Development Of Immunoassay Research Articles

Development of a monoclonal antibody-based lateral flow immunoassay for the detection of benzoic acid in liquid food.

To monitor benzoic acid (BA) residues in liquid food samples, a monoclonal antibody (mAb)-based lateral flow immunoassay (LFA) was developed in this study. First, 2-aminobenzoic acid (2-AA), 3-aminobenzoic acid (3-AA), and 4-aminobenzoic acid (4-AA) were conjugated to BSA and used as immunogens. After cell fusion, mAb 6D8 from 4-AA-BSA performed best with an IC50 value of 0.21 mg L-1 using 3-AA-OVA as a heterogeneous antigen, which represented a 3.4-fold improvement compared with the homogeneous antigen 4-AA-BSA. Subsequently, eight kinds of CGNPs with sizes varying from 20.94 nm to 90.00 nm were synthesized for screening the suitable size to develop a sensitive LFA. Finally, a sensitive LFA based on colloidal gold (23.27 nm) nanoparticles was developed for screening BA with a cut-off value of 4 mg L-1, which could meet the requirement of BA detection in milk, Fanta, Sprite, Coca-Cola, and Smart samples.

Artificial Intelligence-Assisted Digital Immunoassay Based on a Programmable-Particle-Decoding Technique for Multitarget Ultrasensitive Detection.

The development of a multitarget ultrasensitive immunoassay is significant to fields such as medical research, clinical diagnosis, and food safety inspection. In this study, an artificial intelligence (AI)-assisted programmable-particle-decoding technique (APT)-based digital immunoassay system was developed to perform multitarget ultrasensitive detection. Multitarget was encoded by programmable polystyrene (PS) microspheres with different characteristics (particle size and number), and subsequent visible signals were recorded under an optical microscope after the immune reaction. The resultant images were further analyzed using a customized, AI-based computer vision technique to decode the intrinsic properties of polystyrene microspheres and to reveal the types and concentrations of targets. Our strategy has successfully detected multiple inflammatory markers in clinical serum and antibiotics with a broad detection range from pg/mL to μg/mL without extra signal amplification and conversion. An AI-based digital immunoassay system exhibits great potential to be used for the next generation of multitarget detection in disease screening for candidate patients.

Physicochemical and bioinformatic characterization of Oreochromis niloticus vitellogenin as an endocrine disruption biomarker.

Aquatic biota is increasingly being exposed to chemical pollutants due to human activities and the relationship between the level of environmental pollution and fish reproduction is a continuously ongoing issue. The vitellogenin (Vtg) protein synthesis can be induced in the liver of juvenile and male fish after stimulation of the estrogen receptor and therefore, Vtg has been used as a biomarker of xenoestrogen exposure in several fish species. The current study reported the first physicochemical characterization of Vtg from Oreochromis niloticus. Adult male fish were exposed to 17α-ethinylestradiol for Vtg induction. Purified vitellogenin from plasma showed low stability at 25 and 4 °C in saline conditions, and good stability in acidic (low pH) or in heated conditions. The 3D modeling provided useful information on the structure of O. niloticus Vtg showing conserved structural features. According to bioinformatics and experimental results, there are important structural differences between the two chemical forms of Vtg (VtgAb and VtgC) in a phylogenetic context. The present results add information about the development of ecotoxicological immunoassays to study the endocrine disruption in O. niloticus improving the Vtg performance as a biomarker of reproduction in fish.

