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.
Guinn
Barbara-ann
BA
Leukaemia Science Laboratories, Department of Haematological Medicine, Guy's, King's & St. Thomas' School of Medicine, Denmark Hill Campus, Rayne Institute, 123 Coldharbour Lane, London, UK. barbara.guinn@kcl.ac.uk
Collin
Joseph F
JF
Li
Geng
G
Rees
Robert C
RC
Mufti
Ghulam J
GJ
D000368:Aged; D000951:Antigens, Neoplasm / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use; D010582:Bacteriophage lambda / Q000235:genetics; D002469:Cell Separation / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000302:isolation & purification; D015723:Gene Library; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000276:immunology / Q000628:therapy; D015470:Leukemia, Myeloid, Acute / Q000276:immunology / Q000628:therapy; D008297:Male; D012333:RNA, Messenger / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use; D013048:Specimen Handling / Q000379:methods; D010948:Viral Plaque Assay
D000951:Antigens, Neoplasm; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins