We are very grateful to K. Nakagaki, Dr. H. Ishii, Dr. K. Suzuki, A. Yamagata, K. Oofusa for their valuable contributions.

1. Production and purification of sGMR&alpha;
<ol>
<li>Amplify cDNA of sGMR&alpha; from human placenta cDNA library by polymerase chain reaction (PCR).</li>
<li>Add 50 base 5'-UTR sequence of baculovirus polyhedrin end and 27 base coding RGS His-tag to the 3' end to the PCR amplicon by another PCR.</li>
<li>Insert the amplified PCR product into a plasmid pMSG1.1MG.</li>
<li>Inject the construct psGMR/M1.1MG with the helper vector pHA3PIG into eggs of silkworm pnd-w1 strain.</li>
<li>Rear the hatched G0 larvae to moths at 25&deg;C. Screen G1 embryos obtained by mating among siblings or with pnd-w1 for MGFP expression in the eyes to obtain transgenic silkworms bearing the sGMR&alpha; gene.</li>
<li>Extract recombinant sGMR&alpha; from the cocoons of transgenic silkworms with phosphate-buffer containing 500mM NaCl and stir at 4&deg;C for 24h.</li>
<li>Centrifuge sample at 20000 g for 15min to remove a precipitates.</li>
<li>Apply supernatant to a Nickel-affinity column.</li>
<li>Wash the column with 10mM imidazole, 500mM NaCl and 20mM Sodium phosphate, pH6.3, and elute with a linear gradient of imidazole from 10mM to 250mM. Pool the fractions containing the purified sGMR&alpha; and dialyze against phosphate-buffered saline (pH7.4) (PBS).</li>
</ol>
2. Biotinylation of recombinant GM-CSF
<ol>
<li>Remove sorbitol preparation by dialysis against PBS.</li>
<li>Add 1ml of 20mM cold sodium meta-periodate solution at 4&deg;C to the solution on ice.</li>
<li>Incubate the sample for 30min on ice in the dark.</li>
<li>Add glycerol to the solution at a final concentration of 15mM.</li>
<li>Dialyze the solution against 100mM sodium acetate buffer (pH5.5)</li>
<li>Add biotin hydrazide to the solution at a final concentration of 5mM and continuously agitate the solution for 2h at room temperature.</li>
<li>Remove the non-reacted material by dialysis against PBS, pH7.4.</li>
</ol>
3. A cell-free GM-CSF receptor-ligand-binding assay
<ol>
<li>Coat a microtiter plate with 50&mu;l of monoclonal anti-polyHistidine antibody overnight at 4&deg;C.</li>
<li>Wash five times with 500&mu;l of PBST.</li>
<li>Add 100&mu;l of a blocking solution and incubate for 3h at 4&deg;C. </li>
<li>Wash five times with 500&mu;l of PBST.</li>
<li>Add 50&mu;l of sGMR&alpha; in the blocking solution and incubate overnight at 4&deg;C. </li>
<li>Wash five times with 500&mu;l of PBST.</li>
<li>Add 25&mu;l of the samples containing GM-CSF autoantibody and then 25&mu;l of biotinylated GM-CSF. Incubate for 1h at 4&deg;C to form sGMR&alpha;-GM-CSF complex.</li>
<li>Wash five times with 500&mu;l of PBST.</li>
<li>Add 50&mu;l of 0.4mM streptavidin alkaline phosphatase for 1hour at 4&deg;C to detect the biotinylated GM-CSF bound sGMR&alpha;.</li>
<li>Wash five times with 500&mu;l of PBST.</li>
<li>Add 50&mu;l of a chemiluminescent substrate to the solution and incubated for 1h at room temperature.</li>
<li>Using a chemiluminescence plate reader, detect the chemiluminescence activity.</li>
</ol>
4. Representative Results: 
The first step in the GM-CSF receptor signal transduction pathway is the binding of GM-CSF to GM-CSF receptor alpha on the cell surface. Because GM-CSF autoantibody specifically binds GM-CSF and block its binding to the receptor in vitro 16,17, we hypothesized that the autoantibodies inhibit this first reaction by directly binding to GM-CSF. As described previously (Ref. 9-15), we applied transgenic silkworm technology to obtain a large amount of recombinant sGMR&alpha; with high purity.
When the silkworm-derived recombinant sGMR&#x03B1; was loaded on SDS-PAGE under non-reducing conditions, the sGMR&alpha; showed both monomeric (45 kDa) and dimeric (90 kDa) forms, whereas only the monomeric form was detected under reducing conditions (Figure 1B), indicating that the recombinant sGMR&alpha; was a mixture of monomers and disulfide-linked dimmers18.
