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Monoclonal antibodies against nucleophosmin mutants: potentials for the detection of acute myeloid leukemia.

Tan S, Zhang L, Zhong XM, Yang ZL, Zhao LY, Gao YJ, Shao HY, Qin FX, Chen XC, Zhang HJ, Chen H, Wang L - Int J Med Sci (2011)

Bottom Line: The results showed that the pET-32a-NPM-mA vector was successfully constructed and the NPM-mA recombinant protein was used to immunize the mice.Our results show that anti-NPM-mA mAbs were produced.Though they would cross-react with wild type NPM1, the mAbs may still have potential in the detection of NPMc+AMLs.

View Article: PubMed Central - PubMed

Affiliation: 1. Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, 1 Yixueyuan Road, Chongqing, China.

ABSTRACT
Nucleophosmin (NPM1) gene mutations resulting in cytoplasmic delocalization of Nucleophosmin (NPMc+) are the most common genetic alteration in acute myeloid leukemia (AML). Here, we attempted to prepare monoclonal antibodies (mAbs) against NPM1 mutation A (NPM-mA) and investigated the mAbs' clinical utility in immunohistochemical detection of NPMc+AML. The pET-32a-NPM-mA vector with the whole open reading frame of the NPM-mA gene was constructed. E.coli BL21 transformed with the vector were induced to express the NPM-mA recombinant protein. BALB/c mice were immunized with the recombinant NPM-mA. Positive clones were selected by indirect ELISA and the mAbs were obtained. Immunohistochemistry was performed to detect the NPMc+ in bone marrow smears from 10 AML patients with NPM-mA. The results showed that the pET-32a-NPM-mA vector was successfully constructed and the NPM-mA recombinant protein was used to immunize the mice. Two positive clones (2G3 and 3F9) were selected. The mAbs against NPM-mA were raised, but did cross-react with wild type NPM1. The mAbs can be used to detect the cytoplasmic dislocation of NPM1 in all AMLs carrying NPM-mA. Our results show that anti-NPM-mA mAbs were produced. Though they would cross-react with wild type NPM1, the mAbs may still have potential in the detection of NPMc+AMLs.

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Related in: MedlinePlus

PCR amplifying the full sequence of ORF of the NPM-mA gene. The PCR products amplified with a pair of primers against the NPM-mA gene were analyzed by 1% agarose gel electrophoresis.1: DL2000 markers; 2-3: products of PCR.
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Figure 1: PCR amplifying the full sequence of ORF of the NPM-mA gene. The PCR products amplified with a pair of primers against the NPM-mA gene were analyzed by 1% agarose gel electrophoresis.1: DL2000 markers; 2-3: products of PCR.

Mentions: Using a pair of primers specific for NPM-mA gene, a DNA fragment of approximately 900 bp size was amplified from the pEGFP-C1-NPM-mA plasmids by PCR technique (Figure 1), which corresponded to the full length of open reading frame (ORF) of the NPM-mA gene (935 bp).


Monoclonal antibodies against nucleophosmin mutants: potentials for the detection of acute myeloid leukemia.

Tan S, Zhang L, Zhong XM, Yang ZL, Zhao LY, Gao YJ, Shao HY, Qin FX, Chen XC, Zhang HJ, Chen H, Wang L - Int J Med Sci (2011)

PCR amplifying the full sequence of ORF of the NPM-mA gene. The PCR products amplified with a pair of primers against the NPM-mA gene were analyzed by 1% agarose gel electrophoresis.1: DL2000 markers; 2-3: products of PCR.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3100738&req=5

Figure 1: PCR amplifying the full sequence of ORF of the NPM-mA gene. The PCR products amplified with a pair of primers against the NPM-mA gene were analyzed by 1% agarose gel electrophoresis.1: DL2000 markers; 2-3: products of PCR.
Mentions: Using a pair of primers specific for NPM-mA gene, a DNA fragment of approximately 900 bp size was amplified from the pEGFP-C1-NPM-mA plasmids by PCR technique (Figure 1), which corresponded to the full length of open reading frame (ORF) of the NPM-mA gene (935 bp).

Bottom Line: The results showed that the pET-32a-NPM-mA vector was successfully constructed and the NPM-mA recombinant protein was used to immunize the mice.Our results show that anti-NPM-mA mAbs were produced.Though they would cross-react with wild type NPM1, the mAbs may still have potential in the detection of NPMc+AMLs.

View Article: PubMed Central - PubMed

Affiliation: 1. Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, 1 Yixueyuan Road, Chongqing, China.

ABSTRACT
Nucleophosmin (NPM1) gene mutations resulting in cytoplasmic delocalization of Nucleophosmin (NPMc+) are the most common genetic alteration in acute myeloid leukemia (AML). Here, we attempted to prepare monoclonal antibodies (mAbs) against NPM1 mutation A (NPM-mA) and investigated the mAbs' clinical utility in immunohistochemical detection of NPMc+AML. The pET-32a-NPM-mA vector with the whole open reading frame of the NPM-mA gene was constructed. E.coli BL21 transformed with the vector were induced to express the NPM-mA recombinant protein. BALB/c mice were immunized with the recombinant NPM-mA. Positive clones were selected by indirect ELISA and the mAbs were obtained. Immunohistochemistry was performed to detect the NPMc+ in bone marrow smears from 10 AML patients with NPM-mA. The results showed that the pET-32a-NPM-mA vector was successfully constructed and the NPM-mA recombinant protein was used to immunize the mice. Two positive clones (2G3 and 3F9) were selected. The mAbs against NPM-mA were raised, but did cross-react with wild type NPM1. The mAbs can be used to detect the cytoplasmic dislocation of NPM1 in all AMLs carrying NPM-mA. Our results show that anti-NPM-mA mAbs were produced. Though they would cross-react with wild type NPM1, the mAbs may still have potential in the detection of NPMc+AMLs.

Show MeSH
Related in: MedlinePlus