Immunoassay Approach Research Articles

A Dual Nano-Signal Probe-Based Electrochemical Immunosensor for the Simultaneous Detection of Two Biomarkers in Gastric Cancer

Detecting multiple tumor markers is of great importance. It helps in early cancer detection, accurate diagnosis, and monitoring treatment. In this work, gold nanoparticles–toluidine blue–graphene oxide (AuNPs-TB–GO) and gold nanoparticles–carboxyl ferrocene–tungsten disulfide (AuNPs–FMC–WS2) nanocomposites were prepared for labeling Carcinoembryonic antigen (CEA) antibody and Carbohydrate antigen 72–4 (CA72-4) antibody, respectively, and used as two kinds of probes with different electrochemical signals. With the excellent magnetic performance of biotin immune magnetic beads (IMBs), the biofunctional IMBs were firmly deposited on the magnetic glassy carbon electrode (MGCE) surface by applying a constant magnetic field, and then the CEA and CA72-4 antibody were immobilized on the IMBs by the avidin–biotin conjugation. The assay was based on the change in the detection peak current. Under the optimum experimental conditions, the linear range of detection of CEA is of the two-component immunosensor is from 0.01 to 120 ng/mL, with a low detection limit of 0.003 ng/mL, and the linear range of detection of CA72-4 is from 0.05 to 35 U/mL, with a detection limit of 0.016 U/mL. The results showed that the proposed immunosensor enabled simultaneous monitoring of CEA and CA72-4 and exhibited good reproducibility, excellent high selectivity, and sensitivity. In particular, the proposed multiplexed immunoassay approach does not require sophisticated fabrication and is well-suited for high-throughput biosensing and application to other areas.

A broad-spectrum antibody-based lateral flow immunoassay for detection of carbofuran and 3-hydroxy-carbofuran

Simultaneous detection of carbofuran (CBF) and 3-hydroxy carbofuran (3-OH-CBF) in fruits and vegetables is important due to their high toxicity and widespread use in pest control. However, most lateral flow immunoassay (LFA) approaches only detection CBF. To overcome this limitation, two haptens, 6-((2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)oxy)hexanoic acid and 6-((((2,2-dimethyl-2,3-dihydroben zofuran-7-yl)oxy)carbonyl)amino)hexanoic acid, named H1 and H2, were designed for broad-spectrum antibody detection of CBF and 3-OH-CBF. The highly specific monoclonal antibody (mAb) 3F4 based on hapten H1 only recognized CBF, while broad-spectrum mAbs 1D3, 6B7 and 6E8 based on hapten H2 recognized both compounds. The carbon bond to the hydroxyl group of 3-OH-CBF adopts a 28.55° angle relative to the same carbon bond of CBF, explaining the specificity of the H1-based mAb against CBF. After optimizing the antigen-antibody combination, a sensitive colloidal gold-LFA (CG-LFA) strip was constructed based on mAb 1D3 and H2-BSA for CBF and 3-OH-CBF detection following simple sample pretreatment. The established LFA had a limit of detection of 0.49–6.63 ng/mL, and adequate recoveries (69.3–105.7 %) for CBF and 3-OH-CBF in 26 fruit and vegetable samples.

Magnetic microbead-based upconversion immunoassay with laser-induced breakdown spectroscopy readout for the detection of prostate-specific antigen.

