Simple ELISA Research Articles

Detection of biomolecules from nano to atto-gram through plasmonic Elisa via tuning the catalyst

A simple and affordable plasmonic ELISA platform is demonstrated using silver nanocrystals as chromogens, and nanocatalysts in the place of enzyme. Apart from ultralow detection limitup to 10 molecules of human Immunoglobulin G (2.5 × 10−18 g/1.68 × 10−19 M), we exhibit very broad dynamic range covering 11-log which goes up to 100 × 10−9 g/6.72 × 10−9 M. To achieve this broad dynamic range and highly controlled standard deviation, nanocatalysts were tuned in terms of shape, size, metallic ratio, surface modification and other parameters. A comparative study is also done between traditional chromogen to independently developed new silver chromogen, and among different catalysts. Our results indicate plasmonic ELISA with small size nanocatalysts may become a very powerful detection strategy for biomolecules.

Abstract 3746: Identification of cancer specific extracellular vesical biomarkers to aid in diagnosis of cancer

Abstract The limitations of serial tumor biopsies are well known, thus liquid biopsies which allow for repeated plasma-based biomarker analysis are urgently needed. Extracellular vesicles (EVs) represent an extremely stable and emerging proteomic substrate which allows for deep enrichment of biologically relevant signals from plasma, and broad analysis of tumor associated and host response proteins. To identify novel biomarkers, we performed proteomics analysis on EVs purified using size exclusion chromatography and a proprietary buffer system which increases EV and corona protein recovery. Initial DIA mass spectrometry (MS) analysis was performed on EVs from 125 patients (25 each from CRC, breast, NSCLC, ovarian, and normal patients). A subset of 25 normal and 25 ovarian was subjected to a second deeper analysis on a much more powerful DIA platform. Initial analysis identified 204 unique proteins differentially expressed in ovarian cancer patients (>2-fold change; p<0.05). Repeat analysis of the ovarian vs normal cohorts on the Biognosys TrueDiscovery™ platform identified 645 differentially expressed, a significant increase in depth of analysis. These cancer exosome associated proteins represent a broad range of host response signals. Taking ~20 of the top proteins, we extended our analysis using ELISAs to determine if the results by MS were reproducible in a simpler format. The ELISA analysis recapitulated the MS results. This confirms that the TrueDiscovery™ platform can easily be translated into a simple, quick and distributable ELISA platform. Extending these results, a blinded cohort of ovarian and normal EVs was analyzed using a predefined cutoff for a host immune response biomarker. This blinded analysis demonstrated sensitivity of 1.0 and specificity of 0.9, exceptionally high values for a single analyte assay, underscoring the potential value of analyzing the host/immune response proteins in the context of the EV microenvironmentKey biomarkers from cancer patient EVs suggests it is possible to track host immunosuppressive environment via antigen presenting cell and check point antagonist biomarkers. In both cases, highly significant differences are seen comparing EVs from normal patient plasma versus cancer patient plasma. Interestingly, when these biomarkers are evaluated in pre-EV enriched plasma, no significant differences are seen. This underscores the unique nature of the EV microenvironment, and suggests that host immune response and tumor microenvironment may be recapitulated in the EV/host response proteome using our unique methods. These data confirm that the EV microenvironment represents a unique oncological ecosystem with highly specific and sensitive disease biomarkers which are not dysregulated in complete plasma. This novel approach to assessing cancer proteomics has great promise for real time, longitudinal analysis of cancer patient tumor/host immune biology. Citation Format: Todd A. Hembrough, Zachary Opheim, Cheryl Bandoski, Issa Isaac, Martin Mehnert, Alan Ezrin. Identification of cancer specific extracellular vesical biomarkers to aid in diagnosis of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3746.

Prototype Salivary Assay for Quantification of Two Biomarkers for In Vitro Diagnosis of Endometriosis.

Endometriosis, a very common disease in women, is characterized by endometrial structures outside the uterine cavity. The lack of a reliable noninvasive diagnostic test and the often nonspecific symptoms of this pathology are responsible for the delay in definitive diagnosis of this disease. Recently, through a proteomics approach, our research group has identified two potential diagnostic markers for endometriosis in serum (Zn-alpha2-glycoprotein and complement C3 precursor). In this article, we describe the experimental conditions of a simple ELISA for rapid quantification of these two biomarkers in the saliva of patients with endometriosis. Finally, preliminary experiments on a small cohort of patients and controls have confirmed the potential diagnostic value of this assay.

