Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.
Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.
The goal of this article is to describe in detail a methodology to generate and characterize human IgG monoclonal antibodies obtained from human peripheral blood mononuclear cells (PBMCs).
The interest to study human antibodies has grown in many different fields of research. In particular, many research groups are interested in the pathology caused by auto-antibodies1-3. We have cloned and characterized pathogenic auto-antibodies1. The study of auto-antibodies can help to identify their targets and to develop therapeutic strategies, e.g., using competitor antibodies4. Moreover, the study of human antibodies can also be of interest in other fields of research, i.e., to evaluate the immune response after vaccination5, to characterize the antibody profile of individuals that were exposed and became resistant to specific pathogens6 or to study which antibodies are in the natural repertoire7,12.
Several techniques have been developed to generate recombinant human monoclonal antibodies8-12; most of these use phage display and B-cell immortalization. The use of phage display has been extensively applied for the discovery of new antibodies13. However it has a major disadvantage, namely that the heavy and light chain pairs of the human immunoglobulin become dissociated in the process. Production of hybridomas with human B cells or EBV transformation overcomes this drawback.
We use infection of thymic B cells with EBV in combination with polyclonal B cell stimulation via Toll-like receptor 9 (TLR-9)6,12.
In this paper, we describe in detail the technology that we use for the development of IgG human antibodies, with a complete overview of all the steps from PBMC isolation to the in vitro antibody generation. This protocol can be used for the analysis of any type of human IgG profile. In our laboratory, B cells producing IgG antibodies have been successfully separated from the rest of PBMCs after sorting. Fifty sorted B cells8 can then be plated in multi-well plates and immortalized by EBV and TLR-9 activation, for the clonal expansion of single B cells. As feeder cells, fibroblasts from human embryonic lung tissue have been used, cell line wi38, which facilitates the visualization of the immortalized B cells. From these B cells, the sequences of the heavy and light chains of the immunoglobulin can be obtained by PCR, and the antibodies' genes cloned in immunoglobulin G expression vectors and produced in vitro. Using this technique, single antibodies with exactly the same antibody sequence found in the donor can be studied.
Informed consent was obtained from the participants of the study. The study was approved by the institutional ethics committee.
1. Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
2. Staining PBMCs for Sorting CD22+ and IgG+ by Cell Cytometry
3. Sorting of the B Cells CD22+ and IgG+
4. Irradiation of Feeder Cells
Note: Perform the preparation of the feeder cells between 1 – 3 days before sorting. At least 5,000 wi38 cells are needed per well in a 96 round well plate. Perform steps 4.1, 4.2 and 4.4 in a hood.
5. Plating Sorted PBMCs, EBV Infection and Growing
6. ELISA for IgG Antibody Detection
7. RNA Isolation and First Strand cDNA Synthesis of the Producing IgG Clones
8. 1st and 2nd PCR for Amplification of the Heavy and Light Chains of the IgG-producing B-cell Clones
9. Cloning and Sequencing of the Heavy and Light Chains of the Producing IgG clones
10. Production of Antibodies in HEK cells
The sorting gating after staining CD22 and IgG positive cells is shown in Figure 1. In this image the area of the double positive cells – B cells producing IgG antibodies – is selected to sort all these cells in a separate tube. In the analysis, approximately 1% of the total PBMCs correspond to this double positive population. The number of sorted cells obtained will depend on the number of cells obtained in section 1.
The different outcomes after 5 weeks of EBV immortalization and CpG (ODN2006) stimuli are shown in Figure 2. The detection of the growing clones is easy; Wi38 feeder cells have a more elongated fibroblast shape, and the B cell clones appear as very small round cells growing clustered in the middle of the round bottom multi-well plate. At this stage, it is evident that some clones start growing. However, the growing speed can be variable and of course some of the wells containing immortalized cells may not have any cells growing at all.
The supernatant of the growing clones is tested in an ELISA for detecting IgG as shown in Table 2. In this ELISA a standard curve for IgG is tested, together with the supernatant of the clones and the blanks. A positive clone is considered when the value in the ELISA is 3 standard deviations over the blank value. Negative clones’ values are under the 3 standard deviations of the blank. For confirmation, positive clones should be positive in the ELISA at least three times in different supernatants of the same clone and if possible verified by an additional screening method.
The complete sequence of a human IgG antibody obtained applying the steps described in this manuscript is shown in Figure 3. This sequence has been obtained after cloning the immunoglobulin heavy and lambda chain pair from a clonally expanded B cell. Variable and constant regions of the heavy and light chain can be characterized with this technique. After obtaining the sequences, antibodies sequences can be cloned and produced in vitro in HEK cell cultures.
