Name: production of nurr-1 specific polyclonal antibodies free of cross-reactivity against its close homologs, nor1 and nur77
Uploaded: 2015-08-17T14:00:00.000+00:00
Duration: 12 min 30 s
Description: Watch this Scientific Journal Video about Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77 at JoVE.com

This work was supported by NIH grants (NS070577 and NS084869).

Note: The composition of all solutions cited below can be found in the Materials/Equipment Table.
1. Protein A Column Antibody Purification
<ol>
	<li>Equilibrate a protein A spin column and all required buffers to room temperature for 15 min prior to starting the procedure.</li>
	<li>Dilute the immune serum 1:1 with Protein A IgG Binding Buffer. (5 ml of serum is recommended).</li>
	<li>Gently tap a protein A spin column on the bench top to dislodge any resin that may be lodged in the cap. Remove the top cap and gently snap off the bottom closure. Place the column in a 15 ml collection tube and allow storage solution to drain.</li>
	<li>Add 5 ml of Protein A IgG Binding Buffer and allow the solution to drain.</li>
	<li>Apply the diluted immune serum to the column and collect the flow-through. For best results, add a sample volume that contains a concentration of antibodies less than 80% of the column&#8217;s antibody-binding capacity.</li>
	<li>Wash the column with 15 ml of Protein A IgG Binding Buffer or until the absorbance at 280 nm is below 0.1.</li>
	<li>Add 100 &#181;l of Neutralization Buffer to five labeled collection tubes.</li>
	<li>Add 5 ml of IgG Elution Buffer to the Protein A column and collect 1 ml fractions in each of the tubes containing the 100 &#181;l of Neutralization Buffer.</li>
	<li>Measure the absorbance of each fraction at 280 nm and pool fractions with an absorbance greater than 0.5. Keep the pooled fractions on ice until ready for dialysis.</li>
	<li>Regenerate the column by adding 8 ml of IgG Elution Buffer and allow the solution to flow through the column.</li>
	<li>Rinse the column with Protein A IgG Binding solution until the eluent pH returns to 7.4.</li>
	<li>Store the column by adding 5 ml of storage solution (0.02% sodium azide in PBS). When approximately 3 ml remain in the column, cap bottom and secure the top cap. Store the column at 4 &#176;C.</li>
	<li>Determine the length of dialysis tubing needed according to the manufacturer&#8217;s instructions. Using 16 mm flat diameter tubing use 5.47 cm of tube length per ml of solution to be dialyzed. Cut the tubing and rinse with PBS pH 7.4 for 15 to 30 min.</li>
	<li>Fold and clip one end of the dialysis tubing, transfer the pooled IgG containing fractions to dialysis tubing equilibrated in PBS pH 7.4 and close the other end.</li>
	<li>Dialyze at room temperature against 200 volumes of PBS pH 7.4 for 2 hr.</li>
	<li>Change the dialysis buffer (fresh PBS pH 7.4) and dialyze for another 2 hr at room temperature.</li>
	<li>Change the dialysis buffer (fresh PBS pH 7.4) and dialyze overnight at 4 &#176;C.</li>
	<li>Keep the dialyzed antibody solution at 4 &#176;C until ready to proceed with further purification steps.</li>
	<li>Assess the antibody concentration using its absorbance at 280 nm and the molar absorption coefficient of antibodies at 280 nm of 210,000 M-1 cm-112.</li>
</ol>
2. Coupling of LBD Proteins to AminoLink Coupling Resin
<ol>
	<li>Prepare two columns, one for Nor1 and the other for Nur77. Follow the same protocol for both proteins.</li>
