To meet high-throughput screening of the residues of sulfonamides (SAs) with high sensitivity toward sulfamethazine (SM2) in milk samples, a new highly sensitive lateral flow immunoassay (LFA) based ...on amorphous carbon nanoparticles (ACNs) was developed. First, a group-specific monoclonal antibody 10H7 (mAb 10H7) that could recognize 25 SAs with high sensitivity toward SM2 (IC
50
value of 0.18 ng/mL) was prepared based on H1 as an immune hapten and H4 as a heterologous coating hapten. Then, mAb 10H7 was conjugated to ACNs as an immune probe for LFA development. Under the optimized conditions, the LFA could detect 25 SAs with the cut-off value toward SM2 of 2 ng/mL, which could meet the requirement for detection of SAs. In addition, the LFA developed was also used for screening SAs’ residues in real milk samples, with results being consistent with HPLC–MS/MS. Thus, this LFA can be used as a high-throughput screening tool for detection of SAs.
Graphical abstract
Rearrangements of the anaplastic lymphoma kinase (ALK) gene comprise a small subset of non-small cell lung cancer (NSCLC). Patients with NSCLC harboring ALK fusion proteins are sensitive to ALK ...tyrosine kinase inhibitors (TKIs). Various fusion partners of ALK are being discovered with the application of next-generation sequencing (NGS).
Here, we report a female patient with metastatic lung adenocarcinoma harboring LMO7-ALK (L15, A20) rearrangement revealed by NGS. The patient received crizotinib as first-line treatment and has achieved partial response with a progression-free survival over 1 year.
We firstly found that the satisfactory response to crizotinib verified the oncogenic activity of LMO7-ALK fusion. Great progression and wide application of NGS facilitate the findings of rare fusion types.
To improve the detection sensitivity of neomycin (NEO) in milk, we produced a sensitive monoclonal antibody (mAb) against NEO and developed a lateral flow immunoassay based on amorphous carbon ...nanoparticles (ACNPs-LFA). First, we conjugated NEO to carrier protein to prepare mAbs. We obtained six mAbs: mAb 1C6, mAb 1D3, mAb 2D3, mAb 4D5, mAb 5D1, and mAb 5H1. We characterised the mAbs by indirect competitive enzyme-linked immunosorbent assay and selected the most sensitive mAb based on the half-maximal inhibitory concentration (IC
50
). We selected mAb 4D5 (IC
50
= 0.15 ng/mL) for the development of LFA. MAb 4D5 was labelled with ACNPs (ACNPs-mAb 4D5) by electrostatic absorption. Under optimised conditions, 5.4 μg mAb 4D5 coupling with 1 mL ACNPs, NEO-OVA at concentration of 80 μg/mL, 3 μL ACNPs-mAb 4D5 were used to develop LFA. The cut-off value was 8 ng/mL. Therefore, our developed ACNPs-LFA is suitable for on-site detection of NEO residues.
•A novel hapten design strategy focused on atomic charge distribution was proposed.•A high-affinity and specific mAb 8C9 against MQCA was prepared.•A sensitive icELISA for screening MQCA in swine ...muscle and liver was developed.
Production of high-affinity and specific antibodies to small molecules with molecular weight (MW) lower than 200 Da is challenging. Here, we designed a novel hapten, named hapten H6, for the detection of 3-methyl-quinoxaline-2-carboxylic acid (MQCA, MW of 189 Da), a residual marker of olaquindox, one of important veterinary antibiotics. The hapten H6 maintained all structural features of MQCA, especially in mulliken atomic charge distribution. Then, a monoclonal antibody (mAb) named 8C9 was obtained with an IC50 value of 0.2 µg/L, yielding a 15.5- to 88.5-fold improvement compared to previously prepared specific antibodies against MQCA. In addition, mAb 8C9 exhibited ignorable cross-reactivity with other structural analogs. Finally, a highly sensitive and specific indirect competitive ELISA based on mAb 8C9 was developed for the detection of MQCA in swine muscle and liver samples with limit of detection values of 0.04 µg/kg and 0.09 µg/kg, respectively.
