eoe sub articles

EoE Sub Articles

1. Production of &#945;DEC-205 from the hybridoma cell line NLDC-145
<ol>
	<li>For antibody production, thaw cryopreserved NLDC-145 cells producing &#945;DEC-205 at 37 &#176;C in a water bath. Expand the cells at 37 &#176;C and 5% CO2. Two 75 cm2 bottles will be needed to proceed to antibody production. Cell culture procedures should be performed in a safety cabinet to ensure safe working conditions and prevent contamination of the cultures.
	<ol>
		<li>Resuspend 1 mL of thawed cells (1 x 106 - 5 x 106 cells/mL) in 9 mL of ISF-1 medium supplemented with 1% penicillin/streptomycin (pre-warmed to 37 &#176;C) into a cell culture flask (25 cm2). Place the flask horizontally in a cell culture incubator at 37 &#176;C, 5% CO2.</li>
		<li>Culture the cells at 37 &#176;C and 5% CO2, until 70% confluence. This should normally be achieved after 24 - 48 h.</li>
		<li>Once the cells are 70% confluent, transfer the complete NLDC-145 cell suspension (10 mL) into a 15 mL conical centrifuge tube using a pipette controller with a pipette in a volume range from 1-10 mL. Pellet the cells by centrifugation at 250 x g for 10 min at room temperature.</li>
		<li>Pre-warm ISF-1 medium to 37 &#176;C in a water bath and resuspend the pellet in 12 mL of ISF-1 medium supplemented with 1% penicillin/streptomycin. Transfer the resuspended cell suspension into a fresh cell culture flask (75 cm2).</li>
		<li>Culture and expand the cells in ISF-1 medium supplemented with 1% penicillin/streptomycin at 37 &#176;C and 5% CO2, until 70% confluent and 99% viable. This should normally be achieved after 48 - 72 h.</li>
		<li>Split the cells into two 75 cm2 flasks. To do this first flush the cell culture flask bottom/culture surface with the cell suspension to remove all NLDC-145 cells from the surface. Transfer 6 mL of the NLDC-145 cell suspension each into one of two fresh 75 cm2 bottles and add pre-warmed ISF-1 medium supplemented with 1% penicillin/streptomycin up to 12 mL. 
		NOTE: Do not renew the cell culture medium as the NLDC-145 cells will have conditioned the medium. Transfer the cells together with their medium and fill the culture up to the desired volume with fresh medium. This is crucial for viability and maximum antibody production by the NLDC-145 cells.</li>
		<li>Expand the cell cultures at 37 &#176;C and 5% CO2, until about 70% confluent which is generally achieved after 48 - 72 h.</li>
		<li>Once the cells are 70% confluent, transfer 10 mL of the expanded NLDC-145 cell suspension from each of the 75 cm2 bottles into one PETG (polyethylene terephthalate glycol) roller bottle (1,050 cm2). For this, flush the cell culture flask bottom/culture surface with the cell suspension to remove all cells from the surface using a pipette controller and 10 mL pipette.</li>
		<li>Add 140 mL of ISF-1 medium supplemented with 1% penicillin/streptomycin (pre-warmed to 37 &#176;C) directly out of the medium bottle to each of the NLDC-145 containing roller bottles filling them up to the 150 mL mark. 
		NOTE: See note in step 1.1.6.</li>
		<li>Culture the roller bottles at 37 &#176;C, 5% CO2, and 25 rounds/min for three days.</li>
		<li>Add 150 mL of ISF-1 medium supplemented with 1% penicillin/streptomycin (pre-warmed to 37 &#176;C) to each of the NLDC-145 containing roller bottles filling them up to the 300 mL mark.</li>
