Development of Anti-idiotypic Monoclonal Antibody Mimicking SARS-CoV-2 Receptor Binding Domain.

Using the hybridoma technique, we developed a panel of anti-idiotypic monoclonal antibodies (aId-mAb) that mimic The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Receptor-Binding Domain (RBD) molecule against Fragment antigen-binding (Fab) of anti-SARS-CoV-2 (S1, RBD) antibodies. Investigated the in vivo and in vitro effects of these aId-mAbs we developed and examined their antigenic mimicry abilities. Among these 12 antibodies, 6 aId-mAbs (designated FY1B4, FY2A6, H9F3, E6G7, FY7E11, and FY8H3) were selected for further characterization in a series of experiments. First, competitive receptor binding assay results confirmed that six aId-mAbs could specifically bind to the ACE2 receptor in target cells and block the interaction between the RBD molecule and the ACE receptor. Moreover, we examined the immunological activities of these aId-mAbs in female BALB/c and showed that E6G7, H7E11, and H8H3 aId-mAbs induce an antibody response by mimicking RBD and stimulating the immune system. It is considered that these three aId-mAbs will be evaluated as SARS-CoV-2 vaccine candidate molecules in future studies.

Development of an in-house capture ELISA: An attempt to detect CagA antigen in sera of Helicobacter pylori infected patients.

The CagA protein one of the key virulence factors of Helicobacter pylori plays an important role in the pathogenesis of peptic ulcer diseases. Unfortunately the cagA gene status can only be determined by PCR while serology is an alternative approach to detect antigens or antibodies. Our aim is to detect the CagA antigen in sera of infected subjects by the development of an in-house capture ELISA test. Gastric antral biopsies and serum samples were collected from 63 patients. PCR was used to determine the cagA status. Our previously developed recombinant CagA protein and monoclonal antibody were used for setting up the capture ELISA test. H. pylori positive [(38 gastritis, 14 duodenal ulcers (DU), 11 gastric ulcer (GU)] patients were determined by PCR. The cagA gene was detected in 21 (55%) of gastritis, 11 (78%) of DU and 7 (60%) of GU patients. The reagents used in setting up the capture ELISA test following optimization displayed high performance. This study showed that our developed in-house capture ELISA has the potential to detect the CagA antigen at very low concentrations even though not detected in our H. pylori infected patients sera but we are also intended to use it in saliva and stool samples.

SARS-CoV-2 neutralizing antibody development strategies.

In December 2019 a novel coronavirus was detected in Wuhan City of Hubei Province-China. Owing to a high rate of transmission from human to human, the new virus called SARS-CoV-2 differed from others by its unexpectedly rapid spread. The World Health Organization (WHO) described the most recent coronavirus epidemic as a global pandemic in March 2020. The virus spread triggered a health crisis (the COVID-19 disease) within three months, with socioeconomic implications. No approved targeted-therapies are available for COVID-19, yet. However, it is foreseen that antibody-based treatments may provide an immediate cure for patients. Current neutralizing antibody development studies primarily target the S protein among the structural elements of SARS-CoV-2, which mediates the cell entry of the virus through the angiotensin converting enzyme 2 (ACE2) receptor of host cells. This review aims to provide some of the neutralizing antibody development strategies for SARS-CoV-2 and in vitro and in vivo neutralization assays.

Development of mutant human immunoreactive trypsinogen 1 (IRT1) and mutant human immunoreactive trypsinogen 2 (IRT2) for use in immunoassays.

