Noncompetitive Immunoassay Research Articles

Comparison of Survivin Determination by Surface-Enhanced Fluorescence and Raman Spectroscopy on Nanostructured Silver Substrates.

Survivin belongs to a family of proteins that promote cellular proliferation and inhibit cellular apoptosis. Its overexpression in various cancer types has led to its recognition as an important marker for cancer diagnosis and treatment. In this work, we compare two approaches for the immunochemical detection of survivin through surface-enhanced fluorescence or Raman spectroscopy using surfaces with nanowires decorated with silver nanoparticles in the form of dendrites or aggregates as immunoassays substrates. In both substrates, a two-step non-competitive immunoassay was developed using a pair of specific monoclonal antibodies, one for detection and the other for capture. The detection antibody was biotinylated and combined with streptavidin labeled with rhodamine for the detection of surface-enhanced fluorescence, while, for the detection via Raman spectroscopy, streptavidin labeled with peroxidase was used and the signal was obtained after the application of 3,3',5,5'-tetramethylbenzidine (TMB) precipitating substrate. It was found that the substrate with the silver dendrites provided higher fluorescence signal intensity compared to the substrate with the silver aggregates, while the opposite was observed for the Raman signal. Thus, the best substrate was used for each detection method. A detection limit of 12.5 pg/mL was achieved with both detection approaches along with a linear dynamic range up to 500 pg/mL, enabling survivin determination in human serum samples from both healthy and ovarian cancer patients for cancer diagnosis and monitoring purposes.

Structural insights into ternary immunocomplex formation and cross-reactivity: binding of an anti-immunocomplex FabB12 to Fab220-testosterone complex.

Anti-immunocomplex (Anti-IC) antibodies have been used in developing noncompetitive immunoassays for detecting small molecule analytics (haptens). These antibodies bind specifically to the primary antibody in complex with hapten. Although several anti-IC antibody-based immunoassays have been developed, structural studies of these systems are very limited. In this study, we determined the crystal structures of anti-testosterone Fab220 in complex with testosterone and the corresponding anti-IC antibody FabB12. The structure of the ternary complex of testosterone, Fab220, and FabB12 was predicted using LightDock and AlphaFold. The ternary complex has a large (~ 1100 Å2) interface between antibodies. The A-ring of the testosterone bound by Fab220 also participates in the binding of the anti-IC antibody. The structural analysis was complemented by native mass spectrometry. The affinities for testosterone (TES) and three cross-reactive steroids [dihydrotestosterone (DHT), androstenedione (A4), and dehydroepiandrosterone sulfate (DHEA-S)] were measured, and ternary complex formation was studied. The results clearly show the ternary complex formation in the solution. Although DHT showed significant cross-reactivity, A4 and DHEA-S exhibited minor cross-reactivity.

Light‐assisted noncompetitive fluorescence immunoassay for photosensitive antibiotic detection

AbstractThe development of simple, sensitive, and efficient methods for antibiotic detection is of great significance for human health and environmental protection. Currently, although the competitive immunoassays are popular for detecting antibiotic molecules, they need to label antibiotic or specific antibody with signaling molecule. The development of noncompetitive immunoassay for antibiotic detection has always faced significant challenges. Herein, we have developed a light‐assisted noncompetitive immunoassay for the detection of antibiotics based on their photosensitive characteristic. Firstly, the antibody coated on the microplate specifically captures the target antibiotic. Afterward, under light irradiation, the captured photosensitive antibiotic produces reactive oxygen species (ROS), which oxidizes the added ROS dye to generate a fluorescent signal. Therefore, a noncompetitive immunoassay for antibiotic detection is achieved by utilizing the unique property of photosensitive antibiotics to generate ROS instead of labeling signaling molecules. By using antibiotics demeclocycline hydrochloride (DMCO) and polymyxin B (PMB) as research models, the proposed noncompetitive fluorescence immunoassay can specifically detect as low as 5 ng/mL DMCO and 70 ng/mL PMB, respectively. The light‐assisted noncompetitive immunoassay strategy opens a new avenue for simple, sensitive, label‐free, and high‐throughput detection of photosensitive antibiotics or photosensitive drugs.