		<li>Culture the roller bottles now containing 300 mL culture each at 37 &#176;C, 5% CO2, and 25 rounds/min for another three days.</li>
		<li>Add another 100 mL of ISF-1 medium supplemented with 1% penicillin/streptomycin (pre-warmed to 37 &#176;C) to each of the NLDC-145 containing roller bottles, thereby filling them up to the 400 mL mark.</li>
		<li>Culture the roller bottles now containing 400 mL culture each at 37 &#176;C, 5% CO2, and 25 rounds/min for another seven days. 
		NOTE: During this week, the culture gets very dense (up to 95% density) and viability decreases (down to as little as 50%), enabling maximum antibody release.</li>
	</ol>
	</li>
	<li>For the purification of &#945;DEC-205 from the culture supernatant pour the NLDC-145 cell suspension (from both roller bottles) directly into 500 mL autoclaved centrifugation bottles. 
	NOTE: A total volume of 800 mL of culture should be collected.
	<ol>
		<li>Centrifuge the culture for 30 min at 8,600 x g and 4 &#176;C to remove cells and debris.</li>
		<li>Collect the supernatants, discarding the pellets, and pooling the supernatants in a sterile reagent bottle. 
		NOTE: Purification of &#945;DEC-205 can be commenced immediately (step 2.) or the supernatant can be stored short-term at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Purification of the &#945;DEC-205 antibody from the NLDC-145 cell supernatant
NOTE: From the NLDC-145 cell supernatant, &#945;DEC-205 is purified using a protein G Sepharose column (reusable). The column dimensions are 15 mm x 74 mm and 5 mL protein G Sepharose are packed per column.
<ol>
	<li>For preparation and washing of the protein G Sepharose column, put an airtight rubber plug on the upper opening of the protein G Sepharose column. Puncture the rubber plug with two sterile cannulas (20 G x 1 1/2&#34;, 0.90 x 40 mm).
	<ol>
		<li>Connect a 10 mL syringe to one of the two cannulas and a flexible silicon tube (approximately 100 cm long, 2.5 - 3 mm diameter) with a tubing connector to the second cannula. 
		NOTE: The syringe/rubber plug construction is reusable and provides a vacuum resulting in a continuous flow of the large volume of culture supernatant to the protein G Sepharose column. To this end, slightly pull back the plunger of the syringe to ensure continuous fluid flow in the following steps.</li>
		<li>Wash the column with 50 mL of 0.1 M glacial acetic acid (pH 2) to remove potentially remaining antibody from any previous antibody purification. Put the end of the silicon tube in the 0.1 M glacial acetic acid (pH 2) filled reagent bottle. As a result of the induced vacuum, 50 mL of the 0.1 M glacial acetic acid (pH 2) dropwise run through the protein G Sepharose column. 
		NOTE: 0.1 M glacial acetic acid (pH 2) should be stored in a reagent bottle or freshly filled in a beaker.</li>
		<li>Wash the column with 100-200 mL phosphate-buffered saline (PBS). Put the end of the silicon tube into a PBS-filled reagent bottle or beaker. Let 100-200 mL PBS run dropwise through the protein G Sepharose column.</li>
	</ol>
	</li>
	<li>For antibody purification from the NLDC-145 supernatant, load 800 mL of the NLDC-145 supernatant (obtained from step 1.2.2.) onto the column. Put the end of the silicon tube into the NLDC-145 supernatant-filled reagent bottle. Let 800 mL NLDC-145 supernatant run dropwise through the column.
	<ol>
		<li>Wash the column with 500 mL of PBS. Put the end of the silicon tube in a PBS-filled reagent bottle or beaker. Let 500 mL PBS run dropwise through the column.</li>