	<li>Thaw the protein to be coupled to the resin on ice. Determine the protein concentration using its molar extinction coefficient and its absorbance at 280 nm12.
	<ol>
		<li>Alternatively, use a protein determination assay such as the bicinchoninic acid assay13. If the protein is dissolved in an amine-containing buffer dialyze or buffer exchange against the Coupling Buffer. If the protein is in a suitable buffer (no free amines) dilute it 4-fold in Coupling Buffer.</li>
	</ol>
	</li>
	<li>Use 2 ml of resin for 2 mg of protein. All steps that follow are for 2 ml resin in a 10 ml column. Up to 10 mg of protein can be linked per 1 ml of resin (2 ml of 1:1 slurry).</li>
	<li>Prepare a 10 ml column by pushing a frit to the bottom and running PBS pH 7.4 through it. Cap the bottom of the column and add 2 ml of Coupling Buffer.</li>
	<li>Add the desired volume of slurry (4 ml to obtain 2 ml of resin) to the column, remove the bottom cap and let the column drain. Rinse the column with a total of 6 ml (3 resin-bed volume) of Coupling Buffer and allow the column to drain. After the Coupling buffer has drained, replace the bottom cap.</li>
	<li>Add 2-4 ml of protein suspended in Coupling Buffer to the column (keep an aliquot of the protein solution to assess the coupling efficiency by comparing the protein concentration of the eluate in step 2.11 using either the absorbance at 280 nm or the bicinchoninic assay). Cap and mix end over end to have a homogeneous solution.</li>
	<li>Weigh an empty 1.8 ml microcentrifuge tube and move to the fume hood. Carefully transfer NaCNBH3 (about 0.05 g) in the pre-weighed tube. Close the tube and weigh the tube containing NaCNBH3. Do not open the tube outside the hood.</li>
	<li>Based on the weight of NaCNBH3 transferred into the tube, make a 5 M solution by adding 1 M NaOH. The molecular weight of NaCNBH3 is 62.84 g therefore 0.05 g is equal to (0.05/62.84 = 7.96 x 10-4 moles) 7.96 x 10-4 moles. To make a 5 M solution add (7.96 x 10-4 /5) 159 &#181;l of 1 M NaOH.</li>
	<li>Measure the reaction&#8217;s total volume taking the resin volume into consideration. Add 10 &#181;l of 5 M NaCNBH3 per ml of reaction.
	<ol>
		<li>For example: for a 4 ml reaction volume add 40 &#181;l of 5 M NaCNBH3. For 1 ml of resin and 3 ml of added protein, the total volume is 4 ml.</li>
	</ol>
	</li>
	<li>Cap the column and mix end over end. Secure the column on a tube rotator and let the reaction continue overnight at 4 &#176;C.</li>
	<li>In the morning, bring the column to the chemical hood and carefully remove the cap as some gas may have formed. Drain the column into a clean tube and save the eluate to measure the protein concentration using the absorbance at 280 nm method12 or the bicinchoninic acid assay and compare it with the starting protein concentration in step 2.6.</li>
	<li>Wash the resin with 4 ml of Coupling Buffer and let the column drain.</li>
</ol>
3. Blocking Remaining Sites
<ol>
	<li>Wash the resin with 4 ml of quenching buffer. Drain and replace the bottom cap. Add 2 ml of quenching buffer and suspend the resin.</li>
	<li>Assess the total reaction volume by measuring the resin volume and the quenching buffer volume then add 10 &#181;l of 5 M NaCNBH3 per ml of reaction volume.</li>
	<li>Mix gently at room temperature for 30 min end-over-end. After 30 min, bring the column in the fume hood. Carefully remove the top and then remove the bottom cap and let the column drain into a waste tube.</li>