Figure 1. Flow cytometric analysis of CD22+ and IgG+ cells from human peripheral blood mononuclear cells. (A) Selection of the population of living cells is shown in P1. (B) Forward scatter plot. (C) Size scatter plot. (D) The Y axis shows cells separated by anti-CD22-PerCP and on the X axis separated by IgG-PE. P4 square indicates the cell fraction that has been sorted (CD22+, IgG+) and recovered for culture. Please click here to view a larger version of this figure.
Figure 2. Representative image of B cell immortalized clones in a 96 well-plate after 5 weeks of culture. (A) In this well no immortalization was observed after 5 weeks, but wi38 irradiated feeder cells can be observed. (B) A slowly growing clone was observed in this well, with tiny round aggregates in the middle. (C) Fast growing clone showing round aggregates of immortalized B cells. Please click here to view a larger version of this figure.
Figure 3. Human antibody sequences of heavy and light chain pair from a human immortalized B cell clone, F5.2, indicating V CDR1, CDR2 and CDR3 sequences. Please click here to view a larger version of this figure.
1st PCR primers | ||
Forward (5’-3’) | Reverse (3’-5’) | |
IgG | 5′ L-VH 1 ACAGGTGCCCACTCCCAGGTGCAG | 3′ Cγ CH1 GGAAGGTGTGCACGCCGCTGGTC |
5′ L-VH 3 AAGGTGTCCAGTGTGARGTGCAG | ||
5′ L-VH 4/6 CCCAGATGGGTCCTGTCCCAGGTGCAG | ||
5′ L-VH 5 CAAGGAGTCTGTTCCGAGGTGCAG | ||
κ | 5′ L Vκ 1/2 ATGAGGSTCCCYGCTCAGCTGCTGG | 3′ Cκ 543 GTTTCTCGTAGTCTGCTTTGCTCA |
5′ L Vκ 3 CTCTTCCTCCTGCTACTCTGGCTCCCAG | 3′ Cκ 494 GTGCTGTCCTTGCTGTCCTGCT | |
5′ L Vκ 4 ATTTCTCTGTTGCTCTGGATCTCTG | ||
5′ Pan Vκ ATGACCCAGWCTCCABYCWCCCTG | ||
λ | 5′ L Vλ 1 GGTCCTGGGCCCAGTCTGTGCTG | 3′ Cλ CACCAGTGTGGCCTTGTTGGCTTG |
5′ L Vλ 2 GGTCCTGGGCCCAGTCTGCCCTG | ||
5′ L Vλ 3 GCTCTGTGACCTCCTATGAGCTG | ||
5′ L Vλ 4/5 GGTCTCTCTCSCAGCYTGTGCTG | ||
5′ L Vλ 6 GTTCTTGGGCCAATTTTATGCTG | ||
5′ L Vλ 7 GGTCCAATTCYCAGGCTGTGGTG | ||
5′ L Vλ 8 GAGTGGATTCTCAGACTGTGGTG | ||
2nd PCR primers | ||
Forward (5’-3’) | Reverse (3’-5’) | |
IgH | 5′ EcoRI VH1 CAACCGGAATTCGCAGGTGCAGCTGG TGCAG |
3′ NheI JH 1,2,4,5 CTGCTAGCTAGCTGAGGAGACGGT GACCAG |
5′ EcoRI VH1 to 5 CAACCGGAATTCAGAGGTGCAGCTG GTGCAG |
3′ NheI JH 3 CTGCTAGCTAGCTGAGAGACGGTGA CCATTG |
|
5′ EcoRI VH3 CAACCGGAATTCAGAGGTGCAGCTG GTGGAG |
3′ NheI JH 6 CTGCTAGCTAGCTGAGGAGACGGTG ACCGTG |
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5′ EcoRI VH3 23 CAACCGGAATTCAGAGGTGCAGCT GTTGGAG |
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5′ EcoRI VH4 CAACCGGAATTCACAGGTGCAGCT GCAGGAG |
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5′ EcoRI VH 4 34 CAACCGGAATTCACAGGTGCAGCTAC AGCAGTG |
