Resources

Antibody workup

1. [+] General background
   1. Immunostaining is the binding of antibody to an antigen of interest on cells in liquid suspension or in tissue sections. This antibody can be detected by a number of methods:
      1. Chromogenic (horseradish peroxidase, alkaline phosphatase): immunohistochemistry (tissue sections)
      2. Fluorescence: flow cytometry (suspension cells), immunofluorescence (tissue sections)
      3. Mass spectrometry: mass cytometry/cytometry by time of flight (CyTOF), multiplexed ion beam imaging (MIBI)
   2. CyTOF and MIBI require antibodies to be labeled with heavy metal isotopes which are not found naturally in the human body.
   3. CyTOF and MIBI have slightly different isotope detection ranges.
      1. Because CyTOF uses inductively coupled plasma mass spectrometry (ICP-MS), it has an optimal range of roughly 160-170 and a lower limit of 80 (argon doublets, argon is the gas carrier for the mass spectrometer).
      2. Because MIBI uses secondary ion mass spectrometry (SIMS) and does not use a mass filter for argon doublets, it has relatively even sensitivity across 141 to 176 (according to Mike). But due to ionization characteristics it does not efficiently detect cadmium, palladium, or platinum (according to Rashmi).
   4. IonPath and Standard Biotools (Fluidigm) sell pre-conjugated antibodies (links above). Outside of those, we must conjugate ourselves.
   5. Most metal isotope antibody conjugation requires antibody to be purified, without BSA or other carrier protein or tissue culture supernatant, but exceptions and alterative strategies exist.
      1. Most metals require chelation to a polymer, which is then reacted to reduced antibody. BSA and other contaminants competitively inhibit the latter reaction. The exception is Santa Cruz Biotechnology, which uses porcine gelatin which does not interfere.
      2. Monoisotopic and natural abundance platinum (cisplatin) and natural abundance palladium (ethylenediamene palladium chloride) can be reacted directly with reduced antibody.
      3. Theoretically, osmium (osmium tetroxide) can be reacted directly with reduced antibody, but we have not tested it. OsO₄ is a strong oxidizer which reacts with plastic so to control the reaction it would need to be done in glass tubes. The concentration used for labeling cell surfaces is incredibly dilute.
      4. BSA can be removed using kits, but there is typically a great deal of antibody loss.
      5. An alternative strategy is to find your antibody in another format, e.g. labeled with biotin, FITC, digoxigenin, dinitrophenol. Then you can detect it with a secondary antibody labeled with a heavy metal isotope. This requires two-stage staining and may require extra blocking steps.
      6. A corollary is to label your antibody with biotin, FITC, digoxigenin, dinitrophenol, etc. using a kit, e.g. from Biotium. These kits do not suffer from interference by BSA.
2. [−] Antibody clones
   1. Most of the time, the most difficult part of getting good antibody staining is finding a clone that works well under your reaction conditions.
   2. Most flow cytometry/CyTOF antibodies and immunohistochemistry/MIBI antibodies are not cross-platform.
      1. Flow cytometry is primarily done on live (unfixed) cells, which have intact epitopes. However, fixing cells with paraformaldehyde prior to staining often reduces signal, and methanol permeabilization after staining can denature the antigen and/or antibodies such that most signal is lost. These problems are particularly prominent with CD10 and CD13.
      2. Immunohistochemistry is primarily done on formalin-fixed paraffin-embedded (FFPE) sections, which undergo antigen retrieval at 97+° C, where many epitopes are altered by fixation and denaturation.
   3. Factors to consider:

      +++++	Existing antibody in MIBITracker or CyTOF database
      ++++	Offered in a BSA-free non-tissue culture supernatant format. It not, requires purification or conjugate/conjugation to a seconary tag
      ++++	Validated to work consistently in the clinical laboratory, either by NordiQC or Stanford (links above)
      +++	Manufacturer lists FC (CyTOF) or IHC(P) (MIBI) as application.
      ++	Sample/trial size available
      ++	Cheap (BioLegend, Santa Cruz Biotechnology, some Bio-Rad)
      +	Good example image on manufacturer web site
      +	Same clone offered by multiple manufacturers
      −	Manufacturer does not list FC (CyTOF) or IHC(P) (MIBI) as application.