Development of a bead-based multiplex immunoassay for simultaneous quantitative detection of IgG serum antibodies against seven vaccine-preventable diseases

Serosurveillance and seroprevalence studies should be carried out to monitor vaccine-preventable diseases. Multiplex immunoassay (MIA) systems are useful tools for this purpose, allowing the simultaneous quantitative detection of antibodies in one small serum sample, which presents an advantage over conventional methods, such as enzyme-linked immunosorbent assays (ELISAs). Therefore, we developed a multiplex immunoassay for the measurement of antibodies against seven vaccine-preventable infections (measles, rubella, mumps, tetanus, diphtheria, pertussis and Haemophilus influenza type b (Hib) infection). In our multiplex system, heterologous inhibition generally did not exceed 10%, while homologous inhibition varied between 90 and 98%. The intra- and inter-assay variability was ≤11%. The results of in-house MIA showed satisfactory correlation with commercial ELISAs, with Spearman correlation coefficients from 0.90 to 0.98. At the cut-off values defined for our MIA the serostatus can be determined with high sensitivity (89–100%) and specificity (92–98%). Thus, the developed in-house MIA represents a feasible alternative to conventional ELISAs and could be used for large-scale serosurveillance/seroprevalence studies of vaccine-preventable diseases.

Development of receptor binding domain-based double-antigen sandwich lateral flow immunoassay for the detection and evaluation of SARS-CoV-2 neutralizing antibody in clinical sera samples compared with the conventional virus neutralization test

Vaccination is an effective strategy to fight COVID-19. However, the effectiveness of the vaccine varies among different populations in varying immune effects. Neutralizing antibody (NAb) level is an important indicator to evaluate the protective effect of immune response after vaccination. Lateral flow immunoassay (LFIA) is a rapid, safe and sensitivity detection method, which has great potential in the detection of SARS-CoV-2 NAb. In this study, a fluorescent beads-based lateral flow immunoassay (FBs-LFIA) and a latex beads-based LFIA (LBs-LFIA) using double antigen sandwich (DAS) strategy were established to detect NAbs in the serum of vaccinated people. The limit of detection (LoD) of the FBs-LFIA was 1.13 ng mL− 1 and the LBs-LFIA was 7.11 ng mL− 1. The two LFIAs were no cross-reactive with sera infected by other pathogenic bacteria. Furthermore, the two LFIAs showed a good performance in testing clinical samples. The sensitivity of FBs-LFIA and LBs-LFIA were 97.44% (95%CI: 93.15%–99.18%) and 98.29% (95%CI: 95.84%–99.37%), and the specificity were 98.28% (95%CI: 95.37%–99.45%) and 97.70% (95%CI: 94.82%–99.06%) compared with the conventional virus neutralization test (cVNT), respectively. Notably, the LBs-LFIA was also suitable for whole blood sample, requiring only 3 μL of whole blood, which provided the possibility to detect NAbs at home. To sum up, the two LFIAs based on double antigen sandwich established by us can rapidly, safely, sensitively and accurately detect SARS-CoV-2 NAb in human serum.

Production of high-affinity monoclonal antibody and development of immunoassay for 3-methyl-quinoxaline-2-carboxylic acid detection in swine muscle and liver

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 influence of hapten spacer arm length on antibody response and immunoassay development

Antibodies against small molecules with high titer and high affinity are always pursued in the field of vaccines for drugs of abuse, antidotes to toxins and immunoassays in medical, environmental, and food safety. The exposure degree of the target molecule to the immune system is critical to induce a strongly specific antibody response, thus, the spacer arm length between the target molecule and carrier protein plays an important role. However, the influence of spacer arm length on antibody titer, affinity, and assay performance is not yet clear and highly demanded to be addressed. In the present study, we proposed a model study to answer the question by using two typical small molecules, melamine and p–nitroaniline, which were introduced by varied spacer arms with increasing alkane linear length from 2 to 12 carbon atoms brick by brick. The spacer arm lengths of the haptens were obtained by computational chemistry. The titer and affinity of mouse antisera were analyzed and compared, showing that all haptens with spacer arms of 6–8 carbon atoms, i.e. 6.3–8.8 Å in length, induced strong antibodies represented by the highest titer and affinity without exception, while the haptens with spacer arms of 2–4 carbon atoms and 10–12 carbon atoms, i.e. 1.5–3.9 Å and 11.3–13.9 Å in length, failed to induce high–quality antibody response. Moreover, the titer and sensitivity of the subsequently developed immunoassays were significantly affected by using coating haptens with different spacer arm lengths, and coating haptens with a spacer arm of 6.3–8.8 Å in length delivered the optimum detection performance. The antibody recognition mechanism study further confirmed that the hapten spacer arm length had a critical effect on the recognition properties of the induced antibody, which should be interactive with the spacer arm each other. This study showed that the hapten with appropriate spacer arm length is important to antibody response and immunoassay development, providing a valuable and general clue for the rational design of hapten.