Plasma IgG aggregates as biomarkers for multiple sclerosis

We recently reported that multiple sclerosis (MS) plasma contains IgG aggregates and induces complement-dependent neuronal cytotoxicity (Zhou et al., 2023). Using ELISA, we report herein that plasma IgG levels in the aggregates can be used as biomarkers for MS. We enriched the IgG aggregates from samples of two cohorts (190 MS and 160 controls) by collecting flow-through after plasma binding to Protein A followed by detection of IgG subclass. We show that there are significantly higher levels of IgG1, IgG3, and total IgG antibodies in MS IgG aggregates, with an AUC >90%; higher levels of IgG1 distinguish secondary progressive MS from relapsing-remitting MS (AUC = 91%). Significantly, we provided the biological rationale for MS plasma IgG biomarkers by demonstrating the strong correlation between IgG antibodies and IgG aggregate-induced neuronal cytotoxicity. These non-invasive, simple IgG-based blood ELISA assays can be adapted into clinical practice for diagnosing MS and SPMS and monitoring treatment responses.

Diagnostic value of hPG80, as a new multi-cancer blood biomarker, in 16 different cancers: Results of the ONCOPRO prospective study.

3033 Background: hPG80 (also called circulating progastrin) has been described as a promising multi-cancer blood biomarker. Indeed, hPG80 is released from cancer cells to blood at the early steps of tumorigenesis due to WNT pathway activation (You et al eBioMedicine 2020). ONCOPRO study was designed to prospectively assess the hPG80 diagnostic & clinical utility in patients with 16 types of cancers. Methods: ONCOPRO study (NCT03787056) was a large prospective case-control study led in Lyon University Hospital (France), designed to assess the diagnostic, and monitoring value of hPG80 blood titers in 420 patients with 16 newly diagnosed cancers. hPG80 was measured using kit DxPG80.Lab (Biodena care) at diagnosis, and then at each cycle/visit until recurrence for the curative cohorts, or for 3 treatment lines for the non-curative cohorts. The diagnostic value of hPG80 (primary endpoint) was assessed by comparing the hPG80 baseline titers measured in cancer patients to those of 330 healthy volunteers recruited in parallel (age & sex ratios matched with the cancer group), with a sample size calculated for precision of ±0.23 for ROC AUC. Results: Out of 499 cancer patients enrolled between December 2018 and June 2022, 419 were assessable in 16 cancer cohorts (NSCLC & SCLC: n=70; Breast: n=50; Renal: n=50; Prostate: n=50; Liver: n=50; Head&Neck: n=49; Hepatocellular carcinoma (HCC): n=50; Colorectal: n=10; Thyroid: n=10; Pancreas: n=14; Ovarian: n=10; Glioblastoma: n=10; Endometrial: n=10; Bladder: n=10; Superficial oesophago-gastric: n=10; Lymphoma: n=10; Gastric: n=10). The median age of cancer patients was 66 [IQR: 58-73], with 60% men. hPG80 concentration was significantly higher in cancer patients than in healthy subjects (median, 3.8 vs 2.0 pM, P < 0.001). It was not impacted by renal/liver, or CRP level, but slightly linked to age. hPG80 titer was found stable on a 24h-hour period. The global diagnostic accuracy in cancer patients / control was ROC AUC, 0.63 [0.59-0.67] in the whole population. The highest diagnostic accuracy was seen in patients with lung cancer (AUC, 0.75 [0.68-0.82]; Sensitivity (Se)/Specificity (Sp) 0.49/0.90 for hPG80 >7.73 pM) and HCC (AUC, 0.75, 95% CI 0.66-083; Se/Sp 0.48/0.90 for hPG80 > 7.73 pM). Conclusions: This large prospective study confirms that cancer patients have significantly higher blood concentration of hPG80 than healthy subjects. The data about the different cohorts will guide the future development of hPG80. Strong diagnostic accuracy was seen for lung cancers and HCC, with specificity > 90%. The screening programs of these cancers could be enriched by this simple blood hPG80 ELISA assay, for complementing imaging limited specificity. Clinical trial information: NCT03787056 .

