Hybridoma technology remains one of the most reliable and widely used platforms for generating highly specific monoclonal antibodies for use in diagnostics, fundamental research, and clinical practice. Moreover, the combination of unlimited proliferative capacity with the preservation of a key B lymphocyte function, antibody production, enables hybridoma cells to support a comprehensive functional evaluation of cellular responses. Thus, by integrating analysis of proliferation, viability, and productivity, hybridoma-based approaches enable the detection of differential modulatory effects and offer a nuanced assessment of compound bioactivity. This review provides a comprehensive analysis of the functional parameters of hybridoma cells: viability, proliferation, and productivity, as well as the methods used for their evaluation. The main stages of hybridoma cell generation, advances in hybridoma technology, and current applications of hybridoma cells are also reviewed. A key aspect of this review is the differential modulation of functional parameters of hybridoma cells. Modulation of culture conditions and bioactive compounds can differentially influence growth dynamics and specific antibody yield, often revealing an inverse relationship between proliferation and productivity.

The use of traditional colored labels does not fully address the challenges of low sensitivity in rapid tests and insufficient stability of the reagents employed and necessitates additional procedural steps or complex instrumentation to generate an analytical signal. Owing to their unique properties, carbon black nanoparticles (CBNPs) present a promising solution to these issues. However, the current scientific data regarding the reproducibility, scalability, and preservation of structural and functional stability of CBNP-based colored labels remains insufficient for their widespread adoption in routine laboratory practice. Therefore, we have developed and optimized a reproducible method for the functionalization of oxidized CBNP with recognition elements, aimed at producing highly efficient vertical flow immunoassays (VFIAs). Our results demonstrate that the resulting diagnostic reagents maintain their structural and functional properties for one year when stored at +4 °C or −20 °C. We have demonstrated for the first time the reproducibility of the method for producing diagnostic reagents based on oxidized CBNPs with monoclonal antibodies. The novel VFIA based on oxidized CBNPs for the determination of C-reactive protein demonstrates a limit of detection of 2.6 ng/mL compared to similar tests described in the literature. The developed assay allows for the determination of CRP with a visual detection limit of 4 ng/mL in the required concentration range with acceptable intra-assay/inter-day reproducibility and selectivity.

Graphene oxide (GO) nanoparticles hold biomedical promise due to unique properties, but their immunomodulatory effects on phagocytes require evaluation, particularly regarding size- and coating-dependent interactions. Polyethylene glycol (PEG) coatings reduce cytotoxicity, yet long-term impacts of varied coatings remain critical. This study investigated PEGylated GO nanoparticles (P-GO) of two lateral sizes (≈100-300 nm and ≈1-1.5 μm) with linear or branched PEG coatings on THP-1 monocyte viability, apoptosis, metabolism, and wide-spectrum cytokine production. Only the larger branched PEG-coated GO (25 μg/mL) exhibited cytotoxicity after 72 h. Other variants showed no cytotoxicity but modulated THP-1 activity. Larger linear PEG-coated GO induced apoptosis within 24 h. All particles in a concentration of 25 μg/mL were internalized by/adhered to cells, suppressed ROS production, and altered cytokine profiles: TNF-α, MIP-1β, MIP-1α, and G-CSF increased, while HGF and SCGF-β decreased. Larger branched PEG-coated GO suppressed oxidative phosphorylation and glycolysis after 24 h. While a spectrum of effects of PEGylated graphene oxide on THP-1 cell functions was identified, predominantly observed at a dose of 25 μg/mL over a 24 to 72-h exposure period, no clear dependence of P-GO nanoparticle effects on THP-1 cells was observed with respect to PEG coating type (linear vs. branched) or particle size. At 5 μg/mL, P-GO caused minimal functional modulation. Thus, the study underscores the potential of low-concentration P-GO for therapeutic use while cautioning that even non-cytotoxic nanoparticles can profoundly alter immune cell behavior.

Nanoparticles exhibiting peroxidase-like activity (nanozymes) are emerging as promising alternatives to enzyme labels in colorimetric assays, including ELISA. Unlike natural peroxidases, nanozymes demonstrate distinct mechanisms for oxidizing chromogenic substrates such as 3,3′,5,5′-tetramethylbenzidine (TMB), prompting the use of custom substrate formulations in contemporary research. However, analysis of the literature reveals that such compositions often yield abnormally high background signals due to non-specific oxidation of TMB, likely catalyzed by trace metal ions present in assay buffers. This phenomenon narrows the dynamic range, elevates limits of detection (LOD), and restricts the use of higher substrate concentrations that could otherwise enhance assay sensitivity. In this study, we identify the widespread use of sodium acetate buffer as a principal contributor to non-specific oxidation, owing to its limited chelating capacity. Statistical analysis demonstrates that lower pH, elevated TMB concentration, and, to a lesser extent, increased buffer molarity and hydrogen peroxide concentration are the most significant factors promoting non-specific reactions. Additional variables influencing the background include reagent purity and organic co-solvent content. Employing platinum nanozymes as a model system, we propose two effective strategies to mitigate non-specific oxidation: (i) substitution of acetate buffer with citrate buffer and (ii) supplementation of acetate buffer with chelating agents. These approaches reduce LOD by 3–12-fold while maintaining low background signals even under adverse conditions (high TMB concentration and low pH).

Water-soluble fullerene derivatives such as fullerenol C60(OH)24 are promising candidates for nanomedicine applications, yet their effects on innate immune cells remain poorly characterized. We investigated the interaction of fullerenol with human neutrophils isolated from healthy donors, exposed to concentrations of 0.25-200 μg/mL over 24-72 h. Using multi-parameter flow cytometry, we assessed viability, apoptosis, phagocytic activity, and intracellular reactive oxygen species (ROS) production, complemented by cell-free DPPH radical scavenging assays. Fullerenol was taken up by neutrophils in a concentration- and time-dependent manner. No significant cytotoxicity was observed up to 100 μg/mL, while viability declined at 200 μg/mL. Phagocytosis of opsonized E. coli was preserved at lower concentrations, though a statistically significant negative correlation with fullerenol concentration was detected at higher doses. In cell-free assays, fullerenol scavenged DPPH radicals with an EC50 of 48.90 ± 10.02 μg/mL, exhibiting slower kinetics than Trolox or ascorbic acid. Critically, fullerenol suppressed intracellular ROS production by >33% at 50 μg/mL following PMA stimulation of neutrophils. These findings demonstrate that fullerenol C60(OH)24 combines potent intracellular antioxidant activity with a favorable neutrophil safety profile, supporting its potential application in oxidative stress-related conditions.

Regulatory T cells (Tregs) play a key role in immune tolerance and are promising targets for treating immune-mediated diseases. This study investigated the direct effects of PEGylated graphene oxide nanoparticles (LP-GO, BP-GO at 5–25 μg/mL) and fullerenol C60(OH)24 (25–200 μg/mL) on human Treg viability and differentiation in vitro. Tregs were induced from peripheral blood CD4+ T cells using IL-2, TGF-β, and CD2/CD3/CD28 activation beads for 72 h with nanoparticles. Assessments included viability, apoptosis (Zombie aqua/Annexin V), phenotype (CD45+CD4+CD25+CD127dim/−FOXP3+), nanoparticle sorption (intrinsic fluorescence), and IL-10 production. Neither PEGylated graphene oxide nor fullerenol C60(OH)24 affected T-helper (CD4+) viability (95.35–96.15%) nor early/late apoptosis levels. Despite this, we found a decrease in the percentage of CD4+ cells in cultures exposed to 50–200 μg/mL of fullerenol C60(OH)24. The percentage and absolute number of Treg cells decreased with 100–200 μg/mL of fullerenol, while IL-10 levels declined following treatment with 200 μg/mL of the same nanoparticles. Graphene oxide nanoparticles showed virtually no localization within or on cells. However, T helper and Treg cells demonstrated concentration-dependent sorption of fullerenol C60(OH)24 at concentrations of 100–200 μg/mL without a reduction in viability. These findings demonstrate good in vitro biocompatibility of the nanoparticles at pharmacological concentrations up to 25 μg/mL, alongside the inhibition of Treg differentiation with 100–200 μg/mL of fullerenol C60(OH)24.

Due to their unique physicochemical properties, carbon black nanoparticles represent a promising alternative for solving analytical problems. However, diagnostic reagents based on carbon black nanoparticles have not yet found widespread practical application. This review examines the development and application of carbon black nanoparticle conjugates with recognition molecules as diagnostic reagents in test systems that enable non-instrumental interpretation of results. The review critically evaluates the methods for synthesis and characterization of carbon black-based diagnostic reagents. Furthermore, the review summarizes and discusses existing studies comparing the effectiveness of carbon black nanoparticle-based bioconjugates with traditional colorimetric labels. The scientific articles included in the review were carefully analyzed for the presence of an assessment of the reproducibility of methods for obtaining diagnostic reagents based on carbon black nanoparticles and their long-term storage. The main challenges and future prospects of using carbon black nanoparticles in immunoassays are discussed.

3,3',5,5'-Tetramethylbenzidine (TMB) remains one of the most widely utilized chromogenic substrates for horseradish peroxidase (HRP) in colorimetric immunoassays, including enzyme-linked immunosorbent assays (ELISA). Despite its introduction into ELISA workflows over four decades ago, limited research has been conducted to systematically optimize TMB substrate formulations. Recent advancements in the field have proposed innovative approaches to enhance HRP catalysis, such as the use of deep eutectic solvents and ionic liquids, alongside investigations into the chemical properties of TMB and its analogs to identify more efficient alternatives. However, the development of stable and high-performance TMB solutions for clinical diagnostics requires a comprehensive understanding of how formulation parameters influence signal intensity and stability. In this study, we address these gaps by conducting a systematic evaluation of key factors affecting TMB substrate performance, including buffer pH, composition and molarity, specific ion effects, incorporation of organic solvents, and the use of polymer stabilizers. Additionally, novel strategies for signal amplification, identified through an extensive review of literature and patents, were experimentally tested. Based on these findings, we developed an optimized TMB formulation that was benchmarked against existing formulations reported in the literature.

This study investigated the response of human monocytes to co-culture with pegylated (linear or branched) graphene oxide (GO) nanoparticles, specifically examing both small (P-GOs, 100 -200 nm) and larger (P-GOb, 1-5 μm) particles at concentrations of 5, 25, and 50 µg mL–1. Human monocytes (CD14+ cells) were isolated and cultured with these nanoparticles for 72 hours. We measured cell viability, lactate dehydrogenase (LDH) release, and cytokine production. The findings showed that P-GO nanoparticles had little effect on cytokine production, including MIF, GM-CSF, VEGF, IP-10, IL-8, HGF, and SCGF-beta in vitro. At a low concentration (5 μg mL–1), P-GO exhibited minimal influence on cytokines, except forthe LP-GOb variant, which increased M-CSF production. Conversely, 25 and 50 μg mL–1 of P-GO nanoparticles enhanced the release of variouscytokines, including proinflammatory IL-6, IL-1β, IL-1α, IL-18, IL-17, IL-16, IFN-γ, TNF-β, TNF-α, anti-inflammatory IL-1ra, IL-13, IL-10, IL-4, regulatory G-CSF, IL-2, IL-3, IL-5, IL-12 (p40), IL-12 (p70), M-CSF, GM-CSF and chemokines CTACK, Eotaxin, GRO-α, RANTES, MIP-1β, MCP-1, MIP-1α, MCP-3, MIG, SDF-1α, growth factors Basic FGF, PDGF-BB, SCF, and LIF and TRAIL. Although higher concentrations of P-GO nanoparticles resulted in significant cytokine production, monocyte viability remained largely unaffected . LDH release was elevated solely in samples treated with 50 μg mL–1 of LP-GOb. BP-GOs showed minimal influence on cytokine profiles, raising M-CSF levels at the highest concentration. These results indicate that modifying graphene oxide nanoparticles may hold potential for creating graphene-based pharmacological agents.

Nanozymes, while promising alternatives to natural peroxidases in colorimetric assays, are often hindered by lower catalytic efficiencies. Although numerous approaches have been developed to improve signal intensity in nanozyme-based assays, optimization of the reaction medium in which the nanozyme interacts with the substrate remains a significantly underexplored area. The vast majority of studies rely on standard sodium acetate buffers or commercially sourced reagents optimized for horseradish peroxidase, neglecting the unique catalytic properties of different nanozymes. This work presents a systematic optimization of 3,3',5,5'-tetramethylbenzidine (TMB)-based reaction medium composition for four common nanozymes: iron oxide, LaNiO3, Mn-doped CeO2, and platinum nanoparticles. Our findings reveal that while sodium acetate buffer is suitable for LaNiO3, alternative buffers significantly enhance signal intensity (41-68%) for the other nanozymes. Further optimization of ionic strength, organic cosolvent type and concentration, and TMB/H2O2 concentrations yielded improvements in signal intensity, analytical sensitivity, and assay time. This study also identifies common pitfalls encountered during optimization of reaction conditions and proposes potential solutions. We posit that reaction medium should be a standard optimization step in the development of nanozyme-based assays, and the use of commercially sourced reagents with undisclosed compositions should be avoided.