Methods and applications of noncompetitive hapten immunoassays.

Hapten immunoassays have found extensive application across various domains such as disease diagnostics, environmental monitoring, as well as the evaluation of food and pharmaceutical safety. These techniques traditionally rely on competitive assay formats and often face challenges with sensitivity and specificity. This review focuses on the emergent noncompetitive immunoassay technologies that promise to transcend these limitations through innovative approaches. Noncompetitive immunoassays, leveraging novel elements such as anti-idiotype antibodies, anti-immunocomplex (IC) antibodies, and the strategic use of nanomaterial-enhanced signal detection, are setting new benchmarks for analytical performance. These advancements not only enhance the detection capabilities but also significantly improve specificity inherent in traditional methods. Moreover, the integration of novel materials and binding reagents in these assays offers substantial improvements in assay dynamics, providing faster, more accurate, and reliable results. This review consolidates the latest methodologies and their applications, underlining the transformative impact of noncompetitive technologies in the sensitive detection of haptens across various fields.

Cancer Marker Immunosensing through Surface-Enhanced Photoluminescence on Nanostructured Silver Substrates.

Nanostructured noble metal surfaces enhance the photoluminescence emitted by fluorescent molecules, permitting the development of highly sensitive fluorescence immunoassays. To this end, surfaces with silicon nanowires decorated with silver nanoparticles in the form of dendrites or aggregates were evaluated as substrates for the immunochemical detection of two ovarian cancer indicators, carbohydrate antigen 125 (CA125) and human epididymis protein 4 (HE4). The substrates were prepared by metal-enhanced chemical etching of silicon wafers to create, in one step, silicon nanowires and silver nanoparticles on top of them. For both analytes, non-competitive immunoassays were developed using pairs of highly specific monoclonal antibodies, one for analyte capture on the substrate and the other for detection. In order to facilitate the identification of the immunocomplexes through a reaction with streptavidin labeled with Rhodamine Red-X, the detection antibodies were biotinylated. An in-house-developed optical set-up was used for photoluminescence signal measurements after assay completion. The detection limits achieved were 2.5 U/mL and 3.12 pM for CA125 and HE4, respectively, with linear dynamic ranges extending up to 500 U/mL for CA125 and up to 500 pM for HE4, covering the concentration ranges of both healthy and ovarian cancer patients. Thus, the proposed method could be implemented for the early diagnosis and/or prognosis and monitoring of ovarian cancer.

B-318 Development of Non-competitive Immunoassay System for Cyclosporine a Using Alpaca Naïve Library-derived VHH

Abstract Background Cyclosporine A is a potent immunosuppressive drug. It has a narrow therapeutic index, and therefore the measurement of cyclosporine’s blood concentration is essential to obtain optimal therapy. Cyclosporine A, a small cyclic polypeptide, is generally measured by competitive immunoassay, which is theoretically inferior to noncompetitive sandwich immunoassay in terms of sensitivity and specificity. In this study, we report a novel sandwich immunoassay for cyclosporine A using an alpaca naïve library-derived variable domain of heavy chain of heavy chain antibody (VHH) developed against an immunocomplex of cyclosporine A and anti-cyclosporine A mouse monoclonal antibody. Methods We generated a VHH against an anti-cyclosporine A mouse monoclonal antibody complexed with cyclosporine A by alpaca naïve VHH phage display library. A bivalent tandem VHH was generated using the established VHH, and a new sandwich immunoassay for measuring cyclosporine A was constructed using the tandem VHH conjugated with alkaline phosphatase. A prototype fully automated sandwich immunoassay was developed on a fully automated chemiluminescence analyzer using magnetic particles immobilized with the mouse monoclonal antibody and the tandem VHH conjugated with alkaline phosphatase. The prototype assay was further developed by implementing an automated whole blood pretreatment process with strong denaturing agents. The performances of the prototype immunoassays were investigated. Results The established VHH showed a highly specific reaction to the complex of cyclosporine A and the anti-cyclosporine A mouse monoclonal antibody, and a sandwich immunoassay for cyclosporine A was successfully developed using the VHH and the mouse monoclonal antibody. The tandem bivalent VHH exhibited higher reactivity to the complex than the monomeric VHH. The fully automated reagent prototype achieved a measurement range of 30 to 1500 ng/mL. Measurement of cyclosporine A in whole blood samples by the prototype reagent showed a correlation coefficient of 0.96 with mass spectrometry, and a correlation coefficient of 0.95 with a commercial IVD reagent. Conclusion We successfully developed a sandwich reagent prototype for cyclosporine A using VHH established by alpaca naïve VHH phage display library. This is the first report of a fully automated reagent for cyclosporine A measurement including an automated whole blood pretreatment process.

