Neurofilament light chain (Nf-L) is a well-known biomarker for axonal damage, however the corresponding circulating Nf-L analyte in CSF is poorly characterized. We, therefore, isolated new monoclonal antibodies against synthetic peptides, and these monoclonals were characterized for their specificity on brain-specific intermediate filament proteins. Two highly specific antibodies, ADx206 and ADx209, were analytically validated for CSF applications according to well-established criteria. Interestingly, using three different sources of purified Nf-L proteins, a significant impact on interpolated concentrations was observed. With a lower limit of analytical sensitivity of 100 pg/ml using bovine Nf-L as the calibrator, we were able to quantify the Nf-L analyte in each sample and these Nf-L concentrations were highly correlated to the Uman diagnostics assay (Spearman rho= 0.97, P<0.001). In the clinical diagnostic groups, the new Nf-L ELISA could discriminate patients with Alzheimer’s disease (AD, n=20) from frontotemporal lobe dementia (FTD, n=20) and control samples with subjective cognitive decline (SCD, n=20). Henceforth, this novel Nf-L ELISA with well-defined specificity and epitopes can be used to enhance our understanding of harmonizing the use of Nf-L as a clinically relevant marker for neurodegeneration in CSF.

The presence of anti SARS-CoV2-RBD antibody (Anti-RBD) prevents severe COVID-19. We aimed to determine the accuracy of a point-of-care Anti-RBD test implemented in person living with HIV (PLWH), systemic lupus erythematosus (SLE), and chronic kidney disease (CKD). We enrolled 182 non-comorbid subjects and 335 persons with comorbid (PLWH, SLE, CKD) to test anti-RBD assay compared to surrogate viral neutralization (SVNT) as the reference test. We performed linear correlation analysis between anti-RBD vs SVNT and a ROC analyses to ascertain anti-RBD cut-off at 30%, 60%, and 90% inhibition of SVNT to calculate accuracy. Correlations of anti-RBD to SVNT among all groups were excellent with R= 0.7903, R=0.7843, R=0.8153 among non-comorbid, SLE and CKD groups, respectively, with significantly higher correlation among the PLWH group (R=0.8877; p-value=0.0072) compared to non-comorbid group. The accuracy of the anti-RBD test among PLWH and CKD group was similar with the non-comorbid but showed lower sensitivity in the SLE group (p=0.000014). The specificity of the test remained high in all groups. In conclusion, the Anti-RBD test had excellent correlation to SVNT. The persistently high specificity in all groups suggests that this test can be reliably utilized to detect presence of low neutralization capacity, prompting additional vaccination.

Rapid, accurate, and convenient diagnosis is essential for effective disease management. Various detection methods, such as enzyme-linked immunosorbent assay, have been extensively used, with lateral flow immunoassay (LFIA) recently emerging as a major diagnostic tool. Nanoparticles (NPs) with characteristic optical properties are used as probes for LFIA, and researchers have presented various types of optical NPs with modified optical properties. Herein, we review literature on LFIA with optical NPs for the detection of specific targets in the context of diagnostics.

The World Health Organisation declared Covid-19 as a pandemic on 11th March 2020. The main approach to tackle Covid-19 worldwide is to screen and provide supportive care to patients. For screening purpose RT-qPCR based test are used as an initial detections assay. The test is expensive, time consuming and not suitable for mass scale screening/ confirmation requirement. A recent advancement is development of Immunoassay procedures (liquid Phase tests or bed side 10-20minute strip test). In order to help and accelerate bringing life to normal after lock down, Pakistan is in dire need to develop and adopt the immunoassay procedures for mass scale screening and confirmation of COVID-19 infection. It is cheap and easy to perform without a lab requirement.

