This work was supported by grants from the National Institutes of Health (R01 GM092993, R01 NS048357 and R21 NS073684), the National Science Foundation (CAREER Award), the Minnesota Partnership Award for Biotechnology and Medical Genomics, the National Multiple Sclerosis Society (CA1060A), and the Mayo Clinic Center for Clinical and Translational Science (CCaTS). The authors acknowledge support from the Applebaum, Hilton, Peterson and Sanford Foundations, the Moon and Marilyn Park Directorship Fund and the McNeilus family.

Patents for antibodies that promote remyelination and CNS repair are issued and owned by Mayo Clinic. Authors have a potential conflict of interest.

Human natural IgM autoantibodies are appealing candidates for immunotherapies and have demonstrated therapeutic potential for the treatment of various diseases including cancer and CNS disorders 1-7. Advantageously, these antibodies will not elicit an immune response in which the generation of neutralizing antibodies substantially reduces the effective therapeutic dose and efficacy. Importantly, all antibodies with therapeutic potential have been of the IgM isotype and belong to the NAb repertoire 3-5,8,9,37,40,42-45. A major hurdle for the potential clinical application of IgM antibodies is the identification of their antigens, which are undetermined in many cases. Standard techniques employed for IgG antibodies are often not applicable to identify an IgM&#39;s antigen.
This protocol describes the identification of PSA-NCAM as the antigen for the regenerative human IgM antibody HIgM12, effective in animal models of MS and ALS 15-18. The methodology used is principally applicable to all human antibodies with specific V&#954; light chains V&#954;I, V&#954;III and V&#954;IV and mouse antibodies with V&#954;I light chains, irrespective of the antibody&#39;s isotype.
The most critical step in this protocol is the use of IgM antibodies in affinity chromatography applications. More specifically, successful antibodies are required to act as pull-down agents in immunoprecipitations to isolate or to enrich an antigen out of complex tissue- or cell-culture lysates. Enriched antigen fractions may be compared to immunoprecipitations from isotype control antibodies and subsequently analyzed for differences by mass spectrometry. One essential requirement or limitation of this step is the presence of the correct antibody V&#954; light chain and host (human, mouse) to enable antibody binding to protein L agarose. Use of mannan-binding protein bound to agarose (also called mannose-binding lectin) instead of protein L agarose is a potential alternative to immunoprecipitate IgMs without specific V&#954; light chains. However, as stated by Arnold et al. 35, antigen-bound IgM antibodies do not bind to mannan-binding protein, as the target glycan appear to become inaccessible once the IgM has bound to its antigen. Based on these findings mannan-binding protein cannot be used as a binding matrix to immunoprecipitate antigen-loaded IgM molecules 35. Other potential alternatives might be the use of secondary agarose-bound anti-IgM IgG antibodies 46,47 or use of surface-activated magnetic beads 48. IgG antibodies directed against IgMs could be chemically crosslinked to agarose A or agarose G in order to reduce the extent of eluted antibody together with the antigen of interest. A proper comparison between different variants of IgM immunoprecipitation with respect to successfully identified antigens is difficult because most immunoprecipitations were performed to isolate or deplete IgMs without further interest in their antigens. In addition, immunoprecipitations using IgMs were mainly used to confirm an already known or expected single antigen with antibody exposure to purified antigens 49. A disadvantage of agarose-coupled IgGs directed against human IgMs is a very low yield (10 - 15%) of immunoprecipitated serum IgM molecules when compared to the starting material 50. In contrast, surface-activated magnetic beads were successfully applied to antibodies of different isotypes including IgM to immunoprecipitate scrapie-associated fibrils 48. This method is not restricted to specific kappa (V&#954;) or lambda (&#955;) chains or a particular host and may substantially broaden the spectrum of IgM antibodies used in immunoprecipitations.
Another important step in the protocol is the antibody&#39;s ability to function as a detecting antibody (primary antibody) in Western blots or other screening platforms. Successful antibodies must target their antigen with sufficiently high affinity to allow antigen binding in the presence of non-ionic or possibly ionic detergents. High affinity antibodies are common among affinity-maturated IgG antibodies, but less common among antibodies of the IgM isotype, which is one of the reasons why there are relatively few commercially available IgM antibodies as detecting agents in biochemical settings. High affinity binding of antibodies is a requirement for immunoprecipitations of molecular antigens and for Western blotting. Lowering detergent concentrations or the complete absence of detergents in IP buffers and lysis buffers allows antigen targeting by low-affinity antibodies via its Fab domain. In contrast, the antibody&#39;s ability to target protein L agarose via its Fc portion is not substantially affected among isotype controls over a range of different detergent concentrations. However, low detergent concentration in lysis buffer and IP buffer prevents cell membrane disruption and isolation of specific molecules. Similarly, a low detergent concentration in Western blot washing buffers (e.g., PBS-T) allows antigen binding of low-affinity antibodies but at the same time increases non-specific binding and is therefore not an option.
