Development of an indirect competitive enzyme-linked immunosorbent assay for formononetin and its application in a cell-based assay using MC3T3-E1 cells.

Formononetin (FMN) is a methoxy isoflavone found abundantly in leguminous plants and associated foods. Several analytical methods have been developed to detect FMN. However, they are costly, complicated, and time-consuming. This study describes an indirect competitive enzyme-linked immunosorbent assay (icELISA) to determine FMN content in food samples using a monoclonal antibody (mAb) against FMN produced by a newly established hybridoma cell line. Validation studies were conducted, and this assay was found to be sufficiently reliable, with an analytical measurement range of 19.53-1250 ng/mL and a detection limit of 17.42 ng/mL. Furthermore, icELISA was successfully applied for a cell-based assay in which the amount of FMN and ononin uptake was quantified in MC3T3-E1 cells. Hence, icELISA is a simple and reliable method for the detection and quantification of FMN, as well as elucidation of its functions and underlying mechanisms of action.

Development of a Highly Sensitive and Specific Monoclonal Antibody Based on Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Determination of Zearalenone in Food and Feed Samples.

Zearalenone (ZEN) contamination in food and feed is prevalent and has severe effects on humans and animals post-consumption. Therefore, a sensitive, specific, rapid, and reliable method for detecting a single residue of ZEN is necessary. This study aimed to establish a highly sensitive and specific ZEN monoclonal antibody (mAb) and an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the detection of ZEN residues in food and feed. The immunogen ZEN-BSA was synthesized via the amino glutaraldehyde (AGA) and amino diazotization (AD) methods and identified using 1H nuclear magnetic resonance (1H NMR), a high-resolution mass spectrometer (HRMS), and an ultraviolet spectrometer (UV). The coating antigens ZEN-OVA were synthesized via the oxime active ester (OAE), formaldehyde (FA), 1,4-butanediol diglycidyl ether (BDE), AGA, and AD methods. These methods were used to screen the best antibody/antigen combination of a heterologous icELISA. Balb/c mice were immunized with a low ZEN-BSA dose at long intervals and multiple sites. Suitable cell fusion mice and positive hybridoma cell lines were screened using a homologous indirect non-competitive ELISA (inELISA) and an icELISA. The ZEN mAbs were prepared by inducing ascites in vivo. The immunological characteristics of ZEN mAbs were then assessed. The standard curves of the icELISA for ZEN were constructed under optimal experimental conditions, and the performance of the icELISA was validated. The two ZEN-BSA immunogens (conjugation ratios, 11.6:1 (AGA) and 9.2:1 (AD)) were successfully synthesized. Four hybridoma cell lines (2B6, 4D9, 1A10, and 4G8) were filtered, of which 2B6 had the best sensitivity and specificity. The mAb 2B6-based icELISA was then developed. The limit of detection (LOD), the 50% inhibitive concentration (IC50), and the linear working range (IC20 to IC80) values of the icELISA were 0.76 µg/L, 8.69 µg/L, and 0.92-82.24 µg/L, respectively. The cross-reactivity (CR) of the icELISA with the other five analogs of ZEN was below 5%. Three samples were spiked with different concentrations of ZEN and detected using the icELISA. The average intra-assay recoveries, inter-assay recoveries, intra-assay coefficients of variations (CVs), and inter-assay CVs were 93.48-99.48%, 94.18-96.13%, 12.55-12.98%, and 12.53-13.58%, respectively. The icELISA was used to detect ZEN in various samples. The results were confirmed using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) (correlation coefficient, 0.984). The proposed icELISA was highly sensitive, specific, rapid, and reliable for the detection of ZEN in food and feed samples.

Preparation and characterization of a high-affinity monoclonal antibody against nerve growth factor.