Using the cell-free system (Fig. 2A), we evaluated the inhibition of GM-CSF binding to sGMR&alpha; by GM-CSF autoantibodies (Fig. 2B and Fig. 2C). These methods were described in reference 19 by Urano et al. The growth inhibition was closely correlated with the binding inhibition (r=0.988, p=0.002) at various concentrations of the GM-CSF autoantibody (Fig. 3A)19. Similarly, the binding inhibition was significantly correlated with the growth inhibition. The binding inhibition increased in a dose-dependent manner by GM-CSF autoantibodies (Fig. 3B)19. These two parameters for the serum IgG fractions from different patients were correlated each other (r=0.589 p=0.006, Fig. 3C). Neither binding inhibition nor growth inhibition correlated with Kd values, and thus, both parameters were unaffected by binding affinity19. Consequently reproducibility of data between binding and growth inhibition obtained through three independent experiments on three different samples was evaluated. The coefficient of variation indicated that the cell-free system was more excellent than that of bioassay (table 1). 
<img src="/files/ftp_upload/2742/2742fig1.jpg" alt="Figure 1" /> 
Figure 1. Flow chart of the procedure for the production of sGMR&alpha; using transgenic silkworms. A) Structures of the transformation vectors9-11. B) SDS-PAGE and Coomassie Brilliant Blue-staining of the purified sGMR&alpha; under reducing and non-reducing conditions. sGMR&alpha;, soluble GM-CSF receptor alpha; MGFP, monster green fluorescent protein; BmNPVpol5'-UTR, 5'-untranslated region sequence of Bombyx mori nuclear polyhedrosis virus polyhedrin; SV40 polyA, SV40 polyA signal sequence; P3xP3, 3xP3 promoter; Pser1, ser1 promoter; fibL polyA, fibroin L-chain polyA signal sequence; hr3, Bombyx mori nuclear polyhedrosis virus hr3 enhancer.
<img src="/files/ftp_upload/2742/2742fig2.jpg" alt="Figure 2" fo:content-width="6in" /> 
Figure 2. Scheme of the cell-free assay system. A) A competitive binding assay using sGMR&alpha; produced by silkworm. B) Effect of neutralizing and non-neutralizing antibodies on the binding inhibition by cell-free system. C) The difference of binding inhibition between various concentrations of neutralizing antibodies and non-neutralizing antibodies. GM-CSF, granulocyte-macrophage colony-stimulating factor; sGMR&alpha;, soluble GM-CSF receptor alpha; His, RGS-His-tag; AP, alkaline phosphatase.
<img src="/files/ftp_upload/2742/2742fig3.jpg" alt="Figure 3" /> 
Figure 3. GM-CSF binding inhibition to sGMR&alpha; by effect of GM-CSF polyclonal antibodies or the serum IgG fractions from patients with autoimmune PAP. A) Relationship between binding inhibition and growth inhibition by GM-CSF autoantibody. B) Binding inhibition at various concentrations of GM-CSF autoantibody. C) Relationship between IC50 for percent binding inhibition and percent growth inhibition by the serum IgG fractions19. IC50, 50% inhibitory concentration; GM-CSF, granulocyte-macrophage colony-stimulating factor.
<table border="1">
 <tbody>
 <tr>
 <td>Sample</td>
 <td>A</td>
 <td>B</td>
 <td>C</td>
 </tr>
 <tr>
 <td colspan="4">Inter-assay</td>
 </tr>
 <tr>
 <td># of determinations</td>
 <td>3</td>
 <td>3</td>
 <td>3</td>
 </tr>
 <tr>
 <td>Mean value ( % binding inhibition )</td>
 <td>61.6 </td>
 <td>63.8</td>
 <td>69.7</td>
 </tr>
 <tr>
 <td>Mean value ( % growth inhibition )</td>
 <td>21.0</td>
 <td>73.4</td>
 <td>82.8</td>
 </tr>
 <tr>
 <td>Coefficient of variation (%) (% binding inhibition)</td>
 <td>6.0</td>
 <td>5.6</td>
 <td>8.3</td>
 </tr>
 <tr>
 <td>Coefficient of variation (%) (% growth inhibition)</td>
 <td>64.4</td>
 <td>18.3</td>
 <td>10.4</td>
 </tr>
 </tbody>
</table>
Both assays were performed at 5 ng/ml of GM-CSF and a concentration equal to GM-CSF autoantibody.
Table 1. Comparison of coefficient of variations between percent binding and growth inhibitions obtained through three independent experiments.