Dibutyl phthalate (DBP) is widely used as a plasticizer in the production of polymeric materials to give them flexibility, strength and extensibility. However, due to its negative impact on human health, in particular, reproductive function and fetal development, the content of DBP must be controlled in food and environmental objects. The present study aims to develop a sensitive, fast and simple polarization fluorescent immunoassay (FPIA) using monoclonal antibodies derived against DBP (MAb-DBP) for its detection in open waters. New conjugates of DBP with various fluorescein derivatives were obtained and characterized: 5-aminomethylfluorescein (AMF) and dichlorotriazinylaminofluorescein (DTAF). The advantages of using the DBP-AMF conjugate in the FPIA method are shown, the kinetics of binding of this drug with antibodies is studied, the analysis is optimized, and the concentration of monoclonal antibodies is selected for sensitive analysis - 16 nM. The calibration dependence of the fluorescence polarization signal for the detection of DBP was obtained. The observed IC50 (40 ng/mL) and LOD (10 ng/mL) values were improved by a factor of 35 for the previously described FPIA using polyclonal antibodies. This technique was tested by the added/found method, the percentage of DBP discovery in water ranged from 85 to 110%. Using the developed method, real water samples from Lake Onega were tested, and a good correlation was shown between the results of the determination of DBP by the FPIA method and GC-MS/MS. Thus, the FPIA method developed in this work can be used to determine DBP in open water reservoirs.

Simultaneous measurement of different substances from a single sample is an emerging issue to achieve efficient and high-throughput detection in several fields of application. Although immunoanalytical techniques have well-established and prevailing advantages over alternative screening analytical platforms, one of the incoming challenges for immunoassay is exactly multiplexing. The Lateral Flow Immunoassay (LFIA) is a leading immunoanalytical technique for onsite analysis thanks to its simplicity, rapidity and cost-effectiveness. Moreover, LFIA architecture is adaptable to multiplexing and therefore candidates as a possible answer to the pressing demand of multiplexing point-of-need analysis. The review present an overview of diverse approaches for multiplex LFIA, with a special focus on strategies based on new types of magnetic, fluorescent and colored labels

Autoantibodies (Abs) to thyroid antigens are associated with autoimmune thyroid disease (AITD), although sometimes they have no clear clinical significance. The aim of this study was to evaluate the avidity of thyroid Abs in the sera of AITD patients and non-AITD Abs carriers. A multiplex microarray-based assay was developed to detect the avidity of thyroid disease-associated Abs, including Abs to thyroperoxidase (TPO), thyroglobulin (Tg), pendrin, thyroid hormones T3 and T4, pyruvate kinase, and carbonic anhydrase 2. The method is based on indirect multiplex assay with an additional step involving the chaotrope destruction of the autoantibody-antigen complexes. The avidity index (AI) is then calculated. The method was tested on clinical cohort (n =117) and then confirmed by studies with another independent cohort (n =123). For the confirmation study, the median AI for AbTg was 29.9% among healthy Abs carriers, 52.6% for AITD patients, and 92.7% for non-AITD type 1 diabetes (T1D) patients. Median AI for AbTPO were 39.9% in healthy carriers, 73.4% in AITD patients, 83.2% in T1D patients, and 98.5% in AITD patients with thyroid neoplasm. The presence of high-avidity AbTg in T1D patients was also confirmed by a modified ELISA. Identification of high-avidity autoantibodies to thyroid proteins in non-thyroid T1D patients requires further studies.

Background: The duration of the vaccine's protective efficacy against SARS-CoV-2 is unknown. Evaluation of the clinical performance of available tests is required. Objectives: To evaluate the clinical performance of three immunoassays for the detection of IgG antibodies generated by mRNA vaccines against SARS-CoV-2. Methods: Two automated immunoassays (Euroimmun Anti-SARS-CoV-2 ELISA IgG and Abbott SARS-CoV-2 CLIA IgG) and one lateral flow immunoassay (LFIA Test Livzon IgG) were tested. 300 samples distributed in 3 groups were analyzed: 100 subjects over 18 years old and under 45 years old, 100 subjects between 45-65 years old and another 100 over 65 years old. collected before vaccination, at 21 days, 1, 2, 3 and 6 months post-vaccination. Sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio, and agreement (I. Kappa) were calculated for each serological test. Results: Maximum sensitivity for IgG was 98.7%, 98.1%, and 97.8% for the ELISA Euroimmun, CLIA Abbott, and Livzon LFIA assays and maximum specificity for IgG was 99.4%, 99.9%. % and 98.4% ELISA, CLIA and LFIA respectively at 3 months after vaccination with a decrease in antibody levels from the sixth month. The best agreement was observed between ELISA and CLIA 100%; (k = 1.00). The agreement between ELIA, CLIA and LIFIA was 99% (k = 0964) at the second and third month after vaccination. Seroconversion was faster and longer lasting in the younger age groups. Conclusion: Our study showed an equivalent and homogeneous clinical performance for IgG of three immunoassays after vaccination and that the LIFIA assay is the most cost-effective, reliable and accurate for routine use in population studies of seroconversion and seroprevalence.