		<li>For elution, use 20 1.5 mL tubes and pipette 100 &#181;L of 1.5 M Tris-HCl (pH 8.8) into each 1.5 mL tube. Remove the rubber plug from the column and pipette 1 mL of 0.1 M glycine (pH 3) to the upper chamber of the protein G Sepharose column to elute the antibody from the column. Collect the flow-through directly as eluate in one of the prepared 1.5 mL tubes.</li>
		<li>Repeat the elution step (2.2.2.) for all 20 tubes (1.5 mL).</li>
		<li>Determine the optical density of all elution fractions at 280 nm (OD280) using a spectrophotometer in order to identify the antibody-containing fractions. 
		NOTE: Use the first elution fraction as blank.</li>
		<li>Pool all fractions with an OD280 greater than 0.5 (approximately 10 fractions).</li>
		<li>Store the protein G Sepharose column filled with 20% ethanol at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Dialyze the pooled elutions against 1000 mL PBS (in a 2000 mL beaker) at 4 &#176;C overnight using dialysis tubing with a molecular weight cut-off (MWCO) of 12 - 14 kDa.
	<ol>
		<li>Cut the dialysis tubing into pieces of 20 cm. Boil the dialysis tubing in 500-800 mL of 10 mM EDTA (pH 7.5) for 30 min in a beaker using a hot plate to remove contamination. Discard the 10 mM EDTA (pH 7.5) solution and boil the dialysis tubing in deionized water for 10 min. 
		NOTE: The dialysis tubing can be used directly or stored in 0.01% sodium azide (NaN3)/H2O solution at 4 &#176;C until the next usage.</li>
		<li>Close the bottom of the dialysis tubing with an appropriate dialysis tubing closure/single-piece, hinged clamp and carefully pipette the antibody elution into the dialysis tubing. Close the top of the dialysis tubing with a second clamp.</li>
		<li>Fix the upper clamp of the dialysis tubing to a floating stand, put it together with a magnetic stir bar into the PBS-filled beaker, and place the beaker on a magnetic stirrer.</li>
		<li>Dialyze overnight stirring at 4 &#176;C.</li>
	</ol>
	</li>
	<li>To increase the concentration of &#945;DEC-205, load the complete dialysate to a centrifugal concentrator with 10 kDa MWCO. Open one clamp of the tubing and carefully pipette the complete dialysate out of the dialysis tubing into the centrifugal concentrator (10 kDa MWCO). 
	NOTE: Do not touch the concentrator bottom with the pipette tip.
	<ol>
		<li>Centrifuge for 30 min at 693 x g (2,000 rpm) and 4 &#176;C.</li>
		<li>Load the centrifugal concentrator with 10 mL of PBS and centrifuge at 693 x g (2,000 rpm) and 4 &#176;C until the final volume of antibody solution left is 1-1.5 mL. 
		NOTE: If necessary, repeat the centrifugation step of 2.4.1. to adjust to the desired amount.</li>
		<li>Using a spectrophotometer, determine the optical density of the concentrated &#945;DEC-205 solution at 280 nm (OD280). Use PBS as blank.</li>
		<li>Calculate the concentration of &#945;DEC-205 using the following formula: 
		concentration [mg/mL] = OD280/1.4.</li>
		<li>Filter the &#945;DEC-205 solution using a 0.22 &#181;m syringe filter unit. 
		NOTE: Purification of &#945;DEC-205 from the NLDC-145 hybridoma cell supernatant can also be achieved by FPLC (fast protein liquid chromatography). Purified &#945;DEC-205 can be stored at 4 &#176;C or at -18 &#176;C for long-term storage.</li>
	</ol>
	</li>
</ol>
3. Chemical conjugation of OVA to &#945;DEC-205