The “turn-off” mode lateral flow immunoassay (LFA) based on the conventional competitive format and “turn-on” mode LFA mainly determined by inner filter effect (IFE-LFA) were objectively compared ...using T-2 toxin as a model molecule and T-2-BSA and anti-T-2 monoclonal antibody as immunocomplex pairs to improve the sensitivity of the LFA. First, Au nanoparticles (Au NPs), amorphous carbon nanoparticles (ACNPs), quantum dots nanospheres (QDs), and time-resolved fluorescent microspheres (TRFMs) were selected as labels for developing the “turn-off” mode LFAs (Au NPs-LFA, ACNPs-LFA, QDs-LFA, and TRFMs-LFA, respectively). Thereafter, Au NPs and ACNPs were used as absorbers, and QDs and TRFMs were selected as fluorescers, for preparing the “turn-on” mode IFE-LFAs (IFE-LFA based on Au NPs as absorbers and QDs as fluorescers Au NPs-mAb-QDs-BSA, IFE-LFA based on Au NPs as absorbers and TRFMs as fluorescers Au NPs-mAb-TRFMs-BSA, IFE-LFA based on ACNPs as absorbers and QDs as fluorescers ACNPs-mAb-QDs-BSA, and IFE-LFA based on ACNPs as absorbers and TRFMs as fluorescers ACNPs-mAb-TRFMs-BSA). Under optimized conditions, the naked-eye observation cut-off values for the aforementioned eight LFAs were 4, 2, 2, 2, 3, 2, 1.5, and 1 ng/mL, with quantitative limit of detection values of 0.50, 0.23, 0.24, 0.23, 0.47, 0.45, 0.27, and 0.22 ng/mL, respectively. Among all LFAs, ACNPs-mAb-TRFMs-BSA showed the highest sensitivity. The results of this study provide a perspective for improving the sensitivity of the LFAs to meet the requirements of on-site screening for trace substances.
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•“Turn-off” and “turn-on” mode LFAs using the same immune reagent and target were objectively compared.•Dual readout detection and lower immune reagent consumption are significant characteristics of “turn-on” mode LFA.•ACNPs with super absorption performance (250–700 nm) as fluorescence quencher are more conducive to improve “turn-on” mode LFA’s sensitivity.•The capacity of reducing matrix effects by TRFMs as fluorescence donors in “turn-on” mode LFA was better than that of QDs.
To overcome limitations with respect to the low sensitivity of rapid immunoassay methods due to dilution of the extract in extraction solution, ultra-sensitive antibodies are needed. The ...linker-tethering site between the target and carrier protein, the length of the spacer arm, and the atomic charge of the linker-tethering site between the target and spacer arm in haptens design play a pivotal role in the preparation of ultra-sensitive antibodies. In this study, carbendazim (CBZ), a systemic broad-spectrum fungicidal pesticide, was used as model compound. 2-Aminobenzimidazole was chosen as the common skeletal structure and reacted with different groups, such as alkanes and urine groups, to form haptens H1 (previously reported), H2 (novel), and H3 (novel), respectively. Subsequently, eight monoclonal antibodies (mAbs) were generated using these three haptens, which exhibited notable variation in sensitivity. The half maximal inhibitory concentration (IC50) value of mAb 4B11 based on hapten H3 against CBZ was 0.04 ng/mL, which was seven times better than that of mAb 5B10 (IC50 value of 0.28 ng/mL) prepared using hapten H2 and 129 times better than that of mAb 6D5 (IC50 value of 5.15 ng/mL) prepared using hapten H1. Finally, the optimal antigen–antibody combination was employed to establish a sensitive colloidal gold lateral flow immunoassay (CG-LFA) for CBZ detection in vegetables and fruits with convenient sample pretreatment. The developed CG-LFA shows promising practical utility and prospects for application due to its low limit of detection (0.10–0.18 ng/mL) and high recovery ratios (76.0%–96.1%) in six different samples.
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•A novel hapten design strategy focuses on the link site and spacer arm length of the target compound.•An ultra-sensitive monoclonal antibody (mAb) 4B11 against carbendazim was prepared.•A sensitive colloidal gold lateral flow immunoassay in fruits and vegetables was developed.
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.