Simultaneous Detection of SARS-CoV-2 Nucleoprotein and Receptor Binding Domain by a Multi-Area Reflectance Spectroscopy Sensor.

The COVID-19 pandemic has emphasized the urgent need for point-of-care methods suitable for the rapid and reliable diagnosis of viral infections. To address this demand, we report the rapid, label-free simultaneous determination of two SARS-CoV-2 proteins, namely, the nucleoprotein and the receptor binding domain peptide of S1 protein, by implementing a bioanalytical device based on Multi Area Reflectance Spectroscopy. Simultaneous detection of these two proteins is achieved by using silicon chips with adjacent areas of different silicon dioxide thickness on top, each of which is modified with an antibody specific to either the nucleoprotein or the receptor binding domain of SARS-CoV-2. Both areas were illuminated by a single probe that also collected the reflected light, directing it to a spectrometer. The online conversion of the combined reflection spectra from the two silicon dioxide areas into the respective adlayer thickness enabled real-time monitoring of immunoreactions taking place on the two areas. Several antibodies have been tested to define the pair, providing the higher specific signal following a non-competitive immunoassay format. Biotinylated secondary antibodies and streptavidin were used to enhance the specific signal. Both proteins were detected in less than 12 min, with detection limits of 1.0 ng/mL. The assays demonstrated high repeatability with intra- and inter-assay coefficients of variation lower than 10%. Moreover, the recovery of both proteins from spiked samples prepared in extraction buffer from a commercial self-test kit for SARS-CoV-2 collection from nasopharyngeal swabs ranged from 90.0 to 110%. The short assay duration in combination with the excellent analytical performance and the compact instrument size render the proposed device and assay suitable for point-of-care applications.

Noncompetitive immunoassay optimized for pharmacokinetic assessments of biologically active efruxifermin

Efruxifermin (EFX) is a homodimeric human IgG1 Fc-FGF21 fusion protein undergoing investigation for treatment of liver fibrosis due to nonalcoholic steatohepatitis (NASH), a prevalent and serious metabolic disease for which there is no approved treatment. Biological activity of FGF21 requires its intact C-terminus, which enables binding to its obligate co-receptor β-Klotho on the surface of target cells. This interaction is a prerequisite for FGF21 signal transduction through its canonical FGF receptors: FGFR1c, 2c, and 3c. Therefore, the C-terminus of each FGF21 polypeptide chain must be intact, with no proteolytic truncation, for EFX to exert its pharmacological activity in patients. A sensitive immunoassay for quantification of biologically active EFX in human serum was therefore needed to support pharmacokinetic assessments in patients with NASH. We present a validated noncompetitive electrochemiluminescent immunoassay (ECLIA) that employs a rat monoclonal antibody for specific capture of EFX via its intact C-terminus. Bound EFX is detected by a SULFO-TAG™-conjugated, affinity purified chicken anti-EFX antiserum. The ECLIA reported herein for quantification of EFX demonstrated suitable analytical performance, with a sensitivity (LLOQ) of 20.0 ng/mL, to support reliable pharmacokinetic assessments of EFX. The validated assay was used to quantify serum EFX concentrations in a phase 2a study of NASH patients (BALANCED) with either moderate-to-advanced fibrosis or compensated cirrhosis. The pharmacokinetic profile of EFX was dose-proportional and did not differ between patients with moderate-to-advanced fibrosis and those with compensated cirrhosis. This report presents the first example of a validated pharmacokinetic assay specific for a biologically active Fc-FGF21 fusion protein, as well as the first demonstration of use of a chicken antibody conjugate as a detection reagent specific for an FGF21 analog.