3. [+] Validation and quality control
   1. Positive controls
      1. In order to test your antibody, you will need suitable control materials. First, determine what cells express your antigen. Good resources include BioLegend (Tissue distribution) and PathologyOutlines (Positive staining - normal, Positive staining - disease). Typical sources for materials are below.
      2. CyTOF
         1. Cryopreserved peripheral blood mononuclear cells (PBMCs) from Ficoll gradient purification of leukocyte reduction (LR) chambers from normal blood donors from Stanford Blood Center, primarily includes lymphocytes (B cells, T cells, NK cells), and monocytes.
         2. Cryopreserved bone marrow mononuclear cells (BMMCs) from Ficoll gradient purification of normal bone marrow donors from AllCells, primarily includes immature progenitors, lymphocytes (B cells, T cells, NK cells), and monocytes. A tiny fraction of hematopoietic stem cells, dendritic cells, etc. is also present.
         3. Cryopreserved cell lines.
         4. Whole peripheral blood from normal blood donors (~30-100 × 10⁶ cells) from Stanford Blood Center, primarily includes red blood cells, PBMCs, and neutrophils, with a small fraction of basophils and eosinophils.
         5. Whole bone marrow from normal bone marrow donors (100s × 10⁶ cells) from AllCells, primarily includes red blood cells, BMMCs and maturing granulocytes, with a small fraction of maturing basophils and eosinophils.
         6. Whole bone marrow or peripheral blood, normal or diseased (~2-1,000+ × 10⁶ cells, variable) from normal or disease (typically leukemia or myeloma, some lymphoma) post-diagnostic excess material, ask Albert.
         7. Lymph node or other tissue cells disaggregated into cell suspension (~1-5 × 10⁶, variable) from normal or disease post-diagnostic excess material, availability limited, ask Albert.
      3. MIBI
         1. MIBI antibodies are validated by immunohistochemistry (IHC) prior to conjugation, usually at the manufacturer-listed IHC concentration. If it does not work by IHC, it will likely not work by MIBI.
         2. Stanford pathology archive (1,000,000+ blocks available), highly dependent upon specific tissue or disease requested, ask Ferda. Some diseases are very rare, some tissues are not regularly sampled, and lots of blocks have been taken for other research projects and are not available.
            1. For stains done clinically at Stanford, there is a high probability that we can find cases with an IHC slide that you can use for comparison.
         3. Cell pellet embedded in FFPE, blocks managed by individual lab members.
   2. Quality control
      1. [+] Before conjugation, MIBI antibodies are run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to check for BSA or other contaminants which competitively inhibit the conjugation reaction.
         1. SDS is a detergent which denatures proteins — melting them to remove secondary and tertiary structures so that they run more or less linearly. Heating to 95° C also denatures proteins. However, these do not reduce proteins.
         2. Tris(2-carboxyethyl)phosphine) (TCEP), β mercaptoethanol (βME), and dithiothreitol (DTT) reduce proteins — breaking disulfide bonds to remove quarternary structures so that they run as individual chains on SDS-PAGE.
            1. Running reduced antibody causes it to split into the heavy chain (≈50 kDa) and light chain (≈25 kDa) bands. Quantification by densitometry requires summing the two bands.
            2. Running unreduced IgG causes it to run high in the gel, close to its full molecular weight (150 kDa). However, IgM is a pentamer (of tetramers) and IgA is a dimer (of tetramers) so they will run even higher. Quantification may not require summation of multiple bands, but free heavy and light chains may appear at the ≈50 kDa and ≈25 kDa positions.
            3. BSA runs at ≈66.5 kDa.
            4. The Bio-Rad TGX precast gels do not contain SDS, which should be present in the sample and running buffers.
            5. The Bio-Rad TGX Stain-Free precast gels do not require fixation or post-staining, which saves hours of time although more antibody is required (1.5 µg). They can still be fixed and post-stained if desired.
               1. The Stain-Free compound is supposedly comparable to Coomassie Blue with a tryptophan (W) content >1.5%.
               2. Mouse and rabbit IgG heavy and light chain constant regions have tryptophan contents ≥1.8%.
                  mouse IgG1C from 1IGY
                    ICTVPEVSSVFIFPPKPKDTLLITVTPKVTCVVVDISKDDPEVQFSWFVDNVEVHTAQTQPREEQFNSTFRVVSALPIMHQDWLNGKEFKCRVNSAAFPA
                    PIEKTISKTKGKPRAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQSDGQAPENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHE
                    GLHNHHTEKSLSHSPGK
                  mouse IgG2aC from 1IGT
                    APNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPA
                    PIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHE
                    GLHNHHTTKSFSRTPGK
                  mouse IgG2bC from 6KRU
                    APNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPS
                    PIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHE
                    GLKNYYLKKTISRSPGK
                  mouse IgG3C from A0A4U9FKB1
                    PGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPA
                    PIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHE
                    ALHNHHTQKNLSRSPGK
                  mouse IgκC from P01837
                    RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVK
                    SFNRNEC
                  mouse IgλC1 from A0A0G2JE99
                    QPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLS
                    RADCS
                  rabbit IgGC from 2VUO
                    PPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPA
                    PIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHE
                    ALHNHYTQKSISRSPGK
                  rabbit Igκb4C from P01840
                    DPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNR
                    GDC
                  rabbit Igκb5C from P01841
                    ATLAPTVLIFPPAPAQLATGAVTIVCVANKYFPDGTVTWEVDGKPLTTGIETSKTPQNSDDCTYNLSSTLTLQKSNYNSHNEYTCQVAQGAGSVVQSFSR
                    KNC
               3. BSA only has a tryptophan content of 0.49%.
                    MKWVTFISLLLLFSSAYSRGVFRRDTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCK
                    VASLRETYGDMADCCEKQEPERNECFLSHKDDSPDLPKLKPDPNTLCDEFKADEKKFWGKYLYEIARRHPYFYAPELLYYANKYNGVFQECCQAEDKGAC
                    LLPKIETMREKVLASSARQRLRCASIQKFGERALKAWSVARLSQKFPKAEFVEVTKLVTDLTKVHKECCHGDLLECADDRADLAKYICDNQDTISSKLKE
                    CCDKPLLEKSHCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQEAKDAFLGSFLYEYSRRHPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKL
                    KHLVDEPQNLIKQNCDQFEKLGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCCTKPESERMPCTEDYLSLILNRLCVLHEKTPVSEKVTKCC
                    TESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLPDTEKQIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDKCCAADDKEACFAVEGPKLVV
                    STQTALA
            6. For size markers, one can use protein ladder or any random IgG from the "not working" box(es) and BSA. BSA is present in the IHC/MIBI PBS wash buffer, so use 2.5 µL (2.5 µg) rather than preparing separate stocks.
   3. Antibody storage and shelf life discussion from Labome.