Laser-induced breakdown spectroscopy (LIBS) is a promising technique for the readout of immunochemical assays utilizing indirect detection of labels (Tag-LIBS), typically based on nanoparticles. We have previously demonstrated that Tag-LIBS immunoassay employing yttrium-based photon-upconversion nanoparticles (UCNPs) can reach sensitivity similar to commonly used enzyme and fluorescence immunoassays. In this study, we report on further increasing the sensitivity of UCNP-based Tag-LIBS immunoassay by employing magnetic microbeads (MBs) as the solid phase in the determinationof cancer biomarker prostate-specific antigen. Due to the possibility of analyte preconcentration, MBs enabled achieving a limit of detection (LOD) of 4.0pg·mL-1, representing two orders of magnitude improvement compared withequivalent microtiter plate-based assay (LOD of 460pg·mL-1). In addition, utilizing MBs opens up the possibility of an internal standardization of the LIBS readout by employing iron spectral lines, which improves the assay robustness by compensating for LIBS signal fluctuations and bead-bound immunocomplexes lost throughout the washing steps. Finally, the practical applicability of the technique was confirmed by the successful analysis of clinical samples, showing a strong correlation with the standard electrochemiluminescence immunoassay. Overall, MB-based Tag-LIBS was confirmed as a promising immunoassay approach, combining fast readout, multiplexing possibilities, and high sensitivity approaching upconversion luminescence scanning while avoiding the requirement of luminescence properties of labels.

Portable Near-Infrared to Near-Infrared Platform for Homogeneous Quantification of Biomarkers in Complex Biological Samples.

Förster resonance energy transfer (FRET)-based homogeneous immunoassay obviates tedious washing steps and thus is a promising approach for immunoassays. However, a conventional FRET-based homogeneous immunoassay operating in the visible region is not able to overcome the interference of complex biological samples, thus resulting in insufficient detection sensitivity and poor accuracy. Here, we develop a near-infrared (NIR)-to-NIR FRET platform (Ex = 808 nm, Em = 980 nm) that enables background-free high-throughput homogeneous quantification of various biomarkers in complex biological samples. This NIR-to-NIR FRET platform is portable and easy to operate and is mainly composed of a high-performance NIR-to-NIR FRET pair based on lanthanide-doped nanoparticles (LnNPs) and a custom-made microplate reader for readout of NIR luminescence signals. We demonstrate that this NIR-to-NIR FRET platform is versatile and robust, capable of realizing highly sensitive and accurate detection of various critical biomarkers, including small molecules (morphine and 1,25-dihydroxyvitamin D), proteins (human chorionic gonadotropin), and viral particles (adenovirus) in unprocessed complex biological samples (urine, whole blood, and feces) within 5-10 min. We expect this NIR-to-NIR FRET platform to provide low-cost healthcare for populations living in resource-limited areas and be widely used in many other fields, such as food safety and environmental monitoring.

Weakly ionized gold nanoparticles amplify immunoassays for ultrasensitive point-of-care sensors.

Gold nanoparticle-based lateral flow immunoassays (AuNP LFIAs) are widely used point-of-care (POC) sensors for in vitro diagnostics. However, the sensitivity limitation of conventional AuNP LFIAs impedes the detection of trace biomarkers. Several studies have explored the size and shape factors of AuNPs and derivative nanohybrids, showing limited improvements or enhanced sensitivity at the cost of convenience and affordability. Here, we investigated surface chemistry on the sensitivity of AuNP LFIAs. By modifying surface ligands, a surface chemistry strategy involving weakly ionized AuNPs enables ultrasensitive naked-eye LFIAs (~100-fold enhanced sensitivity). We demonstrated how this surface chemistry-amplified immunoassay approach modulates nanointerfacial bindings to promote antibody adsorption and higher activity of adsorbed antibodies. This surface chemistry design eliminates complex nanosynthesis, auxiliary devices, or additional reagents while efficiently improving sensitivity with advantages: simplified fabrication process, excellent reproducibility and reliability, and ultrasensitivity toward various biomarkers. The surface chemistry using weakly ionized AuNPs represents a versatile approach for sensitizing POC sensors.

Growth‐Induced Extinction Development of Gold Nanoclusters as Signal Transducers for Quantitative Immunoassays

AbstractSignal transducers are crucial in bioassay platforms for converting target detection into recordable signals. Commonly used color development for immunoassays involves enzymes and colorimetric substrates. However, due to cost and environmental issues, practical point‐of‐care testing requires alternative signal transducers. Growth‐induced extinction (absorption and scattering) of gold nanoclusters (AuNCs) is proposed as a novel approach for quantitative immunoassays. AuNCs devoid of localized surface plasmon resonance (LSPR) are used as seeds for growth reactions. Through reactions with a growth solution comprised of gold precursor and mild reductant, AuNCs of varying concentrations underwent controlled growth, resulting in nanoparticles of different sizes exhibiting distinct LSPR‐mediated extinction bands. Notably, the seed concentration exhibited a robust correlation with the resulting extinction of the grown particles on a small scale of 110 µL for a 96‐well microplate platform. To demonstrate this signal transduction mechanism, immunosorbent assays are performed using the conjugates of AuNC and detection antibody. The sandwich‐type assay successfully quantified a model antigen, human immunoglobulin G (hIgG), by monitoring LSPR wavelength and absorbance. This assay demonstrated a working range of 0.001–1 µg mL−1 and limit of detection of 1.19 ng mL−1. Signal transducers using the growth of AuNCs offer new alternative candidates for immunoassay platforms.

A Combined LC-MS and Immunoassay Approach to Characterize Preservative-Induced Destabilization of Human Papillomavirus Virus-like Particles Adsorbed to an Aluminum-Salt Adjuvant.

During the multi-dose formulation development of recombinant vaccine candidates, protein antigens can be destabilized by antimicrobial preservatives (APs). The degradation mechanisms are often poorly understood since available analytical tools are limited due to low protein concentrations and the presence of adjuvants. In this work, we evaluate different analytical approaches to monitor the structural integrity of HPV16 VLPs adsorbed to Alhydrogel™ (AH) in the presence and absence of APs (i.e., destabilizing m-cresol, MC, or non-destabilizing chlorobutanol, CB) under accelerated conditions (pH 7.4, 50 °C). First, in vitro potency losses displayed only modest correlations with the results from two commonly used methods of protein analysis (SDS-PAGE, DSC). Next, results from two alternative analytical approaches provided a better understanding of physicochemical events occurring under these same conditions: (1) competitive ELISA immunoassays with a panel of mAbs against conformational and linear epitopes on HPV16 VLPs and (2) LC-MS peptide mapping to evaluate the accessibility/redox state of the 12 cysteine residues within each L1 protein comprising the HPV16 VLP (i.e., with 360 L1 proteins per VLP, there are 4320 Cys residues per VLP). These methods expand the limited analytical toolset currently available to characterize AH-adsorbed antigens and provide additional insights into the molecular mechanism(s) of AP-induced destabilization of vaccine antigens.

Abstract 3630: Measuring EGFr on extracellular vesicles in human plasma samples

Abstract Introduction: Extracellular vesicles (EVs), secreted extracellularly by almost all types of cells, play a pivotal role in intercellular communication and have emerged as valuable indicators in disease progression. Their potential for cancer diagnostics and monitoring is particularly noteworthy. Epidermal growth factor receptor (EGFr) is a major driver of several cancers and serve as an important target or co-target in cancer treatment. The purpose of this study was to develop an assay for measuring EGFr on EVs directly in plasma samples. Additionally three assays were developed to measure the total concentration of plasma EVs. Methods: Recognizing the expected low concentrations of EGFr on EVs, the immunoassays were developed using Single molecule array (Simoa) technology, which has shown up to a 1000-fold improvement in sensitivity as compared to conventional immunoassay approaches. Commercial available calibrator material and antibodies against surface markers (EGFr, CD9, CD63 and CD81) were used to develop four assays: CD81-EGFr, CD81-CD9, CD81-CD63, and CD9-CD63. Plasma samples from healthy individuals (n=13) and small cohorts of various cancer patients (n=30, lung, colorectal, ovarian) were used for development, validation and testing. Results: Each assay was tested for dilution linearity, limit of detection, analytical variation and pre-analytical stability of EVs. EGFr on EVs was lower in cancer patients (median: 489 × 106 EVegfr/mL) compared with healthy individuals (median: 663 × 106 EVegfr/mL). Conversely, total EV concentrations were markedly higher in plasma from cancer patients compared with healthy individuals (CD81-CD9: 9157 vs 4088, CD81-CD63: 3634 vs 1582, CD9-CD63: 9538 vs 1210, median values x106 EV/mL). Positive correlations were observed among the total EV assays (r=0.6, p<0.0001). Conclusion: The developed assays allow measurement of EGFr on EVs and total EVs in human plasma samples. Notable, total EV concentrations were highly increased in cancer as compared with healthy individuals. The assays are now ready for a comprehensive validation using large cohorts of cancer samples. Citation Format: Dorte Aa. Olsen, Torben F. Hansen, Ivan Brandslund, Jonna S. Madsen. Measuring EGFr on extracellular vesicles in human plasma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3630.

