PCR amplification of antibody variable regions using primers that anneal to constant regions

Cloning of antibody variable regions (V) is of great experimental and practical interest, e.g. for genetic manipulation of antibodies of therapeutic or diagnostic value, as well as for developing alternative strategies for animal or human antibody production. Several methods have been so far described for cloning antibody V-regions using Polymerase Chain Reaction (PCR) (reviewed in ref. 1). Since most of these methods are based on the use of degenerated primers, the development of rapid and reliable methods that reduce the risk of introducing primer-dictated mutations is desirable. In this work we describe an improved method for antibody V-region cloning based on Inverse PCR (IPCR) carried out on circularised cDNA (2) using new couples of primers that anneal to antibody constant (C) regions. On the basis of the immunoglobulin mRNA structure, the heavy (H) or light (L) chain IPCR product is expected to consist of: a) a small portion of CH3 or CL, b) the 3' untranslated (3'UT), c) the poly (A) tail, d) the 5' untranslated (5'UT), e) the leader sequence, f) the VH-D-JH or VL-JL region and g) a small portion of CHi or CL. A schematic representation of the strategy exploited for VH amplification is shown in Fig. 1. The method was applied to clone the H and L chain V-regions of the anti-idiotypic monoclonal antibody (mAb) 1G3, an IgG2a (kappa) directed to a paratope-related idiotope of a neutralising anti-Tumor Necrosis Factor (TNF)a antibody (3), and those of the anti-p55 TNF receptor mAb 7H3 (4), belonging to the IgGl (kappa) isotype. V-region cloning was carried out as follows: total RNA from 1G3 or 7H3 hybridoma cell lines was extracted with guanidinium thiocyanate according to the method of Sambrook et al. (5). The cDNA was then synthesised using the oligo (dT) NotI primeradapter and reagents of the Superscript Plasmid System kit (BRL) essentially as described previously (6). cDNA was extracted once with an equal volume of phenol and once with an equal volume of chloroform-isoamylalcohol (24:1 v/v). cDNA was then precipitated by adding a volume of 7.5 M ammonium acetate and three volumes of isopropanol and circularised as follows: the pellet was resuspended in 8 y\ of 10 mM Tris-HCl buffer, pH 8, containing EDTA 1 mM, and mixed with 1 /d of 500 mM Tris—HC1 buffer, pH 7.5, containing 100 mM magnesium chloride, 10 mM dithiothreitol, 5 mM ATP, and 1 /d of T4 DNA ligase (1 U//*l). After a 5 hour incubation at room temperature, 1 /tl of circularised cDNA (denatured 5' at 95 °C) was mixed with 5 /tl of 670 mM Tris-HCl buffer, pH 8.8, containing 166 mM ammonium sulphate, 1 mg/ml bovine serum albumin, ( I O X P C R buffer), 4 /tl of 25 mM magnesium chloride. 8 fd of 1.25 mM dNTPs mixture, 5 /tl of the first primer (30 ng//tl), 5 /tl of the second primer (30 ng//tl), 2.5 units Taq polymerase (Boehringer Mannheim) in a final volume of 50 /tl. The PCR primers, synthesised using an Applied Biosystem synthesiser, were characterised by the following sequences: a) for IgG2a heavy chain amplification: 5'-GTCCACGAGGTGCTGCACAAT (IgG2-CH3, first primer), 5'-GTCACTGGCTCAGGGAAATAACC (IgG2-CHi, second primer); b) for IgGl heavy chain amplification: 5'-CCTGCTCTGTGTTACATGAGGG (IgGI-CH3, first primer), 5'-CCCAGGGTCACCATGGAGTTAG (IgGl-CH1, second primer); for kappa light chain amplification: 5'-AAGATGGATACAGTTGGTGC (K-CLA,