Nerve growth factor (NGF) is produced and released in injured tissues or chronic pain tissues caused by other diseases. Studies have shown that monoclonal antibodies targeting NGF have a good efficacy in the treatment of osteoarthritis (OA), low back pain and chronic pain, which may be a promising therapy. In this study, DNA sequences of NGF-his and NGF-hFc were synthesized using eukaryotic expression system and subcloned into pTT5 expression vector. After that, NGF proteins were expressed by transient expression in HEK293E cells. We immunized mice with NGF-hFc protein and fused mouse spleen cells to prepare hybridomas. NGF-His protein was used to screen out the hybridoma supernatant that could directly bind to NGF. Antibodies were purified from hybridioma supernatant. Futhermore, via surface plasmon resonance (SPR) screening, six anti-NGF mAbs were screened to block the binding of NGF and TrkA receptor in the treatment of chronic pain. Among them, 58F10G10H showed high affinity (KD = 1.03 × 10-9 M) and even better than that of positive control antibody Tanezumab (KD = 1.53 × 10-9 M). Moreover, the specific reactivity of 58F10G10H was demonstrated by TF-1 cell proliferation activity experiments, competitive binding Enzyme-linked immunosorbent assay (ELISA) and the arthritis animal models in mice, respectively. In conclusion, in this study, a method for the preparation of high-yield NGF-HFC and NGF-His proteins was designed, and a high-affinity monoclonal antibody against NGF with potential for basic research and clinical application was prepared.

Fast-dissociating but highly specific antibodies are novel tools in biology, especially useful for multiplex super-resolution microscopy.

Fast-dissociating, highly specific monoclonal antibodies (FDSAs) are single-molecule imaging probes useful for many biological assays including consecutive, multiplexable super-resolution microscopy. We developed a screening assay to characterize the kinetics of antibody-antigen interactions using single-molecule microscopy and established a pipeline to identify FDSAs from thousands of monoclonal candidates. Provided here are detailed protocols to prepare multi-well glass-bottom plates necessary for our assay to identify hybridoma clones secreting FDSAs. Synthesis of fluorescently labeled Fab fragments (Fab probes) from FDSAs is also described. For complete details on the use and execution of this protocol, please refer to Miyoshi et al. (2021).

Development of a novel NS1 competitive enzyme-linked immunosorbent assay for the early detection of Zika virus infection.

Zika virus (ZIKV) is a flavivirus that has emerged as a global health threat after the 2015 outbreak in the Americas, where devastating congenital defects were documented. There are currently no vaccines to prevent ZIKV infections nor commercially available clinical diagnostic tests demonstrated to identify ZIKV without cross-reactive interference of related flaviviruses. Early diagnosis is critical when treating symptomatic patients and in preventing ZIKV transmission. In this context, the development of sensitive and accurate diagnostic methods are urgently needed for the detection of ZIKV acute infection. The aim of this study consisted of obtaining monoclonal antibodies (mAbs) against denatured monomeric ZIKV Nonstructural protein 1 (ZNS1), a useful diagnostic marker for flavivirus early detection, in order to develop a highly specific and sensitive ZNS1 indirect competitive ELISA (icELISA). The production of hybridomas secreting ZNS1 mAbs was carried out through immunizations with denatured monomeric ZNS1. We selected 1F5 and 6E2 hybridoma clones, which recognized the heat-denatured ZNS1 hexameric form by indirect ELISA. Cross-reaction studies indicated that these mAbs specifically bind to a ZNS1 linear epitope, and that they do not cross-react with the NS1 protein from other related flaviviruses. The 1F5 mAb enabled the development of a sensitive and reproducible icELISA to detect and quantify small amounts of ZNS1 disease marker in heat-denatured human sera. Here, we establish a reliable 1F5 based-icELISA that constitutes a promising diagnostic tool for control strategies and the prevention of ZIKV propagation.

Optimization of therapeutic antibodies by predicting antigen specificity from antibody sequence via deep learning.

The optimization of therapeutic antibodies is time-intensive and resource-demanding, largely because of the low-throughput screening of full-length antibodies (approximately 1 × 103 variants) expressed in mammalian cells, which typically results in few optimized leads. Here we show that optimized antibody variants can be identified by predicting antigen specificity via deep learning from a massively diverse space of antibody sequences. To produce data for training deep neural networks, we deep-sequenced libraries of the therapeutic antibody trastuzumab (about 1 × 104 variants), expressed in a mammalian cell line through site-directed mutagenesis via CRISPR-Cas9-mediated homology-directed repair, and screened the libraries for specificity to human epidermal growth factor receptor 2 (HER2). We then used the trained neural networks to screen a computational library of approximately 1 × 108 trastuzumab variants and predict the HER2-specific subset (approximately 1 × 106 variants), which can then be filtered for viscosity, clearance, solubility and immunogenicity to generate thousands of highly optimized lead candidates. Recombinant expression and experimental testing of 30 randomly selected variants from the unfiltered library showed that all 30 retained specificity for HER2. Deep learning may facilitate antibody engineering and optimization.