The role of the DE and EF loop of BKPyV VP1 in the serological cross-reactivity between subtypes

BK virus (BKPyV) is a causative agent of BKPyV-associated nephropathy and graft rejections in kidney transplant patients. It establishes persistent infection in the kidneys, which can lead to reactivation in an immunosuppressed state or transmission to kidney recipients. Complications in the case of donor-derived infections can be caused by differences between the four known BKPyV subtypes, as prior infection with one subtype does not guarantee protection against de novo infection with other subtypes. The recipient and donor pretransplant serotyping is not routinely performed since simple ELISA tests employing antigens derived from the major viral capsid protein 1 (VP1) are hindered by the high cross-reactivity of anti-VP1 antibodies against all subtypes. Identifying subtype-specific epitopes in VP1 could lead to the design of specific antigens and the improvement of serodiagnostics for kidney transplantation. We aimed to study the surface residues responsible for the interactions with the subtype-specific antibodies by focusing on the DE and EF loops of VP1, which have only a small number of distinct amino acid differences between the most common subtypes, BKPyV-I and BKPyV-IV.We designed two mutant virus-like particles (VLPs): we introduced BKPyV-I characteristic amino acid residues (either H139N in the DE loop or D175E and I178V changes in the EF loop) into the base sequence of a BKPyV-IV VP1. This way, we created BKPyV-IV mutant VLPs with the sequence of either the BKPyV-I DE loop or the BKPyV-I EF loop. These mutants were then used as competing antigens in an antigen competition assay with a panel of patient sera, and changes in antibody reactivity were assessed by ELISA.We found that the changes introduced into the BKPyV-IV VP1 EF loop restrict antibody recognition in most samples and that converting the BKPyV-IV DE loop into its BKPyV-I equivalent attracts anti-VP1 BKPyV-I antibodies. Although our results did not lead to the discovery of a subtype-specific epitope on the VP1, they suggested that the arrangement of the EF loop in VP1 might dictate the mode of interaction between virus and anti-VP1 antibodies in general and that the interactions between the antibodies and the viral capsid might be very complex. Consequently, an antigen competition assay as an assay to distinguish between BKPyV serotypes might prove difficult to interpret.

A simple nanobody-based competitive ELISA to detect antibodies against African swine fever virus.

African swine fever virus (ASFV) infection is a big threat to the global pig industry. Because there is no effective vaccine, rapid, low-cost, and simple diagnosis methods are necessary to detect the ASFV infection in pig herds. Nanobodies, with advantages of small molecular weight and easy genetic engineering, have been universally used as reagents for developing diagnostic kits. In this study, the recombinant ASFV-p30 was expressed and served as an antigen to immunize the Bactrian camel. Then, seven nanobodies against ASFV-p30 were screened using phage display technique. Subsequently, the seven nanobodies fused horseradish peroxidase (nanobody-HRP) were secretory expressed and one fusion protein ASFV-p30-Nb75-HRP was selected with the highest sensitivity in blocking ELISA. Using the ASFV-p30-Nb75-HRP fusion protein as a probe, a competitive ELISA (cELISA) was developed for detecting anti-ASFV antibodies in pig sera. The cut-off value of cELISA was determined to be 22.7% by testing 360 negative pig sera. The detection limit of the cELISA for positive pig sera was 1:320, and there was no cross-reaction with anti-other swine virus antibodies. The comparative assay showed that the agreement of the cELISA with a commercial ELISA kit was 100%. More importantly, the developed cELISA showed low cost and easy production as a commercial kit candidate. Collectively, a simple nanobody-based cELISA for detecting antibodies against ASFV is developed and it provides a new method for monitoring ASFV infection in the pig herds.

Immunoaffinity Plastic Blade Spray Mass Spectrometry for Rapid Confirmatory Analysis of Food Contaminants.