NOTE: A ratio of 0.5 mg OVA protein to 2.5 mg &#945;DEC-205 (1:5) is required for optimal chemical conjugation. However, this ratio can vary for other proteins and antibodies and needs to be optimized for alternative conjugates. Reduction of the disulfide bonds of the OVA protein is performed through incubation with 30 mM TCEP-HCl, which exposes the sulfhydryl-groups for chemical conjugation to &#945;DEC-205 and 240 &#181;l of TCEP-HCl are needed in step 3.2. Both steps, TCEP-induced reduction of OVA (step 3.1.) and sulfo-SMCC activation of &#945;DEC-205 (step 3.2.) should preferably be performed in parallel.
<ol>
	<li>Freshly prepare a 125 mM TCEP-HCl solution (pH 7.0). Weigh out the desired amount of TCEP-HCl and dissolve the TCEP-HCl in 0.9 M Tris base (pH 8.8). Use pH indicator strips to test the pH of the 125 mM TCEP-HCl solution (which should be neutral) and adjust the pH with Tris base (pH 8.8).
	<ol>
		<li>Pipette 200 &#181;L OVA protein solution (containing 0.5 mg OVA) into a 1.5 mL sterile tube. Add 240 &#181;l 125 mM TCEP-HCl and 560 &#181;L of sterile ultrapure water to the OVA protein using a pipette to a final concentration of 0.5 mg/mL OVA protein and 30 mM TCEP-HCl (OVA/TCEP-HCl). 
		NOTE: 2.5 mg EndoGrade OVA (lyophilized) is dissolved in 1 mL PBS resulting in a 2.5 mg/mL OVA solution.</li>
		<li>Incubate the resulting OVA/TCEP-HCl at room temperature for 1.5 h. 
		NOTE: Do not extend this incubation step.</li>
	</ol>
	</li>
	<li>To activate &#945;DEC-205 for conjugation, dissolve 2 mg sulfo-SMCC in 100 &#181;L of ultrapure water. 
	NOTE: Sulfo-SMCC is susceptible to hydrolysis. Therefore, larger amounts of undissolved sulfo-SMCC should be handled rapidly or available 2 mg aliquots should be used.
	<ol>
		<li>Dilute &#945;DEC-205 in PBS so that 2.5 mg is contained in 900 &#181;L.</li>
		<li>Mix 2.5 mg of &#945;DEC-205 (900 &#181;L volume; obtained from step 3.2.1.) and 100 &#181;L of sulfo-SMCC (obtained from step 3.2.) in a 1.5 mL tube, resulting in a total volume of 1 mL.</li>
		<li>Incubate the &#945;DEC-205/sulfo-SMCC solution for 30 min at 37 &#176;C and 550 rpm in a heating block.</li>
	</ol>
	</li>
	<li>Following these incubations, excess sulfo-SMCC and TCEP-HCl are immediately removed from the solutions using desalting columns (MWCO 7 kDa; 5 mL column volume).
	<ol>
		<li>Twist off the columns (MWCO 7 kDa) bottom closure, loosen the cap, and place the column into a 15 mL conical tube.</li>
		<li>Centrifuge for 2 min at 1,000 x g at room temperature to remove the liquid.</li>
		<li>Place the column in a fresh tube and remove the cap. Slowly load the antibody/sulfo-SMCC and the OVA/TCEP-HCl, respectively, to the center of the compact resin bed of one column each.</li>
		<li>Centrifuge for 2 min at 1,000 x g at room temperature.</li>
		<li>Discard the columns after use. The solutions containing antibody and OVA primed for conjugation are in the tubes.</li>
		<li>Immediately mix both solutions by pipetting for conjugation of &#945;DEC-205 and OVA.</li>
		<li>Incubate the resulting &#945;DEC-205 and OVA mixture overnight at 4&#176;C.</li>
	</ol>
	</li>
	<li>Following conjugation, excess unbound OVA is removed from the solution and the coupled &#945;DEC-205/OVA is concentrated using a centrifugal protein concentrator (MWCO 150 kDa).
	<ol>
		<li>Pre-rinse the centrifugal protein concentrator (MWCO 150 kDa) by pipetting 12 mL of PBS on the column and centrifuging for 2 min at 2,000 x g at room temperature. 