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5′ EcoRI VH 1 18 CTTCCGGAATTCACAGGTTCAGCT GGTGCAG |
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5′ EcoRI VH 1 24 CTTCCGGAATTCACAGGTCCAGCT GGTACAG |
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5′ EcoRI VH3 33 CTTCCGGAATTCACAGGTGCAGCT GGTGGAG |
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5′ EcoRIVH 3 9 GATCCGGAATTCAGAAGTGCAGCT GGTGGAG |
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5′ EcoRI VH4 39 GATCCGGAATTCACAGCTGCAGCT GCAGGAG |
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5′ EcoRI VH 6 1 GATCCGGAATTCACAGGTACAGCT GCAGCAG |
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κ | 5′ EcoRI Vκ 1 5 CAACCGGAATTCAGACATCCAGATGA CCCAGTC |
3′ BsiWI Jκ 1 to 4 GCCACCGTACGTTTGATYTCCACCTTGGTC |
5′ EcoR1 Vκ 1 9 CTTCCGGAATTCAGACATCCAGTTGAC CCAGTCT |
3′ BsiWI Jκ 2 GCCACCGTACGTTTGATCTCCAG CTTGGTC |
|
5′ EcoR1 Vκ 1D 43 CTTGGCGAATTCAGCCATCCGGATGA CCCAGTC |
3′ BsiWI Jκ 3 GCCACCGTACGTTTGATATCCACT TTGGTC |
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5′ EcoR1 Vκ 2 24 CTTCCGGAATTCAGATATTGTGATGA CCCAGAC |
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5′ EcoR1 Vκ 2 28 CTTCCGGAATTCAGATATTGTGATG ACTCAGTC |
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5′ EcoR1 Vκ 2 30 CTTCCGGAATTCAGATGTTGTGATGA CTCAGTC |
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5′ EcoR1 Vκ 3 11 CTTCCGGAATTCAGAAATTGTGTTG ACACAGTC |
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5′ EcoR1 Vκ 3 15 CTTCCGGAATTCAGAAATAGTGATG ACGCAGTC |
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5′ EcoR1 Vκ 3 20 CTTCCGGAATTCAGAAATTGTGTTGA CGCAGTCT |
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5′ EcoR1 Vκ 4 1 CTTCCGGAATTCAGACATCGTGATG ACCCAGTC |
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λ | 5′ EcoR1 Vλ 1 CTTCCGGAATTCACAGTCTGTGCT GACKCAG |
3′ AvrII Jλ 1 to 3 CTGGTTACCTAGGAGGACGGTSACCT TGGTCCC |
5′ EcoR1 Vλ 2 CTTCCGGAATTCACAGTCTGCCC TGACTCAG |
3′ AvrII Jλ 4 CTGGTTACCTAGGAAAATGATCAGC TGGGTTCC |
|
5′ EcoR1 Vλ 3 CTTCCGGAATTCATCCTATGAGC TGACWCAG |
3′ AvrII Jλ 5 CTGGTTACCTAGGAGGACGGTCAGC TCGGTCCC |
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5′ EcoR1 Vλ 4 to 5 CTTCCGGAATTCACAGCYTGTG CTGACTCA |
3′ AvrII Jλ 6 CTGGTTACCTAGGAGGACGGTCAGCT GGGTGCC |
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5′ EcoR1 Vλ 6 CTTCCGGAATTCAAATTTTATGC TGACTCAG |
3′ AvrII Jλ 7 CTGGTTACCTAGGAGGACGGTCAC TTGGTCCAT |
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5′ EcoR1 Vλ 7 to 8 CTTCCGGAATTCACAGRCTGTG GTGACYCAG |
Table 1. Primers used.