The lack of chromatographic separation in ambient and direct mass spectrometry (MS) ionization techniques jeopardizes the overall selectivity of the developed methods. Incorporating a biosensing element at the ionization source could compensate for that inherent lack of selectivity. Thus, a simplified immunoaffinity-direct MS technique was developed, immunoaffinity blade spray (iBS), featuring a conductive polystyrene blade material. In iBS, the generic coating used in conventional coated blade spray is replaced with a layer of highly specific monoclonal antibodies (mAbs), while the stainless steel is replaced with conductive polystyrene to allow for simple ELISA platelike hydrophobic immobilization of mAbs. Because of its high relevance for climate change-induced food safety issues, the mycotoxin deoxynivalenol (DON) was chosen as a model substance. Following a rapid extraction from wheat flour, DON is immuno-captured, and the blade is positioned in front of the MS for direct iBS-MS/MS analysis. The method's applicability was demonstrated by analyzing spiked and incurred wheat flour samples, omitting the need for time-consuming chromatographic separation. Apart from DON, cross-reacting DON conjugates could be successfully analyzed as well. The direct iBS-MS/MS method is generic and adaptable to detecting any analyte in sample extracts, provided that specific mAbs are available.

Development and application of a recombinant Envelope Domain III protein based indirect human IgM ELISA for Kyasanur forest disease virus

Kyasanur forest virus disease (KFD) is a major public health concern in India. Its etiology KFD virus causes haemorrhagic fever with severe sequelae in humans. Due to continuous spatiotemporal expansion of KFD in last decade, the incidences of positive cases have been increasing in both humans and primates. Early diagnosis is of prime importance for disease management and epidemiological containment. In the present study, the highly immunogenic Envelope Domain III (EDIII) antigen was produced using prokaryotic expression system with an yield of 8 mg/L. The protein was purified using affinity chromatography and confirmed for its immuno-reactivity by western blot and UPLCMS/MS analysis. The recombinant EDIII was used as an antigen for the standardization of ELISA to detect anti KFD IgM antibodies in humans. The ROC curve was prepared to set the optimum cut-off OD for the assay. The comparative evaluation of the assay with a reference MAC ELISA revealed 86.96% concordance, 82.22% sensitivity and 91.48% specificity. Inter-rater agreement was performed with kappa index revealing significant agreement between the assays. This is the first study using safe recombinant protein antigen-based detection of anti KFDV antibodies in humans. This simple and scalable ELISA assay will be applicable for large scale screening of samples for combating the emerging threats of KFD in newer territories.

A Novel Nanobody-Horseradish Peroxidase Fusion Based-Competitive ELISA to Rapidly Detect Avian Corona-Virus-Infectious Bronchitis Virus Antibody in Chicken Serum.

Avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is the causative agent of infectious bronchitis (IB) that has brought great threat and economic losses to the global poultry industry. Rapid and accurate diagnostic methods are very necessary for effective disease monitoring. At the present study, we screened a novel nanobody against IBV-N protein for development of a rapid, simple, sensitive, and specific competitive ELISA for IBV antibody detection in order to enable the assessment of inoculation effect and early warning of disease infection. Using the phage display technology and bio-panning, we obtained 7 specific nanobodies fused with horseradish peroxidase (HRP) which were expressed in culture supernatant of HEK293T cells. Out of which, the nanobody of IBV-N-Nb66-vHRP has highly binding with IBV-N protein and was easily blocked by the IBV positive serums, which was finally employed as an immunoprobe for development of the competitive ELISA (cELISA). In the newly developed cELISA, we reduce the use of enzyme-conjugated secondary antibody, and the time of whole operation process is approximately 1 h. Moreover, the IBV positive serums diluted at 1:1000 can still be detected by the developed cELISA, and it has no cross reactivity with others chicken disease serums including Newcastle disease virus, Fowl adenovirus, Avian Influenza Virus, Infectious bursal disease virus and Hepatitis E virus. The cut-off value of the established cELISA was 36%, and the coefficient of variation of intra- and inter-assay were 0.55–1.65% and 2.58–6.03%, respectively. Compared with the commercial ELISA (IDEXX kit), the agreement rate of two methods was defined as 98% and the kappa value was 0.96, indicating the developed cELISA has high consistency with the commercial ELISA. Taken together, the novel cELISA for IBV antibody detection is a simple, rapid, sensitive, and specific immunoassay, which has the potential to rapidly test IBV antibody contributing to the surveillance and control of the disease.

Development of Highly Sensitive Sandwich ELISA for the Early-Phase Diagnosis of Chikungunya Virus Utilizing rE2-E1 Protein.