487. Analysis of the Association between Specific Antibody Response against Respiratory Syncytial Virus Fusion Protein Conformations and Life-Threatening Infection in Previously Healthy Infants

Abstract Background Specific humoral response against pre-fusion (pre-F) conformation of respiratory syncytial virus (RSV) F protein has been proposed to play a role against severe infection. We aimed to analyze the association between serum IgG titers against pre-F and post-fusion (post-F) conformations of RSV F protein and life-threatening RSV disease (LTD) in previously healthy infants. Methods Prospective cohort study including previously healthy infants < 12 months, hospitalized with a first RSV infection, in 2017-2019. Patients were defined to have LTD when required intensive care and ventilatory support. Pre-F exclusive and post-F-specific antibody responses were determined by post-F competition and non-competitive immunoassays, respectively, and neutralizing activity was measured by plaque reduction neutralization test. Viral load (VL) was assessed by qRT-PCR from nasopharyngeal aspirates collected on admission. Serum samples were collected within 72 h from admission and in convalescence (between 14 to 60 days). Results Seventy-five patients were included, median age 3 months, 60% (n=45) were males; 21 patients developed LTD. Importantly, acute and convalescent post-F and pre-F exclusive IgG titers did not associate with LTD (Fig 1). There was a positive correlation between neutralizing antibody titers and pre-F exclusive IgG titers (p=0.016). Post-F and pre-F exclusive IgG titers negatively correlated with age in acute phase (p< 0.0001 and p= 0.0009). In acute phase, post-F and pre-F exclusive IgG titers were higher in patients ≤ 2 months (Fig 2). Patients > 2 months increased post-F and pre-F exclusive IgG titers in convalescence. Additionally, post-F and pre-F exclusive titers did not correlate with VL, and VL was not related to LTD. Conclusion -Pre-F exclusive and post-F IgG titers did not associate with LTD, even though pre-F exclusive IgG correlated with neutralizing antibody titers. -Infants under two months of age developed a lower humoral response, likely due to an interference by maternal antibodies and/or immunological immaturity. -These findings highlight the importance of fully characterizing the immune response against RSV and its association with disease severity for the development of preventive strategies. Disclosures Barney S. Graham, MD, PhD, BSG: BSG is an inventor on patents for the stabilization of the RSV F protein (WO2014160463A1, Prefusion RSV F proteins and their use).|National Institutes of Health: Inventor on patents for RSV vaccines|National Institutes of Health: inventor on patents for measles and other paramyxovirus vaccines.

PMON224 Newly Observed Interference of Oral Biotin with the Free Testosterone Assay? In a Woman with Postmenopausal Virilization.

Abstract Introduction Approximately 15-20% of US adults take supraphysiologic doses of biotin-containing supplements for common hair and skin problems. When consumed in high doses, competitive and non-competitive immunoassays which use the streptavidin-biotin binding complex can skew results to be either falsely high or falsely low. Case Presentation A 74 y/o Caucasian female who initially presented to her primary care physician concerned for alopecia with frontal balding, acne and moderate hirsutism. Her total testosterone was elevated with 146 ng/dL (7.0–40 ng/dL) with a perplexing normal free testosterone of 4.2 pg/mL (<4.2 pg/mL) in the context of a normal SHBG of 48 nmol/L (14–73 nmol/L). Androstenedione was elevated with 161 ng/dL (17–99 ng/dL) with a normal CMP, prolactin, DHEA-s and 17-hydroxyprogesterone. Transvaginal US was normal. The size of the ovaries was consistent with menopause. Abdominal and pelvic MRI showed normal adrenal glands and ovaries. She was taking between 1 to 3mg of biotin per day. Biotin was discontinued. Repeat testing revealed an elevated free testosterone of 12.7 pg/mL (0.2 - 3.7 pg/mL) along with an elevated total testosterone of 120 ng/dL (2 - 45 ng/dL) and a normal SHBG of 39 nmol/L (14–73 nmol/L) which was now consistent with her clinical picture of postmenopausal hyperandrogenism. Bilateral laparoscopic salpingo-oophorectomy was recommended to exclude an androgen-secreting ovarian tumor or hyperthecosis. Testing was then repeated while taking biotin which again manifested a normal free testosterone of 2.8 pg/mL (<4.2 pg/mL) with an elevated total testosterone of 121.4 ng/dL (7.0–40 ng/dL) and a normal SHBG of 45 nmol/L (14–73 nmol/L). Discussion Biotin may interfere with the total testosterone levels, but little is known of its effects on free testosterone. We report the first case of biotin interference on the free testosterone assay, resulting in falsely low values demonstrated in two different occasions, that improved following withdrawal of this widely used vitamin supplement. Laboratories use a direct analog enzyme immunoassay to calculate the free testosterone however its calibration and specificity has been debatable, and it is still unclear how biotin can affect it. It is critical that laboratorians and clinicians are aware of this biotin interference so that misdiagnosis and inappropriate treatment can be prevented.