Recent Advances in the Immunoassays Based on Nanozymes.

As a rapid and simple method for the detection of multiple targets, immunoassay has attracted extensive attention due to the merits of high specificity and sensitivity. Notably, enzyme-linked immunosorbent assay (ELISA) is a widely used immunoassay, which can provide high detection sensitivity since the enzyme labels can promote the generation of catalytically amplified readouts. However, the natural enzyme labels usually suffer from low stability, high cost, and difficult storage. Inspired by the advantages of superior and tunable catalytic activities, easy preparation, low cost, and high stability, nanozymes have arisen to replace the natural enzymes in immunoassay; they also possess equivalent sensitivity and selectivity, as well as robustness. Up to now, various kinds of nanozymes, including mimic peroxidase, oxidase, and phosphatase, have been incorporated to construct immunosensors. Herein, the development of immunoassays based on nanozymes with various types of detection signals are highlighted and discussed in detail. Furthermore, the challenges and perspectives of the design of novel nanozymes for widespread applications are discussed.

Cutting-Edge Developments in Oncology Research

AbstractThe field of oncology research has made many successful advances, and new discoveries have started making headlines. As an example, the identification of immune checkpoint inhibition mechanisms in carcinogenic cells led to the development of immunoassays, which have helped many cancer convalescents recover. This article covers the most advanced cutting-edge areas of cancer research: exosomes, microbiomes, immunotherapy, nanocarriers, and organoids. Research on exosomes advances cancer detection and treatment modalities, as well as further understanding of mechanisms that regulate carcinogen cell division, proliferation, invasion, and metastasis. Microbiome consents the researchers to understand the disease cancer. Immunotherapy is the third method in the treatment of cancer. Organoid biology will be further expanded with the aim of translating research into customized therapeutic therapies. Nanocarriers enable cancer specific drug delivery by inherent unreceptive targeting phenomena and implemented active targeting strategies. These areas of research may also bring about the advent of the latest cancer treatments in the future. Malignant infections are one of the leading grounds for demise in the society. Patients are treated with surgery, radiation, and chemotherapy. In chemotherapy, the malignant cells are destroyed and the tumor burden is reduced. However, in most cases, resistance to chemotherapy develops. Therefore, there is a constant need for new additional treatment modalities and chemotherapeutic complex rules. Due to the rapid development in cancer research, I can only mention a few goals and treatment options that I have chosen; However, this review specializes in new and admirable significant strategies and compounds.

Flash Characterization of Smartphones Used in Point-of-Care Diagnostics.

Rapidly growing interest in smartphone cameras as the basis of point-of-need diagnostic and bioanalytical technologies increases the importance of quantitative characterization of phone optical performance under real-world operating conditions. In the context of our development of lateral-flow immunoassays based on phosphorescent nanoparticles, we have developed a suite of tools for characterizing the temporal and spectral profiles of smartphone torch and flash emissions, and their dependence on phone power state. In this work, these tools are described and documented to make them easily available to others, and demonstrated by application to characterization of Apple iPhone 5s, iPhone 6s, iPhone 8, iPhone XR, and Samsung Note8 flash performance as a function of time and wavelength, at a variety of power settings. Flash and torch intensity and duration vary with phone state and among phone models. Flash has high variability when the battery charge is below 10%, thus, smartphone-based Point-of-Care (POC) tests should only be performed at a battery level of at least 15%. Some output variations could substantially affect the results of assays that rely on the smartphone flash.