IntroductionChikungunya is caused by an alpha virus transmitted to humans by an infected mosquito. Infection is generally considered to be self-limiting and non-critical. Chikungunya infection may be diagnosed by severe joint pain with fever, but it is difficult to diagnose because the symptoms of chikungunya are common to many pathogens, including dengue fever. Diagnosis mainly depends on viral culture, reverse transcriptase polymerase chain reaction (RT-PCR), and IgM ELISA. Early and accurate diagnosis of the virus can be achieved by the application of PCR methods, but the high cost and the need for a thermal cycler restrict the use of such methods. On the other hand, antibody-based IgM ELISA is considered to be inexpensive, but antibodies against chikungunya virus (CHIKV) only develop after 4 days of infection, so it has limited application in the earlier diagnosis of viral infection and the management of patients. Because of these challenges, a simple antigen-based sensitive, specific, and rapid detection method is required for the early and accurate clinical diagnosis of chikungunya.MethodsThe amino acid sequence of CHIKV ectodomain E1 and E2 proteins was analyzed using bioinformatics tools to determine the antigenic residues, particularly the B-cell epitopes and their characteristics. Recombinant E2-E1 CHIKV antigen was used for the development of polyclonal antibodies in hamsters and IgG was purified. Serological tests of 96 CHIKV patients were conducted by antigen-capture ELISA using primary antibodies raised against rCHIKV E2-E1 in hamsters and human anti-CHIKV antibodies.ResultsWe observed high specificity and sensitivity, of 100% and 95.8%, respectively, and these values demonstrate the efficiency of the test as a clinical diagnostic tool. There was no cross-reactivity with samples taken from dengue patients.DiscussionOur simple and sensitive sandwich ELISA for the early-phase detection of CHIKV infection may be used to improve the diagnosis of chikungunya.

Detection and Quantification of SARS-CoV-2 Receptor Binding Domain Neutralization by a Sensitive Competitive ELISA Assay.

This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include (1) serum heat treatment to prevent non-specific interactions, (2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, (3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and (4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 h for >30 serum samples; this includes the 7.5 h of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the ‘gold standard’ assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious and time consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R2 = 0.975, n = 25), demonstrating high sensitivity.

Development of a Simple ELISA System Using a Jungle Gym Structure as an Antibody-Immobilization Substrate

Development of a Simple ELISA System Using a Jungle Gym Structure as an Antibody-Immobilization Substrate

A Thrombospondin-1 Release Assay (TRA) Coupled to PF4-Treated Cryopreserved Platelets for the Detection of Pathogenic HIT and VITT Antibodies