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
A | 0.076 | 0.077 | 2.003 | 0.080 | 0.138 | 0.102 | 0.188 | 0.338 | 0.040 | 2.041 | 0.051 | 0.081 |
B | 2.011 | 0.085 | 0.074 | 0.069 | 0.081 | 0.122 | 0.372 | 2.133 | 0.119 | 0.097 | 0.072 | 0.072 |
C | 0.068 | 0.179 | 0.091 | 0.073 | 0.077 | 0.097 | 0.606 | 1.882 | 0.081 | 2.071 | 0.094 | 0.075 |
D | 0.063 | 0.070 | 0.065 | 0.071 | 0.082 | 2.071 | 0.339 | 2.089 | 0.076 | 0.086 | 0.066 | 0.069 |
E | 1.921 | 0.077 | 0.065 | 0.085 | 0.095 | 1.968 | 1.910 | 0.122 | 0.072 | 0.070 | 0.065 | 0.066 |
F | 0.113 | 0.068 | 0.066 | 0.082 | 0.088 | 0.090 | 0.460 | 0.070 | 0.079 | 0.952 | 0.098 | 0.065 |
G | 2.041 | 2.108 | 1.472 | 0.665 | 0.331 | 0.194 | 0.123 | 0.094 | 0.080 | 0.072 | 0.070 | 0.072 |
H | 2.146 | 2.132 | 1.634 | 0.665 | 0.341 | 0.178 | 0.132 | 0.094 | 0.082 | 0.080 | 0.074 | 0.068 |
Standards ng/µl | 1000 | 500 | 250 | 125 | 62.5 | 31.25 | 15.6 | 7.8 | 3.9 | 1.95 | 0.975 | 0 |
Table 2. Representative results of IgG screening by ELISA. Clone supernatants are in A1-A10, B1-B10, C1-C10, D1-D10, E1-E10, F1-F10. Blanks are in A11, B11, C11, D11, E11, F11, A12, B12, C12, D12, E12, F12.Standard curve is duplicated: G1-G12 and H1-H12. The corresponding concentration of immunoglobulins in each duplicate is shown below.Positive or IgG producing clones are shown in dark grey. Negative or non-IgG producing clones are shown in light grey
In this manuscript, all the steps for the generation of IgG antibodies from human PBMCs are presented in detail. This protocol includes some advantages over previously published techniques. One of the advantages is that the antibody produced keeps the heavy and light chains corresponding to the original pair in the B cell clone. The identification of IgG antibodies can be done in any type of human donor, and there is no need for exacerbation of the immune response due to vaccination5. The use of the fibroblast cell line wi38 as a feeder cell, allows a more rapid detection of the growing clones, since they are morphologically different and very easy to differentiate, compared to the PBMCs used as feeder cell in previously described works1,6-8. Moreover the use of wi38 as a feeder cell favors the freezing of big amounts of cell aliquots that can be easily thawed and cultured before every experiment.
One of the critical steps in this protocol is the starting material: the PBMCs. If the blood has been waiting too long for centrifugation, or after the extraction of the PBMCs, they are not stored in the appropriate freezing conditions; as such the number of viable cells will be reduced, along with the number of the B cell IgG producing clones obtained. For that reason, an early extraction and a good preservation of the PBMCs are recommended for a successful outcome. The number of IgG producing clones will be limited to the number of PBMCs at the beginning of the experiment. Higher numbers of PBMCs will give higher numbers of IgG producing clones and a diverse antibody production. Another critical step is the cloning of the PCR fragments of the light and heavy chains of the antibody. If the ligation is not successful after several attempts, an extra step of cloning the 2nd PCR product in a TopoTA system is recommended. The digestion of the insert with the appropriate enzymes can be easier in the TopoTA vector, for a subsequent ligation in the pFUSEss expression vectors.
This technique can be transferred to any type of human tissue in which human B cells are enriched3. It can also be applied to the study of other types of immunoglobulins, just by changing the labelled antibodies used for sorting (antibodies against IgM, IgE, IgA or IgD), the primer design for the PCR, and the expressing vectors. The studies of these immunoglobulins can be of interest to understand, initial immune responses, mucosa antibody secretion and allergy profiles, among others.
In conclusion, we have described a technique to produce human recombinant monoclonal IgG antibodies that leaves the heavy and light chains pairs of the human immunoglobulin intact. The technique is useful and easy to perform starting from a blood sample. The monoclonal antibodies obtained through this method are potentially useful in studies on human immune responses. Furthermore, monoclonal antibodies produced with this method might be a good starting point for the development of immuno-therapeutics for different pathological conditions.
The authors have nothing to disclose.
Research contract Miguel Servet (ISCIII CD14/00032) to (G.N.-G.). Fellowship from the Netherlands Organization for Scientific Research “Graduate School of Translational Neuroscience Program” (022005019) to (C.H.).
Grants from the Prinses Beatrix Fonds (Project WAR08-12) and the Association Française contre les Myopathies to (P.M.-M.); as well as by a Veni Fellowship of the Netherlands Organization for Scientific Research (916.10.148) a fellowship of the Brain Foundation of the Netherlands (FS2008(1)-28) and the Prinses Beatrix Fonds (Project WAR08-12) (to M.L.).
We thank Jozien Jaspers for her help in the B-cell sorting by flow cytometry.