Generation and characterization of a high-affinity chimeric anti-OX40 antibody with potent antitumor activity.

OX40 is a costimulatory molecule that belongs to the tumor necrosis factor receptor (TNFR) superfamily. OX40 agonist-based combinations are emerging as promising candidates for novel cancer immunotherapy. Clinical trials have shown that OX40 agonist antibodies could lead to better results in cancer patients. Using a hybridoma platform and three different types of immunization strategies, namely recombinant protein, DNA, and overexpressing cells, we identified a chimeric anti-OX40 antibody (mAb035-hIgG1 from DNA immunization) that shows excellent binding specificity, and slightly stronger activation of human memory CD4+ T cells and similar potent antitumor activity compared with BMS 986178, an anti-OX40 antibody currently being evaluated for the treatment of solid tumors. This paper further systematically investigates the antigen-specific immune response, the number of binders, epitope bins, and functional activities of antibodies among different immunization strategies. Interestingly, we found that different immunization strategies affect the biological activity of monoclonal antibodies.

Dual Site-Specific Chemoenzymatic Antibody Fragment Conjugation Using CRISPR-Based Hybridoma Engineering.

Functionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theranostics, and antibody-drug conjugates (ADC). In addition, therapeutic and theranostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labeling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogeneous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab' fragments bearing two distinct site-specific labeling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab' molecule (DTFab'), which can be easily isolated. To demonstrate feasibility, we functionalized the DTFab' with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theranostics, and next-generation ADCs.

Nanovesicles released by OKT3 hybridoma express fully active antibodies.

Recent findings have shown that nanovesicles preparations from either primary immune cells culture supernatants or plasma contain immunoglobulins, suggesting that a natural way of antibody production may be through exosome release. To verify this hypothesis, we used the OKT3 hybridoma clone, which produces a murine IgG2a monoclonal antibody used to reduce rejection in patients undergoing organ transplantation. We showed exosome-associated immunoglobulins in hybridoma supernatants, by Western blot, nanoscale flow cytometry and immunocapture-based ELISA. The OKT3-exo was also being able to trigger cytokines production in both CD4 and CD8 T cells. These results show that nanovesicles contain immunoglobulin and could be used for immunotherapy. These data could lead to a new approach to improve the effectiveness of therapeutic antibodies by exploiting their natural property to be expressed on nanovesicle membrane, that probably render them more stable and as a consequence more capable to interact with their specific ligand in the best way.

Purification of murine immunoglobulin E (IgE) by thiophilic interaction chromatography (TIC).

In addition to their known implication in allergy studies, IgE antibodies are becoming an increasingly interesting antibody class in cancer research. However, large-scale purification of IgE antibodies still poses substantial challenges, as they cannot be purified using techniques commonly used for other immunoglobulins such as protein A or protein G chromatography. Here, we have developed and optimised a gentle and simple IgE purification method based on thiophilic interaction chromatography (TIC). IgE binds to the thiophilic resin in presence of 1.2 M ammonium sulfate and is eluted in low salt concentration. Monomericity of purified antibodies ranged between 54 and 73%. Preparative size-exclusion chromatography was thereafter performed to further improve the purity, which reached >95% in the final product. The overall recovery was around 30%. The purification method was tested on both hybridoma-produced and recombinantly produced IgE antibodies with reproducible results. In addition, the antigen binding activity of purified IgE antibodies was preserved, as shown by binding ELISA. Purification by TIC is cheap, gentle in terms of pH to preserve IgE folding and function, and universal as any IgE antibody can be purified irrespective of the species of origin or affinity. Potentially, it could be used for purification of other antibody isotypes as well, when gentle conditions are required.

Prokaryotic Expression of the Immunoglobulin's Domains of CRTAM to Characterize a Monoclonal Antibody.

Class-I restricted T cell-associated molecule (CRTAM) is a member of the immunoglobulin superfamily, and it is closely related to nectin-like protein. CRTAM is expressed in activated CD8 T cells, NKT cells, NK cells and in a subpopulation CD4 T cells. In this study, we produce as recombinant proteins, the Ig-domains of CRTAM (IgV-IgC), the IgV, and the IgC. These proteins were successfully purified in the soluble fraction only if the stalk region was included. The recombinant CRTAM recognizes its ligand nectin-like 2 in a cell-free system. We also demonstrate that the IgC domain of CRTAM is recognized by the anti-hCRTAM monoclonal antibody C8 with a 0.62 nM affinity. In conclusion, the stalk region of CRTAM provides solubility for the expression of its Ig-domains as recombinant proteins.