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 an electrochemical immunosensor for the determination of molinate by using phages labeled with CdS nanocrystals as a novel strategy to signal amplification

Research on immunoassays and immunosensors is currently focused mainly on the development of strategies to amplify the resulting signal. In this sense, the development of new hapten tracers that allow simpler, more sensitive, and more reliable immunoassays is desirable. Therefore, we present the development of an electrochemical immunosensor using viral particles (phages) bound with dozens of CdS nanocrystals (CdS NCs) as an outperforming alternative to conventional competitive hapten conjugates. This allows us to obtain an multiplicator effect in signal obtained. Therefore, as proof of concept, an electrochemical immunosensor was developed for the detection of the molinate in river water samples, based on a noncompetitive immunoassay using recombinant M13 phage particles labeled with CdS NCs (CdS NCs-phage). The phage particles have on its surface peptides that recognize the molinate/antibody immune complex. Molinate determination was performed by square wave anodic stripping voltammetry of Cd2+ release from CdS NCs. The limit of detection and the midpoint sensitivity obtained were 34 pg mL-1 and 0.43 ng mL-1, respectively. In addition, other very good analytical parameters were obtained, where percent coefficient of variation (%CV) between 5 % and 25 %, and recovery percentages close to 99 % were found. Results obtained were compared with those determined by HPLC-UV visible, obtaining an excellent correlation between both methods. This electrochemical immunosensor was successfully used to analyze molinate in river water samples without pre-treatment, obtaining excellent recoveries.

Rapid and sensitive noncompetitive immunoassay for detection of aflatoxin B1 based on anti-immune complex peptide

Noncompetitive immunoassays for small molecules are generally considered to be more sensitive than competitive ones. In this study, a phage-peptide against immune complex of aflatoxin B1 (AFB1) and nanobody Nb28 was obtained by phage-display technology. The phage-peptide was labeled with peroxidase and used to develop a direct noncompetitive magnetic-chemiluminescent enzyme-linked immunoassay (Nc-MCLEIA) for AFB1. The 50% signal saturation concentration (SC50) and limit of detection (LOD) of Nc-MCLEIA for AFB1 were 0.089 and 0.006 ng/mL, respectively. Compared with competitive immunoassays developed by the Nb28, the sensitivity and efficiency of Nc-MCLEIA were greatly improved. The recoveries of AFB1 from spiked corn, rice, flour, peanut, peanut oil and corn oil samples ranged from 83.8% to 119.2% with coefficient of variable under 8.9%. Furthermore, parallel analysis of natural corn, rice and flour samples by Nc-MCLEIA and HPLC (high performance liquid chromatography) proved that the Nc-MCLEIA was reliable.