Histopaque-1077 | Sigma-Aldrich | 10771 | solution containing polysucrose and sodium diatrizoate |
FACSAria II cell sorter | BD Biosciences | ||
96 U-bottom micro well plates | Costar | 3799 | |
Advanced Roswell Park Memorial Institute (RPMI) 1640 medium | Gibco, Life Technologies | 12633-020 | |
30% v/v EBV-containing supernatant of the B95-8 cell line | ATCC | CRL-1612 | 3.4 x 108 copies/ml |
CpG2006 | Invivogen | ODN 7909 | |
Wi38 cells | Sigma-Aldrich | 90020107 | |
Interleukin-2 | Roche | 10799068001 | |
ELISA plates | Greiner Bio-One, Microlon | 655092 | |
AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ Fragment Specific (unconjugated) | Jackson ImmunoResearch | 109-006-008 | |
4% non-fat dry milk (Blotting Grade Blocker) | Biorad | 170-6404 | |
Human IgG | Sigma | I 2511 | HUMAN IgG purified Immunoglobulin, 5.6 mg/ml |
Goat F(ab)2 antihuman IgG Fcγ (conjugated with peroxidase (PO)) | Jackson ImmunoResearch | 109-036-008 | |
ELISA reader (Perkin Elmer 2030) | Perkin Elmer | 2030-0050 | |
Peroxidase-conjugated AffiniPure Rabbit Anti-Human IgM, Fc5µ | Jackson ImmunoResearch | 309-035-095 | |
SuperScript III Cells Direct cDNA Synthesis System | Invitrogen | 18080-200 | |
Applied Biosystems (ABI) GeneAm PCR System 2700 | Applied Biosystems | ||
High Pure RNA Isolation Kit | Roche | 11828665001 | |
Reverse transcription system kit | Promega | A3500 | |
Recombinant Taq DNA Polymerase | TAKARA | R001A | |
Primers (2μl) | Sigma | ||
Ultrapure Agarose | Invitrogen | 16500-500 | |
100 bp ladder | Invitrogen | 15628-019 | |
Quantity One 4.5.2 (Gel Doc 2000) | Biorad | 170-8100 | |
QIAquick PCR purification kit | QIAGEN | 28106 | |
BigDye Terminator v3.1 cycle sequencing kit | Applied Biosystems | 4337455 | |
0.1 ml reaction plate (MicroAMP Optical 96-well) | Applied Biosystems | 4346906 | |
Genetic analyser ABI300 | Applied Biosystems | 4346906 | |
DH5α competent cells (E. coli) | Invitrogen | 18263-012 | |
pFUSEss-CHIg-hG1 (4493 bp) | Invivogen | pfusess-hchg1 | |
pFUSEss-CHIg-hG4 (4484 bp) | Invivogen | pfusess-hchg4 | |
pFUSE2ss-CLIg-hk (3875 bp) | Invivogen | pfuse2ss-hclk | |
pFUSE2ss-CLIg-hl2 (3883 bp) | Invivogen | pfuse2ss-hcll2 | |
SOC medium | Invitrogen | 15544-034 | |
LB-based agar medium supplemented with Zeocin (Fast-Media Zeo Agar) | Invivogen | fas-zn-s | |
Terrific Broth (TB)-based liquid medium supplemented with Zeocin (Fast-Media Zeo TB) | Invivogen | fas-zn-l | |
DNA Miniprep kit | Omega Bio Technology | D6942-02 | |
Nanodrop (ND1000 Spectrophotometer) | Nanodrop | ||
LB-based agar medium supplemented with Blasticidin (Fast-Media Blast Agar) | Invivogen | fas-bl-s | |
Terrific Broth (TB)-based liquid medium supplemented with Blasticidin (Fast-Media Blast TB) | Invivogen | fas-bl-l | |
EcoRI | New England Biolabs | R0101S | 20,000 U/ml, in 10x NEBuffer EcoRI |
NheI | New England Biolabs | R0131S | 10,000 U/ml, in 10x NEBuffer 2.1 |
2-Log DNA ladder | New England Biolabs | N3200S | 0.1-10.0 kb, 1,000 μg/ml |
XmaI | New England Biolabs | R0180S | 10,000 U/ml, in 10x CutSmart Buffer |
BsiWI | New England Biolabs | R0553S | 10,000 U/ml, in 10x NEBuffer 3.1 |
AvrII | New England Biolabs | R0174S | 5,000 U/ml, in 10x CutSmart Buffer |
FastAP Thermosensitive Alkaline Phosphatase | Thermo Scientific | EF0651 | 1 U/µL, in 10x FastAP Buffer |
DH5α competent cells | Invitrogen | 18263-012 | |
PE Mouse Anti-Human IgG | BD Pharmingen | 555787 | |
anti-CD22, PerCP-Cy5.5, Clone: HIB22 | Fisher scientific | BDB563942 | |
QIAprep Spin Miniprep Kit | QIAGEN | 27106 | |
BigDye Terminator v3.1 | Applied Biosystems | 4337455 |