Background: Platelet-activating anti-PF4 antibodies that cause thrombocytopenia and thrombosis are implicated in Heparin-induced thrombocytopenia (HIT) and, more recently, in a newly recognized entity, vaccine-induced immune thrombotic thrombocytopenia (VITT). Current frontline PF4-Polyanion ELISAs utilize simple techniques but are highly non-specific. Functional gold-standard assays such as the serotonin-release assay (SRA) and PF4-dependent P-selectin expression assay (PEA) have limited availability due to technical complexity and the requirement for freshly-drawn normal donor platelets which results in significant delays in diagnosis. Thus, there is an unmet need for near-patient, functional assays for HIT/VITT diagnosis.Aim: To develop a highly sensitive and specific functional assay to detect platelet-activating antibodies associated with HIT and VITT using long-term stored platelets. The assay should be technically simple, avoiding current functional HIT testing endpoints such as aggregometry, radioactivity measurement or flow cytometry.Methods: Platelets were subjected to controlled-rate freezing in a trehalose-based buffer. Cryopreserved platelets were then thawed after variable storage periods, incubated with platelet factor 4 (PF4), and sera from HIT or VITT suspected patients. Thrombospondin-1 (TSP1), a protein highly expressed in platelet α-granules, was quantified from activated platelet supernatants in an ELISA-based assay as a measure of platelet activation.Results: In initial studies, p-selectin expression was induced by platelet-activating HIT antibodies using cryopreserved platelets and the TSP1-release assay (TRA) detected HIT antibody-mediated PF4-dependent cryopreserved platelet activation across 20 different platelet lots (data not shown). A representative study using platelets stored for 11 months is shown in Figure 1A. A cohort of thirty-six HIT-suspected patient sera, half with PF4-dependent platelet-activating antibodies (PEA+) and without (PEA-), were used to evaluate the diagnostic accuracy of the TRA (Figure 1B). All 18 PEA+ samples activated platelets strongly with a mean TSP1 concentration of 147 µg/mL measured in the supernatant compared to a mean of 43 µg/mL obtained with non-activating samples. TSP1 release in a subgroup of 7 PEA+/SRA+/ELISA+ and 4 PEA-/SRA-/ELISA+ samples that were matched for ELISA Optical Density was examined (Figure 1C). Results demonstrated that even strong ELISA+ samples that were non-activating (PEA-/SRA-) did not induce platelet activation in the TRA, confirming high specificity of the assay. The TRA was tested for its ability to detect platelet-activating antibodies in four patients who developed VITT after the Janssen vaccine (Ad26.COV2.S). All samples induced high levels of TSP1 release from cryopreserved platelets (Figure 1D).Conclusions: Our results demonstrate that cryopreserved platelets stored for several months are viable and capable of degranulation upon activation. Further, we demonstrate that measurement of TSP-1 released from cryopreserved, PF4-treated platelets is highly accurate for detecting platelet-activating HIT and VITT anti-PF4 antibodies. Specificity and sensitivity were both 100% even with samples that were strongly false-positive in the HIT ELISA. Coupling cryopreserved platelet activation to a simple ELISA endpoint, as demonstrated here, precludes the need for fresh platelets and complex testing techniques. Thus, the TRA has the potential to transform the diagnostic testing paradigm in HIT and VITT by making near-patient in-hospital functional testing available for rapid and accurate diagnosis.Figure Legend:Figure 1. TSP-1 quantification of (A) 3 HIT patient samples (closed circles) relative to 3 normal control samples (open circles) after 11-months of frozen platelet storage. (B) Thirty-six HIT-suspected patient samples: 18 PEA+ samples (closed circles), and 18 PEA- samples (open squares). Data are compared using two-tailed, unpaired Student's t test. **** P<0.0001. (C) Eleven high optical density matched HIT ELISA patient sera: 7 PEA+/SRA+ samples (closed circles) and 4 PEA-/SRA- samples (open squares). (D) TSP-1 quantification of VITT patient sera. NC-normal control. [Display omitted] DisclosuresPruthi: Instrumentation Laboratory: Honoraria; HEMA Biologics: Honoraria; Bayer Healthcare AG: Honoraria; Genentech: Honoraria; CSL Behring: Honoraria; Merck: Honoraria. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Sub Picogram Detection of Interleukin 6 in Complex Biofluids Using Lubricant Infused Surface Technology

Overexpression of interleukin 6 (IL-6) in human blood could indicate the severity of many medical conditions such as inflammatory diseases, infections, and different types of cancer including lung, colorectal, prostate, and breast cancers. More importantly, in patients with COVID-19, the risk of respiratory failure could be examined via measuring the level of IL-6 expression. Early diagnosis of the aforementioned diseases requires precise detection of IL-6 at very low concentrations (≤5 pg mL-1). While different biosensing approaches such as electrochemical methods, chemiluminescence immunoassays, and immunofluorescence assays (IFA) have shown satisfactory limit of detections (LODs) for IL-6, they could only perform well in buffer or processed serum, and their sensitivities are significantly reduced in more complex biofluids such as human whole plasma or blood. Here, we introduce a novel IFA platform capable of detecting IL-6 in human whole plasma with an LOD of 0.5 pg mL-1 which is significantly lower than the reported LODs in buffer and plasma via IFA methods. The new IFA system comprises IL-6 capture antibodies covalently micropatterned on a lubricant infused matrix. The lubricant infused surface coating that is utilized in our system can effectively supress non-specific adsorption of all biomolecules, thereby diminishing the background noise and enhancing the sensitivity of the biosensor in complex biofluids. The micro-sized islands of the IL-6 capture antibodies throughout the lubricated surface provide a large number of sample analytes to be measured for high throughput purposes. Moreover, we have developed a new type of bioink in which the capture antibodies are conjugated with an epoxy-based silane coupling agent allowing for covalently attachment of the capture antibodies to the fluorosilanized surface via microcontact printing technique. In addition, the assay is performed on low-cost poly(methyl methacrylate) (PMMA) substrate using simple sandwich-based ELISA.

