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 ...

<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 Görüntüleme.  Niigata University Medical and Dental Hospital. Hücre reseptör bağlayıcı tahlil reseptörlerine granülosit-makrofaj koloni uyarıcı faktör (GM-CSF) bağlama tahmin etmek için tasarlanmıştır. Bu mükemmel tekrarlanabilirlik ile GM-CSF otoantikor çözünen GM-CSF reseptör alfa bağlayıcı biotinlenmiş GM-CSF yarışmalı inhibisyonu değerlendirmek için bize sağlar.