Exploration of Specific Nanobodies As Immunological Reagents to Detect Milk Allergen of β-Lactoglobulin without Interference of Hydrolytic Peptides.

Milk proteins are widely used for food supplementation, despite the potential risk of food allergy, especially against β-lactoglobulin (BLG), which makes BLG surveillance critical. Possible interaction of detecting antibodies with BLG-derived peptides will result in unprecise inspection. Thus, in this study, it was proposed to generate nanobodies (Nbs) and validate the immunological detection of intact BLG rather than hydrolytic peptides. Nbs were successfully retrieved and characterized with high stability and target specificity. A competitive enzyme-linked immunosorbent assay (cELISA) was developed with a linear range from 39 to 10,000 ng/mL and a detection limit (LOD) of 4.55 ng/mL, with a recovery of 86.30%-95.09% revealed by analysis of spiked samples. Meanwhile, a sandwich ELISA (sELISA) was established with Nb82 and BLG polyclonal antibody (pAb-BLG) providing a linear range from 29.7 to 1250 ng/mL and an LOD of 13.82 ng/mL with a recovery of 87.82%-103.97%. The interaction of selected Nbs with BLG-derived peptides was investigated by Nb structure modeling and BLG docking. No binding on hydrolytic peptides was revealed, confirming the precision of Nb-mediated immunoassays. In summary, this study successfully identified BLG-specific Nbs for immunoassay development and guaranteed the monitoring of intact BLG without interference of hydrolytic peptides, providing experimental evidence that our Nbs recognize intact food allergen.

Kinetic Analysis of Prostate-Specific Antigen Interaction with Monoclonal Antibodies for Development of a Magnetic Immunoassay Based on Nontransparent Fiber Structures.

Prostate cancer is the second most common cancer diagnosed in men worldwide. Measuring the prostate-specific antigen (PSA) is regarded as essential during prostate cancer screening. Early diagnosis of this disease relapse after radical prostatectomy requires extremely sensitive methods. This research presents an approach to development of an ultrasensitive magnetic sandwich immunoassay, which demonstrates the limit of PSA detection in human serum of 19 pg/mL at a dynamic range exceeding 3.5 orders of concentration. Such attractive performance stems, inter alia, from the kinetic analysis of monoclonal antibodies (mAbs) against free PSA to select the mAbs exhibiting best kinetic characteristics and specificity. The analysis is carried out with a label-free multiplex spectral-correlation interferometry compatible with inexpensive single-use glass sensor chips. The high sensitivity of developed PSA immunoassay is due to electronic quantification of magnetic nanolabels functionalized by the selected mAbs and three-dimension porous filters used as an extended solid phase. The assay is promising for PSA monitoring after radical prostatectomy. The proposed versatile approach can be applied for the rational design of highly sensitive tests for detection of other analytes in many fields, including in vitro diagnostics, veterinary, food safety, etc.

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 immunoassay based on a specific antibody for sensitive detection of nicosulfuron in environment

Nicosulfuron, one of the most widely used selective herbicides in corn field, can effectively control annual and perennial grass weeds, sedges, and some broadleaf weeds. The residual phytotoxicity of nicosulfuron in soil and water has become increasingly prominent. Therefore, an efficient method for detection of nicosulfuron was critical to ensure the sustainable and healthy development of agriculture and the ecological environment. In this paper, five nicosulfuron haptens which contained carboxyl group or aldehyde groups were designed and synthesized, and an indirect competitive immunoassay was developed for the first time. The assay showed an IC50 of 8.42 ng/mL and had negligible cross reactivities toward other sulfonylurea herbicides. In the spike and recovery studies, the recovery rate from soil samples was 95 %–104 %, and that of wheat roots was 92 %–98 %, which showed a good correlation with LC-MS analysis for nicosulfuron. The immunoassay was then used to quantify nicosulfuron concentration which could cause the obvious phytotoxic symptoms to wheat. Obvious symptoms of nicosulfuron phytotoxicity in wheat root was observed at the concentration of 0.068 ± 0.006 mg/kg (ELISA result) which was consistent with 0.072 ± 0.007 mg/kg obtained by LC-MS. The developed immunoassay method is an effective tool for environment contamination monitoring.

