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Updated: Mar 24, 2026
Generation of Murine Monoclonal Antibodies by Hybridoma Technology
Published on: January 2, 2017
Preparation of Monoclonal Antibodies and a Simple Myeloperoxidase-Immunosorbent Assay for Detecting Human
Zhi-Ping Bian1, Xiong-Zhi Li1, Heng-Fang Wu2
11 Research Institute of Cardiovascular Disease, First Affiliated Hospital of Nanjing Medical University , Nanjing, China .
Abstract:
Myeloperoxidase (MPO), a leukocyte hemoprotein released from neutrophils, is thought to be a potential participant in plaque formation and plaque rupture. Therefore, MPO is regarded as an early marker predicting the risk for atherosclerosis, especially for coronary artery disease and acute coronary syndrome. We generated hybridoma clones 1E3 and 3E8 secreting monoclonal antibodies (mAbs) specific to human MPO. BALB/c mice were immunized with MPO protein purified from human neutrophils. Splenocytes from these mice were fused with the mouse myeloma cell line SP2/0. Based on isotyping of the mAbs, both clones 1E3 and 3E8 were referred to the IgG1 subclass. The specificities of 1E3 and 3E8 were assessed by enzyme-linked immunosorbent assay (ELISA), and only 3E8 was confirmed by western blot. We developed a simple MPO-immunosorbent assay (MPO-ISA) on microplate based on both the immune activity and peroxidase activity of MPO. The mAb secreted by clone 3E8 was chosen as coating antibody to capture the plasma MPO without interfering with the peroxidase activity of MPO. Then, tetramethylbenzidine substrate was added to the microwell directly, catalyzed by captured MPO, and a colored product was formed. The simple MPO-ISA test has a sensitivity of 3.68 ng/mL. The linear concentration of MPO-ISA for commercial MPO standard ranged to 250 ng/mL. The average recovery rate is 101.02%. The imprecision within-day was <10% at three different MPO levels. The imprecision between-day was <10% at low and middle MPO levels and varied to 14.61% at the high MPO level. We found that the established MPO-ISA can detect the plasma MPO from human and cavy, but not from mouse and rat. Compared with the commercial human MPO ELISA assay, the MPO-ISA can be used to detect the natural human MPO protein, but not recombinant MPO polypeptides. The generated mAbs and MPO-ISA test may be useful tools to assess risk for inflammation and cardiac events.
Insights
Researchers developed a new assay to detect myeloperoxidase (MPO), an early marker for atherosclerosis and cardiac events. This simple MPO-immunosorbent assay (MPO-ISA) shows promise for assessing inflammation and cardiovascular risk.
Area of Science:
- Biochemistry
- Immunology
- Cardiovascular Research
Background:
- Myeloperoxidase (MPO) is a leukocyte enzyme implicated in atherosclerosis development and rupture.
- MPO serves as an early predictive marker for coronary artery disease and acute coronary syndrome.
Purpose of the Study:
- To generate monoclonal antibodies (mAbs) specific to human MPO.
- To develop a novel, simple MPO-immunosorbent assay (MPO-ISA) for detecting MPO in biological samples.
Main Methods:
- Hybridoma technology was used to produce mAbs 1E3 and 3E8 against human MPO.
- An MPO-ISA was designed utilizing mAb 3E8 for MPO capture and its inherent peroxidase activity for detection.
- Assay validation included sensitivity, linearity, recovery, and imprecision assessments.
Main Results:
- Monoclonal antibody 3E8 was confirmed for specificity and used in the MPO-ISA.
- The MPO-ISA demonstrated a sensitivity of 3.68 ng/mL and linearity up to 250 ng/mL.
- The assay successfully detected plasma MPO in humans and cavies, distinguishing natural MPO from recombinant forms.
Conclusions:
- The generated mAbs and the MPO-ISA are valuable tools for MPO detection.
- This assay can aid in assessing risks associated with inflammation and cardiac events.