Recognition of synthetic polyanionic ligands underlies "spontaneous" reactivity of Vγ1 γδTCRs.

Although γδTCRs were discovered more than 30 yr ago, principles of antigen recognition by these receptors remain unclear and the nature of these antigens is largely elusive. Numerous studies reported that T cell hybridomas expressing several Vγ1-containing TCRs, including the Vγ1Vδ6 TCR of γδNKT cells, spontaneously secrete cytokines. This property was interpreted as recognition of a self-ligand expressed on the hybridoma cells themselves. Here, we revisited this finding using a recently developed reporter system and live single cell imaging. We confirmed strong spontaneous signaling by Vγ1Vδ6 and related TCRs, but not by TCRs from several other γδ or innate-like αß T cells, and demonstrated that both γ and δ chains contributed to this reactivity. Unexpectedly, live single cell imaging showed that activation of this signaling did not require any interaction between cells. Further investigation revealed that the signaling is instead activated by interaction with negatively charged surfaces abundantly present under regular cell culture conditions and was abrogated when noncharged cell culture vessels were used. This mode of TCR signaling activation was not restricted to the reporter cell lines, as interaction with negatively charged surfaces also triggered TCR signaling in ex vivo Vγ1 γδ T cells. Taken together, these results explain long-standing observations on the spontaneous reactivity of Vγ1Vδ6 TCR and demonstrate an unexpected antigen presentation-independent mode of TCR activation by a spectrum of chemically unrelated polyanionic ligands.

Identification of multiple potent neutralizing and non-neutralizing antibodies against Epstein-Barr virus gp350 protein with potential for clinical application and as reagents for mapping immunodominant epitopes.

Prevention of Epstein-Barr virus (EBV) infection has focused on generating neutralizing antibodies (nAbs) targeting the major envelope glycoprotein gp350/220 (gp350). In this study, we generated 23 hybridomas producing gp350-specific antibodies. We compared the candidate gp350-specific antibodies to the well-characterized nAb 72A1 by: (1) testing their ability to detect gp350 using enzyme-linked immunosorbent assay, flow cytometry, and immunoblot; (2) sequencing their heavy and light chain complementarity-determining regions (CDRs); (3) measuring the ability of each monoclonal antibody (mAb) to neutralize EBV infection in vitro; and (4) mapping the gp350 amino acids bound by the mAbs using competitive cell and linear peptide binding assays. We performed sequence analysis to identify 15 mAbs with CDR regions unique from those of murine 72A1 (m72A1). We observed antigen binding competition between biotinylated m72A1, serially diluted unlabeled gp350 nAbs (HB1, HB5, HB11, HB20), and our recently humanized 72A1, but not gp350 non-nAb (HB17) or anti-KSHV gH/gL antibody.

ReactELISA method for quantifying methylglyoxal levels in plasma and cell cultures.

Methylglyoxal (MG) is a toxic glycolytic by-product associated with increased levels of inflammation and oxidative stress and has been linked to ageing-related diseases, such as diabetes and Alzheimer's disease. As MG is a highly reactive dicarbonyl compound, forming both reversible and irreversible adducts with a range of endogenous nucleophiles, measuring endogenous levels of MG are quite troublesome. Furthermore, as MG is a small metabolite it is not very immunogenic, excluding conventional ELISA for detection purposes, thus only more instrumentally demanding LC-MS/MS-based methods have demonstrated convincing quantitative data. In the present work we develop a novel bifunctional MG capture probe as well as a high specificity monoclonal antibody to finally setup a robust reaction-based ELISA (ReactELISA) method for detecting the highly reactive and low-level (nM) metabolite MG in human biological specimens. The assay is tested and validated against the current golden standard LC-MS/MS method in human blood plasma and cell-culture media. Furthermore, we demonstrate the assays ability to measure small perturbations of MG levels in growth media caused by a small molecule drug buthionine sulfoximine (BSO) of current clinical relevance. Finally, the assay is converted into a homogenous (no-wash) AlphaLISA version (ReactAlphaLISA), which offers the potential for operationally simple screening of further small molecules capable of perturbing cellular MG. Such compounds could be of relevance as probes to gain insight into MG metabolism as well as drug-leads to alleviate ageing-related diseases.