CLL-032: Approach Strategies in the Diagnosis and Evolution of Chronic Lymphocytic Leukemia

Context: CLL is a neoplasm of mature B-cells that is typically manifested within the peripheral blood. The risk of progression to overt CLL for low-count MBL, however, is unknown, and, to date, no cases of low-count MBL progressing to CLL have been reported. Objective: It was discovered in the last few years that the production of some percentage of mutant p-53 proteins, with the increased stability in type B lymphocytes, leads to the carcinogenesis process. The aim of this study was to apply the sandwich enzyme-linked immunosorbent assay (sandwich ELISA), as screening method in discovery early stagy of CLL with isoform p-53 protein, resisted in the oncologic conventional treatment. Design: The monoclonal antibody PAb 240 recognizes an epitope that is structurally hidden in the wild-type conformation of p-53 and becomes exposed by denaturing the p-53 protein or the mutant conformations of p-53, where point mutations in the P-53 gene alter the terminal structure of the p-53 protein. Setting: In the context of a heterogeneous malignant disease, such as CLL-B, this simple and inexpensive ELISA method, such as employed in this study, proves useful for identifying patients to be considered as candidates for personalized therapeutic strategies, based on the mutation of the TP- 53 gene and the presence of p-53 isoform protein. Conclusions: The presented research has an impact on the clinical management of patients and requires adequate therapeutic adaptation in a personalized medicine. Personalized treatments will be applied by combining diagnostic tools, immunohistochemistry (IHC), polymerase chain reaction (PCR), single-chain peptide microarray (SSPMa), next-generation sequencing (NGS), knowledge databases and therapeutic drugs.

Novel Surrogate Neutralizing Assay Supports Parvovirus B19 Vaccine Development for Children with Sickle Cell Disease.

Children with sickle cell disease (SCD) suffer life-threatening transient aplastic crisis (TAC) when infected with parvovirus B19. In utero, infection of healthy fetuses may result in anemia, hydrops, and death. Unfortunately, although promising vaccine candidates exist, no product has yet been licensed. One barrier to vaccine development has been the lack of a cost-effective, standardized parvovirus B19 neutralization assay. To fill this void, we evaluated the unique region of VP1 (VP1u), which contains prominent targets of neutralizing antibodies. We discovered an antigenic cross-reactivity between VP1 and VP2 that, at first, thwarted the development of a surrogate neutralization assay. We overcame the cross-reactivity by designing a mutated VP1u (VP1uAT) fragment. A new VP1uAT ELISA yielded results well correlated with neutralization (Spearman’s correlation coefficient = 0.581; p = 0.001), superior to results from a standard clinical diagnostic ELISA or an ELISA with virus-like particles. Virus-specific antibodies from children with TAC, measured by the VP1uAT and neutralization assays, but not other assays, gradually increased from days 0 to 120 post-hospitalization. We propose that this novel and technically simple VP1uAT ELISA might now serve as a surrogate for the neutralization assay to support rapid development of a parvovirus B19 vaccine.

A Reliable Indirect ELISA Protocol for Detection of Human Antibodies Directed to SARS-CoV-2 NP Protein.

A few months ago, the availability of a reliable and cost-effective testing capacity for COVID-19 was a concern for many countries. With the emergence and circulation of new SARS-CoV-2 variants, another layer of challenge can be added for COVID-19 testing at both molecular and serological levels. This is particularly important for the available tests principally designed to target the S gene/protein where multiple mutations have been reported. Herein, the SARS-CoV-2 NP recombinant protein was utilized to develop a simple and reliable COVID-19 NP human IgG ELISA. The optimized protocol was validated against a micro-neutralization (MN) assay, in-house S-based ELISA, and commercial chemiluminescence immunoassay (CLIA). The developed assay provides 100% sensitivity, 98.9% specificity, 98.9% agreement, and high overall accuracy with an area under curve equal to 0.9998 ± 0.0002 with a 95% confidence interval of 0.99 to 1.00. The optical density values of positive samples significantly correlated with their corresponding MN titers. The assay specifically detects IgG antibodies to the SARS-CoV-2 NP protein and does not cross-detect IgG to the viral S protein. Moreover, it does not cross-react with antibodies related to other coronaviruses (e.g., the Middle East respiratory syndrome coronavirus or human coronavirus HKU1). The availability of this reliable COVID-19 NP IgG ELISA protocol is highly valuable for its diagnostic and epidemiological applications.

Deciphering Intracellular Signaling Pathways in Tumoral Pathologies.

