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 border="0" cellpadding="0" cellspacing="0" width="1072">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:235px;">Fisher Scientific</td>
			<td style="width:119px;">MT-10-013-CVRF</td>
			<td style="width:503px;">HIGH GLUCOSE, with glutamine, with sodium pyruvate, 500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>10,000 U/mL, 100 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-2 Supplement (100X)</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>5 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27 Supplement (50X)</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>serum free, 10 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum - Optima</td>
			<td>Atlanta Biologicals</td>
			<td>S12450</td>
			<td>500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">STERILE WATER FOR IRRIGATION</td>
			<td>Baxter Healthcare</td>
			<td>#2F7114</td>
			<td>USP-1000 ml, 12/CA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine hydrobromide</td>
			<td>Sigma</td>
			<td>P7886-50MG</td>
			<td>D-Lys-(D-Lys)n-D-Lys &#183; xHBr, Molecular Weight 30,000-70,000 g/mol, 50 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bovine serum albumin fraction V</td>
			<td>Sigma</td>
			<td>A-3294-100G</td>
			<td>heat shock fraction, protease free, pH 7,&#160; purity 98%, 100 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G5767-500G</td>
			<td>C6H12O6, 
			 
			Molecular Weight: 180.16 g/mol, ACS reagent, 500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Recombinant Rat FGF basic Protein</td>
			<td>R&#38;D systems</td>
			<td>3339-FB-025</td>
			<td>BSA as a carrier protein, 25 ug, lyophilized, &#62;95%, 16.2 kDa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Rat PDGF-AA Protein</td>
			<td>R&#38;D systems</td>
			<td>1055-AA-50</td>
			<td>carrier free, 50 ug, lypphilized, &#62;97%, E.coli-derived, 12.5 kDa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Tris[hydroxymethyl]aminomethane (Tris bas</td>
			<td>Biorad</td>
			<td>#1610719</td>
			<td>1 kg, 99.8% pure, powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td>Biorad</td>
			<td>#1610302</td>
			<td>1 kg, powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>Fisher Scientific</td>
			<td>BP-381-5</td>
			<td>C2H5NO2, Molecular weight: 75.07 g/mol, White crystalline Powder, 5 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Sigma</td>
			<td>S7653-5KG</td>
			<td>NaCl, Molecular Weight: 58.44 g/mol, 5 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate monobasic</td>
			<td>Sigma</td>
			<td>S0751-500G</td>
			<td>NaH2PO4, Molecular Weight: 119.98 g/mol, 500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Falcon 60mm 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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">75cm&#178; tissue culture flask</td>
			<td>Corning Life Sciences</td>
			<td>430720U</td>
			<td>75cm&#178; U-Shaped Canted Neck Cell Culture Flask with Plug Seal Cap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce&#8482; 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 height="21">
			<td height="21" style="height:21px;">4-20% Mini-PROTEAN TGX Precast Gel</td>
			<td>Biorad</td>
			<td>456-1094</td>
			<td>10-well, 50 &#181;l, for use with Mini-PROTEAN electrophoresis cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immobilon-P Membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td>PVDF, 0.45 &#181;m, 26.5 cm x 3.75 m roll</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Polysialic Acid-NCAM Antibody, clone</td>
			<td>Millipore</td>
			<td>MAB5324</td>
			<td>50 microliters, Host: mouse, monoclonal IgM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XT sample buffer (Western blot)</td>
			<td>Biorad</td>
			<td>#1610791</td>
			<td>10 ml, 4 x premixed protein sample buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-mercaptoethanol</td>
			<td>Biorad</td>
			<td>#1610710</td>
			<td>25 ml, 98 % pure, 14.2 M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endoneuraminidase-N (Endo-N)</td>
			<td>ABC Scientific</td>
			<td>ABC0020</td>
			<td>50 microliters, 200 &#181;g/ml, 3500 U/mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 mm glass coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-545-102</td>
			<td>Circles No. 1; Thickness: 0.13 to 0.17mm; Size: 25mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES buffer solution</td>
			<td>ThermoFisher Scientific</td>
			<td>15630-080</td>
			<td>100 ml, 1M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hanks&#39; 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 height="21">
			<td height="21" style="height:21px;">Papain</td>
			<td>Worthington Biochemical Cooperation</td>
			<td>LS003119</td>
			<td>lyophilized powder, 100 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesiumsulfate</td>
			<td>Sigma</td>
			<td>M-2643-500G</td>
			<td>powder, 500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine</td>
			<td>Gibco</td>
			<td>#25030-081</td>
			<td>100 ml, 200 mM&#160;</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/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/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/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/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/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/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 VÃŽÂº Light Chain-containing Human IgM Antibodies: Polysialic Acid PSA Attached to NCAM as a Case Study at JoVE.com

Antibody Binding Specificity for Kappa VÃƒÅ½Ã‚Âº 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.

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 ...