Although there are extensive literature reports on the use of gold nanoparticle (AuNP) based homogeneous assays for detection of biomolecules, very few experimental description and procedures involving their preparation are described. In this study, AuNPs conjugated to Bovine Serum Albumin or Envelope protein from Dengue II were developed as a homogeneous immunoassay for antibody detection. We report here optimization of key parameters to prepare an immunoassay like conjugation protein concentration, centrifugation time, electrolyte addition and assay temperature. We determined that saturating protein concentrations improved AuNPs surface coverage and uniformity of the assay and addition of sodium chloride improved sensitivity of the antibody detection method and assay stability. Furthermore, we showed that dynamic light scattering can be used to monitor changes in gold nanoparticles in the preparation and detection steps. Additionally, numerical simulations of the plasmonic optical response of AuNPs were carried out to scan for size-dependent response of the AuNPs. The AuNPs homogeneous immunoassay developed was further used in the detection of antibodies in vitro to detect Dengue virus infection.

The measurement of sP4 often varies due to different laboratory methodologies and individual canine characteristics. In this investigation, sP4 outcomes obtained from a commercial POC analyzer, designed for efficient sP4 assessment in bitches, were compared with results derived from CMIA, the reference laboratories in Thailand. Thorough documentation encompassed various parameters: mean, standard deviation (SD), 95% confidence interval (CI), minimum and maximum sP4 concentration values. Additionally, we meticulously recorded the Pearson’s correlation coefficient, Lin’s concordance correlation coefficient, and the bias correction factor. Interestingly, there was no significant difference (p &gt; 0.05) in the means obtained by the POC and CMIA. The Pearson’s correlation coefficient between POC and CMIA stood at 0.957, with Lin’s concordance correlation coefficient for POC recorded as 0.949. Furthermore, the bias correction factor was established at 0.991. This investigation followed established CMIA guidelines, modified to incorporate the mean and 95% CI as criteria for optimal breeding time using the POC analyzer. In conclusion, the commercial POC immunologic analyzer emerges as a valuable tool, aiding in precisely determining the optimal timing for natural mating or artificial insemination in bitches.

Botulinum neurotoxin (BoNT) intoxication can lead to the disease botulism, characterized by flaccid muscle paralysis that can cause respiratory failure and death. Due to the significant morbidity and mortality costs associated with BoNTs high toxicity, developing highly sensitive, rapid, and field-deployable assays are critically important to protect the nation’s food supply against either accidental or intentional contamination. We report here that the B-cell based biosensor assay (CANARY^® Zephyr) detects BoNT/A in buffer and various food matrices rapidly in ≤ 40 min, in small volumes ≈ 50 μL, with minimal processing of samples, and is extremely portable (suitcase-sized equipment). BoNT/A was detected at limits of detection (LOD) < 0.075 ng ± 0.02 in assay buffer while milk matrices (non-fat, 2 %, whole milk) increased the LOD to < 0.175 – 0.314 ng. Limits of detection for the assay in complex foods were < 1 ng ± 0.0 (neutralized acidic juices-carrot, orange and apple); < 16.7 ng ± 7.7 (liquid egg); and varied from < 0. 39 – 3.125 ng for solid complex foods (ground beef, green bean baby puree, smoked salmon). These results show that the CANARY^® Zephyr assay can be a highly useful tool in clinical, environmental, and food safety surveillance programs.