<ol>
	<li>Arai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+neutralizing+capacity+of+GM-CSF+reflects+disease+severity+in+a+patient+with+pulmonary+alveolar+proteinosis+successfully+treated+with+inhaled+GM-CSF.">Serum neutralizing capacity of GM-CSF reflects disease severity in a patient with pulmonary alveolar proteinosis successfully treated with inhaled GM-CSF.</a> Respir Med. 98, 1227-1230 (2004).</li><li>Tazawa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-macrophage+colony-stimulating+factor+and+lung+immunity+in+pulmonary+alveolar+proteinosis.">Granulocyte-macrophage colony-stimulating factor and lung immunity in pulmonary alveolar proteinosis.</a> Am J Respir Crit Care Med. 171, 1142-1149 (2005).</li><li>Inoue, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+a+large+cohort+of+patients+with+autoimmune+pulmonary+alveolar+proteinosis+in+Japan.">Characteristics of a large cohort of patients with autoimmune pulmonary alveolar proteinosis in Japan.</a> Am J Respir Crit Care Med. 177, 752-762 (2008).</li><li>Uchida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+autoantibodies+specifically+eliminate+granulocyte-macrophage+colony-stimulating+factor+activity+in+the+lungs+of+patients+with+idiopathic+pulmonary+alveolar+proteinosis.">High-affinity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis.</a> Blood. 103, 1089-1098 (2004).</li><li>Sakagami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+GM-CSF+autoantibodies+and+reproduction+of+pulmonary+alveolar+proteinosis.">Human GM-CSF autoantibodies and reproduction of pulmonary alveolar proteinosis.</a> N Engl J Med. 361, 2679-2681 (2009).</li><li>Raines, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+molecular+cloning+of+a+soluble+human+granulocyte-macrophage+colony-stimulating+factor+receptor.">Identification and molecular cloning of a soluble human granulocyte-macrophage colony-stimulating factor receptor.</a> Proc Natl Acad Sci U S A. 88, 8203-8207 (1991).</li><li>Williams, W., VonFeldt, J., Rosenbaum, H., Ugen, K., Weiner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+a+soluble+form+of+the+granulocyte-macrophage+colony-stimulating+factor+receptor+alpha+chain+from+a+myelomonocytic+cell+line.+Expression,+biologic+activity,+and+preliminary+analysis+of+transcript+distribution.">Molecular cloning of a soluble form of the granulocyte-macrophage colony-stimulating factor receptor alpha chain from a myelomonocytic cell line. Expression, biologic activity, and preliminary analysis of transcript distribution.</a> Arthritis Rheum. 37, 1468-1478 (1994).</li><li>Prevost, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-macrophage+colony-stimulating+factor+(GM-CSF)+and+inflammatory+stimuli+up-regulate+secretion+of+the+soluble+GM-CSF+receptor+in+human+monocytes:+evidence+for+ectodomain+shedding+of+the+cell+surface+GM-CSF+receptor+alpha+subunit.">Granulocyte-macrophage colony-stimulating factor (GM-CSF) and inflammatory stimuli up-regulate secretion of the soluble GM-CSF receptor in human monocytes: evidence for ectodomain shedding of the cell surface GM-CSF receptor alpha subunit.</a> J Immunol. 169, 5679-5688 (2002).</li><li>Iizuka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+recombinant+mouse+monoclonal+antibody+in+transgenic+silkworm+cocoons.">Production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons.</a> FEBS J. 276, 5806-5820 (2009).</li><li>Iizuka, M., Tomita, M., Shimizu, K., Kikuchi, Y., Yoshizato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+enhancement+of+recombinant+protein+synthesis+in+transgenic+silkworms+by+a+5'-untranslated+region+of+polyhedrin+gene+of+Bombyx+mori+Nucleopolyhedrovirus.">Translational enhancement of recombinant protein synthesis in transgenic silkworms by a 5'-untranslated region of polyhedrin gene of Bombyx mori Nucleopolyhedrovirus.</a> J Biosci Bioeng. 105, 595-603 (2008).</li><li>Zou, W., Ueda, M., Yamanaka, H., Tanaka, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+combinatorial+protein+library+displayed+on+yeast+cell+surface+using+DNA+random+priming+method.">Construction of a combinatorial protein library displayed on yeast cell surface using DNA random priming method.</a> J Biosci Bioeng. 92, 393-396 (2001).</li><li>Tamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+transformation+of+the+silkworm+Bombyx+mori+L.+using+a+piggyBac+transposon-derived+vector.">Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.</a> Nat Biotechnol. 18, 81-84 (2000).</li><li>Tomita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+silkworms+produce+recombinant+human+type+III+procollagen+in+cocoons.">Transgenic silkworms produce recombinant human type III procollagen in cocoons.</a> Nat Biotechnol. 21, 52-56 (2003).</li><li>Ogawa, S., Tomita, M., Shimizu, K., Yoshizato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+transgenic+silkworm+that+secretes+recombinant+proteins+in+the+sericin+layer+of+cocoon:+production+of+recombinant+human+serum+albumin.">Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: production of recombinant human serum albumin.</a> J Biotechnol. 128, 531-544 (2007).</li><li>Tomita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+germline+transgenic+silkworm+that+secretes+recombinant+proteins+in+the+sericin+layer+of+cocoon.">A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon.</a> Transgenic Res. 16, 449-465 (2007).</li><li>Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+pulmonary+alveolar+proteinosis+as+an+autoimmune+disease+with+neutralizing+antibody+against+granulocyte/macrophage+colony-stimulating+factor.">Idiopathic pulmonary alveolar proteinosis as an autoimmune disease with neutralizing antibody against granulocyte/macrophage colony-stimulating factor.</a> J Exp Med. 190, 875-880 (1999).</li><li>Tanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lungs+of+patients+with+idiopathic+pulmonary+alveolar+proteinosis+express+a+factor+which+neutralizes+granulocyte-macrophage+colony+stimulating+factor.">Lungs of patients with idiopathic pulmonary alveolar proteinosis express a factor which neutralizes granulocyte-macrophage colony stimulating factor.</a> FEBS Lett. 442, 246-250 (1999).</li><li>Brown, C., Pihl, C., Murray, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomerization+of+the+soluble+granulocyte-macrophage+colony-stimulating+factor+receptor:+identification+of+the+functional+ligand-binding+species.">Oligomerization of the soluble granulocyte-macrophage colony-stimulating factor receptor: identification of the functional ligand-binding species.</a> Cytokine. 9, 219-225 (1997).</li><li>Urano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-free+assay+to+estimate+the+neutralizing+capacity+of+granulocyte-macrophage+colony-stimulating+factor+autoantibodies.">A cell-free assay to estimate the neutralizing capacity of granulocyte-macrophage colony-stimulating factor autoantibodies.</a> J Immunol Methods. 360, 141-148 (2010).</li><li>Hayashida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+a+second+subunit+of+the+receptor+for+human+granulocyte-macrophage+colony-stimulating+factor+(GM-CSF):+reconstitution+of+a+high-affinity+GM-CSF+receptor.">Molecular cloning of a second subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF): reconstitution of a high-affinity GM-CSF receptor.</a> Proc Natl Acad Sci U S A. 87, 9655-9659 (1990).</li><li>Hansen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+GM-CSF+receptor+complex+reveals+a+distinct+mode+of+cytokine+receptor+activation.">The structure of the GM-CSF receptor complex reveals a distinct mode of cytokine receptor activation.</a> Cell. 134, 496-507 (2008).</li></ol>

We have nothing to disclose.

The cell-free assay estimated the neutralizing capacity of GM-CSF autoantibodies with excellent reproducibility and rapidity. The binding inhibition by GM-CSF autoantibodies or the patient's serum IgG fractions was evaluated by this assay. The data showed a correlation between the binding inhibition of the cell-free assay and the growth inhibition of a bioassay using TF-1 cells, respectively. The bioassay has been widely utilized, but harbored difficulties in comparing data between different facilities and different time...

<table>
	<tr>
		<td>Name of reagent</td>
		<td>Company</td>
		<td>Catalog #</td>
		<td>Comments</td>
	</tr>
	<tr>
		<td>human placenta cDNA library</td>
		<td>Takara</td>
		<td></td>
		<td></td>
	</tr>
	<tr>
		<td>Nickel affinity column</td>
		<td>GE Healthcare</td>
		<td>17-5247-01</td>
		<td></td>
	</tr>
	<tr>
		<td>biotin hydrazide (EZ-Link Biotin Hydrazide)</td>
		<td>PIERCE</td>
		<td>21339</td>
		<td></td>
	</tr>
	<tr>
		<td>rhGM-CSF (leukine)</td>
		<td>Genzyme Corporation</td>
		<td></td>
		<td></td>
	</tr>
	<tr>
		<td>Nunc Immobilizer Amino</td>
		<td>Nalge Nunc International</td>
		<td>436007</td>
		<td></td>
	</tr>
	<tr>
		<td>Monoclonal Anti-polyHistidine antibody produced in mouse</td>
		<td>Sigma-Aldrich</td>
		<td>H1029</td>
		<td>0.2ml</td>
	</tr>
	<tr>
		<td>blocking solution (StabilCoat)</td>
		<td>Surmodics</td>
		<td>SC01-1000</td>
		<td>1000ml</td>
	</tr>
	<tr>
		<td>ZyMAX Streptavidin-AP Conjugate</td>
		<td>Invitrogen</td>
		<td>43-8322</td>
		<td></td>
	</tr>
	<tr>
		<td>CDP-Star Ready-to-Use With Sapphire-II</td>
		<td>Applied Biosystems</td>
		<td>T2214</td>
		<td></td>
	</tr>
	<tr>
		<td>chemiluminescence plate reader</td>
		<td>BERTHOLD TECHNOLOGIES</td>
		<td>TriStar LB 941</td>
		<td></td>
	</tr>
</table>

