Introduction

Monoclonal antibodies (MAbs) are clinically useful for the diagnosis and treatment of various diseases. Some neutralizing MAbs have already been developed, such as anti-severe acute respiratory syndrome (SARS) (1), anti-human immunodeficiency virus (HIV) (2,3), and anti-hepatitis C virus antibodies (4), and they are expected to be used as potential antibody-based therapeutics. To produce human MAbs, mouse MAbs have been prepared using the hybridoma technique (5,6) and thereafter have been humanized using genetic technology. In order to efficiently produce human MAbs, we have recently attempted to determine human immoglobulin (Ig) variable genes directly from single antigen-specific B cells using the reverse transcription PCR (RT-PCR) method. Although several studies (7,8,9,10,11,12) have reported the successful characterization of Ig variable genes using RT-PCR from a single B cell, conventional methods can only amplify 40–50 different Ig variable genes, a limitation that is due to the set of the designed primers employed. Therefore, this method is inappropriate if the targeted Ig variable gene is incompatible with the designed primers. In this respect, 5′-rapid amplification of cDNA ends (5′-RACE) (13) may overcome this problem because a common primer could be used as a 5′-primer. However, conventional 5′-RACE is not suitable for the isolation of genes from single cells because the amount of the cDNA product derived from a single cell is extremely low and it may therefore be lost during the complex steps of the purification procedures for 5′-RACE.

To overcome this problem, we have developed a novel method called single-cell 5′-RACE. In this method, first-strand cDNA is synthesized directly from a single cell and a 3′-tailing reaction is performed without removing any residual dNTPs. In the same tube, a second-strand cDNA is synthesized with an adaptor designed to suppress nonspecific products by panhandle formation during PCR (14,15,16,17,18). Here we present our findings regarding the successful application of single-cell 5′-RACE to amplify the Ig variable genes from human single B cells. Our novel method will be useful for the characterization of any genes from single cells in both humans and animals.

Materials and Methods

Tailing Reaction Test

To examine the 3′-tailing efficiency, 3′-tailing of a primer oligonucleotide (Cm-1st; all oligonucleotide primer sequences are shown in (Table 1)) was performed with 5 U terminal deoxy-nucleotidyl-transferase (Invitrogen, Carsbad, CA, USA) in the presence of 1.25 mM dATP, dTTP, dGTP, or dCTP in a 10-µL reaction mixture containing 25 pmol Cm-1 st primer, 10 mM MgCl₂, 1 mM dithiothreitol (DTT), and 10 mM Tris-HCl, pH 7.5, for 1 h at 37°C. Then, the preheated products were analyzed on 15% polyacrylamide gel with 7 M urea.

B Cell Preparation

Human peripheral blood lymphocytes were prepared from 10 mL of heparinized peripheral blood from a healthy donor by centrifugation at 400× g on a Ficoll-Hypaque gradient (19). Human B cells were isolated by removing non-B cells from lymphocytes by automated magnetic cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) using CD2, CD 14, CD 16, and glycophorin A microbeads (all from Miltenyi Biotec), according to the manufacturer's instructions.

Single-Cell Reverse Transcription and 5′-RACE

Single B cells were picked up under microscopy using TransferMan® NK2 (Eppendorf, Hamburg, Germany) and then transferred to the tubes containing the reverse transcription solution. Both cell lysis and reverse transcription were carried out with 40 U SuperScript™ III (Invitrogen) using 0.25 pmol each Cm-RT, Cg-RT, Cl-RT, and Ck-RT primers in 2.5 µL reaction mixture containing 2 U RNaseOUT™ (Invitrogen), 0.5 mM each dNTP, 1.5 mM MgCl₂, 37.5 mM KCl, 10 mM DTT, 0.25% Nonidet™ P-40 (NP40), and 0.1 mg/mL bovine serum albumin (BSA) in 25 mM Tris-HCl, pH 8.3, for 1 h at 55°C. The 3′-tailing of cDNA was performed by adding to the reverse transcription reaction 1 µL 10× tailing reaction reagents containing 100 mM MgCl₂, 10 mM DTT, 100 mM Tris-HCl, pH 7.5, and 20 mM dGTP, followed by 7.5 U terminal deoxynucleotidyl-trans-ferase and water to 10 µL. The reaction was kept for 1 h at 37°C. Next, total dGTP-tailed cDNA was used without purification as a template for second-strand cDNA synthesis. Second-strand synthesis was carried out with 1.25 U LA Taq™ (Takara, Tokyo, Japan) and 25 pmol oligo(dC) adaptor in a 50-µL reaction mixture containing 0.2 mM each dNTP in 1× GC Buffer II (Takara): 1 cycle of denaturation at 94°C for 3 min, followed by 23 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 60°C, and elongation for 3 min at 72°C. Five microliters of the cDNA were then used as a first PCR template with adaptor primer 1 (AP1) and either Cm-1 st and Cg-1 st for the amplification from the Ig heavy chain or Cl-1st and Ck-1st from the light chain, followed by nested PCR using adaptor primer 2 (AP2) and either Cm-nest or Cg-nest for the amplification from the heavy chain or Cl-nest and Ck-nest from the light chain to enhance the specificity. The amplification of each product was carried out using LA Taq according to the manufacturer's instructions: 1 cycle of denaturation at 94°C for 3 min, followed by 25–30 cycles of denaturation for 20 s at 94°C, annealing for 20 s at 60°C, and elongation for 1 min 30 s at 72°C. The PCR product was then analyzed using a Cm-nest, Cg-nest, Cl-nest, or Ck-nest primer by either direct sequencing or sequencing after conventional subcloning into pT7Blue (Novagen, San Diego, CA, USA).