Production and characterization of a monoclonal antibody for Pefloxacin and mechanism study of antibody recognition.

In this report, an artificial antigen (PFLX-BSA: Pefloxacin connected bovine serum albumin) was successfully prepared. The monoclonal antibody against pefloxacin was produced and characterized using a direct competitive ELISA. The linear range of detection was 0.115-6.564 µg/L. The limit of detection defined as IC15 was 0.170 ± 0.05 µg/L and the IC50 was 0.902 ± 0.03 µg/L. The antibody variable region genes were amplified, assembled, and sequenced. A three-dimensional structural model of the variable region was constructed to study the mechanism of antibody recognition using molecular docking analysis. Three predicted essential amino acids, Thr53, Arg97 of heavy chain and Thr52 of light chain, were mutated to verify the theoretical model. Three mutants lost binding activity significantly against pefloxacin as predicted. These may provide useful insights for studying antigen-antibody interaction mechanisms to improve antibody affinity maturation in vitro.

Development of Monoclonal Antibodies Specifically Recognizing the Nonstructural Protein 12 of Type 2 Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens that has caused tremendous economic losses to the swine industry worldwide. Although extensive research has been focused on PRRSV, little is known about the structure and biological functions of individual nonstructural viral proteins, especially the nonstructural protein 12 (Nsp12). In this study, we generated and identified the monoclonal antibody (mAb) against PRRSV Nsp12. Six strains of hybridoma cells named 2B10, 2B12, 5E1, 5G6, 5E7, and 8B2 secreting anti-Nsp12 mAbs were obtained by the hybridoma technique. All the mAbs were specifically reacted with PRRSV by indirect immunofluorescence assay and four of them (2B12, 5E1, 5G6, and 5E7) were specifically reacted by Western blot. Furthermore, the 5E7 specifically recognized multiple type 2 PRRSV strains, including highly pathogenic and classical PRRSV strains, but not type 1 PRRSV strain. Taken together, the mAbs against Nsp12 provide a valuable tool to specifically recognize type 2 PRRSV as a diagnostic reagent and study the biological function of Nsp12 in the future.

Multiple Tolerization Reduces Antibody Binding Against Tolerogen Cells: Implications for the Production of Monoclonal Antibodies.

We report an immunization technique that can update the production of monoclonal antibodies (mAbs): the multiple tolerization subtractive immunization (MTSI). A total of 10 BALB/C mice were used. Animals in group 1 received one inoculation of RWPE-1 cells (nontumoral), followed by cyclophosphamide, and then received serial inoculations of nonirradiated PC3 cells (tumoral). Animals in group 2 received our MTSI protocol, as follows: one inoculation of RWPE-1 cells, followed by cyclophosphamide (Cy). This whole tolerization step was repeated three other times, with 14-day intervals between the last Cy exposure and the next RWPE-1 cell inoculation. Finally, the animals received the same nonirradiated PC3 cell exposure as group 1. Blood was taken from each animal, and their polyclonal sera individually tested against the nontumoral RWPE-1 cells in flow cytometry. We found out that, after the MTSI was employed, the serum of the immunized animals, in group 2, contained considerably less antibodies that reacted against the tolerogenic cells, compared with the serum of the animals that underwent regular subtractive immunization. We showed that, by repeating the tolerization cycles, the polyclonal antibodies produced by mice have a reduced specificity toward common/immunodominant epitopes present at nontumoral cells, and thus this technique can be readily used by others in studies involving murine mAb protocols.

Generation and characterization of monoclonal antibodies against the N-terminus of alpha-2-antiplasmin.