a cell free assay system estimating the neutralizing capacity of gm-csf antibody using recombinant soluble gm-csf receptor

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease severity in patients with autoimmune pulmonary alveolar proteinosis (PAP)1-3. As abrogation of GM-CSF bioactivity in the lung is the likely cause for autoimmune PAP4,5, it is promising to measure the neutralizing capacity of GM-CSF autoantibodies for evaluating the disease severity in each patient with PAP.
Until now, neutralizing capacity of GM-CSF autoantibodies has been assessed by evaluating the growth inhibition of human bone marrow cells or TF-1 cells stimulated with GM-CSF6-8. In the bioassay system, however, it is often problematic to obtain reliable data as well as to compare the data from different laboratories, due to the technical difficulties in maintaining the cells in a constant condition.
OBJECTIVE: To mimic GM-CSF binding to GM-CSF receptor on the cell surface using cell-free receptor-binding-assay.
METHODS: Transgenic silkworm technology was applied for obtaining a large amount for recombinant soluble GM-CSF receptor alpha (sGMR&alpha;) with high purity9-13. The recombinant sGMR&alpha; was contained in the hydrophilic sericin layers of silk threads without being fused to the silk proteins, and thus, we can easily extract from the cocoons in good purity with neutral aqueous solutions14,15. Fortunately, the oligosaccharide structures, which are critical for binding with GM-CSF, are more similar to the structures of human sGMR&alpha; than those produced by other insects or yeasts.
RESULTS: The cell-free assay system using sGMR&alpha; yielded the data with high plasticity and reliability. GM-CSF binding to sGMR&alpha; was dose-dependently inhibited by polyclonal GM-CSF autoantibody in a similar manner to the bioassay using TF-1 cells, indicating that our new cell-free assay system using sGMR&alpha; is more useful for the measurement of neutralizing activity of GM-CSF autoantibodies than the bioassay system using TF-1 cell or human bone marrow cells.
CONCLUSIONS: We established a cell-free assay quantifying the neutralizing capacity of GM-CSF autoantibody.

Watch this Scientific Journal Video about A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor at JoVE.com

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

We designed a cell-free receptor binding assay in order to estimate the binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to the receptors. It enables us to evaluate competitive inhibition of biotinylated GM-CSF binding to soluble GM-CSF receptor alpha by GM-CSF autoantibody with excellent reproducibility.

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

a-cell-free-assay-system-estimating-neutralizing-capacity-gm-csf

Research

JoVE Journal

Immunology and Infection

12.6K Views.  Niigata University Medical and Dental Hospital. We designed a cell-free receptor binding assay in order to estimate the binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to the receptors. It enables us to evaluate competitive inhibition of biotinylated GM-CSF binding to soluble GM-CSF receptor alpha by GM-CSF autoantibody with excellent reproducibility.

Video: A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in na&#239;ve human populations.

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The increasing number of SARS-CoV-2 infections raises the need for better antibody detection assays. Current antibody detection methods compromise sensitivity for speed or are sensitive but time-consuming. A large proportion of SARS-CoV-2-neutralizing antibodies target the receptor-binding domain (RBD), one of the primary immunogenic compartments of SARS-CoV-2. We have recently designed and developed a highly sensitive, bioluminescent-tagged RBD (NanoLuc HiBiT-RBD) to detect SARS-CoV-2 antibodies. The following text describes the procedure to produce the HiBiT-RBD complex and a fast assay to evaluate the presence of RBD-targeting antibodies using this tool. Due to the durability of the HiBiT-RBD protein product over a wide range of temperatures and the shorter experimental procedure that can be completed within 1 h, the protocol can be considered as a more efficient alternative to detect SARS-CoV-2 antibodies in patient serum samples.

The outlined protocol describes the procedure for producing the HiBiT-receptor-binding domain protein complex and its application for fast and sensitive detection of SARS-CoV-2 antibodies.

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...