Monoclonal Antibodies in Immunodiagnosis and Immunotherapy最新文献

CD300a and CD300A, among the CD300 immunoglobulin (Ig)-like receptor family members in mice and humans, respectively, are expressed on myeloid cell lineage. The interaction of CD300a and CD300A with their ligands phosphatidylserine and phosphatidylethanolamine, respectively, exposed on the plasma membrane of dead cells mediate an inhibitory signal in myeloid cells. We previously reported that a neutralizing antimouse CD300a monoclonal antibody (mAb) enhanced efferocytosis by macrophages and ameliorated acute ischemic stroke (AIS) in mice. Unlike mouse CD300a, human CD300A has a single nucleotide polymorphism (SNP, rs2272111) encoding a nonsense mutation of glutamine (CD300A^Q111) instead of arginine (CD300A^R111) at residue 111. In this study, we show that the SNP frequency is 32%-35% for the heterozygous allele and 4%-5% for the homozygous alleles, except Africa. In addition, we developed a humanized antihuman CD300A mAb, named TNAX103, that recognizes both CD300A^R111 and CD300A^Q111. We show that TNAX103 interfered with the binding of CD300A^R111 and CD300A^Q111 to dead cells. In addition, the injection of TNAX103 decreased neurological scores and prolonged survival in humanized mice after middle cerebral artery occlusion. These results suggest that TNAX103 may be potentially useful for the treatment of patients expressing either CD300A^R111 or CD300A^Q111 with AIS.

Nucleophosmin (NPM1) is abundant in the nucleolus, and it shuttles between the nucleolus, nucleus, and cytoplasm to facilitate its roles in ribosome biogenesis, chromatin remodeling, DNA repair, and cell cycle regulation. It is overexpressed in various types of cancer and is related to malignancy. Furthermore, its localization is important for its cellular function and the malignant transformation of cancer cells. In this study, we describe our novel rat monoclonal antibody (mAb) 4H7, which recognizes NPM1. Our results indicated that this mAb recognizes endogenous human NPM1 in several cancer cell lines and is suitable for immunoprecipitation, immunofluorescence staining, and immunoblotting. Therefore, mAb 4H7 is expected to be useful for the functional analysis of human NPM1 in cancer and for the diagnosis of malignant transformation via expression and localization assays.

The human norovirus (HuNov) major capsid VP1comprises an S (shell) and a P (protruding) domain; the latter is responsible for virus attachment and infection. The dimeric formation of P (containing P1 and P2 subdomains) is indispensable for forming a receptor-binding pocket, enabling HuNov to dock to attachment factor histo-blood group antigens (HBGAs) on the host cell. Thus, the P-specific antibody may hamper the engagement of P and HBGA, thereby inhibiting virus infection. In this study, we developed and characterized two HuNov P-specific murine monoclonal antibodies (MAbs), namely, 5C6 and 1H12. They can bind to P protein with high affinity, as evidenced by the results of indirect fluorescent assay, western blot, and Biolayer interferometry assay. Particularly, the MAb 1H12 recognizes the P2 subdomain, whereas the 5C6 targets the distal P1. These MAbs may contribute to the exploration of novel epitopes on HuNov VP1 and to the development of new antivirals.

Congenital hypothyroidism (CH) is a major health issue that can lead to intellectual disability if not detected and treated earlier. The preliminary screening program for neonatal CH in Indonesia gave a provisional incidence of 1:2513. Newborn screening using a dried blood spot sample is the standard method for CH detection, but it has limitations. Despite the proven benefits of CH screening, Indonesia still faces significant challenges in implementing a nationwide program. This study aimed to develop a more sensitive and accessible screening method by creating monoclonal antibodies (mAbs) against the thyroid-stimulating hormone (TSH).TSH protein was isolated from newborn cord blood and confirmed by Western blot analysis. Mice were immunized with purified TSH, and hybridoma cell lines were generated through cell fusion. Hybridoma supernatants were screened for TSH-specific antibodies using ELISA. The mAb with the highest titer was purified by dialysis. Western blot analysis confirmed the presence of TSH in the isolated protein fraction at 28 kDa. Immunized mice showed a significant increase in antibody titer compared with the control group. Hybridoma clones secreting high-titer antibodies against TSH were identified. This research successfully isolated TSH and produced mAbs against it. They enable the development of rapid, point-of-care diagnostic tests, such as lateral flow immunoassays, which can provide results within minutes. It will lay the groundwork for the development of innovative CH screening tools that can significantly improve the early diagnosis and treatment of this condition, particularly in resource-limited settings.

Paracoccidioidomycosis (PCM) is a chronic endemic mycosis in Latin America, predominantly caused by Paracoccidioides brasiliensis (Pb18) and Paracoccidioides lutzii (Pl01). Diagnosing PCM is challenging due to species-specific antigenic differences, therefore new biomarkers for accurate and rapid detection are needed. This study explores multiple tolerization subtractive immunization (MTSI) to generate monoclonal antibodies against rare or weakly expressed epitopes of Pb18 and Pl01, potentially improving PCM diagnosis. These strains were cultured to obtain cell-free antigens (CFA). MTSI involved immunizing BALB/c mice with CFA from Pb18 as a tolerogen and Pl01 as an immunogen, using Freund's adjuvant and cyclophosphamide to induce immune tolerance. The immune response was monitored via Enzyme-linked immunosorbent assay (ELISA) and Western blotting. Hybridomas were generated by fusing splenocytes from immunized mice with myeloma cells, after which clonal selection was conducted based on reactivity to Pl01 antigens. The study explores the presence of various proteins, including gp43 and Hsp60, in the protein profile of CFAs. Additionally, polyclonal antibody reactivity to Pb18 antigens was significantly reduced, suggesting that MTSI effectively promoted immunological tolerance. Followig the screening of hybridomas, clones with good reactivity to Pl01 and less reactive to Pb18 were selected. The monoclonal clones C1 and E6 exhibited potential specificity for Pl01 antigens. The effective generation of P. lutzii-specific antibodies by MTSI demonstrates this technology's promise for the development of accurate PCM diagnostic instruments. These antibodies have the potential to enhance patient outcomes and reduce the incidence of false-negative diagnoses, which could lead to better disease management.

Coagulation factor XIII (FXIII) is an enzyme that strengthens hemostatic clots, and its deficiency can cause life-threatening bleeding. We immunized mice with human plasma-derived FXIII to generate monoclonal antibodies (mAbs) against the B subunit (FXIII-B), which stabilizes the A subunit (FXIII-A) of FXIII, and analyzed their properties. The epitopes of the seven mouse antihuman FXIII-B mAbs obtained were found to be the 3rd, 5th, 6th, 9th, and 10th Sushi domains. One of these mAbs, mAb 5-6C, recognized the 10th Sushi domain and inhibited the fibrin cross-linking reaction without affecting the amine incorporation activity of FXIII. We previously reported that the 10th Sushi domain is the site where FXIII-B binds to fibrin and functions to bring FXIII-A closer to the substrate fibrin. Except for mAb 5-6C, mouse mAbs with high yields were used to measure the amount of FXIII-B antigen by an immunochromatography test (ICT), which showed a high correlation with enzyme-linked immunosorbent assay-obtained results. In addition, we developed a prototype ICT to detect anti-FXIII-B autoantibodies using mAb 1-3C, which showed good results in measuring the amount of FXIII-B antigen. Thus, mouse mAbs may be useful for clinical applications. mAb 5-6C targeting the 10th Sushi domain may also be useful for inhibiting thrombosis progression when humanized as antibody medicines.