Development of Lateral Flow Test-System for the Immunoassay of Dibutyl Phthalate in Natural Waters

The use of a large amount of toxic synthetic materials leads to an increase in the pollution of environmental objects. Phthalates are compounds structurally related to esters of phthalic acid that are widely used in the manufacturing of synthetic packaging materials as plasticizers. Their danger is conditioned by leaching into the environment and penetrating into living organisms with negative consequences and effects on various organs and tissues. This work presents the first development of lateral flow immunoassay to detect dibutyl phthalate, one of the most common representatives of the phthalates group. To form a test zone, a hapten-protein conjugate was synthesized, and gold nanoparticles conjugated with antibodies to dibutyl phthalate were used as a detecting conjugate. The work includes the preparation of immunoreagents, selectivity investigation, and the study of the characteristics of the medium providing a reliable optical signal. Under the selected conditions for the analysis, the detection limit was 33.4 ng/mL, and the working range of the determined concentrations was from 42.4 to 1500 ng/mL. Time of the assay-15 min. The developed technique was successfully applied to detect dibutyl phthalate in natural waters with recovery rates from 75 to 115%.

ODP178 Development and Validation of a Noncompetitive Immunoassay Optimized for the Assessment of Pharmacokinetics of Biologically Active Efruxifermin in Humans

Abstract Efruxifermin (EFX) is a novel fusion biotherapeutic consisting of two modified human fibroblast growth factor (FGF21) variants tethered via short linkers to a hIgG1 Fc domain. EFX is under clinical development by Akero Therapeutics, Inc. for treatment of non-alcoholic steatohepatitis (NASH). Fusion to the IgG Fc moiety extends the circulating half-life of EFX, enabling once-weekly subcutaneous administration. In addition, point mutations in the FGF21 moiety of EFX, relative to native human FGF21, protect against degradation of the C-terminus by the endogenous endopeptidase fibroblast activation protein (FAP), and increase affinity for the FGF21 co-receptor b-Klotho, which together enhance in vivo PD activity of EFX. Accordingly, to support clinical PK a sensitive and specific assay is needed for determination of biologically active (i. e., C-terminal intact) EFX serum concentrations that is minimally cross-reactive with endogenous FGF21 or potential inactive metabolites of EFX. Moreover, the assay needs to have reliability with serum samples from NASH patients, in whom hyperlipidemia and insulin resistance are prevalent. To achieve the analytical aims, we undertook development of a noncompetitive immunosorbent assay design, wherein a biotinylated version of a rat mAb directed against EFX's intact C-terminus serves as the capture reagent, while an affinity purified chicken anti-EFX pAb labeled with Sulfo-TAG functions as the detection reagent. This novel ECLIA design permits measurement of an electrochemiluminescence signal for maximizing assay sensitivity. In brief, serum samples are diluted 1: 10 in buffer and added to the MSD plate coated with rat mAb. After incubation and washing, bound EFX is detected using an affinity purified chicken anti-EFX PAB labeled with SULFO-TAG. After washing and addition of read buffer, the signal responses are measured in a SQ120 reader and concentration in test samples is estimated by dose-interpolation using a weighted 5-PL curve fit with an EFX standard curve ranging from 10–2,000 ng/mL. The new assay allows measurement of serum EFX at concentrations as low at 20 ng/mL. Upon pre-study validation, the assay has met analytical performance criteria outlined in BMV regulatory guidances. We conclude the optimized ECLIA will be useful for reliable assessment of EFX PK in patients with NASH. Presentation: No date and time listed

Development of novel immunoassays for pro-opiomelanocortin (POMC)-derived peptides as surrogate markers of adrenocorticotrophin levels for use in the diagnosis of Cushing’s syndrome

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Development of fluorescent lateral flow immunoassay for SARS-CoV-2-specific IgM and IgG based on aggregation-induced emission carbon dots.

