Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.
In this video, we describe a simple and efficient method to visualize and analyze an antibody repertoire at global level. It's possible to describe entire B cell repertoires in individuals and their dynamic changes in response to immune stimulation. This method can be applied to analyze pathological samples from patients of emerging viral infections and to obtain the data with unprecedented insight into the pathogenesis of the infection.
One of the most exciting areas for the application is a vaccine control. Vaccine efficacy can be evaluated by analyzing the dynamics of global antibody repertoire in vaccine-administered individuals. The individual are new to this method may need to optimize the PCR conditions because the efficiency of antibody gene amplification depends on the types of starting materials.
Fla-cher for identification of immunoglobulin amplicons. It is important to demonstrate these actions visually. Demonstrating the procedure will be Miss Sayuri Yamaguchi and Mr.Hanbing Xue, a technician and graduate student from my laboratory.
To begin this procedure, dissect the tissue from an eight-week-old C57 black six mouse and pass it through a stainless steel mesh with two milliliters of PBS to obtain dispersed cells. Transfer the cell suspension to a two-milliliter microcentrifuge tube and centrifuge at 600 times g and at four degrees Celsius for five minutes. Discard the supernatant and add 800 microliters of ACK lysing buffer to the pellet.
Incubate on ice for two minutes to lyse the red blood cells in the tissue. Next, wash the tissue cells three times using three milliliters of PBS for each wash. Centrifuge again at 600 times g and four degrees Celsius for five minutes.
Discard the supernatant and add 800 microliters of phenol/guanidine isothiocyanate reagent to the pellet and vortex thoroughly. Incubate at 25 degrees Celsius for five minutes. Then, add 200 microliters of chloroform and shake manually for 15 seconds.
Incubate at 25 degrees Celsius for two minutes. Centrifuge at 12, 000 times g and at 25 degrees Celsius to separate the phases. Transfer the upper aqueous phase to a fresh tube.
Add one volume of 70%ethanol. Vortex briefly and apply this mixture to the silica spin column. Elute the RNA with 30 to 100 microliters of water.
After this, use a fluorometer to quantitate the initial RNA concentration. Store the purified RNA at minus 80 degrees Celsius. First, synthesize the first-strand cDNA from two to 10 micrograms of total RNA template using the 5'RACE CDS primer and the SMART-PCR oligonucleotide according to the manufacturer's instructions.
For the mouse immunoglobulin, PCR amplify cDNA with high-fidelity DNA polymerase, using the universal forward primer and immunoglobulin class-specific reverse primers, as outlined in the text protocol. For the human immunoglobulin, perform the first PCR using the universal forward primer and immunoglobulin class-specific reverse primers with tag sequences. Include the index sequences for each sample by second PCR using index sequence primers.
Next, electrophorese either of the PCR products on a 2%agarose gel. Visualize the DNA bands on a UV transilluminator and excise the gel slice containing the broad band between 600 and 800 base pairs. Add 10 microliters of membrane-binding solution per 10 milligrams of gel slice.
Mix and incubate at 50 to 65 degrees Celsius until the gel slices completely dissolve. Then, transfer the gel solution onto the silica membrane spin column. Wash the column once with washing buffer and elute the DNA with 50 microliters of nuclease-free water.
Using a fluorometer, quantify the purified amplicons and pool the amplicons from each immunoglobulin class in equal amounts for NGS sequencing. After this, use a microcapillary-based electrophoresis with DNA sizing chip to determine the size and concentration of the libraries. Store the libraries at minus 20 degrees Celsius.
First, generate a sample spreadsheet of the sequencing run as outlined in the text protocol. Next, thaw a reagent cartridge and the libraries. Make a 0.2 normal solution of sodium hydroxide and dilute the libraries to obtain the desired molar concentrations.
Then, rinse and dry the flow cell and add 600 microliters of the diluted and denatured library solution into the well of the reagent cartridge, and start the sequencing run. A perspective of a murine antibody repertoire as a whole can be obtained from cells or tissues such as the spleen, bone marrow, lymph node, or blood. Representative results of the IgM, IgG1, IgG2c, and immunoglobulin light chain repertoires from a naive mouse spleen are shown here.
In the V(D)J rearrangement by 3D V(D)J plot, the size of each ball represents the relative number of reads. In other words, it represents the number of antibody mRNAs in whole B cells. The 3D mesh consists of 110 immunoglobulin heavy chain V genes, 12 immunoglobulin heavy chain D genes, and four immunoglobulin heavy chain J genes, which are aligned to reflect their order on the chromosome.
The 2D VJ plot shows the profile of VJ rearrangement in the IGL repertoire. The length of each bar represents the relative number of reads. The X-axis represents 101 immunoglobulin light chain V-kappa genes, and three immunoglobulin light chain V-lambda genes, and the Y-axis represents four immunoglobulin light chain J-kappa genes, and three immunoglobulin light chain J-lambda genes.
A perspective of a human antibody repertoire as a whole can be analyzed from various tissues including peripheral blood mononuclear cells or pathological tissues. The representative results of IgM, total IgG, total IgA, IgD, IgE, and IgL repertoires from normal peripheral blood mononuclear cells are shown here. Like the murine model, the repertoire profile of V(D)J rearrangement is shown on 3D V(D)J plot in which the size of each ball represents the relative number of reads.
The 3D mesh consists of 56 immunoglobulin heavy chain V genes, 27 immunoglobulin heavy chain D genes, and six immunoglobulin heavy chain J genes, aligned in the order they appear on the chromosome. The profile of VJ rearrangement in the IgL repertoire is depicted in the 2D VJ plot in which the length of each bar represents the relative number of reads. The X-axis represents 41 immunoglobulin light chain V-kappa genes, and 32 immunoglobulin light chain V-lambda genes, and the Y-axis represents five immunoglobulin light chain J-kappa genes, and five immunoglobulin light chain J-lambda genes.
In the study material of this protocol, a small number of sorted cells by flow cytometry or even cryosections of pathological samples are usable. However, careful optimization of PCR conditions will be necessary. Because the output information of this protocol is huge, bioinformatics literacy would help finding the structure of antibody network.
That would lead unpreceded to deep understanding of immune system.