Fullerenols are polyhydroxylated derivatives of fullerene (C60(OH)n) with antioxidant, antiviral, and antibacterial properties and potential biomedical applications due to their solubility and biocompatibility. However, comprehensive assessment of their cytotoxicity is required, particularly regarding their effects on immune system cells. This study investigated the effects of fullerenol C60(OH)24 (MST-Nano, St. Petersburg, Russia) on the viability, apoptosis, and metabolism of THP-1 human monocytic leukemia cells. Cells were treated with concentrations ranging from 0.25 to 1000 µg/mL and incubated for 24, 48, and 72 h. Viability, apoptosis, and nanoparticle association were assessed by flow cytometry; glycolysis and mitochondrial respiration were measured after 24 h on a Seahorse XFe96 analyzer (Agilent Technologies, Santa Clara, CA, USA). Results showed that the effects of fullerenol depend on concentration and exposure time. At 24 h, 750 µg/mL increased viability, while 1000 µg/mL induced apoptosis. After 48 and 72 h, apoptosis increased at concentrations ≥750 µg/mL, with reduced viability. Nanoparticle association correlated with concentration and inversely correlated with viability but was independent of incubation time. Metabolic analysis revealed decreased glycolysis at 750 µg/mL after 24 h, while mitochondrial respiration was unaffected. Thus, our study demonstrated that fullerenol nanoparticles were safe for the THP-1 monocytic cell line up to 500 µg/mL.

Fullerenols are water-soluble spherical carbon nanoparticles derived from fullerenes. Since the discovery of this class of nanomaterials in the late 20th century, a substantial body of literature has accumulated on their properties and potential applications in many areas including biomedicine. This review analyzes existing data on the size of fullerenol nanoparticles and their internalization by cells. Results of in vitro and in vivo studies assessing toxicity of fullerenol are discussed. A dedicated section explores the potential medical applications of fullerenol, considering its pronounced antioxidant properties, high stability, and ability to exert antiviral and antitumor effects. The review primarily focuses on fullerenol C60(OH)n, but is not strictly limited to it. Despite a significant number of publications, there are no known cases of fullerenol-based drugs successfully advancing to clinical trials. In this review, the authors critically evaluate published studies, aiming to identify aspects that require further investigation in the context of fullerenol's potential biomedical applications.

We report the first analytical application of albumin nanoparticles loaded with luminescent europium complexes for immunoassay development. These nanoparticles, synthesized via a desolvation method, exhibited a uniform spherical morphology with a hydrodynamic diameter of 263 nm and strong, long-lived luminescence at 615 nm (λex = 360 nm). Surface functionalization with streptavidin enabled specific binding to biotinylated proteins. The nanoparticles were applied as labels in a sandwich time-resolved solid-phase immunoassay for human IgG detection in black 96-well plates. Unlike commercial DELFIA assays, the method eliminates the need for signal enhancement steps, as the nanoparticles intrinsically contain high concentrations of europium complexes. Optimization studies revealed that the sharp emission peaks of europium can compromise assay reproducibility; however, employing surface scanning and increasing measurement replicates per well partially mitigated this effect. Time-resolved detection reduced background by two orders of magnitude and increased signal intensity nearly tenfold in IgG-positive samples. The assay demonstrated minimal cross-reactivity with IgA and IgM (~2%) and enabled IgG detection at serum dilutions up to 1:100,000. Comparative analysis showed strong concordance with commercial immunoassays and no concentration-dependent bias. The primary limitation observed was suboptimal intra-assay reproducibility (CV > 20% in four of six tested sera).

Lanthanide complexes with organic ligands exhibit unique photophysical properties, including long-lived emission lifetimes, large Stokes shifts, and sharp emission bands, making them highly attractive for bioanalytical and biomedical applications. Here, we report the first successful use of the desolvation method to synthesize bovine serum albumin (BSA) nanoparticles loaded with europium complexes. This simple approach involved the dropwise addition of an ethanolic solution of europium complex precursors into an aqueous BSA solution. We systematically optimized synthesis parameters to produce nanoparticles with high brightness and low polydispersity. Under optimized conditions, the resulting nanoparticles exhibited a uniform size of 150-160 nm (PDI<0.15) as determined by dynamic light scattering and electron microscopy. Each nanoparticle encapsulated approximately 350 europium complexes, achieving quantum yields of 0.77-1.18 % with excitation/emission maxima at 360/615 nm. The synthesis yield was ∼80 %, and the nanoparticles demonstrated excellent size stability across physiological pH conditions and after three months of storage. However, luminescence intensity decreased over time and under certain buffer conditions. Cytotoxicity studies using Vero cells showed cell viability above 80 % at nanoparticle concentrations up to 1 mg/mL. The key challenges to be addressed are the improvement of synthesis reproducibility and the stability of luminescent properties.