	<li>Wash the column with 5 resin volumes of Wash solution. Monitor the washes for the presence of residual proteins by measuring the eluate&#8217;s absorbance at 280 nm. Uncoupled proteins are washed out by the high salt concentration.</li>
	<li>Wash the resin with 6 ml of degassed PBS pH 7.4 containing 0.02% sodium azide. Keep the column at 4 &#176;C until needed.</li>
</ol>
4. Purification of Nurr1 Specific Antibodies
<ol>
	<li>Rinse the Nur77 LBD coupled column with 10 ml of PBS pH 7.4 and let the column drain. Cap the bottom of the column and place the drained Nur77 LBD coupled column in a new 15 ml collection tube.</li>
	<li>Add 5 ml of protein A purified anti-Nurr1 antibodies, cap the column and place on a rotator at 4 &#176;C for 1 hr. Remove the top and bottom caps and collect the flow through. Set aside on ice until ready to add to the Nor1 LBD coupled column.</li>
	<li>Rinse the Nor1 LBD coupled column with 10 ml of PBS pH 7.4 and let the column drain. Cap the bottom of the column and place the Nor1 LBD coupled column in a 15 ml collection tube.</li>
	<li>Add the flow through from the Nur77 LBD coupled column, cap the column and place on a rotator at 4 &#176;C for 1 hour. Remove the top and bottom caps and collect the flow through.</li>
	<li>Measure the antibody concentration by measuring the absorbance at 280 nm.</li>
</ol>
5. ELISA-based Analysis of Purified Anti-Nurr1 Antibody
<ol>
	<li>Add 100 &#181;l of protein (2.5 &#181;g/ml) to the wells of a 96 well plate. If testing 3 different proteins, add 100 &#181;l of protein 1 to wells A1 to H4, 100 &#181;l of protein 2, to wells A5 to H8 and 100 &#181;l of protein 3 to wells A9 to H12. Cover the plate and incubate overnight at 4 &#176;C.</li>
	<li>Wash the coated wells twice with 300 &#181;l per well of PBS pH 7.4 and add 300 &#181;l of ELISA blocking buffer to the entire antigen coated wells and incubate 2 hours at room temperature.</li>
	<li>While the plate is blocking prepare a desired starting dilution of purified anti-Nurr1 antibody (ranging from 10 to 100 &#181;g/ml) in ELISA blocking buffer.</li>
	<li>After 2 hr of blocking, replace the ELISA block buffer with 100 &#181;l of fresh ELISA block buffer to all wells.</li>
	<li>Add 100 &#181;l of diluted purified anti-Nurr1 antibody to all the wells in row A. Serially dilute the antibodies by transferring 100 &#181;l of solution from row A to the wells in row B followed by transferring 100 &#181;l of solution from row B to row C continuing down to row G. After mixing the solutions in wells in row G discard the extra 100 &#956;l. Wells in row H only receive the initial 100 &#181;l of blocking buffer. Incubate at room temperature for 1 hr.</li>
	<li>Wash the plate three (3) times with 300 &#181;l per well of 0.05% Tween 20 in PBS pH 7.4 and blot the plate to remove excess wash buffer.</li>
	<li>Add 100 &#181;l of Horse Radish Peroxidase (HRP) conjugated Goat anti-rabbit IgG diluted in ELISA block buffer (1:8,000 dilution; the typical range is from 1:5,000 to 1:25,000). Incubate at room temperature for 1 hr.</li>
	<li>Wash the plate three (3) times as described in 5.6. Rinse all wells by filling them with 300 &#956;l of deionized water. Blot excess water by inverting the plate onto absorbing paper.</li>
	<li>Add 100 &#181;l of 3, 3&#8217;, 5, 5&#8217; tetramethylbenzidine (warmed to room temperature) to all wells and incubate 15 to 30 min&#160;in the dark at room temperature.</li>
	<li>Stop the reaction by adding 50 &#181;l of 2 N H2SO4.</li>
	<li>Read absorbance at 450 nm subtracting background at 650 nm.</li>
</ol>