BACKGROUND: Few studies have examined the use of immunoassay urine drug testing of cancer patients in palliative care clinics. OBJECTIVES: We examined the frequency of immunoassay urine drug test (UDT) abnormalities and the factors associated with aberrancy at a safety-net hospital palliative medicine clinic. METHODS: A retrospective review of the electronic medical records of consecutive eligible patients seen at the outpatient palliative medicine clinic in a resource-limited safety net hospital system was conducted between September 1, 2015 and December 31, 2020. We collected longitudinal data on patient demographics, UDT findings, and potential predictors of aberrant results. RESULTS: Of the 913 patients in the study, 500 (55%) underwent UDT testing, with 455 (50%) having the testing within the first three visits. Among those tested within the first three visits, 125 (27%) had aberrant UDT results; 44 (35%) of these 125 patients were positive for cocaine. In a multivariable regression model analysis of predictors for aberrant UDT within the first 3 visits, non-Hispanic White race (odds ratio [OR]=2.13; 95% confidence interval [CI]: 1.03-4.38; p=0.04), history of illicit drug use (OR=3.57; CI: 1.78-7.13; p< 0.001), and history of marijuana use (OR=7.05; CI: 3.85-12.91; p< 0.001) were independent predictors of an aberrant UDT finding. CONCLUSION: Despite limitations of immunoassay UDT, it was able to detect aberrant drug taking behaviors in a significant number of patients seen at a safety net hospital palliative care clinic, including cocaine use. These findings support universal UDT monitoring and utility of immunoassay-based UDT in resource limited settings.

In this study, two heterogeneous fluorescence immunoassays using CdSe/ZnS quantum dot (QD) to label anti-progesterone antibody (P4Ab) for the determination of progesterone (P4) were performed in the wells of a 96-well microtiter plate. First, P4Ab was conjugated to hydrophilic CdSe/ZnS QDs via ethyl-3-(dimethylaminopropyl) carbodiimide(EDC)-N-hydroxysuccinimide) chemistry(NHS) (QDs-P4Ab conjugates). The QDs-P4Ab conjugate was employed as a second antibody in a sandwich assay, where the P4Ab was immobilized onto the 3-aminopropyltrimethoxysilane (APTMS) sol-gel membrane of the wells of a 96-well microtiter plate, and P4 was bound between the immobilized P4Ab and the QDs-P4Ab conjugate. In this assay, the fluorescence intensity of the QDs increased with increasing P4 concentrations. This assay had a detection limit of 553.9 pg/ml and a sensitivity of 18,251.96 pg/ml with a linear range of 2,184.6 – 117,082 pg/ml. In the direct binding assay, P4 was directly bound to the QDs-P4Ab conjugates immobilized onto the APTMS sol-gel membrane of the wells of a 96-well microtiter plate. In this direct binding assay the fluorescence intensity of the QDs decreased with increasing P4 concentrations, and this assay had a linear range of 28.95 – 26,607.7 pg/ml with a detection limit of 3.32 pg/ml and a sensitivity of 987.24 pg/ml. These fluorescence immunoassays have been successfully applied for the determination of P4 in real human serum, and the results were well correlated with those of a certified radioimmunoassay (RIA) method.

The COVID-19 pandemic has created huge damage to society and brought panics around the world. Such panics can be ascribed to the seemingly deceptive features of the COVID-19: compared to other deadly viral outspreads, it has medium transmission and mortality rates. As a result, the severity of this virus was deeply underestimated by the society at the beginning of the outbreak. Based on this, in this review, we define the viruses with features similar to those of COVID-19 as the Panic Zone viruses. To contain those viruses, accurate and fast diagnosis followed by effective isolation and treatment of patients are pivotal at the early stage of virus breakouts. This is especially true when there is no cure or vaccine available for a transmissible disease, which is the case for current COVID-19 pandemic. As of April 2020, more than one hundred kits for the COVID-19 diagnosis on the market are surveyed in this review. It is of critical importance to rationally use these kits for the efficient management and control of the Panic Zone viruses. Therefore, we discuss guidelines to select diagnostic kits at different outbreak stages of the Panic Zone viruses, COVID-19 in particular. While it is of utmost importance to use detection kits with low false negativity at the early stage of an outbreak, the low false positivity gains its importance at later stages of the outbreak. Finally, since a massive attack from a viral pandemic requires a massive defense from the whole society, we urge both government and private sectors to research and develop affordable point-of-care (POC) detection kits, which can be used massively by the general public (and therefore called as massive POC) to contain Panic Zone viruses in future.