Understanding the dynamic changes in antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for evaluating the effectiveness of the vaccine and the stage for the recovery of the COVID-19 disease. A rapid and accurate method for the detection of SARS-CoV-2-specific antibodies is still urgently needed. Here, we developed a novel fluorescent lateral flow immunoassay (LFA) platform for the detection of SARS-CoV-2-specific IgM and IgG by the aggregation-induced emission carbon dots conjugated with the SARS-CoV-2 spike protein (SSP). The aggregation-induced emission carbon dots (AIE-CDs) are one of the best prospect fluorescent probe materials for exhibiting high emission efficiency in both aggregate and solid states. The AIE-CDs were synthesized and displayed dual fluorescence emission, which provides a new perspective for the design of a high sensitivity testing system. In this work, the novel LFA platform adopted the AIE carbon dots, which are used to detect SARS-CoV-2-specific IgM and IgG conveniently. Furthermore, this sensor had a low LOD of 100 pg/ml. Therefore, this newly developed strategy has potential applications in the areas of public health for the advancement of clinical research.

Development of a GNP-based lateral flow immunoassay for the detection of isoprothiolane in rice samples

Isoprothiolane (IPT) is widely used to control rice blast, but can cause unacceptable harm to organisms making its detection in rice crucial. In this work, a hapten based on the structure of IPT was produced to prepare a monoclonal antibody (mAb) with high sensitivity and specificity. The half maximum inhibitory concentration of the anti-IPT mAb was 0.81 ng/mL as determined by ELISA, and the cross reactivity with analogues was negligible. A GNP-based lateral flow immunoassay (LFIA) method was subsequently developed to quantitatively detect IPT in brown and polished rice. The optimized LFIA, in brown rice samples, showed a visual limit of detection, cut-off value and calculated limit of detection of 2 mg/kg, 10 mg/kg and 81 ug/kg, respectively and 0.2 mg/kg, 4 mg/kg and 66 ug/kg in polished rice samples. Recovery rates in spiked brown and polished rice were 95.21 %–103.06 % and 91.05 %–104.13 %, with a coefficient of variation of 1.25 %–4.17 % and 2.93 %–8.42 %, respectively. No significant difference in recovery rates were seen between LFIA and LC-MS/MS indicating that the prepared GNP-based LFIA had good potential for the detection of IPT in rice.

Adsorption and Electropolymerization of p-Aminophenol Reduces Reproducibility of Electrochemical Immunoassays.

This paper investigates the electrochemical behavior of p-aminophenol (PAP) on commercially available carbon screen-printed electrodes (CSPEs) and gold screen-printed electrodes (GSPEs) at neutral and basic pHs for the development of inexpensive immunoassays. The electrochemical oxidative signal from PAP results from its adsorption to the electrode. The formation of self-assembled monolayers on gold electrodes prevented PAP adsorption but also reduced its oxidative current, confirming that adsorption increases signal production. On bare electrodes, PAP adsorption results in oxidative current variability depending on the electroactive surface area of the screen-printed electrode. This variability could not be remedied by cleaning and reusing the same GSPE. Decreasing the PAP concentration to 3.8 μM greatly improved the consistency of the measurements, suggesting that the adsorption of PAP is concentration-dependent. Multiple PAP oxidations on the same electrode caused polymerization, limiting PAP in continuous monitoring applications. Infrared and Raman spectroscopy allow the distinction between adsorbed PAP and electropolymerized PAP on the surface of a gold wafer. The results from this study suggest that the use of PAP production in immunoassays with SPEs must be fine-tuned, and electrodes must be cleaned or disposed of between measurements.