A novel approach for rapid high-throughput selection of recombinant functional rat monoclonal antibodies.

BACKGROUND: Most monoclonal antibodies against mouse antigens have been derived from rat spleen-mouse myeloma fusions, which are valuable tools for purposes ranging from general laboratory reagents to therapeutic drugs, and yet selecting and expressing them remains a time-consuming and inefficient process. Here, we report a novel approach for the rapid high-throughput selection and expression of recombinant functional rat monoclonal antibodies with different isotypes. RESULTS: We have developed a robust system for generating rat monoclonal antibodies through several processes involving simultaneously immunizing rats with three different antigens expressing in a mixed cell pools, preparing hybridoma cell pools, in vitro screening and subsequent cloning of the rearranged light and heavy chains into a single expression plasmid using a highly efficient assembly method, which can decrease the time and effort required by multiple immunizations and fusions, traditional clonal selection and expression methods. Using this system, we successfully selected several rat monoclonal antibodies with different IgG isotypes specifically targeting the mouse PD-1, LAG-3 or AFP protein from a single fusion. We applied these recombinant anti-PD-1 monoclonal antibodies (32D6) in immunotherapy for therapeutic purposes that produced the expected results. CONCLUSIONS: This method can be used to facilitate an increased throughput of the entire process from multiplex immunization to acquisition of functional rat monoclonal antibodies and facilitate their expression and feasibility using a single plasmid.

Development of an HSV-1 neutralization test with a glycoprotein D specific antibody for measurement of neutralizing antibody titer in human sera.

BACKGROUND: Investigating the neutralizing antibody (NAb) titer against HSV-1 is essential for monitoring the immune protection against HSV-1 in susceptible populations, which would facilitate the development of vaccines against herpes infection and improvement of HSV-1 based oncolytic virotherapy. RESULTS: In this study, we have developed a neutralization test based on the enzyme-linked immunospot assay (ELISPOT-NT) to determine the neutralizing antibody titer against HSV-1 in human serum samples. This optimized assay employed a monoclonal antibody specifically recognizing glycoprotein D to detect the HSV-1 infected cells. With this test, the neutralizing antibody titer against HSV-1 could be determined within one day by automated interpretation of the counts of cell spots. We observed good correlation in the results obtained from ELISPOT-NT and plaque reduction neutralization test (PRNT) by testing 22 human serum samples representing different titers. Moreover, 269 human serum samples collected from a wide range of age groups were tested, the average neutralizing antibody titer (log2NT50) was 8.3 ± 2.8 and the prevalence of NAbs was 83.6 % in this cohort, it also revealed that the average neutralizing antibody titer in different groups increased with the age, and no significant difference in neutralizing antibody titers was observed between males and females. CONCLUSIONS: These results prove that this novel assay would serve as an accurate and simple assay for the assessment of the neutralizing antibody titers against HSV-1 in large cohorts.

Salt induces expression of RH3.2A, encoding an H3.2-type histone H3 protein in rice (Oryza sativa L.).

Histone H3 is one of the four histones, along with H2A, H2B, and H4, which form the eukaryotic nucleosome octamer core. In this study, a new gene RH3.2A encoding an H3.2-type histone H3 protein from rice (Oryza sativa L.) was reported. RH3.2A was cloned through RT-PCR from salt-treated rice seedlings. This gene encoded a protein of 136 amino acid residues that were similar to some plant histone H3 proteins reported previously. However, the cDNA sequence of RH3.2A and other rice H3 genes were different. Alignment of RH3.2A encoding protein with other plant histone H3 proteins revealed that three amino acid residues (32, 88, and 91) were markedly different between H3.1-type and H3.2-type proteins. The mRNA expression analysis of RH3.2A revealed that RH3.2A gene was upregulated by salt stress in rice roots and ABA treatment in seedlings. The potential role of RH3.2A during salt stress was discussed.

Isolation and characterization of two cDNAs encoding translation initiation factor 1A from rice(Oryza sativa L.).

The eukaryotic initiation factor 1A(eIF1A) is essential for transferring of the initiator Met-tRNA to 40S ribosomal subunits to form the 40S pre-initiation complex. In present study, we describe the cloning and characterization of two eIF1A genes from rice, which were designated as Oryza sativa eukaryotic initiation factor 1A genes OseIF1A-1, OseIF1A-2, respectively. Both rice elF1As shared high identities in amino acids with eIF1A proteins from other eukaryotes. The mRNA expression analysis revealed that OseIF1A-2 mRNA was much more accumulated than OseIF1A-1 in all tissues but each gene is expressed in root, stem, leaf and flowering spike in high and nearly equal level, and in immature spike in lower level. These results, together with their different location in unrooted phylogenetic tree inferred from amino acid sequences of all known eIF1As, suggested that there are two types of eIF1A genes with different function or different regulation in rice.