Prussian blue nanoparticles (PBNPs), also called nanozymes, are very attractive as an alternative to horseradish peroxidase in immunoassay development due to their simple and low-cost synthesis, stability and high catalytic activity. Today, there is a method for highly effective PBNP synthesis based on the reduction of an FeCl3/K3[Fe(CN)6] mixture by hydrogen peroxide. However, there is a lack of research showcasing the use of these highly effective PBNPs for specific target detection in clinical settings, as well as a lack of comprehensive comparisons with conventional methods. To address this gap, we prepared diagnostic reagents based on highly effective PBNPs by modifying them using gelatin and attaching anti-C-reactive protein (CRP) monoclonal antibodies through cross-linking with glutaraldehyde. As a result, a solid-phase colorimetric immunoassay in a sandwich format (nanozyme-linked immunosorbent assay [NLISA]) using highly effective PBNPs as a label for CRP detection has been demonstrated for the first time. The assay demonstrated a detection limit of 21.8 pg/mL, along with acceptable selectivity, precision (CV < 25

Objective. To study the influence of glycodelin on T-helpers and Tregs level in the process of forming an immune response to the introduction of allogeneic bone marrow (BM) cells in a dynamic experiment on Wistar rats. Materials and methods. The original experimental model "host versus transplant reaction" on male Wistar rats without preliminary conditioning in recipients was used in the study. Animals were administered recombinant glycodelin against the background of allogeneic intraperitoneal transplantation of BM cells in a dynamic experiment. The level of peripheral T-helpers (CD4+) and Tregs (CD4+CD25+FOXP3+), and the expression of FOXP3 in the spleen and mesenteric lymph nodes were assessed. The material was collected on the 3rd and 21st days of the experiment. Results. Glycodelin was shown to reduce the absolute number of T-helpers in the peripheral blood (on the 3rd and 21st days) and to increase the proportion of Tregs on the 21st day of the experiment against the background of the introduction of allogeneic BM cells. It was found out, that glycodelin reduced the level of Tregs in the white pulp of the spleen on the 3rd day of the experiment, while the number of these cells on the 21st day increased, reducing the number of T-helpers at the same time. At the level of the mesenteric lymph nodes, glycodelin reduced the level of T-helpers on the 21st day of the experiment, simultaneously increasing the number of Tregs. In general, a unidirectional and distributed effect of glycodelin on the immune response at the level of T-helpers was observed, that was a decrease of T-helpers, but an increase of Tregs on the 21st day of the experiment. Conclusion. Thus, glycodelin had an immunomodulatory effect on T-helpers and Tregs formation. The vector of the obtained effects was immunosuppressive in nature and contributed to the suppression of the immune response to allogeneic cells.

Objective. The range of peptide drugs is expanding, but no drug with immunosuppressive activity has yet been found. Considering the fact that the trophoblastic β1-glycoprotein (PSG) is a fetoplacental protein with immunosuppressive activity, short peptide fragments of this protein were studied in the formation of an immune response in a situation of allogeneic cell transplantation. To study the effect of PSG peptides (YECE, YQCE, YVCS, and YACS) on the levels of peripheral and local T-regulatory cells (Treg) during the formation of an immune response to the introduction of allogeneic bone marrow cells (BM) in a dynamic experiment on Wistar rats. Materials and methods. We used an original host-versus-graft model in male Wistar rats without preconditioning of recipients. Animals were injected with PSG peptide fragment composition against a background of allogeneic intraperitoneal transplantation of BM cells in a dynamic experiment, in which the following parameters were evaluated: the level of peripheral "true" Tregs (CD4+CD25+FOXP3+), CD4+CD25-FOXP3+ cells, and FOXP3 expression in mesenteric lymph nodes. Material was collected on days 3 and 21 of the experiment. Results. PSG peptide administration against a background of allogeneic BM cells was found to reduce the absolute and relative amount of Treg in the peripheral blood of rats on days 3 and 21 of the experiment. PSG peptides against the background of the introduction of allogeneic BM cells reduced the absolute and relative amounts of CD4+CD25-FOXP3+ cells on the day 3 of the experiment. The introduction of PSG peptides against the background of the introduction of BM cells resulted in a relative decrease in FOXP3 expression in the T zone of mesenteric lymph nodes on the day 21 of the experiment. Conclusions. Thus, the PSG peptides did not have the expected effect on the level of peripheral and local Treg cells; moreover, the presence of the peptides led to a decrease in the number of these cells.