<ol>
	<li>Hawk, J. D., Abel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+NR4A+transcription+factors+in+memory+formation.">The role of NR4A transcription factors in memory formation.</a> Brain Res. Bull. 85 (1-2), 21-29 (2011).</li><li>Law, S. W., Conneely, O. M., DeMayo, F. J., O'Malley, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+new+brain-specific+transcription+factor,+NURR1.">Identification of a new brain-specific transcription factor, NURR1.</a> Mol. Endocrinol. 6 (12), 2129-2135 (1992).</li><li>Zetterstr&#246;m, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+neuron+agenesis+in+Nurr1-deficient+mice.">Dopamine neuron agenesis in Nurr1-deficient mice.</a> Science. 276 (5310), 248-250 (1997).</li><li>Kadkhodaei, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nurr1+is+required+for+maintenance+of+maturing+and+adult+midbrain+dopamine+neurons.">Nurr1 is required for maintenance of maturing and adult midbrain dopamine neurons.</a> J. Neurosci. 29 (50), 15923-15932 (2009).</li><li>Decressac, M., Volakakis, N., Bj&#246;rklund, A., Perlmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NURR1+in+Parkinson+disease--from+pathogenesis+to+therapeutic+potential.">NURR1 in Parkinson disease--from pathogenesis to therapeutic potential.</a> Nat Rev Neurol. 9 (11), 629-636 (2013).</li><li>Pe&#241;a de Ortiz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+Expression+of+the+Orphan+Nuclear+Receptor+Gene+hzf-3/nurr1+during+Spatial+Discrimination+Learning.">Hippocampal Expression of the Orphan Nuclear Receptor Gene hzf-3/nurr1 during Spatial Discrimination Learning.</a> Neurobiol Learn Mem. 74 (2), 161-175 (2000).</li><li>Vecsey, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+inhibitors+enhance+memory+and+synaptic+plasticity+via+CREB:CBP-dependent+transcriptional+activation.">Histone deacetylase inhibitors enhance memory and synaptic plasticity via CREB:CBP-dependent transcriptional activation.</a> J. Neurosci. 27 (23), 6128-6140 (2007).</li><li>Col&#243;n-Cesario, W. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockdown+of+Nurr1+in+the+rat+hippocampus:+implications+to+spatial+discrimination+learning+and+memory.">Knockdown of Nurr1 in the rat hippocampus: implications to spatial discrimination learning and memory.</a> Learn Mem. 13 (6), 734-744 (2006).</li><li>McQuown, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDAC3+is+a+critical+negative+regulator+of+long-term+memory+formation.">HDAC3 is a critical negative regulator of long-term memory formation.</a> J. Neurosci. 31 (2), 764-774 (2011).</li><li>Hawk, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NR4A+nuclear+receptors+support+memory+enhancement+by+histone+deacetylase+inhibitors.">NR4A nuclear receptors support memory enhancement by histone deacetylase inhibitors.</a> J. Clin. Invest. 122 (10), 3593-3602 (2012).</li><li>Bridi, M. S., Abel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NR4A+orphan+nuclear+receptors+mediate+transcription-dependent+hippocampal+synaptic+plasticity.">The NR4A orphan nuclear receptors mediate transcription-dependent hippocampal synaptic plasticity.</a> Neurobiol Learn Mem. 105, 151-158 (2013).</li><li>Gill, S. C., von Hippel, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+protein+extinction+coefficients+from+amino+acid+sequence+data.">Calculation of protein extinction coefficients from amino acid sequence data.</a> Anal. Biochem. 182 (2), 319-326 (1989).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal. Biochem. 150 (1), 76-85 (1985).</li><li>Zetterstr&#246;m, R. H., Williams, R., Perlmann, T., Olson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+expression+of+the+immediate+early+transcription+factors+Nurr1+and+NGFI-B+suggests+a+gene+regulatory+role+in+several+brain+regions+including+the+nigrostriatal+dopamine+system.">Cellular expression of the immediate early transcription factors Nurr1 and NGFI-B suggests a gene regulatory role in several brain regions including the nigrostriatal dopamine system.</a> Brain Res. Mol. Brain Res. 41 (1-2), 111-120 (1996).</li><li>Xiao, Q., Castillo, S. O., Nikodem, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+messenger+RNAs+for+the+orphan+nuclear+receptors+Nurr1+and+Nur77+(NGFI-B)+in+adult+rat+brain+using+in+situ+hybridization.">Distribution of messenger RNAs for the orphan nuclear receptors Nurr1 and Nur77 (NGFI-B) in adult rat brain using in situ hybridization.</a> Neuroscience. 75 (1), 221-230 (1996).</li></ol>