Assessment of immunoprecipitation with subsequent immunoassays for the blood-based diagnosis of Alzheimer's disease.

The Aβ42/40 ratio and the concentration of phosphorylated Tau181 in blood plasma represent attractive biomarkers for Alzheimer's disease. As a means for reducing potential matrix effects, which may interfere with plasma immunoassays, we have previously developed a pre-analytical sample workup by semi-automated immunoprecipitation. Here we test the compatibility of pre-analytical immunoprecipitations with automated Aβ1-40, Aβ1-42 and phosphorylated Tau181 immunoassays on the Lumipulse platform and compare the diagnostic performance of the respective immunoprecipitation immunoassay approaches with direct plasma measurements. 71 participants were dichotomized according to their Aβ42/40 ratios in cerebrospinal fluid into the diagnostic groups amyloid-positive (n = 32) and amyloid-negative (n = 39). The plasma Aβ1-42/1-40 ratio and phosphorylated Tau181 levels were determined on the Lumipulse G600II platform (Fujirebio) by direct measurements in EDTA-plasma or after Aβ- or Tau-immunoprecipitation, respectively. Pre-analytical immunoprecipitation of Aβ turned out to be compatible with the Lumipulse Aβ assays and resulted in a numerical, yet statistically not significant increase in the area under the ROC curve for plasma Aβ1-42/1-40. Additionally, we observed a significant increase in the standardised effect size (Cohen's D). Pre-analytical immunoprecipitation of Tau resulted in increased differences between the diagnostic groups in terms of median and mean phosphorylated Tau 181 levels. Furthermore, we observed a greater Cohen's d (p < 0.001) and a larger area under the ROC curve (p = 0.038) after Tau-IP. Our preliminary findings in a small, preselected sample indicate that pre-analytical immunoprecipitation may have the potential to improve the diagnostic performance of plasma biomarker immunoassays for Aβ1-42/1-40 and phosphorylated Tau181 to predict brain amyloid deposition.

Pathogenic Escherichia coli (E. coli) food borne outbreak: Detection methods and controlling measures

Food-borne illnesses and diseases are major threats to human health. Common food borne pathogens include bacteria such as Escherichia coli Serogroup (O157), Bacillus cereus, Clostridium botulinum, Campylobacter sp., C. perfringens, some Listeria monocytogenes, Salmonella spp., Shigella spp., Staphylococcus aureus, and Vibrio spp. etc. Pathogenic E. coli infection usually causes severe diarrhea. Escherichia coli is the most common cause of acute urinary tract infections as well as urinary tract sepsis. Diarrheagenic E. coli pathotypes can be passed in the feces of humans and other animals. Safeguarding public health during mass gatherings is a big challenge. Despite the recent advances in food preservation techniques and food safety, significant disease outbreaks linked to food borne pathogens such as bacteria, fungi, and viruses still occur worldwide indicating that these pathogens still constitute significant risks to public health. However, at present the conventional method for E. coli detection requires several days, especially in cases where E. coli concentrations are low. Enrichment is a commonly used method for bacterial isolation to increase the cell counts of target bacteria. However, traditional culturing method is also a slow and laborious process requiring a series of steps and may require the use of adjunct methods (e.g. biochemical, serological, nucleic acid-based methods for conclusive identification) and can take up to a week for bacteria. The culture-based approaches coupled with other methods such as PCR, immunoassays, bacteriophages, NGS, Biosensors, and MALDI TOF MS are increasingly being used for the detection and identification of food borne pathogens. Nucleic acid-based methods such as Real-Time PCR and vPCR combined with sequencing approaches are more widely used than immunoassay and NGS-based approaches for pathogen detection. Nanobiotechnology is the latest approach for the detection of pathogens. Bacteriophages are one such unique biological entity that showed excellent host selectivity and have been actively used as recognition probes for pathogen detection. Antibiotics are efficient, powerful, and the most commonly used treatment against pathogenic E. coli. Other ways to treat or prevent E. coli contamination, such as probiotics, antimicrobial nanoparticles, and radiation treatment had been reported.

