The overall goal of this custom array method for alloantibody detection is to delineate humane leukocyte antigen or HLA epitopes in organ transplantation. This method can help answer key questions in the organ transplantation field about the specific immune epitopes involved in transplant rejection. The main advantage of this technique is that it's fully customizable, allowing incorporation of personalized antigen sets that can reflect individual organ donors'HLA sequences.
The technique has the potential to detect specific HLA epitopes for alloantibodies. To retrieve sequences from the International Immunogenetics Project human leukocyte antigen project database, in the ambiguous allele combinations page, open the database allele query form and input the subject's first allele name into the search for box. Click Search for Alleles Now and locate the matching allele name displayed on the screen.
Click Allele and copy and paste the protein sequence into a text document under an allele name header line to arrange the allele name in sequence in a FASTA format. When all of the sequences have been identified, copy and paste the sequences from the FASTA format into the enter your input sequences box on the Cluster Omega website and click submit your job using the standard parameter settings. Then click submit.
An alignment of the sequences will be displayed. To mark the donor-specific mismatches, copy and paste the alignment text into a new text document and use a distinguishing font color to mark each mismatched donor-specific residue within the aligned sequences that belong uniquely to the donor. Underline all of the letters in the donor sequences that extend 14 residues up and downstream of the marked donor-specific mismatches and use the underlined sequences as templates to sequentially derive short 15 amino acid sequences in a series that overlap by four residues between any two immediately adjacent sequences in the series.
Copy and paste these 15 amino acid sequences into a spreadsheet in a column format accompanied by notation columns that include the corresponding names of the donor alleles. To design a custom array layout, generate a spreadsheet of peptide sequences with corresponding allele callings with 20 rows and 30 columns. Then input the sequences into each of the array cells and use the spot synthesizer to run an automated peptide array synthesis.
When designing an array, it is critical to use individual donors'HLA sequences as a template for deriving the antigen set. When the arrays have been synthesized, block the membranes with 20 milliliters of 5%nonfat milk dissolved in Tris-buffered saline with 0.1%tween 20 or TBST for the appropriate incubation period with rocking. Next, wash the membrane three times with 20 milliliters of fresh TBST for five minutes per wash followed by incubation with 20 microliters of crude-recipient serum in 2.5%milk in TBST for two to three hours at room temperature.
At the end of the incubation, wash the membrane three times for 10 minutes per wash and incubate the membranes with goat anti-human horse radish peroxidase conjugated IgG secondary antibody for two hours. Remove the unbound secondary antibody with three 10-minute washes in TBST and develop the membranes in five milliliters of freshly-prepared luminol solution plus five milliliters of peroxide solution. After one minute, visualize the enhanced chemiluminescence signals on a suitable imager.
After saving the developed images, incubate the membranes with 20 milliliters of commercial stripping buffer at 37 degrees Celsius for 20 minutes followed by three 10-minute washes in TBST. Then block, re-probe, and visualize the membranes as just demonstrated with a serum sample from the same patient obtained at a different time point. To manually annotate the positive antigen peptides, locate the spots with positive antibody signals in the saved developed image files and determine each of their grid positions.
Then retrieve the peptide sequences from the master worksheet to identify any potentially shared epitope sequences based on their overlapping segments of reactive peptides. To structurally model antigen epitopes, obtain prototype HLA crystal structures from the protein data bank and display the prototype HLA structure in Pymol. Highlight the reactive peptide sequences to map the discovered anti-donor epitopes to the 3D structures of the prototype HLA.
Then click Display and Background and select White, saving the data as a PNG file. After sequencing multiple alignments of this representative donor patient's alleles as just demonstrated, an alignment file was obtained for each of the alleles. The template sequences of the donor that were sufficiently long enough to cover all of the mismatched residues were then identified and underlined and three series of 15-mer peptides were derived for the first allele.
After all of the donor's alleles were analyzed, a total of 202 peptides were loaded into an array for the specific probing of the donor's post-transplant serum. Comparing the results with those obtained from a subsequent re-probing of the donor's pre-transplant serum revealed the antibody signals associated with the post-transplant specimen of which several donor-specific residues mismatched from the recipient appeared to be involved in inciting antibody reactivity. When the antibody reactive epitopes on HLA-DQA1 and DQB1 were modeled to a co-crystal heterodimer structure, a prominent segment of the structure known as the beta one strand was identified as a hotspot that had a much higher chance of being targeted by alloantibodies among the transplant subjects in a representative five-case cohort.
Once mastered, the technique can be completed in seven to eight hours if it is performed properly. After its development, the technique paved the way for researchers in the field of transplant rejection to explore the personalized detection of donor-specific alloantibodies.