		NOTE: If necessary, repeat the centrifugation step (3.4.1.) until a volume of about 5 mL has passed through the column.</li>
		<li>Before loading the &#945;DEC-205/OVA onto the centrifugal protein concentrator, save a 20 &#181;L sample of the un-concentrated &#945;DEC-205/OVA for western blot analysis. Store this aliquot at 4 &#176;C until analysis.</li>
		<li>Load the &#945;DEC-205/OVA onto the centrifugal protein concentrator by pipetting. 
		NOTE: Avoid any contact with the bed of the upper chamber of the centrifugal concentrator.</li>
		<li>Fill the concentrator to 15 mL with PBS and centrifuge the concentrator for 5 min at 2,000 x g at room temperature.</li>
		<li>Save a sample of the flow-through (flow-through I) for western blot analysis and discard the remaining flow-through.</li>
		<li>Fill the concentrator to 10 mL with PBS and centrifuge the concentrator for at least 8 min at 2,000 x g at room temperature.</li>
		<li>Save a sample for the second flow-through (flow-through II) for western blot analysis and discard the remaining flow-through.</li>
		<li>Once the desired enrichment is achieved (around 1.5 mL of the &#945;DEC-205/OVA solution should be left in the upper chamber) gently aspirate the concentrated sample. 
		NOTE: If too much fluid is left in the upper chamber, centrifugation can be repeated but should be kept as short as possible.</li>
	</ol>
	</li>
	<li>Determine the protein concentration of the resulting &#945;DEC-205/OVA using a microvolume spectrophotometer. Use PBS as blank.</li>
	<li>Filter the &#945;DEC-205/OVA using a 0.22 &#181;m syringe filter unit. 
	NOTE: For later analysis and in vivo experiments, &#945;DEC-205/OVA can be stored at 4 &#176;C or -18 &#176;C.</li>
</ol>
4. Verification of the chemical conjugation by ELISA
<ol>
	<li>Perform ELISA for further verification of successful chemical conjugation resulting in &#945;DEC-205/OVA.</li>
	<li>Coat an appropriate 96-well ELISA plate with 100 &#181;L/well of 3 ng/&#181;L rabbit &#945;OVA antibody in coating buffer (0.1 M sodium bicarbonate (NaHCO3) pH 9.6 diluted in H2O).
	<ol>
		<li>Incubate the plate overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Following coating, wash the plate three times with PBS, e.g., using an ELISA washer.</li>
	<li>Block the plate by pipetting 200 &#181;L of blocking buffer (10% FCS in PBS) in each well of the plate and incubate the plate for 30 min at room temperature.</li>
	<li>Serially dilute &#945;DEC-205/OVA (obtained from step 3.6.) 1:2 in blocking buffer (10% FCS in PBS) to obtain dilutions ranging from 6 &#181;g/mL down to 93.8 ng/mL &#945;DEC-205/OVA and add 100 &#181;L/well of these decreasing amounts of &#945;DEC-205/OVA to the wells.
	<ol>
		<li>Incubate the plate for 1 h at room temperature.</li>
	</ol>
	</li>
	<li>Wash the plate three times with PBS, e.g., using an ELISA washer.</li>
	<li>Add 100 &#181;L of the goat &#945;rat-IgG+IgM(H+L)-HRPO antibody (diluted to 1:2,000 in blocking buffer (10% FCS in PBS)) to each well of the plate.
	<ol>
		<li>Incubate for 1 h at room temperature.</li>
	</ol>
	</li>
	<li>Wash the plate three times using PBS, e.g. using an ELISA washer.</li>
	<li>Add 50 &#181;L of HRPO-substrate to the wells. When observing a clear color reaction, stop the reaction through addition of 150 &#181;l stopping solution (1M H2SO4) per well.</li>
	<li>After 5 min, read absorption at 450 nm using an ELISA reader.</li>
</ol>