Around 70% of circulating alpha-2-antiplasmin (α2AP), the main natural plasmin inhibitor, is N-terminally cleaved between residues Pro12 and Asn13 by antiplasmin-cleaving enzyme. This converts native Met-α2AP into the more potent fibrinolysis inhibitor Asn-α2AP. The Arg6Trp (R6W) polymorphism affects the N-terminal cleavage rate of Met-α2AP in a purified system, with ~8-fold faster conversion of Met(R6)-α2AP than Met(W6)-α2AP. To date, assays to determine N-terminally intact Met-α2AP in plasma have been limited to an ELISA that only measures Met(R6)-α2AP. The aim of this study was to generate and characterize monoclonal antibodies (mAbs) against Met(R6)-α2AP, Met(W6)-α2AP and all α2AP forms (total-α2AP) in order to develop specific Met(R6)-α2AP and Met(W6)-α2AP ELISAs. Recombinant Met(R6)-α2AP, Met(W6)-α2AP and Asn-α2AP were expressed in Drosophila S2 cells. Using hybridoma technology, a panel of 25 mAbs was generated against a mixture of recombinant Met(R6)-α2AP and Met(W6)-α2AP. All mAbs were evaluated for their specific reactivity using the three recombinant α2APs in one-site non-competitive ELISAs. Three mAbs were selected to develop sandwich-type ELISAs. MA-AP37E2 and MA-AP34C4 were selected for their specific reactivity against Met(R6)-α2AP and Met(W6)-α2AP, respectively, and used for coating. MA-AP15D7 was selected for its reactivity against total-α2AP and used for detection. With the novel ELISAs we determined Met(R6)-α2AP and Met(W6)-α2AP levels in plasma samples and we showed that Met(R6)-α2AP was converted faster into Asn-α2AP than Met(W6)-α2AP in a plasma milieu. In conclusion, we developed two specific ELISAs for Met(R6)-α2AP and Met(W6)-α2AP, respectively, in plasma. This will enable us to determine N-terminal heterogeneity of α2AP in plasma samples.

TX99 Is a Neutralizing Monoclonal Antibody Against Mouse TIGIT.

T cell immunoglobulin and ITIM domains (TIGIT) is an inhibitory immunoreceptor expressed on NK cells, effector and memory T cells, and regulatory T cells (Tregs). The ligands for TIGIT are CD155 (PVR) and CD112 (PVRL2, nectin-2), which are broadly expressed on hematopoietic cells and nonhematopoietic cells. TIGIT negatively regulates antitumor responses, but promotes autoimmune reaction. Although neutralizing anti-human TIGIT mAbs are under clinical trials for cancers, how the blockade of TIGIT interaction with the ligands shows tumor immunity still remains unclear. Although analyses of mouse tumor model using a neutralizing anti-mouse TIGIT (mTIGIT) mAbs should be useful to address this issue, there are limitations to this type of studies due to unavailability of neutralizing anti-mTIGIT mAbs. In this study, we generated five clones of anti-mTIGIT mAbs, designated TX99, TX100, TX103, TX104, and TX105. We show that TX99 and TX100 showed the strongest binding to TIGIT. We also show that TX99 interfered with the interaction between TIGIT and CD155 and increased NK cell-mediated cytotoxicity against CD155-expressing RMA-S cells. Thus, TX99 is a unique neutralizing mAb that can be used for studies of mTIGIT functions.

Quantification of Histidine-Rich Protein 3 of Plasmodium falciparum.

Malaria is a life-threatening infectious disease and continues to be a major public health crisis in many parts of the tropical world. Plasmodium falciparum is responsible for the majority of mortality and morbidity associated with malaria. During the intraerythrocytic cycle, P. falciparum releases three proteins with high histidine content as follows: histidine-rich protein 1 (HRP1), histidine-rich protein 2 (HRP2), and histidine-rich protein 3 (HRP3). Currently, most of the diagnostic tests of P. falciparum infection target HRP2, and a number of monoclonal antibodies (mAbs) against HRP2 have been developed for use in HRP2 detection and quantification. When parasites have HRP2 deletions, the detection of HRP3 could augment the sensitivity of the detection system. The combination of both HRP2 and HRP3 mAbs in the detection system will enhance the test sensitivity. In the HRP quantitative enzyme-linked immunosorbent assay (ELISA), both HRP2 and HRP3 contribute to the result, but the relative contribution of HRP2 and HRP3 was unable to investigate, because of the nonavailability of HRP3 specific antibody ELISA. Hence an ELISA test system based on HRP3 is also essential for detection and quantification. There is not much documented in the literature on HRP3 antigen and HRP3 specific mAbs and polyclonal antibodies (pAbs). In the present study, recombinant HRP3 was expressed in Escherichia coli and purified with Ni-NTA agarose column. The purified rHRP3 was used for the generation and characterization of monoclonal and pAbs. The purification of monoclonal and pAbs was done using a mixed-mode chromatography sorbent, phenylpropylamine HyperCel™. With the purified antibodies, a sandwich ELISA was developed. The sandwich ELISA method was explored to detect and quantify HRP3 of P. falciparum in the spent medium. The generated mAbs could be potentially used for the detection and quantification of P. falciparum HRP3.