The authors have nothing to disclose.

The success of this protocol relies on the availability of pure proteins for characterization of the antibodies raised against a specific member of the protein family of interest. There is no need to purify the antibodies using protein A/G until it is clearly established that there are significant cross-reactivities by ELISA and Western blotting.
As stated in the text, it is important to avoid suspending the protein(s) to be cross-linked to the column in an amine-containing buffer such as Tris...

The transcription factor Nurr1 and its homologs (Nur77 and Nor1) belong to the nuclear receptor subfamily 4A (NR4A)1. They are also orphan receptors because their endogenous ligands are not identified yet. Nurr1 was first cloned in 1992 and although known to be expressed in the brain2, its essential role for development and maintenance of midbrain dopamine neurons was revealed by knockout mouse studies3. In addition, its important role for maintenance of midbrain dopamine neurons was recently demonstrated by a conditional knockout study4. Due to these elegant studies showing Nurr1&#8217;s critical roles for midbrain dopamine neurons, many subsequent studies have largely focused on the midbrain dopamine neurons and dopamine-related neurodegenerative disorder, Parkinson&#8217;s disease (PD)5.
Notably, Nurr1 is expressed not only in the midbrain dopamine (mDA) neurons, but also in diverse brain areas2, suggesting that it may have functional roles in many non-DA areas, which is strongly supported by more recent studies showing that Nurr1 plays important roles in various brain functions. For examples, it was shown that memory-inducing activities such as learning, or other hippocampus-dependent tasks result in up-regulation of Nurr1 expression in the hippocampus6,7. In addition, knock down of Nurr1 expression in the hippocampus was sufficient to impair long-term memory and/or synaptic plasticity8-11, strongly suggesting that Nurr1 plays diverse roles in many brain areas. Thus, to further understand the cell-type-specific and subcellular expression of Nurr1, it is desirable to use Nurr1-specific antibodies, which do not exhibit any cross-reactivity to its homologs Nur77 or Nor1. This paper describes a pre-adsorption protocol to generate Nurr1-specific antibodies and present additional data showing its specificity.

<table><tbody><tr><td>Purified Ligand Binding Domain from Nurr1, Nor 1 and Nur77</td><td></td><td></td><td>Column Storage solution</td></tr><tr><td>AminoLink Coupling Resin and Kit</td><td>Thermo Scientific</td><td>44890</td><td></td></tr><tr><td>Protein A spin column</td><td>Thermo Scientific</td><td>89978</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>96 well Flate-bottom plates, High Flange, 330 &amp;mu;l</td><td>Thermo Scientific</td><td>3455</td><td></td></tr><tr><td>10% Normal Goat Serum</td><td>KPL</td><td>50-675-69</td><td></td></tr><tr><td>Milk Diluent/ Blocking Concentrate&amp;nbsp;</td><td>KPL</td><td>50-82-01</td><td></td></tr><tr><td>IgG Elution Buffer</td><td>Thermo Scientific</td><td>21004</td><td></td></tr><tr><td>Micro BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23235</td><td></td></tr><tr><td>Horse Radish Peroxidase conjugated Goat anti-Rabbit IgG (H+L)</td><td>Thermo Scientific</td><td>31460</td><td></td></tr><tr><td>Ni-NTA Resin</td><td>Thermo Scientific</td><td>88221</td><td></td></tr><tr><td>3, 3&#039;, 5, 5&#039; Tetramethylbenzydine</td><td>Thermo Scientific</td><td>34028</td><td></td></tr><tr><td>2N H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Macron Fine Chemicals</td><td>H381 05</td><td></td></tr><tr><td>Protein A IgG Binding Buffer</td><td>Thermo Scientific</td><td>21001</td><td></td></tr><tr><td>IgG Elution Buffer</td><td>Thermo Scientific</td><td>21004</td><td></td></tr><tr><td>Column Storage solution</td><td></td><td></td><td>Phosphate Buffered Saline containing 0.02% sodium azide</td></tr><tr><td>Spectra/Por 7 pre-treated dialysis tubing</td><td>Spectrum Labs</td><td>132128</td><td></td></tr><tr><td>10 ml Disposable columns</td><td>Thermo Scientific</td><td>29924</td><td></td></tr><tr><td>AminoLink Coupling Buffer</td><td></td><td></td><td>0.1M sodium phosphate, 0.05% NaN3, pH 7.0</td></tr><tr><td>Quenching buffer</td><td></td><td></td><td>1M Tris. HCI, 0.05% NaN3, pH&lt;br/&gt; 7.4</td></tr><tr><td>Wash Solution</td><td></td><td></td><td>1M NaCl, 0.05% NaN3</td></tr><tr><td>ELISA Blocking buffer</td><td></td><td></td><td>1% Normal Horse serum (NHS), 1% Normal Goat Serum (NGS) in 1X KPL milk diluent</td></tr><tr><td>Storage Solution</td><td></td><td></td><td>PBS pH 7.4 containing 0.02% sodium azide</td></tr><tr><td>Micropipettes: 10 &amp;mu;l; 20 &amp;mu;l; 200 &amp;mu;l; 1000 &amp;mu;l</td><td></td><td></td><td></td></tr></tbody></table>