Single-step noncompetitive immunocomplex immunoassay for rapid aflatoxin detection

Owing to the high carcinogenicity of aflatoxins, these toxic secondary metabolites pose a severe risk to human and animal health and can have major economic implications. Herein, we report the development of a noncompetitive immunoassay for aflatoxins based on a monoclonal capture antibody and a unique anti-immunocomplex (anti-IC) antibody fragment (scFv) isolated from a synthetic antibody repertoire. The anti-IC scFv recognizes the immunocomplex and enables the development of noncompetitive sandwich-type assays despite the small size of the analyte. The single-step assay developed in this work, with a detection limit of 70 pg mL−1, could detect aflatoxins within 15 min. The assay was applied to the analysis of spiked food samples, and the results showed that the method could provide a rapid and simple tool for aflatoxin detection. Moreover, the work demonstrates the potential of anti-IC antibodies and non-competitive immunoassays for the analysis of small molecule contaminants.

Influence of Autoantibodies to Estradiol and Progesterone on the Blood Serum Hormones Concentrations in Postmenopausal Healthy Women and Breast Cancer Patients

Background: It is known that antibodies to steroid hormones are associated with some human diseases (systemic lupus erythematosus, thrombosis, recurrent pregnancy loss, autoimmune dermatitis et al.). There were no any data about action of specific autoantibodies on the sex hormones in hormone-dependent cancer patients. Objectives: The purpose of this study was to investigate the probable links between estradiol (Es) and progesterone (Pg) levels and autoantibodies of A-class specific to these hormones (IgA-Es and IgA-Pg) in the blood serum of healthy women and breast cancer patients. Patients and Methods: There were examined 521 nearly diagnosed breast carcinoma patients from Regional Clinical Oncology Dispensary (Kemerovo, Russian Federation) and 143 healthy women from Kemerovo (Russian Federation). The serum concentration of Es and Pg were analyzed by competitive immunoassay using specific monoclonal antibodies. The serum IgA-Pg and IgA-Es were analyzed by solid-phase non-competitive immunoassay using Pg and Es conjugated with bovine serum albumin as adsorbed antigens. The data analysis was performed using software STATISTICA 8.0 (Stat Soft Inc., USA).Results: We discovered that low ratios Pg/Es<4.3 were associated with breast cancer. Personal IgA-Pg/IgA-Es ratios negatively correlated with Es levels and positively correlated with Pg levels and Pg/Es ratios in compared groups. The influence of IgA-Pg/IgA-Es ratio on the hormones levels and their ratio in heathy women was more strong than in breast cancer patients. Conclusions: Our clinical results correspond to the known experimental data about influence of specific antibodies on the levels of steroid hormones in immunized animals. Human autoantibodies to Es and Pg could stimulate or inhibit promotion of carcinogenesis by influence on Pg/Es ratio depending on individual IgA-Pg/IgA-Es ratio.

Development of a Linear Immobilization Carrier-Based Immunoassay for Aflatoxin.

We explored the feasibility of developing immunoassay technology with a linear carrier, to develop a simpler and cheaper rapid immunoassay technology. We selected aflatoxins as an example for research, as they are a group of highly toxic and carcinogenic compounds representing a worldwide threat to human health and life. With a non-competitive immunoassay, we detected and evaluated the effect of 28 different linear materials on antibody immobilization. Mercerized cotton and Dyneema line were chosen from the linear materials for further comparison using a competitive immunoassay, because both showed high-signal values and relatively low background noise. The results showed the sensitive IC50 of mercerized cotton as the reaction carrier was 0.33 ng/mL, and the linear range was 0.16~3.25 ng/mL. The sensitivity using Dyneema line as the reaction carrier was 1.16 ng/mL. The competitive curves of four sample matrices were established to evaluate the stability of the detection system; these were basically consistent with those without sample matrices. In conclusion, both mercerized cotton and Dyneema, will be suggested for the novel development of linear immobilization carrier-based immunoassays for other analytes, and especially to construct inexpensive and easy-to-obtain biological and environmental analytical technologies and biosensors.

New Approach to Generate Ratiometric Signals on Immunochromatographic Strips for Small Molecules.

