 4C5 was obtained, with a half inhibitory concentration (IC50) value of 0.26 ng mL-1 determined by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). A colloidal gold-based immunochromatographic strip (CG-ICS) was established to monitor tacrolimus in human whole blood based on the mAb 4C5. Through visual observation, it was found that the visual limit of detection (vLOD) and cut-off value of qualitative detection were 1.0 and 20.0 ng mL-1, respectively. The calculated LOD (cLOD) value of quantitative detection was 0.16 ng mL-1, and the linear range was 0.48-7.57 ng mL-1. In addition, the results of the CG-ICS for analyzing real positive samples of human whole blood were basically consistent with those of LC-MS/MS. Therefore, the CG-ICS was suitable for rapid and accurate clinical monitoring of tacrolimus.

Utilization of a lateral flow colloidal gold immunoassay strip based on surface-enhanced Raman spectroscopy for rapid detection of glycinin.

Glycinin is an important storage protein in soybean, but it can also lead to allergic reactions in humans. In this study, based on a low-cost, simple, rapid and portable lateral flow immunoassay test strip, combined with high sensitivity surface enhanced Raman spectroscopy (SERS) technology, a sandwich lateral flow immunochromatographic test strip for rapid detection of soybean allergen glycinin is established. In the experiment, colloidal gold was covalently conjugated with rabbit-derived polyclonal antibodies of glycinin and Raman probe molecule 4-aminothiophenol(4-PATP) to prepare the immunoprobe. The respective optimal PATP and optimal antibody labeling amounts of colloidal gold solution were 1.05 × 10-2mol/L and 4.6 × 10-8mol/L. The detection limit of the test strip for glycinin was 4.87 ng/mL. The recovery rate ranged from 91 to 107 % and the CV was between 3 and 10 %. The test strip underwent no cross-reaction with ß-conglycinin, sesame protein, peanut protein, wheat protein or whey protein. The results of the experiment showed that this method exhibits high sensitivity and specificity.

Study on the performance of novel nanomaterials for detection of biomarkers such as PCT based on immunochromatography sensitivity.

In this study, by comparing the UV-vis spectral characteristics of colloidal gold and colloidal gold enhancer, and their differences as immunochromatographic tracers in the qualitative detection of PCT, IL-6, Hp and quantitative determination of PCT performance, the factors that may affect the sensitivity were discussed. The results show that the absorbance at 520 nm of CGE diluted 20-fold and colloidal gold diluted 2-fold were comparable, and the sensitivity of CGE immunoprobe for qualitative detection of PCT, IL-6 and Hp was higher than that of colloidal gold immunoprobe, and the reproducibility and accuracy of both immunoprobes for quantitative detection of PCT were good. Indicating that the high sensitivity of CGE immunoprobe detection is mainly due to the absorption coefficient of CGE at 520 nm is about 10 times that of colloidal gold immunoprobe, CGE has stronger light absorption capacity and stronger quenching effect on rhodamine 6 G on the nitrocellulose membrane surface of the test strip.

Development of a colloidal gold immunochromatographic strip for rapid and sensitive detection of nicotine.

Nicotine (NCT) is commonly contained in e-cigarette oils, and long-term nicotine intake can exacerbate cardiovascular disease, neurodegenerative disease, and lung cancer. Therefore, there is a need to develop a fast and sensitive method for detecting NCT in e-cigarette oils. We prepared anti-NCT monoclonal antibodies (mAbs), which belong to the IgG2 subclass. The optimum mAb had a half-maximal inhibitory concentration (IC50) of 6.81 ng/mL for NCT. We developed a colloidal gold immunochromatographic strip (CG-ICS) to detect NCT content with a visual cut-off and limit of detection of 20 ng/mL and 0.40 ng/mL, respectively. The recovery rates of NCT from spiked samples ranged between 100.36% and 109.96% based on CG-ICS. These results were consistent with those obtained from high performance liquid chromatography (HPLC). We confirmed the reliability of CG-ICS using NCT-positive samples. Therefore, CG-ICS represents an effective and rapid on-site screening tool for the detection of NCT in e-cigarette oils.

Development of Ic-ELISA and Colloidal Gold Lateral Flow Immunoassay for the Determination of Cypermethrin in Agricultural Samples.

Cypermethrin (CYP) is an insecticide in the pyrethroid family and is used widely in agriculture and for public health purposes. However, CYP has been shown to have negative impacts on reproduction, immunity and nerves in mammals. In this study, a monoclonal antibody (mAb) against CYP was prepared and used to establish an indirect competitive immunosorbent assay (ic-ELISA) and colloidal gold lateral flow immunoassay (LFIA) for the quantitative and qualitative determination of CYP residues in agricultural products. The half inhibition concentration of the ic-ELISA was 2.49 ng/mL, and the cut-off value and visual limit of detection of the LFIA were 0.6 and 0.3 µg/mL, respectively. The recovery rates of the ic-ELISA ranged from 78.8% to 87.6% in tomato, cabbage and romaine lettuce. The qualitative results of LFIA and quantitative results of ic-ELISA and HPLC were in good agreement in blind samples. Overall, the established ic-ELISA and LFIA proved to be accurate and rapid methods for the determination of CYP in agricultural products.

Development of a colloidal gold immunochromatographic test strip for the rapid detection of iprodione.

Iprodione is a dicarboximide fungicide that is widely used in agriculture around the world. A reliable and rapid detection method is needed for the on-site monitoring of iprodione residues in a variety of agricultural products. Herein, a colloidal gold immunochromatographic test strip was developed based on a selected coating antigen and a specific monoclonal antibody against iprodione. The particle size of colloidal gold, the preparation technique of the conjugate pad, the composition of the loading buffer, and the extraction solvent were comprehensively optimized for the test strip. A cut-off value of 0.9 mg kg-1 (50 ng mL-1) and a visual limit of detection of 0.09 mg kg-1 (5 ng mL-1) were achieved in a complex matrix of tobacco. No cross-reactivity was observed for iprodione metabolite and four other widely used pesticides during tobacco growth. Furthermore, the developed colloidal gold immunochromatographic test strip was applied to determine iprodione residues in tobacco samples, and the obtained results were in good agreement with those obtained by liquid chromatography tandem mass spectrometry. Additionally, the test strip was found to be stable afterlong-term storage at 37 °C for two months. The developed colloidal gold immunochromatographic test strip showed excellent accuracy, sensitivity, specificity, and stability, therefore, it is suitable for the rapid detection of iprodione residues in complex matrices.