production of nurr-1 specific polyclonal antibodies free of cross-reactivity against its close homologs, nor1 and nur77

The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The nuclear receptor related 1 protein (Nurr1 or NR4A2) plays a key role in the maintenance of the dopaminergic system. Dopamine dysfunctions associated with the Nurr1 gene include Parkinson&#8217;s disease, schizophrenia and manic depression among others. Furthermore, recent evidence indicates that Nurr1 is also expressed in other brain areas such as the hippocampus and plays critical roles for learning and memory. The other members of the family are nerve growth factor IB (Nur77 or NR4A1) and neuron-derived orphan receptor 1 (NOR1 or NR4A3). To help investigate the precise functional roles of Nurr1 in dopaminergic and other brain region-related neuronal dysfunctions antibodies devoid of cross-reactivities against Nur77 and NOR1 were needed. Since the proteins are more divergent in their LBDs than in their DNA binding domains immunization with purified LBDs should yield antibodies specific for Nurr1 with minimal reactivities against Nur77 and/or NOR1. Although anti-Nurr1 antibodies were successfully generated these showed significant immunoreactivity against the other members of the family. Affinity chromatography over immobilized Protein A followed by pre-adsorption against immobilized Nur77 and NOR1 LBDs yielded Nurr1 specific antibodies free of cross-reactivity. Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption. The goal of the protocol is to increase polyclonal antibodies specificity through pre-adsorption against cross-reactive antigens.

A comparison of Protein A column purified Nurr1-specific antibodies with Protein A purified anti-Nurr1 antibodies followed by passage through Nur77 LBD and Nor1 LBD columns is shown in Figure 1. As can be seen, Protein A-purified Nurr1 antibody exhibited a strong binding to Nurr1 LBD. However, it also showed significant binding to Nur77 LBD and to Nor1 LBD. When Protein A-purified Nurr1 antibodies were further purified against Nur77 LBD and Nor1 LBD, the final affinity purified Nurr1 antibodies exhibited...

Watch this Scientific Journal Video about Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77 at JoVE.com

Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77

Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption.

The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The ...

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7.4K Views.  McLean Hospital. Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption.

Video: Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Medicine

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

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

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

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

Immunology and Infection

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug&#39;s pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.
In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-na&#239;ve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The ...

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate (NMDA) Receptor in Blood

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune encephalitis. Detection of the specific autoantibody against the NMDA receptor in the blood or cerebrospinal fluid (CSF) is essential for the accurate diagnosis of this condition. The NMDA receptor is an ion channel protein complex that contains four subunits, including two mandatory NMDA receptor subunit 1 (NR1) and one or two NMDA receptor subunit 2A (NR2A), NMDA receptor subunit 2B (NR2B), NMDA receptor subunit 2C (NR2C), or NMDA receptor subunit 2D (NR2D). The epitope of anti-NMDA receptor autoantibody was reported to be present at the extracellular N-terminal domain of the NR1 subunit of the NMDA receptor. The goal of this study is to develop a simple cell-based immunofluorescence assay that can be used as a screening test to detect the presence of autoantibodies against NR1 subunit of the NMDA receptor in the blood to facilitate the clinical and basic research of anti-NMDA receptor autoimmune encephalitis.