A common task in the immunodetection of structurally close compounds is to analyze the selectivity of immune recognition: it is required to understand the regularities of immune recognition and to elucidate the basic structural elements which provide it. Triazines are compounds of particular interest for such a research due to their high variability and the necessity of their monitoring to provide safety of agricultural products and foodstuffs. We evaluated the binding of 20 triazines with polyclonal (pAb) and monoclonal (mAb) antibodies obtained using atrazine as the immunogenic hapten. A total of >3000 descriptors was used in QSAR analysis of binding activities (pIC50). Comparison of the two enzyme immunoassay systems showed that the system with pAb is much easier to describe using 2D QSAR methodology, while the system with mAb can be described using the 3D QSAR COMFA. Thus, for the 3D QSAR model of the polyclonal antibodies, the main statistical parameter q² (‘leave-many-out’) is equal 0.498, and for monoclonal antibodies q² is equal 0.566. Obviously, in the case of pAb, we deal with several targets, while in the case of mAb the target is one, and therefore it is easier to describe it using specific fields of molecular interactions distributed in space.

Births occurring before thirty-seven weeks of gestation are referred to as preterm births (PTBs), which is the primary cause of neonatal mortality and long-term disabilities. The unknown mechanism behind PTB makes diagnosis difficult, yet early detection is necessary for controlling and averting related consequences. The primary focus of this work is to provide an overview of the known risk factors associated with preterm labor and the conventional and advanced procedures for early detection of PTB, including multi-omics and artificial intelligence (AI)/machine learning (ML)-based approaches. It also discusses the principles of detecting various proteomic biomarkers based on lateral flow immunoassay and microfluidic chips, along with the commercially available point-of-care testing (POCT) devices and associated challenges. After briefing the therapeutic and preventive measures of PTB, the review summarizes with an outlook.

: Early and accurate detection of infectious diseases is a key step for surveillance, epidemiology, and control, notably timely disease diagnosis, patient management and follow-up. In this study, we aimed to develop hand-held ultra-fast duplex PCR assays coupled to amplicon detection by lateral flow (LF) immunoassay to deliver a rapid, and simple molecular diagnostic test for con-comitant detection and identification of the main Leishmania parasites encountered in Tunisia and the Old World. We selected two DNA targets to amplify L. major/L. tropica and L. infantum/L. tropica group of species DNAs, respectively. We optimized the experimental conditions of a du-plex ultra-fast PCR. The amplification is performed by a portable Palm convection PCR machine within 18 min and the products are detected by a LF cassette within 10 minutes. The test allows the identification of the infecting species according to the position and number of test lines re-vealed. Tested on a selection of DNAs of representative Leishmania strains of the three studied species (N=37), the ultra-fast duplex PCR-LF showed consistent, stable, and reproducible results. The analytical limit of detection of the test was 0.4pg for L. major, 4pg for L. infantum and 40pg for L. tropica.

Hemolysin II (HlyII) – one of the pathogenic factors of Bacillus cereus, a pore-forming β-barrel toxin – possesses a C-terminal extension of 94 amino acid residues, designated as the C-terminal domain of HlyII (HlyIICTD), which plays an important role in the functioning of the toxin. Our previous work described a monoclonal antibody (HlyIIC-20), capable of strain-specific inhibi-tion of hemolysis caused by HlyII, and demonstrated the dependence of the efficiency of hemol-ysis on the presence of proline at position 324 in HlyII outside the conformational antigenic de-terminant. In this work, we studied 16 mutant forms of HlyIICTD. Each of the mutations, ob-tained by multiple site-directed mutagenesis leading to the replacement of amino acid residues lying on the surface of the 3D structure of HlyIICTD, led to a decrease in the interaction of HlyIIC-20 with the mutant form of the protein. Changes in epitope structure confirm the high conformational mobility of HlyIICTD required for the functioning of HlyII. Comparison of the effect of the introduced mutations on the effectiveness of interaction between HlyIICTD and HlyIIC-20 and a control antibody recognizing a non-overlapping epitope enabled identifying the amino acid residues N339, K340 included in the conformational antigenic determinant rec-ognized by HlyIIC-20.