Role of tissue inhibitor of matrix metalloproteinase-1 in Iraqi patients with acute myeloid leukemia

Abstract: BACKGROUND: Leukemia is characterized by an uncontrolled expansion or proliferation of hematopoietic cells that are unable to develop appropriately into mature blood cells. Tissue inhibitor of metalloproteinases (TIMP) is glycoprotein with 28 Da Molecular weight. It has proteolytic and proliferative activity show pleiotropic effects in the bone marrow regulates cell responsible for survival and growth also healthy hematopoietic progenitor cells and involve in cancer progression. OBJECTIVES: The aim of this study was to measure TIMP in Iraqi acute myeloid leukemia patients as well as the correlation between tissue inhibitor of matrix metalloproteinase-1 and blast cells. PATIENTS MATERIALS AND METHODS: The study involved 50 patients from Iraqi National Hematology Center/Al-Mustansiriyah University and Baghdad Teaching Hospital with acute myeloid leukemia and 50 control participants who were physically similar. The patients’ ages ranged from 20 to 70 years. Tissue inhibitor of matrix metalloproteinase concentration in plasma was measured using a sandwich enzyme immunoassay approach that is quantitative. RESULTS: The present study demonstrates a statistically significant increase in the level of tissue inhibitor of matrix metalloproteinase-1 patients with acute myeloid leukemia. The level of TIMP-1 in serum AML patients was 443.7 ± 0.3 pg/mL while in healthy control serum was 149.5 ± 0.088 pg/mL. The current result showed a positive significant correlation between TIMP-1 level and blast Cells percentage (r = 0.495; P = 0.031), while the correlation between leukocytes number and platelets number was insignificant (r = 0.388; P = 0.078, r = −0.444; P = 0.155). CONCLUSION: TIMP-1 levels increased in the CML patient compared with healthy control also there was a significant correlation between TIMP-1 and Blast cell level while no correlation between level of TIMP-1 and number of leukocytes and platelets. The level of TIMP in patients untreated and undergoing chemotherapy does not change.

Exploring the inflammatory profile of homelessness population: a comprehensive analysis of individuals in two temporary shelters in Lisbon.

Homeless people are continuously facing adverse living conditions as poor access to basic nutrition, hygiene conditions and healthcare services, being at increased risk of severe infectious diseases as HIV and hepatitis as well as cardiovascular diseases and mental disorders. The characterization of homeless people's health is fundamental to identify their health care needs. Considering that the aforementioned diseases are associated with chronic inflammatory processes, the main goal of this study was to characterize the inflammatory profile of a homeless population through quantification in saliva of a panel of inflammatory cytokines. The inflammatory profile was assessed in 114 individuals residing in two temporary shelters located in Lisbon and that accepted to participated in the study. Inflammatory proteins were quantified using a Multiplex Immunoassay approach. Data analysis was performed using the GraphPad Prism software and statistical significance among the groups was assessed using the nonparametric Mann-Whitney test. Even though some protein levels might be masked by drug treatment, data analysis showed high levels of INF-ϒ, IL-10 and TNF-α in the infectious disease group, critical cytokines for the immune response against viruses and bacteria. Also, cytokines like IL-1β and IL-6 were detected at statistically significant levels in the cardiovascular disease group and all cytokines included in this study were quantified in the mental disorders group. These findings may help the healthcare services in the evaluation of treatment efficacy and disease monitoring, and in the development of effective public healthcare strategies and policy interventions to improve quality of life of the homeless population.