Amniotic variant of glycodelin (GdA) has pronounced immunomodulatory properties, participating in the formation of immune tolerance during pregnancy. We investigated the effect of glycodelin on the level of T regulatory lymphocytes (Treg) and the level of acute phase proteins (α-2-macroglobulin (α-2M), orosomucoid, C-reactive protein (CRP)) upon administration of allogeneic red bone marrow (BM) cells to Wistar rats in a dynamic experiment in vivo. It was found that the introduction of GdA in animal whith allogeneic BM led to an increase in the proportion of peripheral Treg among CD4+ lymphocytes at the end of the experiment (on the 21st day) in comparison with the group that was injected with BM. It was shown that glycodelin reduced the level of CRP and α-2M, but increased the level of orosomucoid in the serum of experimental animals at the beginning of the experiment (day 3), however, by the end of the experiment (day 21), normalization of protein values was observed in all groups of experimental animals acute phase to the level of intact animals. Thus, glycodelin is able to realize an immunosuppressive effect on allogeneic cells through an increase in the level of Treg and orosomucoid, as well as a decrease in the concentration of CRP and α-2M.

Pregnancy-specific β1-glycoprotein (PSG), one of the most important proteins of pregnancy, has a pronounced immunosuppressive effect. Short peptides of PSG, the so-called SLiMs (short linear motifs), are promising molecules for mild immunosuppression. We studied in vitro effect of short PSG peptides (YACS, YQCE, YVCS, and YECE) on differentiation and cytokine profile of human T-regulatory lymphocytes (Treg). T helpers isolated from the peripheral blood and polarized into the Treg phenotype with a T-cell activator (anti-CD2/3/28) and the cytokines IL-2 and transforming grown factor β (TGFβ) were used. PSG peptides were shown to have no direct modulatory effect on Treg differentiation in a culture of CD4+ cells polarized to the Treg phenotype. At the same time, PSG peptides had no effect on the viability and number of CD4+ cells in the in vitro culture. PSG peptides also had no effect on the levels of TNFα, IL-8, IL-2, macrophage inflammatory protein 1β, IL-17, IL-10, IL-6, granulocyte-macrophage CSF, monocyte chemoattractant protein 1, IL-13, IL-5, IL-7, IL-12(p70), IL-1β, granulocyte CSF, IL-4, but decreased IFNγ levels. The observed ability of the YQCE peptide to reduce the production of this proinflammatory Th1 cytokine by T helper cells can be interpreted as a positive effect. Our findings can be used for further development of safe peptide drugs based on SLiMs sequences.

LaNiO3 perovskite nanoparticles, especially nanospheres (LNNS), show great promise in biomedical assays due to their peroxidase-like catalytic properties. However, LNNS-based diagnostic reagents have not been tested in nanozyme enzyme-linked immunosorbent assay (NLISA) or other enzyme-linked immunosorbent assays, and there is limited data on their synthesis. To fill this gap, it is necessary to develop a method for creating LNNS conjugates with monoclonal antibodies and to investigate the reproducibility, scalability, and applicability of these diagnostic reagents in NLISA. We have successfully developed a method for producing novel diagnostic reagents utilizing LaNiO3 nanospheres. Our research demonstrates the application of these nanospheres in a NLISA specifically designed for the detection of C-reactive protein (CRP) in real serum samples. This method is both reproducible and scalable, allowing for the efficient production of nanospheres that are functionalized with monoclonal antibodies targeting CRP, with a mean diameter of approximately 270 nm. Based on the promising results obtained from our experiments, we have developed and optimized a sandwich-format NLISA for CRP detection. This assay achieved a lower limit of detection at 0.178 mu g L-1, with a dynamic range from 12.5 to 0.195 mu g L-1 and a linear detection range extending from 0.195 to 6.25 mu g L-1, showcasing its potential for clinical applications. The new NLISA method, utilizing LaNiO3 nanospheres in a sandwich format for the detection of CRP, significantly enhances sensitivity compared to similar use horseradish peroxidase-based ELISA. In this study for the first time, the functionalization of lanthanum nickelate nanospheres with recognition elements has been demonstrated. This advancement also sheds light on the technological challenges involved in synthesizing diagnostic reagents, identifying areas that need further exploration.