<table><tbody><tr><td>Blocking buffer (ELISA)</td><td></td><td></td><td>10 % FBS in PBS</td></tr><tr><td>Cell culture flask T25</td><td>Greiner Bio-One</td><td>690175</td><td>we use standard CELLSTAR filter cap cell culture flasks; alternatively use suspension culture flask (690195 )</td></tr><tr><td>Cell culture flask T75</td><td>Greiner Bio-One</td><td>658175</td><td>we use standard CELLSTAR&amp;nbsp; filter cap cell culture flasks; alternatively use suspension culture flask (658195)&amp;nbsp;</td></tr><tr><td>Cell culture flask T175</td><td>Greiner Bio-One</td><td>661175</td><td>we use standard CELLSTAR filter cap cell culture flasks; alternatively use suspension culture flask (661195)</td></tr><tr><td>Centrifugal concentrator MWCO 10 kDa</td><td>Sartorius</td><td>VS2001</td><td>Vivaspin 20 centrifugal concentrator</td></tr><tr><td>Centrifugal protein concentrator MWCO 100 kDa, 5 - 20 ml</td><td>Thermo Fisher Scientific</td><td>88532</td><td>Pierce Protein Concentrator, PES 5 -20 ml; we use the Pierce Concentrator 150K MWCO 20mL (catalog number 89921), which is however no longer available&amp;nbsp;</td></tr><tr><td>Centrifuge bottles</td><td>Nalgene</td><td>525-2314</td><td>PPCO (polypropylene copolymer) with PP (polypropylene) screw closure, 500 ml; obtained from VWR, Germany</td></tr><tr><td>Coating buffer (ELISA)</td><td></td><td></td><td>0.1 M sodium bicarbonate (NaHCO3) in H2O (pH 9.6)</td></tr><tr><td>Desalting columns MWCO 7 kDa</td><td>Thermo Fisher Scientific</td><td>89891</td><td>Thermo Scientific Zeba Spin Desalting Columns, 7K MWCO, 5 mL</td></tr><tr><td>Detection reagent ELISA (HRPO substrate)</td><td>Sigma-Aldrich/Merck</td><td>T8665-100ML</td><td>3,3&amp;prime;,5,5&amp;prime;-Tetramethylbenzidine (TMB) liquid substrate system</td></tr><tr><td>ELISA 96-well plate</td><td>Thermo Fisher Scientific</td><td>442404</td><td>MaxiSorp Nunc-Immuno Plate</td></tr><tr><td>Fetal calf serum</td><td>PAN-BIOtech</td><td>P40-47500</td><td>FBS Good forte</td></tr><tr><td>ISF-1 medium</td><td>Biochrom/bioswisstec</td><td>F 9061-01</td><td></td></tr><tr><td>NLDC-145 hybridoma</td><td>ATCC</td><td>HB-290</td><td>if not already at hand, the hybridoma cells can be acquired from ATCC</td></tr><tr><td>Ovalbumin</td><td>Hyglos (via BioVendor)</td><td>321000</td><td>EndoGrade OVA ultrapure with &amp;lt;0.1 EU/mg</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>Gibco Penicillin/Streptomycin 10.000 U/ml; alternatively Gibco Penicillin/Streptomycin 5.000 U/ml (15070-063) can be used</td></tr><tr><td>PETG polyethylene terephthalate glycol cell culture roller bottles</td><td>Nunc In Vitro</td><td>734-2394</td><td>standard PDL-coated, vented (1.2X), 1050 cm&amp;sup2;, 100 - 500 ml volume; obtained from VWR, Germany&amp;nbsp;&amp;nbsp;</td></tr><tr><td>pH indicator strips</td><td>Merck</td><td>109535</td><td>pH indicator strips 0-14</td></tr><tr><td>Polyclonal goat &amp;alpha;rat-IgG+IgM-HRPO antibody&amp;nbsp; (ELISA)</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>112-035-068</td><td>obtained from Dianova, Germany; used at 1:2000 for ELISA</td></tr><tr><td>Polyclonal rabbit &amp;alpha;OVA (ELISA)</td><td>Abcam</td><td>ab181688</td><td>used at 3 ng/&amp;micro;l</td></tr><tr><td>Protein G Sepharose column</td><td>Merck/Millipore</td><td>P3296</td><td>5 ml Protein G Sepharose, Fast Flow are packed onto an empty column PD-10 (Merck, GE 17-0435-01)</td></tr><tr><td>Rubber plug</td><td>Omnilab</td><td>5230217</td><td>DEUTSCH &amp;amp; NEUMANN rubber stoppers (lower &amp;Phi; 17 mm; upper &amp;Phi; 22 mm)</td></tr><tr><td>Silicone tube</td><td>Omnilab</td><td>5430925</td><td>DEUTSCH &amp;amp; NEUMANN (inside &amp;Phi; 1 mm; outer &amp;Phi; 3 mm)</td></tr><tr><td>Slim-Fast</td><td></td><td></td><td>we have used regular Slim-Fast Chocolate freely available at the pharmacy as in this western blot approach it yielded better results than milk powder</td></tr><tr><td>Stopping solution (ELISA)</td><td></td><td></td><td>1M H2SO4</td></tr><tr><td>Sulfo-SMCC</td><td>Thermo Fisher Scientific</td><td>22322</td><td>Pierce Sulfo-SMCC Cross-Linker; alternatively use catalog number A39268 (10 x 2 mg)</td></tr><tr><td>Syringe filter unit 0.22 &amp;micro;m&amp;nbsp;</td><td>Merck/Millipore</td><td>SLGV033RS</td><td>Millex-GV Syringe Filter Unit, 0.22 &amp;micro;m, PVDF, 33 mm, gamma sterilized&amp;nbsp;</td></tr><tr><td>Syringe 10 ml</td><td>Omnilab</td><td></td><td>Disposable syringes Injekt&amp;reg; Solo B.Braun</td></tr><tr><td>Sterican&amp;reg; cannulas</td><td>B. Braun</td><td></td><td>Sterican&amp;reg; G 20 x 1 1/2&quot;&quot;; 0.90 x 40 mm; yellow</td></tr><tr><td>TCEP-HCl</td><td>Thermo Fisher Scientific</td><td>A35349</td><td></td></tr><tr><td>Tubing connector</td><td>Omnilab</td><td></td><td>Kleinfeld miniature tubing connectors for silicone tube</td></tr></tbody></table>