Characterizing a Silver Nanoparticle-Based Electrochemical Biosensor for Shiga Toxin Detection.

Shiga toxins (1, 2) regularly cause outbreaks and food recalls and pose a significant health risk to the infected population. Therefore, new reliable tools are needed to rapidly detect Shiga toxin cost-effectively in food, water, and wastewater before human consumption. Enzyme immunoassay and polymerase chain reaction approaches are the gold standard detection methods for the Shiga toxin. However, these methods require expensive instruments along with expensive reagents, which makes them hard to convert into point-of-use and low-cost systems. This study introduces an electrochemical biosensing method that utilizes silver nanoparticles (AgNPs) as electrochemical tags and commercially available low-cost screen-printed carbon electrodes for detection. This study introduces the modification of reference electrodes on commercially available screen-printed carbon electrodes to detect AgNPs dissolved in nitric acid. This biosensor achieved a 2 ng/mL lowest measured concentration for Shiga toxin-1 in less than 3 h. These biosensor results also showed that the AgNP-based sensor has better linearity (for graph between peak current vs concentration) and lower standard deviation compared to gold nanoparticles (AuNP)-based electrochemical biosensors.

MOF-Based Biosensors for the Detection of Carcinoembryonic Antigen: A Concise Review.

Cancer has been considered one of the most serious diseases in recent decades. Early diagnosis of cancer is a crucial step for expedited treatment. Ideally, detection of cancer biomarkers, which are usually elevated because of cancer, is the most straightforward approach to detecting cancer. Among these biomarkers, the carcinoembryonic antigen (CEA) is considered one of the most important tumor markers for colorectal cancer. The CEA has also been recognized as a biomarker for other types of cancers, including breast, gastric, ovarian, pancreatic, and lung cancers. Typically, conventional CEA testing depends on immunoassay approaches, which are known to be complex, highly expensive, and time consuming. Accordingly, various types of biosensors have been designed for the detection of cancer biomarkers. The main prerequisites of these biosensors are high sensitivity, fast response, and low cost. Many nanostructures have been involved in the design of biosensors, such as nanoparticles of certain metals and metal oxides that are further functionalized to contribute to the sensing of the biomarkers. Alternatively, metal organic frameworks (MOFs), which are extended crystalline structures comprising metal clusters surrounded by organic linkers, have been shown to be highly promising for the development of biosensors. The 3D structure of MOFs results in a combination of high surface area and high interconnected porosity, which are believed to facilitate their function in the design of a biosensor. This review briefly classifies and describes MOF-based biosensor trials that have been published recently for the aim of detecting CEA.

Engineering a Point-of-Care Paper-Microfluidic Electrochemical Device Applied to the Multiplexed Quantitative Detection of Biomarkers in Sputum.

Health initiatives worldwide demand affordable point-of-care devices to aid in the reduction of morbidity and mortality rates of high-incidence infectious and noncommunicable diseases. However, the production of robust and reliable easy-to-use diagnostic platforms showing the ability to quantitatively measure several biomarkers in physiological fluids and that could in turn be decentralized to reach any relevant environment remains a challenge. Here, we show the particular combination of paper-microfluidic technology, electrochemical transduction, and magnetic nanoparticle-based immunoassay approaches to produce a unique, compact, and easily deployable multiplex device to simultaneously measure interleukin-8, tumor necrosis factor-α, and myeloperoxidase biomarkers in sputum, developed with the aim of facilitating the timely detection of acute exacerbations of chronic obstructive pulmonary disease. The device incorporates an on-chip electrochemical cell array and a multichannel paper component, engineered to be easily aligned into a polymeric cartridge and exchanged if necessary. Calibration curves at clinically relevant biomarker concentration ranges are produced in buffer and artificial sputum. The analysis of sputum samples of healthy individuals and acutely exacerbated patients produces statistically significant biomarker concentration differences between the two studied groups. The device can be mass-produced at a low cost, being an easily adaptable platform for measuring other disease-related target biomarkers.

A Review of Detection Methods for Vancomycin-Resistant Enterococci (VRE) Genes: From Conventional Approaches to Potentially Electrochemical DNA Biosensors.

