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Optimised SEREX technique for the identification of leukaemia associated antigens

Optimised SEREX technique for the identification of leukaemia associated antigens
Optimised SEREX technique for the identification of leukaemia associated antigens
The serological analysis of antigens by recombinant expression cloning (SEREX) has been used by many laboratories to immunoscreen lambda phage cDNA libraries produced from a range of tumour cell types. We and others have found it difficult to extract an optimal quality and quantity of mRNA for the preparation of cDNA libraries which represent the genes transcribed in haematological samples. The difficulty is believed to be due to residual haem groups in the isolated RNA sample which inhibit the activity of reverse transcriptase used in the later production of cDNA. During our preparation of a cDNA library for SEREX studies, we optimised the isolation of mRNA from samples from patients with haematological malignancies. We compared the efficacy of different methods of mRNA extraction using a range of haematological sample sizes and describe the most efficient techniques to maximise mRNA yield and quality for cDNA library production. The phage library we prepared contained a range of cDNA insert sizes, including high molecular weight sequences which, following immunoscreening with autologous patient sera, led to the isolation of 17 novel antigens. Using the methodology described, we have shown SEREX to be effective for the isolation of leukaemia-associated antigens, which may act as targets for immunotherapy
mRNA isolation, haematological malignancy, SEREX, tumour antigen
0022-1759
207-214
Guinn, Barbara-ann
728d28c9-a23d-413a-ba1d-4531005705d7
Collin, Joseph F.
25bc20db-f8c2-48fe-85e1-373eb91a94eb
Li, Geng
0bf8cbba-ed32-4c1f-9641-a8f2709d149c
Rees, Robert C.
8a19c735-749f-421c-9118-a62d7e395e1e
Mufti, Ghulam J.
940de420-bc41-4006-8517-f2c926ba70aa
Guinn, Barbara-ann
728d28c9-a23d-413a-ba1d-4531005705d7
Collin, Joseph F.
25bc20db-f8c2-48fe-85e1-373eb91a94eb
Li, Geng
0bf8cbba-ed32-4c1f-9641-a8f2709d149c
Rees, Robert C.
8a19c735-749f-421c-9118-a62d7e395e1e
Mufti, Ghulam J.
940de420-bc41-4006-8517-f2c926ba70aa

Guinn, Barbara-ann, Collin, Joseph F., Li, Geng, Rees, Robert C. and Mufti, Ghulam J. (2002) Optimised SEREX technique for the identification of leukaemia associated antigens. Journal of Immunological Methods, 264 (1-2), 207-214. (doi:10.1016/S0022-1759(02)00095-9).

Record type: Article

Abstract

The serological analysis of antigens by recombinant expression cloning (SEREX) has been used by many laboratories to immunoscreen lambda phage cDNA libraries produced from a range of tumour cell types. We and others have found it difficult to extract an optimal quality and quantity of mRNA for the preparation of cDNA libraries which represent the genes transcribed in haematological samples. The difficulty is believed to be due to residual haem groups in the isolated RNA sample which inhibit the activity of reverse transcriptase used in the later production of cDNA. During our preparation of a cDNA library for SEREX studies, we optimised the isolation of mRNA from samples from patients with haematological malignancies. We compared the efficacy of different methods of mRNA extraction using a range of haematological sample sizes and describe the most efficient techniques to maximise mRNA yield and quality for cDNA library production. The phage library we prepared contained a range of cDNA insert sizes, including high molecular weight sequences which, following immunoscreening with autologous patient sera, led to the isolation of 17 novel antigens. Using the methodology described, we have shown SEREX to be effective for the isolation of leukaemia-associated antigens, which may act as targets for immunotherapy

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More information

Published date: June 2002
Keywords: mRNA isolation, haematological malignancy, SEREX, tumour antigen

Identifiers

Local EPrints ID: 71599
URI: http://eprints.soton.ac.uk/id/eprint/71599
ISSN: 0022-1759
PURE UUID: 6f53dcfb-8240-4ed7-a917-579eff2fe4bc

Catalogue record

Date deposited: 15 Dec 2009
Last modified: 19 Jul 2019 23:44

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