Preparation and Identification of Monoclonal Antibodies Against Tree Shrews CD3ε

doi:10.13523/j.cb.20180408

China Biotechnology

2018, Vol. 38

Issue (4): 54-62 DOI: 10.13523/j.cb.20180408

Preparation and Identification of Monoclonal Antibodies Against Tree Shrews CD3ε

Jing-wen XU,Xue-mei ZHANG,Zhong-xiang WU,Wen-bing ZHU,Xi JIANG,Wei GONG,Li-wei YAN,Jie SONG,Hui LI,Shao-zhong DONG(

)

Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Disease, Yunnan Innovation Team of Standardization and Application Research in Tree Shrew. Kunming 650118, China

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Abstract Objective:

To develop monoclonal antibodies (mAbs) against tree shrews CD3ε, and identify their biological characteristics.

Method:

BALB/c mice were immunized with GST-CD3ε protein as an immunogen. Immunized mice spleen cells were fused with SP2/0 cells with the hybridoma technique. GST- CD3ε proteins and GST proteins were used as coating antigens to establish the indirect ELISA. After screening and three rounds of cloning process, the strain of hybridomas secreting anti-CD3ε mAbs were obtained. More anti- CD3ε mAbs were prepared by mice ascites, which were purified by Protein A resin. Then anti- CD3ε mAbs were identified with indirect ELISA, superposable ELISA, antibody titer evaluation, Western blot and FACS.

Result:

Five hybridomas were obtained, and named 78I, 87I, 92D1, 75II and 35C8.The titer of five ascites were 1:10 ⁶, 1:10 ⁶, 1:10 ⁴, 1:10 ⁶ and 1:10 ³. The dissociation constant (Kd) of these ascites were 1.8×10 ^-5, 2.9×10 ^-5, 4.9×10 ^-5, 7.3×10 ^-5 and 3.6×10 ^-5. Monoclonal antibody epitope analysis revealed that 78I, 87I, and 75II recognized the same antigenic epitope, whereas 92D1 and 35C8 recognized another antigenic epitope of GST- CD3ε. Western blot analysis showed that HRP-labeled 92D1 recognized GST-CD3ε and tree shrews’ PBMC, and had an antibody cross-reactivity with PBMC from rats, mice and monkeys. After FACS detection, 92D1 labeled with PE-Cy5.5 can specifically identify the PBMC of tree shrews.

Conclusion:

Murine anti-tree shrews’ CD3ε mAbs were successfully prepared, which laid the foundation for the further application in immune detection of tree shrews.

Key words： Tree shrews CD3ε Monoclonal antibodies

Received: 13 December 2017 Published: 08 May 2018

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	Jing-wen XU
	Xue-mei ZHANG
	Zhong-xiang WU
	Wen-bing ZHU
	Xi JIANG
	Wei GONG
	Li-wei YAN
	Jie SONG
	Hui LI
	Shao-zhong DONG

Cite this article:

Jing-wen XU,Xue-mei ZHANG,Zhong-xiang WU,Wen-bing ZHU,Xi JIANG,Wei GONG,Li-wei YAN,Jie SONG,Hui LI,Shao-zhong DONG. Preparation and Identification of Monoclonal Antibodies Against Tree Shrews CD3ε. China Biotechnology, 2018, 38(4): 54-62.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180408 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I4/54

Table 1 Primer sequences for the construction of expression vectors of the recombinant proteins

Fig.1 Cloned gene and double enzyme digestion analysis of recombinant plasmid
(a) Cloned CD3ε gene. M: DNA marker DL10000; 1: The PCR products of CD3ε (b) Double enzyme digestion analysis of recombinant plasmid pGEX-5X-CD3ε M: DNA marker DL10000; 2 & 3: pGEX-5X-CD3ε digested with EcoR I and Xho I

Fig. 2 Preparation of recombinant GST-CD3ε proteins and GST tagging proteins
(a) Induction of recombinant GST-CD3ε proteins M: Protein marker; 1: Total proteins of pGEX-5X-CD3ε before induction; 2: Ultrasound supernatant proteins of pGEX-5X-CD3ε after induction (b) Induction of GST tagging proteins M: Protein marker; 3: Total proteins of pGEX-5X-1 before induction; 4: Total proteins of pGEX-5X-1 after induction; 5: Ultrasound supernatant proteins of pGEX-5X-1 after induction (c) Purification of recombinant GST-CD3ε proteins and GST tagging proteins 6: Purified GST-CD3ε proteins; 7: Purified GST proteins

Fig. 3 Purification of anti-CD3ε ascites mAbs
(a) Purification of 78I and 87I ascites mAbs. M: Protein marker; 1: Purified 78I mAbs; 2: Purified 87I mAbs (b) Purification of 75II and 92D1 ascites mAbs M: Protein marker; 3: Purified 75II mAbs; 4: Purified 92D1 mAbs (c) Purification of 35C8 ascites mAbs M: Protein marker; 5: Purified 35C8 mAbs

Fig. 4 Affinity test of anti-CD3ε mAbs

Table 2 The result of superposable ELISA test of anti-CD3ε mAbs

Fig.5 Identification of specificity of anti-CD3ε mAbs by Western-blot
(a) Recognition of recombinant GST-CD3ε proteins and GST tagging proteins by 92D1-HRP 1: Purified GST; 2: Purified GST-CD3ε (b) Recognition of tree shrews’ PBMC total proteins by 92D1-HRP 3: Tree shrews’ PBMC total proteins 20μg; 4: Tree shrews’ PBMC total proteins 10μg; 5: Tree shrews’ PBMC total proteins 5μg (c) Antibody cross-reaction of 92D1-HRP 6: Tree shrews’ PBMC total proteins 10μg; 7: Rats’ PBMC total proteins 10 μg; 8: Mice’s PBMC total proteins 10μg; 9: Monkeys’ PBMC total proteins 10μg

Fig.6 Flow cytometry analysis the PBMC of tree shrews using the anti-CD3ε mAb 92D1
(a) Blank control:Tree shrews’ PBMC with no antibody (b) Tree shrews’ PBMC with PE-Cy5.5-92D1


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