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood

We ectopically expressed NR1 subunit of NMDA receptor tagged with green fluorescent protein in human embryonic cells (HEK293) as antigen to detect autoantibodies against NMDA receptor in the blood of patients suspected with autoimmune encephalitis. This simple method may be suitable for screening purposes in clinical settings.

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune ...

Neuroscience

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells is regulated by their expression of germline-encoded inhibitory and activating receptors. The engagement of these receptors by their cognate ligands on target cells determines whether the intercellular interaction will result in NK cell killing. This protocol details the design and optimization of two complementary mass cytometry (CyTOF) panels. One panel was designed to phenotype NK cells based on receptor expression. The other panel was designed to interrogate expression of known ligands for NK cell receptors on several immune cell subsets. Together, these two panels allow for the profiling of the human NK cell receptor-ligand repertoire. Furthermore, this protocol also details the process by which we stain samples for CyTOF. This process has been optimized for improved reproducibility and standardization. An advantage of CyTOF is its ability to measure over 40 markers in each panel, with minimal signal overlap, allowing researchers to capture the breadth of the NK cell receptor-ligand repertoire. Palladium barcoding also reduces inter-sample variation, as well as consumption of reagents, making it easier to stain samples with each panel in parallel. Limitations of this protocol include the relatively low throughput of CyTOF and the inability to recover cells after analysis. These panels were designed for the analysis of clinical samples from patients suffering from acute and chronic viral infections, including dengue virus, human immunodeficiency virus (HIV), and influenza. However, they can be utilized in any setting to investigate the human NK cell receptor-ligand repertoire. Importantly, these methods can be applied broadly to the design and execution of future CyTOF panels.

Here we design two complementary mass cytometry (CyTOF) panels and optimize a CyTOF staining protocol with the aim of profiling the natural killer cell receptor and ligand repertoire in the setting of viral infections.

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells ...

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response without any interference such as low protein expression, enzyme inactivity, or structural changes. This makes APN an attractive candidate in the development of vaccines that selectively target the mucosal epithelium. Previous studies have shown that APN is a receptor protein for both enterotoxigenic Escherichia coli (E. coli) F4 and transmissible gastroenteritis virus. Thus, APN shows promise in the development of antibody-drug conjugates or novel vaccines based on APN-specific antibodies. In this study, we compared production of APN-specific monoclonal antibodies (mAbs) using traditional hybridoma technology and recombinant antibody expression method. We also established a stably transfected Chinese hamster ovary (CHO) cell line using pIRES2-ZSGreen1-rAbs-APN and an E. coli expression BL21(DE3) strain harboring the pET28a (+)-rAbs-APN vector. The results show that antibodies expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and mAbs produced using hybridomas could recognize and bind to the APN protein. This provides the basis for further elucidation of the APN receptor function for the development of therapeutics targeting different APN-specific epitopes.

The recombinant antibody protein expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and monoclonal antibodies produced using traditional hybridoma technology can recognize and bind to the porcine aminopeptidase N (APN) protein.

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response ...

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions.
The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

A Hybridoma Technology for Producing Monoclonal Antibodies against a Metallopeptidase

Source: Xia, P., et al. <a href="https://app.jove.com/v/62437">Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium.</a> J. Vis. Exp. (2021).
This video demonstrates an assay to produce monoclonal antibodies against porcine aminopeptidase N (APN), a membrane-bound metallopeptidase, using a traditional hybridoma technology. Upon immunizing a mouse with APN, the spleen cells are harvested and fused with immortal myeloma cells to produce hybridomas. The hybridomas are then selected and proliferated using the HAT (hypoxanthine-aminopterin-thymidine) medium to produce monoclonal antibodies specific to different APN epitopes.

Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77

Summary

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Chapters in this video

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

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

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Generation of Monoclonal Antibodies Against Natural Products

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate (NMDA) Receptor in Blood

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

A Hybridoma Technology for Producing Monoclonal Antibodies against a Metallopeptidase