enzyme-linked immunosorbent assay to verify chemically-coupled antibody-antigen conjugates

Source: Volckmar, J. et al., <a href="https://app.jove.com/v/62018">Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo.</a> J. Vis. Exp. (2021)
This video demonstrates the detection of chemically conjugated antibody-antigen complexes via the sandwich enzyme-linked immunosorbent assay, or ELISA, technique. The captured antibody binds to the specific epitope of the antigen of the antibody-antigen conjugate, which can be later visualized using an enzyme-linked secondary antibody.

Enzyme-Linked Immunosorbent Assay to Verify Chemically-Coupled Antibody-Antigen Conjugates

Source: Volckmar, J. et al., Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In ...

To elicit an immune response, antigens bind to the antibodies that are specific for the endocytic receptors present on the surface of dendritic cells, or DCs &#8212; the antigen-presenting cells &#8212; which help in the uptake of antigens.
The formation of an antibody-antigen complex is a crucial step and can be accomplished in vitro by chemically coupling the antigen to a DC-targeting monoclonal antibody.
To verify the efficiency of the conjugation process, begin with an ELISA plate precoated with the captured antibodies. Treat the plate with a blocking solution to prevent the non-specific binding of the antigens to the coated surface.
Add chemically crosslinked, antibody-antigen conjugates to the plate, and incubate. The specific epitope of the antigen interacts with the captured antibody&#39;s paratope forming an antibody-antigen-antibody complex.
Next, overlay the plate with a third set of antibodies linked to an enzyme. These antibodies bind specifically to the primary antibodies present in the conjugate, enabling its detection.
Add a buffer containing the chromogenic substrate to visualize the conjugates. The enzyme attached to the antibody reacts with the substrate, generating the colored product.
Treat the plate with an acidified inactivation solution, which inactivates the enzyme and terminates the reaction. Analyze the plate spectrophotometrically. The presence of a colored solution verifies the presence of antibody-antigen conjugates and confirms successful conjugation.

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157 vistas. Fuente: Volckmar, J. et al., Conjugación química de un anticuerpo dirigido DEC-205 purificado con proteína de longitud completa para atacar células dendríticas de ratón in vitro e in vivo. J. Vis. Exp. (2021) Este video muestra la detección de complejos anticuerpo-antígeno químicamente conjugados a través de la técnica de ensayo de inmunoabsorción enzimática sándwich, o ELISA. El anticuerpo capturado se une al epítopo específico del antígeno del conjugado anticuerpo-antígeno, ...

Video: Ensayo de inmunoabsorción enzimática para verificar conjugados anticuerpo-antígeno acoplados químicamente - Concepto

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

Visualización interior de la membrana bacteriana para la selección de una biblioteca de fragmentos de anticuerpos

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

Investigación

JoVE Journal

Bioquímica

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Caracterización de las respuestas de isotipo de inmunoglobulina dependiente del timo y timo-independiente en ratones usando el análisis enzima-ligado del inmunosorbente

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Enzyme-Linked Immunosorbent Assay to Verify Chemically-Coupled Antibody-Antigen Conjugates

Transcripción

Enzyme-Linked Immunosorbent Assay to Verify Chemically-Coupled Antibody-Antigen Conjugates