Vancomycin-resistant Enterococci (VRE) genes are bacteria strains generated from Gram-positive bacteria and resistant to one of the glycopeptides antibiotics, commonly, vancomycin. VRE genes have been identified worldwide and exhibit considerable phenotypic and genotypic variations. There are six identified phenotypes of vancomycin-resistant genes: VanA, VanB, VanC, VanD, VanE, and VanG. The VanA and VanB strains are often found in the clinical laboratory because they are very resistant to vancomycin. VanA bacteria can pose significant issues for hospitalized patients due to their ability to spread to other Gram-positive infections, which changes their genetic material to increase their resistance to the antibiotics used during treatment. This review summarizes the established methods for detecting VRE strains utilizing traditional, immunoassay, and molecular approaches and then focuses on potential electrochemical DNA biosensors to be developed. However, from the literature search, no information was reported on developing electrochemical biosensors for detecting VRE genes; only the electrochemical detection of vancomycin-susceptible bacteria was reported. Thus, strategies to create robust, selective, and miniaturized electrochemical DNA biosensor platforms to detect VRE genes are also discussed.

Analysis and identification of suspected snake venom samples using nano-ultra-high performance liquid chromatography-high resolution mass spectrometry

Snake venom is a complex mixture secreted from the glands of poisonous snakes， which contains proteins， peptides， lipids， nucleosides， sugars， amino acids， amines， metal ions， and other components. According to the toxicological classification， snake venoms can be classified as neurotoxins， anticoagulants and procoagulant toxins， cardiac toxins， other toxin proteins， and enzymes. Proteins and peptides are the key components of snake venom. The establishment of rapid， accurate analysis and identification methods for proteins in snake venom is a prerequisite for snake venom-related forensic identification， intoxication events， and pharmaceutical development. Until now， the classical analysis and identification methods have mainly been biochemical or immunoassays for DNA or proteins， such as polymerase chain reaction， agglutination test， enzyme-linked immunosorbent assay， fluorescent immunoassay， and various biosensing approaches. These methods have some limitations such as a high false-positive ratio， low sensitivity， poor anti-interference ability， and limited species discrimination capability. In recent years， with the rapid development of mass spectrometry （MS） techniques， the proteomics of snake venom has also attracted much attention and has contributed to the identification of snake species， in which non-targeted and targeted proteomics represent two main divisions. However， species identification via proteomics is in its infancy in forensic science. First， the tandem MS spectra of peptide sequences are highly complex， which poses a great challenge for the strict and accurate matching of peptides based on the rational speculation of MS fragmentation rules and theoretical calculations in non-targeted proteomics. Second， for the confirmation and identification of unknown substances， reference substances are commonly needed， but those for snake venom are lacking. Proteomics in snake venom identification is still in progress to improve the identification confidence and clarify the identification rules. In this work， a method based on nano-ultra-high performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry （Nano LC-MS/HRMS） and size exclusion chromatography （SEC） was developed for identifying proteins and their source species， with strict rules for five suspected snake venom samples and their contamination in one case. Three SEC elution peaks were obtained from each of the five samples， which were lyophilized and treated with trypsin in solution， and then separated and analyzed by Nano LC-MS/HRMS. First， the Full MS/dd MS2 mode was used for the non-targeted acquisition of peptide information in the samples， and after submission to the Swiss-Prot database， the protein databases of Serpentes， Colubroidea， Elapidae， Elapinae， and Naja were contracted stepwise and compared. A total of 32 proteins from Naja atra were identified under the conditions of both peptide spectrum match and false discovery rate less than 1%， and number of characteristic peptides greater than or equal to two. All of these were derived from ten families of Naja atra， mainly three-finger toxins， metalloproteinases， and phospholipase A2. Proteins D3TTC2， D5LMJ3， Q7T1K6， Q9DEQ3， and Q9YGI4 were the most common among the five samples. Finally， the parallel reaction monitoring mode was adopted to select two unique peptides for each protein for targeted verification. It was considered that a protein in the samples was truly identified when it met the strict standard "the Δm/z of at least 75% y+ and b+ ions of each unique peptide was less than 5 ppm". After these consequently procedures， we identified that all five samples contained the venom of the Naja atra. Our identification method is a systematic and strict example that can provide effective technical support for the forensic identification of snake venom poisoning， as well as for pharmaceutical development toward snake venoms.