Immunoreactive Trypsinogen (IRT) is a protein-based pancreatic proenzyme that has an important role in protein digestion in humans. In human body, once IRT present in the small intestine, the proteolytic cleavage activates trypsinogen into trypsin. When IRT is in the active form, it is capable to cleave antibodies, other proteins and even itself while it is desired to use in immunoassays. According to the literature, there are three important IRT isoforms called Immunoreactive Trypsinogen 1 (IRT1), Immunoreactive Trypsinogen 2 (IRT2), and Immunoreactive Trypsinogen 3 (IRT3). However, trypsinogen 1 (cationic trypsinogen, IRT1) and trypsinogen 2 (anionic trypsinogen, IRT2) are the major isoforms in human pancreatic juice and used in the diagnosis of cystic fibrosis (CF). In this study, it is aimed to restrain its proteolytic activity with K23D mutation, which changes lysine (K) residue at the 23rd position to aspartic acid (D). Because we wanted to produce a hassle-free human recombinant immune reactive trypsinogen proenzyme which has similar antigenic properties with the native form. It is also aimed that the mutant IRTs do not exhibit proteolytic activity for the development of durable detection kits with a longer shelf life for both two isoforms. The innovation was actualized in order to use IRTs as a standard antigen in Immunoassays such as ELISA kits. The gene was synthesized as mutated and expressed in P. pastoris X-33 strain. The loss of proteolytic activity has been proven with the BAEE test. Antigenic properties of K23D IRTs and the effect of proteolytic inactivation on their performance in immunoassays were assessed with ELISA and Western Blot. In ELISA results K23D mutated IRTs showed higher signals than Wild-Type forms.

Generation of anti-idiotypic antibodies that mimic HBsAg and vaccination against hepatitis B virus.

The advent of hybridoma technology has opened up a new avenue in vaccine development, and antigen-mimicking properties of anti-idiotypic antibodies have provided promising alternatives in the generation of experimental anti-idiotypic vaccines. In the present study, mice were immunized with anti-hepatitis B virus (HBV) mouse monoclonal and anti-HBV goat polyclonal antibody to produce anti-idiotypic antibodies. Two mouse monoclonal antibodies (6C9, 6H9) were obtained from the fusions, and the immunogenic properties and specificity of antibodies were analyzed. BALB/c mice were immunized with varying concentrations of anti-idiotypic antibodies (25, 50, 75, and 100 micrograms of anti-Id), and it was shown that anti-idiotypic antibodies generated hepatitis B surface antigen (HBsAg), as well as a BSA-specific antibody response. A simple method for the purification of monoclonal antibodies by dialyzing antibody against water has also been reported.

Development of mouse hybridomas by fusion of myeloma cells with lymphocytes derived from spleen, lymph node, and bone marrow.

Since its discovery by Kohler and Milstein in 1975, hybridoma technology has found a wide use in almost every field of biology and medicine. A general and simple approach for developing monoclonal antibodies is to use splenocytes from immunized mice. In the present study, 10 fusion experiments were carried out to analyze the hybridization efficiencies of mouse myeloma cells with lymphocytes derived from spleen, lymph node, and bone marrow and we found a higher yield of antigen specific antibody producing hybridoma lines when the lymph nodes were used.

Production and characterization of monoclonal antibodies against hepatitis B viruses and application of a quick sandwich ELISA.

Hepatitis B virus (HBV) infection is a major health problem worldwide. The diagnosis of acute and chronic hepatitis B infection is based on the detection of hepatitis B surface antigen (HBsAg). We report here the development of hybrid cell producing monoclonal antibodies (MAbs) specific for HBsAg using hybridoma technology. BALB/c mice were immunized with a mixture of HBsAg subtype "ad" and subtype "ay." Spleen and lymph nodes were used as a source of high-titer antibody producing lymphocytes and removed and fused with myeloma cells of F0 origin separately. In the five fusion experiment, enzyme-linked immunosorbent assay (ELISA) tests showed that among 1594 hybridomas only 5 hybrids (9D12, 2B7, 4G5, 2G3, and 6E7) reacted with HBsAg. These MAbs were characterized for use in the development of diagnostic kits based on sandwich ELISA test system. The MAbs were conjugated with horseradish peroxidase (HRP) and used in the quick sandwich ELISA system. This system is a quite practical and time-saving test system when compared with common and commercial sandwich ELISA for diagnosis of hepatitis B surface antigen in human serum.