The presence of an antigen protein core is another requirement for this method. Proteins with posttranslational modifications (e.g., glycoproteins, lipoproteins) and unmodified proteins, but not sole lipids or carbohydrates, are detectable in Western blots. It is not possible to immunoprecipitate lipids (e.g., sphingolipids) from tissue or cell lysates in the presence or absence of detergents. The physicochemical properties of detergents and lipids are too similar to allow selective lipid-antibody interactions, while at the same time detergents are essential to disrupt membranes in order to allow selective isolation of specific molecules. To our best knowledge, immunoprecipitations solely comprised of carbohydrate, in the absence of a protein core, have not been previously reported. This becomes particularly relevant because IgM antibodies frequently target carbohydrates and glycolipids, which are not necessarily linked to a protein unit. Other chromatographic techniques are required for the separation and immunologic identification of lipids and carbohydrates in the absence of a protein core. For example, thin layer chromatography (TLC) of cellular lipids with subsequent antibody detection on the TLC plate (immuno-TLC) can be implemented 51,52.
Other anti-PSA antibodies have been described in previous studies and results compared with HIgM12 as well as the commercially available anti-PSA IgM (clone 2-2B). The most commonly used antibody in the field of PSA is an IgG antibody (clone 735) available as mouse monoclonal or rabbit polyclonal antibody 53. This anti-PSA IgG antibody detects PSA on SynCAM and NCAM on different CNS cell types including microglial cells 41. In contrast to the anti-PSA IgG antibody, human IgM antibodies HIgM12, HIgM42 and the anti-PSA mouse IgM (clone 2-2B) are unable to target PSA on SynCAM (but on NCAM) at various embryonic and early postnatal stages in mice, while non-polysialylated SynCAM is easily detectable 15. The IgM antibodies used are also not able to detect PSA on microglial cells (Figures 3 + 4). A possible explanation for differences observed between antibody isotypes may be low PSA-SynCAM levels compared to levels of PSA-NCAM combined with the potentially lower affinity binding of IgM antibodies compared to the anti-PSA IgG. To test this hypothesis, we performed immunoprecipitations in NCAM KO animals at embryonic stage E17 with vast amounts of SynCAM present and used HIgM12 as a &#34;pull-down agent&#34;15. In E17 WT littermate controls HIgM12 immunoprecipitated PSA-NCAM to an extent that showed similar intensities of &#34;pulled down&#34; PSA-NCAM compared to the IgMs heavy chain as detected by densitometry in subsequent Western blot using eluted antigens. This outcome suggested at least a sufficient affinity of HIgM12 to its target PSA. In contrast to WT littermate controls HIgM12 did not detect PSA attached to SynCAM in NCAM KO animals. Identical results were obtained in immunoprecipitations using the human anti-PSA IgM HIgM42. HIgM12 and HIgM42, as well as the mouse anti-PSA IgM (clone 2-2B) were unable to target PSA on SynCAM in Western blots from WT and NCAM KO animals at embryonic and postnatal developmental stages. Given the low amount of HIgM12 required (0.1 &#181;g/ml) to specifically detect PSA attached to NCAM in Western blots using small amounts of CNS tissue (0.1 &#181;g CNS tissue per well) 15 it appears to be unlikely that low affinities alone are responsible for complete lack of PSA-SynCAM detection by HIgM12 in three different methods.
We conclude that there are significant differences between anti-PSA IgM antibodies and the frequently published anti-PSA IgG antibody (clone 735). It is not clear why commercially available anti-PSA IgM antibodies were not used more frequently in the past to confirm results obtained with antiPSA IgG antibody 735. This is particularly interesting because HIgM12 has already proven efficacy in different disease models. While other anti-PSA IgM antibodies may have similar therapeutic effects it remains unclear whether the anti-PSA IgG antibody (clone 735) has a similar therapeutic outcome in models of MS and ALS.
In brief, methods described here were used primarily to identify the antigen of the regenerative human antibody HIgM12 to support potential clinical trials for MS and possibly other neurodegenerative diseases. The identification of antigens for antibodies with biological activity is as essential step to understand their mechanism of action. This becomes particularly relevant in the context of numerous monoclonal antibodies currently being tested for safety and efficacy in human trials. While the number of clinically tested IgM antibodies to date is small, recent advances in hybridoma technology together with an increasing number of studies highlighting the therapeutic potential of this antibody class 1-10,37,40,42-45 urges the development of new or modified methods applicable to identify non-protein IgM antigens.

Antibodies of the IgM isotype demonstrate great therapeutic potential for the treatment of various diseases including cancer and CNS disorders 1-7. The Vollmers&#39; group identified numerous antibodies in cancer patients for potential use as tumor-specific biomarkers or active therapeutics that are able to kill malignant cells by inducing apoptotic pathways 4,8,9. Interestingly, all identified antibodies with therapeutic potential are of the IgM isotype and belong to the group of &#34;natural autoantibodies&#34; (NAbs).
Similarly, the Rodriguez&#39;s group identified mouse and human antibodies that stimulate remyelination in chronically demyelinated spinal cord lesions in models of multiple sclerosis (MS). Identical to antibodies with anti-cancer effects, all remyelination-promoting antibodies are NAbs and of the IgM isotype 1,6,7,10. The precise antigens for most identified IgMs are still undetermined including the human remyelination-promoting antibody rHIgM22, currently in Phase I clinical trials for MS patients 11. Despite repeated efforts by experts in the field of lipid and carbohydrate research both in the academic setting and in partnership with industry 11, attempts to identify rHIgM22&#39;s antigen have not been successful. The failure of standard techniques used to identify antigens of IgG antibodies to work with IgM antibodies identifies a critical need to refine methods specific for these antibodies that most likely target carbohydrate or lipid antigens.
The focus of this manuscript is the human regenerative antibody HIgM12 and the experimental procedures used to identify its antigen. Antibody HIgM12 was identified from patients with Waldenstrom&#39;s macroglobulinemia, stimulates neurite outgrowth in vitro 12-14 and targets polysialic acid (PSA) attached to the neural cell adhesion molecule (PSA-NCAM) 15,16. HIgM12 extends life-span in a mouse model of ALS 17 and improves functional outcome in Theiler&#39;s murine encephalomyelitis virus (TMEV)-infected mice. Specifically, HIgM12 stimulates spontaneous horizontal and vertical motor activity in chronically demyelinated mice and increases numbers of small and medium diameter spinal cord axons eight weeks after a single, low dose of intraperitoneal injected antibody 18.
The neural cell adhesion molecule (NCAM) is a glycoprotein of the immunoglobulin (Ig) superfamily expressed on the cell surface of neurons, glia, skeletal muscle, and natural killer cells 19-25. The three major NCAM isoforms termed NCAM180, NCAM140, and NCAM120, are alternative splice variants of a primary transcript that vary only in their cytoplasmic domain. Within the CNS, NCAM is the major polysialylated molecule (&#62; 95%) with long, negatively charged sialic acid homopolymers. Polysialic acid with n &#62; 10 is termed PSA but shorter oligomeric structures exist that are also biologically relevant. Other polysialylated proteins expressed in the CNS are SynCAM1 26, Neuropilin-2 (NRP-2) 27,28 and a sodium channel subunit 25 (for review see 29).
The methods described here permit antigen identification for human and mouse immunoglobulins containing specific kappa (V&#954;) light chains (V&#954;I, V&#954;III or V&#954;IV light chains for human antibodies and V&#954;I light chains for mouse antibodies) irrespective of the antibody&#39;s isotype (e.g., IgG, IgM, IgA, IgD or IgE). This limitation is based on use of protein L agarose for immunoprecipitations with antibodies of the IgM isotype. Alternative strategies may include mannose-binding lectins and secondary IgG anti-IgM antibodies covalently linked to agarose beads, which may broaden the applicability of this method to more IgM antibodies including those with lambda (&#955;) light chains (see discussion). Ratios of serum IgM V&#954; light chains compared to IgM &#955; light chains derived from healthy individuals are 1.5:1 30.
Based on the chromatographic methodology used here to separate, enrich, and immunologically detect certain molecules 31, all antigens are required to include at least one small protein domain. The antibody&#39;s specific binding epitope can be within or outside the protein domain (e.g., in glycoproteins, lipoproteins). Initial biochemical steps used to identify the specific antigen for HIgM12, respective to narrow down the list of potential candidates, are the most crucial steps in this method. Cell type specific preparations and cell morphology-based characterizations are described for glial cells but this methodology can be extrapolated to accommodate other cell types within or outside the CNS.
There is an urgent need to develop new or modified techniques applicable for the increasing number of IgM antibodies with therapeutic potential for different human disorders particularly in those cases (the majority of IgM antigens) where the antibody targets are carbohydrate or lipid structures.

<table><tbody><tr><td>DMEM</td><td>Fisher Scientific</td><td>MT-10-013-CVRF</td><td>High glucose, with glutamine, with sodium pyruvate, 500 ml</td></tr><tr><td>DMEM/F-12, HEPES</td><td>Life Technologies</td><td>11330-032</td><td>500 ml, L-glutamine, sodium pyruvate, high glucose</td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td>10,000 U/ml, 100 ml</td></tr><tr><td>N-2 Supplement (100x)</td><td>Life Technologies</td><td>17502-048</td><td>5 ml</td></tr><tr><td>B-27 Supplement (50x)</td><td>Life Technologies</td><td>17504-044</td><td>serum free, 10 ml</td></tr><tr><td>Fetal Bovine Serum - Optima</td><td>Atlanta Biologicals</td><td>S12450</td><td>500 ml</td></tr><tr><td>Sterile water for irrigation</td><td>Baxter Healthcare</td><td>#2F7114</td><td>USP-1,000 ml, 12/CA</td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Sigma</td><td>P7886-50MG</td><td>D-Lys-(D-Lys)n-D-Lys &amp;middot; xHBr, Molecular Weight 30,000-70,000 g/mol, 50 mg</td></tr><tr><td>bovine serum albumin fraction V</td><td>Sigma</td><td>A-3294-100G</td><td>heat shock fraction, protease free, pH 7,&amp;nbsp; purity 98%, 100 g</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G5767-500G</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;, Molecular Weight: 180.16 g/mol, ACS reagent, 500 g</td></tr><tr><td>Trypsin from bovine pancreas</td><td>Sigma</td><td>T9935-100mg</td><td>essentially salt-free, lyophilized powder, 9,000 BAEE units/mg protein</td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma</td><td>D5025-150KU</td><td>Type IV, lyophilized powder, 2,000 Kunitz units/mg protein</td></tr><tr><td>Recombinant Rat FGF basic Protein</td><td>R&amp;amp;D systems</td><td>3339-FB-025</td><td>BSA as a carrier protein, 25 &amp;#181;g, lyophilized, &gt; 95%, 16.2 kDa</td></tr><tr><td>Recombinant Rat PDGF-AA Protein</td><td>R&amp;amp;D systems</td><td>1055-AA-50</td><td>carrier free, 50 &amp;#181;g, lyophilized, &gt; 97%, &lt;em&gt;E. coli&lt;/em&gt;-derived, 12.5 kDa</td></tr><tr><td>Neurobasal-A</td><td>Life Technologies</td><td>10888-022</td><td>500 ml, No Glutamine, No Aspartic Acid, No Glutamic Acid</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148-1KG</td><td>crystalline, 1 kg, reagent grade, Molecular Weight 30.03 g/mol (as monomer)</td></tr><tr><td>Tris[hydroxymethyl]aminomethane (Tris bas</td><td>Biorad</td><td>#1610719</td><td>1 kg, 99.8% pure, powder</td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Biorad</td><td>#1610302</td><td>1 kg, powder</td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP-381-5</td><td>C&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;NO&lt;sub&gt;2&lt;/sub&gt;, Molecular weight: 75.07 g/mol, White crystalline Powder, 5 kg</td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S7653-5KG</td><td>NaCl, Molecular Weight: 58.44 g/mol, 5 kg</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>S0751-500G</td><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, Molecular Weight: 119.98 g/mol, 500 g</td></tr><tr><td>Phosphate-buffered Solution 1x (PBS)</td><td>Cellgro</td><td>MT-21-040-CV</td><td>Without Calcium and Magnesium, 6 x 500 ml</td></tr><tr><td>Falcon 60 mm TC-Treated Cell Culture Dish</td><td>Corning Life Sciences</td><td>#353002</td><td>60 mm Cell Culture Dish, TC-treated polystyrene, 20/pack, sterile</td></tr><tr><td>Falcon 60 mm x 15 mm Petri dish</td><td>Corning Life Sciences</td><td>#351007</td><td>Not TC-Treated Bacteriological Petri Dish, 20/Pack</td></tr><tr><td>6 well cell culture plates</td><td>Corning Costar</td><td>CLS3516</td><td>6 well, flat bottom (Individually wrapped), gamma-irradiated, growth area 9.5 c</td></tr><tr><td>75 cm&amp;sup2; tissue culture flask</td><td>Corning Life Sciences</td><td>430720U</td><td>75 cm&amp;sup2; U-Shaped Canted Neck Cell Culture Flask with Plug Seal Cap</td></tr><tr><td>Pierce&amp;trade; Protein L Plus Agarose</td><td>ThermoFisher Scientific</td><td>#20520</td><td>2 ml, crosslinked 6% beaded agarose (CL-6B), binding capacity: 10 to 20 mg IgG</td></tr><tr><td>4 - 20% Mini-PROTEAN TGX Precast Gel</td><td>Biorad</td><td>456-1094</td><td>10-well, 50 &amp;micro;l, for use with Mini-PROTEAN electrophoresis cells</td></tr><tr><td>Immobilon-P Membrane</td><td>Millipore</td><td>IPVH00010</td><td>PVDF, 0.45 &amp;micro;m, 26.5 cm x 3.75 m roll</td></tr><tr><td>Anti-Polysialic Acid-NCAM Antibody, clone</td><td>Millipore</td><td>MAB5324</td><td>50 microliters, Host: mouse, monoclonal IgM</td></tr><tr><td>4x Laemmli Sample Buffer</td><td>Biorad</td><td>#1610747</td><td>10 ml, 4x premixed protein sample buffer</td></tr><tr><td>2-mercaptoethanol</td><td>Biorad</td><td>#1610710</td><td>25 ml, 98 % pure, 14.2 M</td></tr><tr><td>Endoneuraminidase-N (Endo-N)</td><td>ABC Scientific</td><td>ABC0020</td><td>50 microliters, 200 &amp;micro;g/ml, 3,500 U/mg</td></tr><tr><td>25 mm glass coverslips</td><td>Fisher Scientific</td><td>12-545-102</td><td>Circles No. 1; Thickness: 0.13 to 0.17 mm; Size: 25 mm</td></tr><tr><td>HEPES buffer solution</td><td>ThermoFisher Scientific</td><td>15630-080</td><td>100 ml, 1 M</td></tr><tr><td>Hanks&#039; Balanced Salt Solution 1x (HBSS)</td><td>Cellgro</td><td>MT-21-022-CV</td><td>500 ml, without Calcium, Magnesium, Phenol Red</td></tr><tr><td>Papain</td><td>Worthington Biochemical Cooperation</td><td>LS003119</td><td>lyophilized powder, 100 mg</td></tr><tr><td>Magnesiumsulfate</td><td>Sigma</td><td>M-2643-500G</td><td>powder, 500 g</td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>#25030-081</td><td>100 ml, 200 mM&amp;nbsp;</td></tr></tbody></table>

antibody binding specificity for kappa (v&#954;) light chain-containing human (igm) antibodies: polysialic acid (psa) attached to ncam as a case study

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. 
This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

This section illustrates examples of results that can be obtained by studying human IgM-specific PSA-antigens in the CNS. The use of human IgM antibodies containing specific V&#954; light chains in biochemical settings and by fluorescent immunocytochemistry is shown.
HIgM12 immunoprecipitates its antigen from cerebral brain lysates and acts as a detecting agent (primary antibody) in Western blots. Neither isotype control antibody nor agarose L beads immunoprecipitate a similar antigen, which demonstrates the specificity of the pull down (Figure 1). Western blots using CNS lysates from WT and NCAM KO mice demonstrate the specificity of HIgM12-binding to PSA-containing NCAM (Figure 2A). Enzymatic digestion of CNS tissue from WT mice using ENDO-NF identifies PSA attached to NCAM as the specific binding epitope for HIgM12 (Figure 2B). Using the method outlined herein, IBA-1-positive rat microglial cultures of high purity are obtained (Figure 3). Commercially available anti-PSA antibody (clone 2-2B) does not label cell surface or internal PSA pools in fixed and permeabilized IBA-1-positive microglial cells by fluorescent microscopy. Consistent with previously published literature, which reports no cell-surface expression of PSA-NCAM in microglia 16,41 HIgM12 does not label cell surface antigens in purified rat microglia. Interestingly, HIgM12 labeling of fixed and permeabilized microglia results in an intracellular, perinuclear pattern that is not restricted to specific organelles (Figure 3). Results are in contrast to those obtained using an anti-PSA IgG antibody (clone 735) 26, which targets PSA-SynCAM in microglia at the level of the Golgi 41.
In contrast to microglia, HIgM12 and A2B5 target cell surface antigens in highly enriched rat OPC cultures under live cell conditions (Figure 4). OPC cultures contain ~ 5% contaminating microglial cells. Figure 5 illustrates a virtually identical cell-surface staining pattern between HIgM12 and anti-PSA mAb (clone 2-2B) in GFAP-positive astrocytes (Figure 5A). Immunofluorescent labeling of ENDO-NF-digested WT astrocytes compared to buffer control-treated WT astrocytes using HIgM12 demonstrates the presence of astrocytic PSA (Figure 5B). The presence of PSA-positive astrocytes in vivo still has to be confirmed.
<img alt="Figure 1" src="/files/ftp_upload/54139/54139fig1.jpg" /> 
Figure 1: HIgM12 acts as a &#34;pull-down&#34; Agent in Immunoprecipitations. Immunoprecipitations from total brain lysate of three-month-old mice using HIgM12, human isotype control HIgM126 or agarose L beads only as &#34;pull down&#34; agents. Eluted proteins were run in Western blots with membranes probed against HIgM12. Molecular weights of eluted proteins from HIgM12-coated beads were in the range of 160 - 200 kDa in addition to the IgMs heavy chain (~ 65 - 73 kDa). This figure has been modified from J. Neurochem.15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54139/54139fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54139/54139fig2.jpg" />
Figure 2: HIgM12 Targets the Polysialic Acid (PSA) Moiety on NCAM. A. Brain homogenates from P7, P13, P16 and P27 NCAM total KO mice and heterozygous littermate controls were run in Western blots with membranes probed against HIgM12, PSA and &#946;-actin as loading control. B. 10 &#181;g of adult WT mouse brain lysate in 1% NP40 in PBS, pH 6.5 was treated with endoneuraminidase N (ENDO-N) (exo)neuraminidase with &#945;2-3 polysialic acid polymer specificity (Sialidase) or PBS overnight at 37 &#186;C, run as doublets in Western blots and probed against HIgM12, PSA and &#946;-actin as loading control. This figure has been modified from J. Neurochem.15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54139/54139fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54139/54139fig3.jpg" /> 
Figure 3: The Absence of PSA on Rat Microglia is Mirrored by a Lack of HIgM12 Labeling. Rat microglia were cultured for 48 hr on poly-D-lysine coated glass coverslips and either labeled live on ice with HIgM12 or after fixation with HIgM12 or anti-PSA mAb (clone 2-2B). Cells were triple labeled with microglia marker IBA-1 (green), HIgM12/PSA (red) and DAPI (blue). Images show lack of microglial cell surface binding using HIgM12 (upper row) and lack of anti-PSA binding (clone 2-2B) to the cell surface and internal stores in primary rat microglia (lower row). Based on bipolar morphology and absence of IBA-1 staining, the small PSA-positive cell (lower row) was suggested to be an OPC. In fixed cells, HIgM12 staining resulted in a punctated, perinuclear pattern that was not restricted to specific organelles and was considered non-specific binding (middle row). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54139/54139fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54139/54139fig4.jpg" /> 
Figure 4: HIgM12 Targets Cell Surface PSA-NCAM on Rat OPCs. Rat OPCs and microglia were cultured for 4 days in proliferation medium on poly-D-lysine coated glass coverslips and triple labeled live on ice with HIgM12 or A2B5 (green), internal macrophage/monocyte marker CD68 (clone ED-1) (red) and DAPI (blue). Images show cell populations majorly positive for OPC marker A2B5 (upper row) and HIgM12 (lower row) with few CD68-positive microglia (~ 5%). Phase contrast and DAPI images were added to reveal cell integrity and cell numbers for each population. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54139/54139fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54139/54139fig5.jpg" /> 
Figure 5: HIgM12 and anti-PSA mAb co-label an Astrocytic Subpopulation in Mouse Mixed Glia. A. Mouse mixed glial cells from WT and NCAM total KO animals containing astrocytes, oligodendrocyte-lineage cells and microglia were cultured for 3 days and labeled live on ice with HIgM12 (green), anti-PSA mAb (clone 2-2B) (red) and subsequently for astrocytic marker GFAP (purple) and DAPI (blue). HIgM12 and anti-PSA reveal a virtual identical staining pattern on GFAP-positive WT astrocytes but lack binding to cultured astrocytes from NCAM KO mice. This figure has been modified from J. Neurochem.15. B. Mixed glia were cultured identically as described under A. and treated overnight with endoneuraminidase-NF (kindly provided by Dr. Martina Muehlenhoff) or PBS control. Cells were labeled live on ice with HIgM12 (green) and subsequently stained for internal markers GFAP (red) and DAPI (blue). HIgM12 targets cell surface antigens on PBS-control treated mixed glial cultures but not endoneuraminidase-NF treated mixed glia. This figure has been modified from JNSCI16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54139/54139fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
All images were taken at 60X magnification using the same instrument settings and processed identically.
<img alt="Table 1" src="/files/ftp_upload/54139/54139table1.jpg" /> 
Table 1: Buffer and Solution Recipes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54139/Table1.xls">Please click here to download this table.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>CNS tissue</td>
			<td>ENDO-NF/PBS</td>
			<td>Lysis buffer</td>
			<td>Total volume</td>
		</tr>
		<tr>
			<td>1. WT mouse</td>
			<td>67 &#181;g (0.45 &#181;l)</td>
			<td>2 &#181;l (12 &#181;g) ENDO-NF</td>
			<td>7.55 &#181;l</td>
			<td>10 &#181;l</td>
		</tr>
		<tr>
			<td>2. WT mouse</td>
			<td>67 &#181;g (0.45 &#181;l)</td>
			<td>2 &#181;l PBS</td>
			<td>7.55 &#181;l</td>
			<td>10 &#181;l</td>
		</tr>
		<tr>
			<td>3. WT mouse</td>
			<td>67 &#181;g (0.45 &#181;l)</td>
			<td>no PBS or ENDO-NF</td>
			<td>9.55 &#181;l</td>
			<td>10 &#181;l</td>
		</tr>
		<tr>
			<td>4. NCAM KO mouse</td>
			<td>67 &#181;g (0.45 &#181;l)</td>
			<td>2 &#181;l (12 &#181;g) ENDO-NF</td>
			<td>7.55 &#181;l</td>
			<td>10 &#181;l</td>
		</tr>
		<tr>
			<td>5. NCAM KO mouse</td>
			<td>67 &#181;g (0.45 &#181;l)</td>
			<td>2 &#181;l PBS</td>
			<td>7.55 &#181;l</td>
			<td>10 &#181;l</td>
		</tr>
		<tr>
			<td>6. NCAM KO mouse</td>
			<td>67 &#181;g (0.45 &#181;l)</td>
			<td>no PBS or ENDO-NF</td>
			<td>9.55 &#181;l</td>
			<td>10 &#181;l</td>
		</tr>
	</tbody>
</table>
Table 2: ENDO-NF digestion of CNS tissue from P7 WT and NCAM KO mice.