Lumit: A Homogeneous Bioluminescent Immunoassay for Detecting Diverse Analytes and Intracellular Protein Targets.

Traditional immunoassays to detect secreted or intracellular proteins can be tedious, require multiple washing steps, and are not easily adaptable to a high-throughput screening (HTS) format. To overcome these limitations, we developed Lumit, a novel immunoassay approach that combines bioluminescent enzyme subunit complementation technology and immunodetection. This bioluminescent immunoassay does not require washes or liquid transfers and takes less than 2h to complete in a homogeneous "Add and Read" format. In this chapter, we describe step-by-step protocols to create Lumit immunoassays for the detection of (1) secreted cytokines from cells, (2) phosphorylation levels of a specific signaling pathway node protein, and (3) a biochemical protein-protein interaction between a viral surface protein and its human receptor.

Detection of COVID-19: A Smartphone-Based Machine-Learning-Assisted ECL Immunoassay Approach with the Ability of RT-PCR CT Value Prediction.

The unstoppable spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has severely threatened public health over the past 2 years. The current ubiquitously accepted method for its diagnosis provides sensitive detection of the virus; however, it is relatively time-consuming and costly, not to mention the need for highly skilled personnel. There is a clear need to develop novel computer-based diagnostic tools to provide rapid, cost-efficient, and time-saving detection in places where massive traditional testing is not practical. Here, we develop an electrochemiluminescence (ECL)-based detection system whose results are quantified as reverse transcriptase polymerase chain reaction (RT-PCR) cyclic threshold (CT) values. A concentration-dependent signal is generated upon the introduction of the virus to the electrode and is recorded with a smartphone camera. The ECL images are used to train machine learning algorithms, and a model using artificial neural networks (ANNs) for 45 samples was developed. The model demonstrated more than 90% accuracy in the diagnosis of 50 unknown real samples, detecting up to a CT value of 32 and a limit of detection (LOD) of 10-12 g mL-1 in the testing of artificial samples.

Rational Design and Development of SARS-CoV-2 Serological Diagnostics by Immunoprecipitation-Targeted Proteomics.

Current design of serological tests utilizes conservative immunoassay approaches and is focused on fast and convenient assay development, throughput, straightforward measurements, and affordability. Limitations of common serological assays include semiquantitative measurements, cross-reactivity, lack of reference standards, and no differentiation between human immunoglobulin subclasses. In this study, we suggested that a combination of immunoaffinity enrichments with targeted proteomics would enable rational design and development of serological assays of infectious diseases, such as COVID-19. Immunoprecipitation-targeted proteomic assays allowed for sensitive and specific measurements of NCAP_SARS2 protein with a limit of detection of 313 pg/mL in serum and enabled differential quantification of anti-SARS-CoV-2 antibody isotypes (IgG, IgA, IgM, IgD, and IgE) and individual subclasses (IgG1-4 and IgA1-2) in plasma and saliva. Simultaneous evaluation of the numerous antigen–antibody subclass combinations revealed a receptor-binding domain (RBD)-IgG1 as a combination with the highest diagnostic performance. Further validation revealed that anti-RBD IgG1, IgG3, IgM, and IgA1 levels were significantly elevated in convalescent plasma, while IgG2, IgG4, and IgA2 were not informative. Anti-RBD IgG1 levels in convalescent (2138 ng/mL) vs negative (95 ng/mL) plasma revealed 385 ng/mL as a cutoff to detect COVID-19 convalescent plasma. Immunoprecipitation-targeted proteomic assays will facilitate improvement and standardization of the existing serological tests, enable rational design of novel tests, and offer tools for the comprehensive investigation of immunoglobulin subclass cooperation in immune response.