The heterogeneity of diseases such as cancer makes it necessary to use high-throughput screening techniques to obtain the maximum number of parameters and characteristics of tumors. These obtained biomarkers can be used for the prediction, prognosis, and treatment or search for new therapeutic targets. In this sense, microarray technology allows exhaustive analysis in a short time and from a great variety of biological samples, becoming a fundamental tool in biomedical research projects. Here, operational process of protein microarrays based on the antibody-antigen interaction is described, emphasizing their application in intracellular signaling pathways in tumoral pathologies. In addition, a final validation using nucleic acid programmable protein array (NAPPA) technology in a simple ELISA assay was included to decipher functional characterization of featured proteins from microarray screening.

Targeting NLRP3 Inflammasome-Induced Therapy Resistance in ALL

Background: Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood cancer and the most frequent cause of death from cancer before 20 years of age. Survival rates for patients with relapsed ALL remain less than 50% due to the emergence of chemoresistance, particularly steroid resistance. Several lines of evidence suggest the importance of therapy-related activation of the NLRP3 inflammasome in ALL treatment resistance. The NLRP3 inflammasome has been shown to convey steroid resistance, through epigenetic mechanisms leading to NLRP3 promoter hypomethylation and upregulation of NLRP3 expression, and through inflammasome-dependent modulation of the expression of the glucocorticoid receptor. Objectives: The primary objective of this study is to define the kinetics of activation of the NLRP3 inflammasome in ALL patients following systemic chemotherapy. A secondary objective is to determine whether inhibition of the NLRP3 inflammasome enhances the ALL response to conventional chemotherapy in established, patient-derived xenograft (PDX) preclinical models of pediatric ALL. Specifically, we will investigate whether a newly developed synthetic triterpenoid, CDDO-2P-Im (which we have shown to inhibit activation of NLRP3), can suppress the in vivo growth of steroid-resistant human ALL PDX lines and augment their response to the steroid dexamethasone (DEX), and other conventional chemotherapy agents. Methods: We obtained 1-3 ml of peripheral blood from newly diagnosed ALL patients (0-40 years of age) before and after induction chemotherapy and from normal controls without significant inflammatory or infectious diseases under the UH Hospital Cleveland Medical Center IRB-approved protocol STUDY20190453. Either the Ella Simple Plex protein assay or ELISA were used to determine the expression levels of inflammasome associated proteins such as IL-1β, IL-18, caspase-1 and ASC. Western blot was used to analyze the inflammasome signaling proteins such as casepase-1, IL-1β, IL-18, ASC, NLRP3, AIM2, and LonP1 in the cell lysates. Cell viability assay with Cell-Titer-Glo was performed with different leukemia cell lines, such CEM-1, CEM-7, NALP-3 to determine effects of CDDO-2P-Im and DEX on the leukemia growth. Flow cytometry with Annex-V combined with 7-AAD staining was used for apoptosis assay to detect the effect of CDDO-2P-Im and chemotherapy reagents such as DEX. Leukemia PDX models were established to evaluate the in vivo effects of DEX combined with CDDO-2P-Im. Results: Currently 22 ALL patients and 13 controls were included, with plasma samples from 7 ALL patients. The post chemotherapy expression levels of IL-18, which is one of the important inflammasome proteins in the plasma, are significantly elevated compared with pre-chemotherapy. The expression of NLRP3 mRNA and that of downstream proteins caspase-1 and IL-18 in ALL culture supernatants are upregulated after leukemia cells are exposed to doxorubicin and dexamethasone at 12 and 18 hours. The newly synthesized triterpenoid analog CDDO-2P-Im, which inhibits the activation of NLRP3 inflammasome, inhibited the in vitro growth of ALL cells in culture, induced apoptosis of both steroid-sensitive and steroid-resistant leukemia cells, enhanced the anti-leukemia effect of dexamethasone in steroid-resistant leukemia cells and significantly prolonged the survival of mice bearing human PDX lines. Conclusions: Our preliminary data indicate that chemotherapy activates the NLRP3 inflammasome and downstream signals in patients undergoing therapy for ALL. Importantly, the novel triterpenoid analog, CDDO-2P-Im blocks NLRP3 activation, suppresses ALL viability and exhibits significant single agent activity in steroid-resistant ALL. Disclosures No relevant conflicts of interest to declare.