In peptide amphiphile, The positively charged amino acid arginine can inspire the ordered self-assembly of gold nanocomposites (AuNPs), transfer positive charge to AuNPs, and weaken the aggregation ...of AuNPs by electrostatic repulsion, whereas hydrophobic fatty acid chains regulate the self-assembly of AuNPs through hydrophobic interaction, which may be a novel strategy to overcome disordered arrangement and aggregation of AuNPs to obtain an ultra-sensitive electrochemical immunosensor for determining the total aflatoxin amount. In this study, a peptide amphiphile (C14R5), composed of five arginine residues as the hydrophilic chain and myristic acid as the hydrophobic chain, inspired AuNPs to form monodispersed hollow raspberry-like AuNPs (rasAuNPs). rasAuNPs could captured and immobilized large amounts of aflatoxin antigens via the Au–S bonds, resulting in binding to more anti-aflatoxin antibodies. In the absence of aflatoxins, the enriched antigens bound to abundant antibodies, resulting in a low blank signal current. By contrast, in the presence of aflatoxins, enough antibodies could bind to the targets and less antibodies could recognize the antigens, increasing the detection signal intensity. Under the optimal conditions, the developed sensor demonstrated a wide linear range (0.13–29.06 pg mL−1) and a low limit of detection for total aflatoxins (0.05 pg mL−1) using a mixed standard (AFB1: AFB2: AFG1: AFG2 with a weight ratio of 1:1:1:1) in peanut, peanut milk, and maize powder samples. Hence, this novel strategy improves the sensitivity of electrochemical sensors and can be easily applied to detect other small molecule compound for the purpose of food safety.
The developed electrochemical immunosensor remarkably enhanced the signal intensity as compared to the conventional AuNPs modified electrode. Display omitted
•C14R5 inspired AuNPs to form orderly self-assembled monodispersed hollow rasAuNPs.•The rasAuNPs modified on electrodes could capture more antigens and antibodies than that of AuNPs.•The sensitivity of electrochemical immunosensor based on the rasAuNPs modified electrodes could be significantly improved.
Abstract
To reduce the false positive results caused by cross reactivity of the antibodies with other structural analogues, it is crucial to prepare a high specificity and sensitivity antibody ...against target for developing an accurate immunoassay. In this study, tilmicosin (TM) was selected as a model molecule. Firstly, two-dimensional similarity, electrostatic potential energy, mulliken atomic charges and overlapping of different haptens with TM were calculated using Gaussian 09W and Discovery studio, and the newly designed TM-HS was selected as the optimal hapten. Furthermore, a monoclonal antibody (mAb 12C8) was produced with the half maximal inhibitory concentration (IC
50
) of 0.36 ng/mL, and negligible cross-reactivity (CR) with other antibiotics. Finally, a lateral flow immunoassay (LFA) for the detection of TM based on amorphous carbon nanoparticles (ACNPs) labeled mAb 12C8 was developed by the reflectance value under natural light. The recoveries of TM ranged from 83.18% to 103.25% with a coefficient of variation (CV) < 12.47%. The results showed that the cut-off value of TM in milk samples was 1 ng/mL, and the limits of detection (LODs) for chicken muscle, bovine muscle, porcine muscle and porcine liver samples were 5.23, 5.98, 6.85 and 7.31 μg/kg, respectively. In addition, 40 real samples were tested by the LFA, and the detection results were consisted with that of high-performance liquid chromatography-UV detector (HPLC–UV). Those results indicated that the developed LFA is an accurate and useful tool for on-site screening of TM in milk and animal tissues.
BackgroundRearrangements of the anaplastic lymphoma kinase (ALK) gene comprise a small subset of non-small cell lung cancer (NSCLC). Patients with NSCLC harboring ALK fusion proteins are sensitive to ...ALK tyrosine kinase inhibitors (TKIs). Various fusion partners of ALK are being discovered with the application of next-generation sequencing (NGS).Case presentationHere, we report a female patient with metastatic lung adenocarcinoma harboring LMO7-ALK (L15, A20) rearrangement revealed by NGS. The patient received crizotinib as first-line treatment and has achieved partial response with a progression-free survival over 1 year.ConclusionsWe firstly found that the satisfactory response to crizotinib verified the oncogenic activity of LMO7-ALK fusion. Great progression and wide application of NGS facilitate the findings of rare fusion types.