The COVID-19 pandemic continues to affect individuals across the globe, with some individuals experiencing more severe disease than others. The relatively high frequency of re-infections and breakthrough infections observed with SARS-CoV-2 highlights the importance of extending our understanding of immunity to COVID-19. Here we aim to shed light on the importance of antibody titers and epitope utilization in protection from re-infection. Health care workers are highly exposed to COVID-19 and are therefore also more likely to become re-infected. We utilized quantitative, multi-antigen, multi-epitope SARS-CoV-2 protein microarrays to measure IgG and IgA titers against various domains of the nucleocapsid and spike proteins. Potential re-infections in a large, diverse health care worker cohort (N=300) during the second wave of the pandemic were identified by assessing the IgG anti-N titers before and after the second wave. We assessed epitope coverage and antibody titers between the 'single infection’ and ‘re-infection’ groups. Clear differences were observed in the breadth of the anti-N response before the second wave, with the epitope coverage for both IgG (p=0.019) and IgA (p=0.015) being significantly increased in those who did not become re-infected compared to those who did. Additionally, the IgG anti-N (p=0.004) and anti-S titers (p=0.018) were significantly higher in those not re-infected. These results highlight the importance of the breadth of elicited antibody epitope coverage following natural infection in protection from re-infection and disease in the COVID-19 pandemic.

The continuing mutability of the SARS-CoV-2 virus can result in failures of diagnostic assays. To address this, we describe a generalizable bioinformatics-to-biology pipeline developed for calibration and quality assurance of inactivated SARS-COV-2 variant panels provided to Radical Acceleration of Diagnostics programs (RADx)-radical program awardees. Heuristic genetic analysis based on variant-defining mutations demonstrated the lowest genetic variance in the Nucleocapsid protein (Np)- C-terminal domain (CTD) across all SARS-COV-2 variants. We then employed the Shannon entropy method on (Np) sequences collected from the major variants, verifying the CTD with lower entropy (less prone to mutations) than other Np regions. Polyclonal and monoclonal antibodies were raised against this target CTD antigen and used to develop an Enzyme-linked immunoassay (ELISA) test for SARS-CoV-2. Blinded Viral Quality Assurance (VQA) panels comprising of UV-inactivated SARS CoV-2 variants (XBB.1.5, BF.7, BA.1, B.1.617.2, and WA1) and distractor respiratory viruses (CoV 229E, CoV OC43, RSV A2, RSV B, IAV H1N1, and IBV) were assembled by the RADx-rad Diagnostics core and tested using the ELISA described here. The assay tested positive for all variants with high sensitivity (Limit of Detection: 1.72-8.78 ng/mL) and negative for the distractor virus panel. Epitope mapping for the monoclonal antibodies identified a twenty amino acid antigenic peptide on the Np-CTD that an in-silico program also predicted for the highest antigenicity. This work provides a template for a bioinformatics pipeline to select genetic regions with a low propensity for mutation (low Shannon entropy) to develop robust ‘pan-variant’ antigen-based assays for viruses prone to high mutational rates.