Watch this Scientific Journal Video about Antibody Binding Specificity for Kappa VÎº Light Chain-containing Human IgM Antibodies: Polysialic Acid PSA Attached to NCAM as a Case Study at JoVE.com

Antibody Binding Specificity for Kappa (V&#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (V&#954;) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations.

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents ...

antibody-binding-specificity-for-kappa-v-light-chain-containing-human

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

14.0K Views.  Mayo Clinic. We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (Vκ) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations. 

Video: Antibody Binding Specificity for Kappa (Vκ) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes through their two different arms. bsAbs have been actively studied for their ability to directly recruit immune cells to kill tumor cells. Currently, the majority of bsAbs are produced in the form of recombinant proteins, either as Fc-containing bsAbs or as smaller bsAb derivatives without the Fc region. In this study, GPC3-S-Fab, an antibody fragment (Fab) based bispecific antibody, was designed by linking the Fab of anti-GPC3 antibody GC33 with an anti-CD16 single domain antibody. The GPC3-S-Fab can be expressed in Escherichia coli and purified by two affinity chromatographies. The purified GPC3-S-Fab can specifically bind to and kill GPC3 positive liver cancer cells by recruiting natural killer cells, suggesting a potential application of GPC3-S-Fab in liver cancer therapy.

Here, we present a protocol to produce a bispecific antibody GPC3-S-Fab in Escherichia coli. The purified GPC3-S-Fab has potent cytotoxicity against GPC3 positive liver cancer cells.

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes ...

Cancer Research

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Biochemistry

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Biology

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

Here, we present a protocol for the purification of MHC class I and class II peptide complexes from mouse and human cell lines providing high-quality immunopeptidomics data. The protocol focuses on sample preparation using commercially available antibodies.

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

Bioengineering

Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by Fc&#947;RIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the Fc&#947;RIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

A protocol for studying Fc&#947;RIIIa-driven events by therapeutic antibodies in human natural killer cells is described here. This artificial stimulation platform permits the interrogation of downstream effector functions such as degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in binding.

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and ...

Determining Binding Affinity (KD) of Radiolabeled Antibodies to Immobilized Antigens

Determining binding affinity (KD) is an important aspect of the characterization of radiolabeled antibodies (rAb). Typically, binding affinity is represented by the equilibrium dissociation constant, KD, and can be calculated as the concentration of antibody at which half the antibody binding sites are occupied at equilibrium. This method can be generalized to any radiolabeled antibody or other protein and peptide scaffolds. In contrast to cell-based methods, the choice of immobilized antigens is particularly useful for validating binding affinities after long-term storage of antibodies, distinguishing binding affinities of fragment antigen-binding region (Fab) arms in bispecific antibody constructs, and determining if there is variability in antigen expression between different cell lines. This method involves immobilizing a fixed amount of antigen to specified wells on a breakable 96-well plate. Then, nonspecific binding was blocked in all wells with bovine serum albumin (BSA). Subsequently, the rAb was added in a concentration gradient to all wells. A range of concentrations was chosen to allow the rAb to reach saturation, i.e., a concentration of antibody at which all antigens are continuously bound by the rAb. In designated wells without immobilized antigen, nonspecific binding of the rAb can be determined. By subtracting nonspecific binding from total binding in the wells with immobilized antigen, specific binding of the rAb to the antigen can be determined. The KD of the rAb was calculated from the resulting saturation binding curve. As an example, binding affinity was determined using radiolabeled amivantamab, a bispecific antibody for epidermal growth factor receptor (EGFR) and cytoplasmic mesenchymal-epithelial transition (cMET) proteins.

Determining Binding Affinity KD of Radiolabeled Antibodies to Immobilized Antigens

Here, a method is described to determine the binding affinity (KD) of radiolabeled antibodies to immobilized antigens. KD is the equilibrium dissociation constant that can be determined from a saturation binding experiment by measuring the total, specific, and nonspecific binding of a radiolabeled antibody at various concentrations to its antigen.

Antibody Binding Specificity for Kappa (Vκ) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

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