The self-calibration capability of ratiometric signals has been widely considered to enhance the accuracy, sensitivity, and anti-interference ability of immunoassays. Exploring a new approach to generate ratiometric signals can provide more options for various requirements. Herein, we integrated the negative-readout competitive and positive-readout noncompetitive immunoassays into a single assay by employing different color tracers, labeled peptidomimetic and anti-immunocomplex peptides, to create a new unconstrained ratiometric signal approach. Using an immunochromatographic strip (ICS) and a fungicide benzothiostrobin as the analytical platform and analyte, respectively, we showed that this approach can be extensively applied to fluorescence and colorimetry readouts, which have also been proven for strong anti-interference ability to an external light environment. Moreover, the enormous intuitional color changes of ratiometric fluorescent and colorimetric ICSs (RFICS and RCICS) enabled the formation of the color reference cards (like the pH paper) for visual judgment. After adaptation with a portable smartphone, the quantitative detection limits for RFICS and RCICS were 0.17 and 0.44 ng mL-1, respectively. In addition, the ICSs showed good accuracy for the detection of benzothiostrobin in spiked samples.

Development of competitive and noncompetitive immunoassays for clothianidin with high sensitivity and specificity using phage-displayed peptides

Clothianidin is a widely used pesticide that has been banned from outdoor use by the European Union due to its toxicity. To improve the sensitivity and specificity of existing clothianidin immunoassays, we developed competitive and noncompetitive immunoassays for clothianidin based on phage-displayed peptides. Cyclic 8-, 9-, and 10-residue peptide libraries were constructed using an optimized phagemid pComb-pVIII to prevent the loss of theoretical library diversity. Twenty-eight peptidomimetics and two anti-immunocomplex peptides were isolated through a blended panning process and used to develop competitive and noncompetitive phage enzyme-linked immunosorbent assays (P-ELISAs), respectively. After optimization, the half inhibition concentration (IC50) and half saturation concentration (SC50) of competitive and noncompetitive P-ELISAs were 3.83 ± 0.23 and 0.45 ± 0.02 ng/mL, respectively. Competitive P-ELISA showed 2.6–18.2% cross-reactivity with imidaclothiz, nitenpyram and imidacloprid. Importantly, noncompetitive P-ELISA, which has the best specificity and great sensitivity for clothianidin, showed no cross-reactivity with the analogs. The average recoveries of competitive and noncompetitive P-ELISAs were 73.8–104.1% and 76.6–102.2%, respectively, while the relative standard deviations were ≤ 11.0%. In addition, the results of P-ELISAs in the analysis of blind samples were consistent with those of high-performance liquid chromatography.

An enhanced open sandwich immunoassay by molecular evolution for noncompetitive detection of Alternaria mycotoxin tenuazonic acid

Open sandwich enzyme-linked immunosorbent assay (OS-ELISA), a novel noncompetitive immunoassay format, has shown great potential in rapid detection for small molecules compared with traditional competitive format. Here, an enhanced OS-ELISA towards the mycotoxin tenuazonic acid (TeA) was developed for the first time based on heavy chain variable region (VH) and light chain variable region (VL) from the hybridoma cells (3F10) producing anti-TeA monoclonal antibody (mAb). The established OS-ELISA exhibited a limit of detection of 0.08 ng/mL, and was 13 times more sensitive than mAb-based indirect competitive ELISA (ic-ELISA). The proposed assay was also applied to detect TeA contents in juice, flour and tomato ketchup samples with satisfactory recoveries of 87.6%–111.3%. Finally, the great accuracy of the established OS-ELISA method was validated by the standard ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS).

Binding affinity-guided design of a highly sensitive noncompetitive immunoassay for small molecule detection

Small molecules are immunochemically classified as hapten that lacking of at least two epitopes, usually using competitive format for establishing immunoassays. However, theoretically, noncompetitive immunoassay format is more sensitive and has a wider analytical range. In the present study, a novel hapten of halofuginone was synthesized and used to produce a monoclonal antibody (mAb). By analyzing the binding kinetics, we found that the affinity of analyte–enzyme to mAb was much greater than that of analyte, which could result in a low sensitivity of competitive assay format. Based on this, we established a novel noncompetitive immunoassay by using a replacement approach. The noncompetitive format has obvious advantages in sensitivity and analytical range, which promoted approximately 3.5– and 5–fold, respectively, compared to the competitive immunoassay. Ultimately, the newly designed noncompetitive immunoassay in this work will provide insights as well as alternative method to traditional small molecule competitive assays.