Pathogenicity of many bacteria including Bacillus cereus and Staphylococcus aureus depends on pore-forming toxins (PFTs), which cause lysis of host cells by forming pores in membranes of eukaryotic cells. Bioinformatic analysis revealed in over 600 PFTs a region homologous to the Lys171-Gly250 sequence in hemolysin II (HlyII) from B. cereus, which we designated as “homologous peptide”. Three β-barrel PFTs were used for a detailed comparative analysis. Two of them –HlyII and cytotoxin K2 (CytK2), – are synthesized in Bacillus cereus sensu lato; the third – S. aureus α-toxin (Hla) – is the most investigated representative of the family. Protein modeling showed certain amino acids of homologous peptide to be located on the surface of the monomeric forms of these β-barrel PFTs. We obtained monoclonal antibodies against both a cloned homologous peptide and a 14-membered synthetic peptide DSFNTFYGNQLFMK as part of homologous peptide. The HlyII, CytK2 and Hla regions recognized by the obtained antibodies, as well as an antibody capable of suppressing the hemolytic activity of CytK2, were identified in the course of the work. Antibodies capable of recognizing PFTs of various origins can be useful tools for both identification and suppression of the cytolytic activity of PFTs.

Determination of the presence in the blood of antibodies specific to the causative agent of a particular disease (serodiagnosis) is an effective approach in medical analytical chemistry. Serodiagnostics performed in the lateral flow immunoassay format (immunochromatography) meet the modern requirements for point-of-care testing and are supported by existing technologies of large-scale diagnostic tests production—thus raising increased attention in a tense epidemiological situation. For traditional lateral flow serodiagnostics formats, a large number of nonspecific immunoglobulins in the sample significantly reduces the degree of detectable binding. To overcome these limitations, an assay based on the formation of immobilized antigen— specific antibody—labeled antigen complexes detection was proposed. However, the requirements for its implementation, providing maximum sensitivity, have not been established. This article describes the mathematical model for the above assay. The influence of the ratio of reagent concentrations on the analysis results is considered. It is noted that the formation of specific antibody complexes with several labeled antigens is the main limiting factor in reducing the detection limit, and methods are proposed to minimize this factor. Recommendations for the choice of the assay conditions, following from the analysis of the model, are confirmed experimentally.

Identifying anti-spike antibodies that exhibit strong neutralizing activity against current dominant circulating variants and antibodies that are escaped by these variants have important implications in the development of therapeutic and diagnostic solutions as well as in improving understanding of the humoral response to SARS-CoV-2 infection. We characterized seven anti-RBD monoclonal antibodies for their binding activity, pairing capability and neutralization activity to SARS-CoV-2 and three variant RBDs (UK, SA and BR P.1) via lateral flow immunoassays. The results allowed us to group these antibodies into three distinct epitope bins. Our studies showed that two antibodies had broadly potent neutralizing activity against SARS-CoV-2 and these variant RBDs and that one antibody did not neutralize the SA and BR P.1 RBDs. The antibody escaped by the SA and BR P.1 RBDs retained binding activity to SA and BR P.1 RBDs but was unable to induce neutralization. Further, we demonstrated that the lateral flow immunoassay can be a rapid and effective tool for antibody characterization, including epitope classification and antibody neutralization kinetics. From these studies, the potential contributions of the mutations (N501Y, E484K and K417N/T) contained in these variants’ RBDs on antibody pairing capability, neutralization activity and therapeutic antibody targeting strategy are discussed.

The trafficking of illegal drugs by criminal networks at borders, harbors, or airports is an increasing issue in public health as these routes ensure the main supply of illegal drugs. The prevention of drug smuggling, including the installation of scanners and other analytical devices to detect ultra-small traces of drugs within a reasonable time frame, remains a challenge. The presented immunosensor is based on a monolithic affinity column with a large excess of immobilized hapten, which traps fluorescently labeled antibodies as long as the analyte cocaine is absent. In the presence of the drug, some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled complementary metal-oxide-semiconductor (CMOS) camera. Liquid handling is performed with high-precision syringe pumps and microfluidic chip-based mixing devices and flow cells. The biosensor achieved limits of detection of 23 pM (7 ppt) of cocaine with a response time of 90 seconds and a total assay time below 3 minutes. With surface wipe sampling, the biosensor was able to detect 300 pg of cocaine. This immunosensor belongs to the most sensitive and fastest detectors for cocaine and offers near-continuous analyte measurement.

The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (K_d), can be considered as one of the most important characteristics for any antibody-antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC₅₀ approaches the K_d value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to K_d. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols.