This work was supported by a grant from the German Federal Ministry of Education and Research (Project BiGRUDI 
[Biologische Gefahrenlagen: Risikobewertung, ultraschnelle Detektion 
und Identifizierung von bioterroristisch relevanten Agenzien, 13N9594]). We thank B. Diemar (Charit&eacute;-Universit&auml;tsmedizin 
Berlin, Institut f&uuml;r Pharmakologie) for excellent technical assistance.

1. Protocol - IgY-extraction by means of PEG-precipitation
Fig. 1 gives a schematic diagram of the IgY-extraction procedure (see Table 2). The protocol was first described in Polson et al. 1980. 
All steps should be performed using latex gloves.
<ol>
 <li>The eggshell is carefully cracked and the yolk is transferred to a "yolk spoon" in order to remove as much egg white as possible. </li>
 <li>The yolk is transferred to a filter paper and rolled to remove remaining egg white, then the yolk skin is cut with a lancet or a similar instrument (pipette tip). 
 The yolk is poured into a 50 ml tube and the egg volume is registered (V1).</li>
 <li>Twice the egg yolk volume of PBS is mixed with the yolk (&sum;V1+V2), thereafter 3.5 % PEG 6000 (in gram, pulverized) of the total volume is added and vortexed, 
 followed by 10 min rolling on a rolling mixer. That step of the extraction procedure separates the suspension in two phases. One phase consists of "yolk solids and fatty substances" (original quotation of Polson et al. 1980) and a watery phase containing IgY and 
 other proteins.</li>
 <li>The tubes are centrifuged at 4&deg;C for 20 min (10,000 rpm according to 13,000 x g, Heraeus Multifuge 3SR+, fixed angle rotor). The supernatant (V3) 
 is poured through a folded filter and transferred to a new tube.</li>
 <li>8.5 % PEG 6000 in gram (calculated according to the new volume) are added to the tube, vortexed and rolled on a rolling mixer as in step 3.</li>
 <li>Repeat step 4 with the difference that the supernatant is discarded.</li>
 <li>The pellet is carefully dissolved in 1 ml PBS by means of a glass stick and the vortexer. PBS is added to a final volume of 10 ml (V4). 
 The solution is mixed with 12 % PEG 6000 (w/v, 1.2 gram) and treated as in step 3 (vortex, rolling mixer). </li>
 <li>Repeat step 6 and dissolve the pellet carefully in 800 &mu;l PBS (glass stick and vortex). Wait for the air bubbles to disappear and then transfer 
 (pipette) the extract to a dialysis capsule. Rinse the tube with 400 &mu;l PBS and add the volume to the dialysis device (V5). (For preparation of dialysis 
 devices and membranes see appendix.)</li>
 <li>The extract is dialysed over night in 0.1 % saline (1,600 ml) and gently stirred by means of a magnetic stirrer. The next morning, the saline is replaced by PBS and dialysed for another three hours.</li>
 <li>Thereafter the IgY-extract is pulled from the dialysis capsule by a pipette and transferred to 2ml tubes. The final volume is around 2 ml (V6).</li>
 <li>The protein content (mg/mL) of the samples is measured photometrically at 280 nm (1:50 diluted with PBS) and calculated according to the Lambert-Beer law with an extinction coefficient of 1.33 for IgY (see Fig. 2), Fig. 4 shows the quality of the preparation (purity and recovery are around 80 %).</li>
 <li>It is advisable to store the samples in aliquots at -20&deg;C (do not freeze the samples at -70&deg;C).</li>
 <li>The quality of the final preparations is analysed by simple SDS-PAGE as described in JoVE protocol <a href="https://www.jove.com/details.php?id=1916">https://www.jove.com/details.php?id=1916</a></li>
</ol>
 2. Appendix- before use the dialysis bag must be prepared in the following way according to the recommendations of the manufacturer:
<ol>
 <li>
20 dialysis bags are cut in pieces of 30 cm and given in a 2000 ml glass beaker.
 </li>
 <li>
1,750 ml of a 5 mM EDTA-solution are added.
 </li>
 <li>
A glass funnel is placed above the bags to ensure that the bags are covered by the EDTA solution. The solution is heated and boiled for 5 min (hot plate). The solution is decanted and the bags are washed three times with distilled water.
 </li>
 <li>
Once again 1,750 ml EDTA-solution are added, boiled for 5 min and washed three times with distilled water as above.
 </li>
 <li>
Finally the dialysis bags are boiled for 10 min in distilled water and stored at 4&deg;C. Take out the dialysis bags by means of sterilized tweezers.
 </li>
 </ol>

3. Representative results

 <img src="/files/ftp_upload/3084/3084fig1.jpg" alt="Figure 1" /> 
 Figure 1. Schematic diagram of IgY-extraction by means of polyethylene glycol precipitation according to the original description of 
 Polson et al. 1980. <a href="https://www.jove.com/files/ftp_upload/3084/3084largefig1.jpg">To view a larger image click here.</a>

 <img src="/files/ftp_upload/3084/3084fig2.jpg" alt="Figure 2" /> 
 Figure 2. Development of total-IgY in egg yolk of immunised hens in dependence of age. Given is the weekly mean of total IgY/egg and SD (n = 4 laying hens). From [Pauly et al. 2009]).

 <img src="/files/ftp_upload/3084/3084fig3.jpg" alt="Figure 3" /> 
 Figure 3. 
 Laying capacity monitoring of four hens immunised with different antigens. The arrows indicate immunisation date (from [Pauly et al. 
 2009]).<a href="https://www.jove.com/files/ftp_upload/3084/3084largefig3.jpg">To view a larger image click here.</a>

 <img src="/files/ftp_upload/3084/3084fig4.jpg" alt="Figure 4" /> 
 Figure 4. Polyacrylamide-Gelelectrophoresis of individual IgY preparations from different eggs under reducing conditions 
 Lane 1 - Molecular weight marker, Lane 2 - IgY-standard, Lane 3-5 - different IgY samples prepared as described in the protocol. These are final preparations according to step 10 of the protocol. 
 HC - heavy chains, LC - light chaines, ? - minor impurities corresponding to molecular weight around 35 kDa (probably the C-terminal fragment of vitellogenin II precursor, [Klimentzou et al., 2006]).
<table border="1">
<tbody>
 <tr>
 <td>&nbsp;</td>
 <td>Chicken 21 (Antigen A)</td>
 <td>Chicken 22 (Antigen A)</td>
 <td>Chicken 19 (Antigen B)</td>
 <td>Chicken 23 (Antigen C)</td>
 </tr>
 <tr>
 <td>Egg number (total IgY)1, 2-years period</td>
 <td>625 (38 g)</td>
 <td>626 (38 g)</td>
 <td>545 (33 g)</td>
 <td>608 (37 g) </td>
 </tr>
 <tr>
 <td>Egg number (total IgY), first year</td>
 <td>345 (20 g)</td>
 <td>304 (16 g)</td>
 <td>326 (18 g)</td>
 <td>308 (17 g)</td>
 </tr>
 <tr>
 <td>Egg number (total IgY), second year</td>
 <td>280 (18 g)</td>
 <td>322 (21 g)</td>
 <td>219 (15 g)</td>
 <td>300 (20 g)</td>
 </tr>
 <tr>
 <td>% of max. possible egg number (first year)2</td>
 <td>106</td>
 <td>93</td>
 <td>100</td>
 <td>95</td>
 </tr>
 <tr>
 <td>% of max. possible egg number (second year)</td>
 <td>86</td>
 <td>99</td>
 <td>67</td>
 <td>92</td>
 </tr>
 <tr>
 <td>number of processed eggs</td>
 <td>565</td>
 <td>620</td>
 <td>283</td>
 <td>401</td>
 </tr>
 </tbody>
</table>

1The amount of total IgY is calculated by multiplying the number of eggs by a mean value of 60 mg protein per egg according to the data of Figure 2.



 2The max. possible egg number per year is 325 according to an information of the breeder.


Table 1. Two years statistics of egg laying capacity of chickens immunized with different antigens. Laying capacity in percent of the maximum of four hens after immunisation with different antigens as well as the IgY-outcome during two years (see also Fig. 3), [Pauly et al. 2009]).
<table border="1">
<tbody>
 <tr>
 <td>Egg Nr.</td>
 <td>Hen Nr. Laying Date</td>
 <td>V1[ml] yolk </td>
 <td>V2[ml] PBS</td>
 <td>1. PEG prec. [g]</td>
 <td>Vol [ml] after </td>
 <td>2. PEG prec. [g]</td>
 <td>Pellet</td>
 <td>3. PEG prec. [g]</td>
 <td>Pellet</td>
 <td>Vol [ml]</td>
 <td>Total protein mg/ml</td>
 <td>Corrected total protein mg/ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>2xV1 &sum;V1 + V2 </td>
 <td>3,5% [w/v] &sum;V1 + V2 </td>
 <td>prec., centrif. and filtration V3 </td>
 <td>8,5% [w/v] V3</td>
 <td>dissolved in ml PBS V4</td>
 <td>12% [w/v] V4</td>
 <td>Dissolved in ml PBS V5</td>
 <td>After dialysis against PBS V6</td>
 <td>A280/ml</td>
 <td>A280/ml:1.33</td>
 </tr>
 <tr>
 <td>1</td>
 <td>H293 / 22.10.</td>
 <td>15</td>
 <td>30 45</td>
 <td>1.58</td>
 <td>31</td>
 <td>2.63</td>
 <td>10</td>
 <td>1.2</td>
 <td>1.2</td>
 <td>1.7</td>
 <td>35.0</td>
 <td>26.3</td>
 </tr>
 <tr>
 <td>2</td>
 <td>H293 / 23.10.</td>
 <td>13</td>
 <td>26 39</td>
 <td>1.37</td>
 <td>25</td>
 <td>2.12</td>
 <td>10</td>
 <td>1.2</td>
 <td>1.2</td>
 <td>1.7</td>
 <td>28.8</td>
 <td>21.6</td>
 </tr>
</tbody>
</table>
Table 2. Exemplary protocol of PEG precipitation

<ol>
	<li>Akita, E. M., Nakai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+four+purification+methods+for+the+production+of+immunoglobulins+from+eggs+laid+by+hens+immunized+with+an+enterotoxigenic+E.+coli+strain.">Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain.</a> J. Immunol. Methods. 160, 207-214 (1993).</li><li>De Meulenaer, B., Huyghebaert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+purification+of+chicken+egg+yolk+immunoglobulins:+a+review.">Isolation and purification of chicken egg yolk immunoglobulins: a review.</a> Food Agricult. Immunol. 13, 275-288 (2001).</li><li>Klimentzou, P., Paravatou-Petsotas, M., Zikos, C. h., Beck, A., Skopeliti, M., Czarnecki, J., Tsitsilonis, O., Voelter, W., Livaniou, E., Evangelatos, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+immunochemical+evaluation+of+antibodies+Y+for+the+poorly,+immunogenic+polypeptide+prothymosin+alpha.">Development and immunochemical evaluation of antibodies Y for the poorly, immunogenic polypeptide prothymosin alpha.</a> Peptides. 27, 183-193 (2006).</li><li>Pauly, D., Dorner, M., Zhang, X., Hlinak, A., Dorner, B., Schade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+laying+capacity,+immunoglobulin+Y+concentration,+and+antibody+titer+development+in+chickens+immunized+with+ricin+and+botulinum+toxins+over+a+two-year+period.">Monitoring of laying capacity, immunoglobulin Y concentration, and antibody titer development in chickens immunized with ricin and botulinum toxins over a two-year period.</a> Poultry Science. 88, 281-290 (2009).</li><li>Polson, A., von Wechmar, M. B., van Regenmortel, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+viral+IgY+antibodies+from+yolks+of+immunized+hens.">Isolation of viral IgY antibodies from yolks of immunized hens.</a> Immunol. Commun. 9, 475-493 (1980).</li><li>Schade, R., Behn, I., Erhard, M., Hlinak, A., Staak, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chicken egg yolk antibodies, production and application : IgY-Technology. , (2001).</li><li>Schade, R., Calzado, E. G., Sarmiento, R., Chacana, P. A., Porankiewicz-Asplund, J., Terzolo, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+egg+yolk+antibodies+(IgY-technology):+a+review+of+progress+in+production+and+use+in+research+and+human+and+veterinary+medicine.">Chicken egg yolk antibodies (IgY-technology): a review of progress in production and use in research and human and veterinary medicine.</a> Altern. Lab Anim. 33, 129-154 (2005).</li><li>Witkowski, P. T., Bourquain, D. R., Hohn, O., Schade, R., Nitsche, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+gun-supported+DNA+immunisation+of+chicken+for+straightforward+production+of+poxvirus-specifiv+IgY+antibodies.">Gene gun-supported DNA immunisation of chicken for straightforward production of poxvirus-specifiv IgY antibodies.</a> J. Immunol. Methods. 341, 146-153 (2009).</li></ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by Landesamt fur Gesundheit und Soziales Berlin (H 0069/03).

The choice of a suitable IgY purification method is influenced by scale of purification (laboratory or industrial), cost effectiveness, technology (laboratory equipment), and impact on the environment (waste management). Various IgY extraction methods were reviewed in detail by De Meulenaer &amp; Huyghebaert (2001, for review see also Schade et al. 2005). In general, these methods can be divided into three principal groups:

<ol> 
 <li>
 Precipitation methods: involving ammonium or sodium sulphate, polyethyleneglycol (PEG), caprylic acid and caragenean. 
 </li>
 <li>
 Chromatographic methods: affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, thiophylic interaction chromatography, and gel-filtration chromatography.
 </li>
 <li>
 Ultrafiltration.
 </li>
</ol> 
The purity of an IgY preparation can be increased by a combination of methods; for example, PEG precipitation can be combined with affinity chromatography. In some instances, depending on the final application, a water extract of IgY is sufficient to achieve good results (Akita &amp; Nakai 1993). According to our experience, the IgY-sample we obtain by PEG-precipitation worked very well in a lot of different immunological assays. By standardizing the PEG-precipitation method one technical assistant is able to process approximately 84 eggs per week, which corresponds to an amount of total IgY between 4 and 7 g per week. A simple calculation: A laying hen is immunised with an antigen. After a few boost immunisations the hen produces specific antibodies (Ab) with a titre of 1:50,000 during a period of 30 days. 30 eggs x 2 ml IgY extract (see protocol and Fig. 3) corresponds to 60 ml total IgY, which in turn corresponds to an Ab-dilution of 3,000 l (the capacity of a bucket is 10 l), that means with 3,000 l of Ab solution 300 buckets can be filled. So, in view of such huge Ab-quantity it is no problem to store an Ab-pool of constant quality and specificity. This technique reduces the charge/lot variability otherwise observed in polyspecific Ab. 

 Despite of the aspect of quantity, IgY Ab have further advantages compared with mammalian IgG: No activation of mammalian complement system, no cross-reactivity with HAMA (human anti mouse antibody), rheumatoid factors or human blood group antigens (lack of heteroagglutinins). 


An outstanding advantage of IgY-Ab is the so-called phylogenetic distance. Phylogenetic distance is the reason for the frequently described differences between the Ab specificities of mammals and chickens, even when identically immunised. In addition to differences between the mammalian and the avian immune system themselves, differences in phylogenetic development in these two animal classes contributes to the different Ab specificities. Several authors have reported that chickens often produce Abs against phylogenetically highly conserved mammalian proteins or peptides more efficiently than do rabbits (for review see Schade et a. 2005). As a consequence, a conserved antigen can remain"masked" to the rabbit immune system, and thus cause only a weak or a"silent" response. Furthermore, if chickens and rabbits are immunised with the same mammalian antigen, very often the chickens respond with an Ab-specificity that can rarely be achieved in rabbits. Also the aspect of animal welfare is important since the Ab are non-invasively extracted from egg yolk. In combination with Gene-Gun (plasmid) immunisation the IgY-production is completely non-invasive (Witkowski et al. 2009).
 
 
In conclusion, the production of Ab in hens and the IgY-extraction by means of PEG-precipitation is very cost-effective and results in highly specific Ab with stable titres up to 1:1,000,000. Due to the phylogenetic distance between Aves and Mammalia, chicken are able to produce specific Ab against highly conserved mammalian antigens in contrast to e.g. rabbits. The extraordinary amount of Ab obtained by IgY-technology opens the door also for using IgY-Ab in human- and veterinary medicine for therapeutic/prophylactic purposes.

<ol>
<li>
Dialyse Bag Visking, Type 27/32, cut off -14 kD, Carl Roth GmbH, Karlsruhe, Germany
</li><li>
Falcon tubes Blue Max, 50 ml Polypropylene Conical Tube, Becton Dickinson Labware, Franklin Lakes NY, USA 
</li><li>
Folded Filter MN 615 &frac14;, &empty; 150 mm, Carl Roth GmbH, Karlsruhe, Germany Heraeus 
</li><li>
Magnetic stirrer, Variomag Mono, H+P Labortechnik AG, Oberschlei&szlig;heim, Germany 
</li><li>
Micro-Dialyse-Capsule 5,0 ml, Carl Roth GmbH, Karlsruhe, Germany 
</li><li>
Multifuge 3SR+, Thermo Scientific, Langenselbold, Germany 
</li><li>
Photometer UV-160A, Shimadzu Deutschland GmbH, Duisburg, Germany 
</li><li>
Polyethylene Glycol 6000, Rotipuran Ph. Eur., Carl Roth GmbH, Karlsruhe, Germany 
</li><li>
Premixed PBS-Buffer 10x, Roche Diagnostics GmbH, Mannheim, Germany 
</li><li>
 Roller Mixer SRT2, Stuart, Staffordshire, UK 
</li><li> 
Vibrofix VF2, Janke &amp; Kunkel, IKA-Labortechnik, Staufen i.Br., Germany 
</li><li>
Yolk spoon, Fackelmann (houshold effects), Germany 
</li>
</ol>

igy technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (peg) precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

Watch this Scientific Journal Video about IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation at JoVE.com

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

igy-technology-extraction-chicken-antibodies-from-egg-yolk

Research

JoVE Journal

Immunology and Infection

64.3K Views.  Robert Koch-Institute. פרוטוקול זה מתאר מיצוי מסוים של IgY הכולל חלמון ביצה באמצעות משקעים פוליאתילן גליקול ונותן מידע כללי על טכנולוגיה IgY.

Video: IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Biochemistry

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen&#039;s Eggs

This article reports a simple method to isolate the outer and inner perivitelline sublayers of chicken eggs while minimizing structural alteration and to optimize protein solubilization of each sublayer for proteomic analyses.

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. ...

Standardization of a Cytometric Bead Assay Based on Egg-Yolk Antibodies

Immunoassays are important tests for the detection of numerous molecular targets. Among the methods currently available, the cytometric bead assay has gained prominence in recent decades. Each microsphere that is read by the equipment represents an analysis event of the interaction capacity between the molecules under test. Thousands of these events are read in a single assay, thus ensuring high assay accuracy and reproducibility. This methodology can also be used in the validation of new inputs, such as IgY antibodies, for the diagnosis of diseases. These antibodies are obtained through immunizing chickens with the antigen of interest and then extracting the immunoglobulin from the yolk of the animals' eggs; therefore, this is a painless and highly productive method for obtaining the antibodies. In addition to a methodology for the high-precision validation of the antibody recognition capacity of this assay, this paper also presents a method for extracting these antibodies, determining the best coupling conditions for the antibodies and latex beads, and determining the sensitivity of the test.

This protocol describes a methodology for the preparation of latex beads for assays using IgY antibodies for antigen detection.

Immunoassays are important tests for the detection of numerous molecular targets. Among the methods currently available, the cytometric bead assay has ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Bioengineering

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Biology

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Polyclonal antibodies are defined as a collection of antibodies directed against different antigenic determinants of an antigen or several antigens (1). While polyclonal antibodies are powerful tools for identifying biological molecules, there is one important limitation - they are unable to distinguish between antigens that share antigenic determinants. For example, when bovine serum albumin is used to immunize an animal, B cells with different surface Ig will respond to different antigenic determinants on bovine serum albumin. The result is a mixture of antibodies in the antiserum. Because bovine serum albumin shares some epitopes with human serum albumin in evolutionarily conserved regions of the protein, this anti-bovine serum albumin antiserum will also react with human serum albumin. Therefore, this antiserum will not be useful for distinguishing between bovine and human serum albumins.
Several approaches have been taken to overcome the specificity issue of polyclonal antisera. One is by absorbing the unwanted antibodies by passing the antiserum through a chromatography column of immobilized antigens (2). This method is tedious and frequently unable to completely remove the unwanted antibodies. Another approach is to isolate individual antibody-producing B cells and expand them in culture. However, like most normal untransformed cells, B cells do not survive in long-term culture.
To overcome the inability of B cells to survive in culture, one approach is to prepare a myeloma-B cell hybridoma. In 1847, Henry Bence-Jones discovered that patients with multiple myeloma, a lymphoid tumor, produced a large quantity of antibodies (3). B cells in these patients have become malignant and grow uncontrollably. Since the malignant B cells are derived from a single clone, they are identical and produce only a single type of antibody (i.e., a monoclonal antibody, or mAb). However, most of these myeloma cells produce antibodies of unknown specificities. In 1975, by fusing a myeloma cell to a B cell, Cesar Milstein and Georges Kohler succeeded in producing a hybridoma that can be cultured indefinitely in vitro and produces an unlimited number of monoclonal antibodies of known antigenic specificity (4). The rationale behind their approach is to combine the immortal properties of the myeloma cell and the antibody producing properties of the B cell. Their technique revolutionized antibody production and provides a powerful means for identification and purification of biological molecules using monoclonal antibodies.
Generally, preparing a monoclonal antibody requires several months. The general procedure includes the following steps:
<ol>
	<li>Immunization and screening of antibody titer</li>
	<li>Fusion of antibody-producing B cells and myeloma cells</li>
	<li>Selective growth of the hybridoma</li>
	<li>Screening the hybridomas for producing the desired monoclonal antibody</li>
	<li>Cloning by limiting dilution - a process whereby cells are diluted to a concentration to statistically allow for less than 1 cell to be added to the wells of a 96-well plate. Some wells will end up with 0 cells and some will have 1 cell. The wells with seeded with 1 cell will eventually grow into a monoclonal population of cells.</li>
	<li>Growth of the hybridoma and preparation of monoclonal antibody</li>
</ol>
This protocol focuses on the last step - growth of the hybridoma and preparation of the monoclonal antibody. The antibody is purified from the culture supernatant by ammonium sulfate precipitation (often referred to as salting out) - a commonly used method of removing proteins from a solution. Proteins in solution form hydrogen bonds, along with other hydrophilic interactions, with water through their exposed polar and ionic groups. When concentrations of small, highly charged ions (such as ammonium or sulfate) are added, these groups compete with the proteins for binding to water. This removes the water molecules from the protein and decreases its solubility, resulting in precipitation of the protein.

Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4
1 Microbiology, Immunology, and Cancer Biology Graduate Program, ...

Education

JoVE Science Education

Advanced Biology

Immunology

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

Source: Susannah C. Shissler1, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation (IP, also known as a &#39;pull-down&#39; assay) is a widely used technique that has applications in a variety of fields. First conceived in 1984, it was refined in 1988 (1, 2). The fundamental goal of IP is purification and isolation of a specific protein using an antibody against that protein. The word &#34;immuno&#34; refers to the use of an antibody while the word &#34;precipitation&#34; refers to pulling down a specific substance from a solution. The target protein might be endogenous or recombinant. Most recombinant proteins have an epitope tag (i.e. myc or flag) attached to them to simplify subsequent purification. Typically, it is easier to optimize recombinant protein IP because the antibodies against recombinant epitope tags are very strong and effective. Antibodies against endogenous proteins have extremely variable efficacy - making it much more difficult to optimize these IPs. A necessary step after immunoprecipitation is verification of purification. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots (Figure 1). An important control is the use of a different antibody during the Western blot to verify pull down of the correct protein. The combination of IP with subsequent techniques is a powerful analysis tool. The goal after purification may be characterization of the protein itself by NMR, mass spectrometry, and in vitro assays, or analysis of the protein&#39;s interacting partners (i.e. protein, DNA, RNA) (3, 4, 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10502/10502fig1.jpg" /> 
Figure 1: Overview of Immunoprecipitation Procedure. Immunoprecipitation is the isolation of a specific protein using an antibody. After production of lysate from cells, there are two major steps- pre-clearing and pull down. During pre-clearing step, the cell-lysates are pre-cleared of proteins that bind to antibodies non-specifically using an isotype control antibody. In pull down step, the target protein is pulled down using a protein-specific antibody. The isolated protein is then analyzed by Western blot. Isotype antibodies and protein specific antibodies have the same constant domain, but different antigen binding domains. A key component of this protocol is Protein A/G agarose beads that bind the constant domain of antibodies- allowing immunoprecipitation of the target protein. <a href="https://www.jove.com/files/ftp_upload/10502/10502fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Antibodies are the key component of an immunoprecipitation that differentiate it from other forms of protein purification (i.e. nickel affinity column purification). Antibodies are molecules made by B cells that can recognize specific protein epitopes. Antibodies have two domains: constant (Fc) and antigen binding (Fab) (Figure 1). The constant domain identifies the type of antibody and dictates function in vivo. Usually, the constant domains of antibodies used for IP are mouse, rat, or rabbit IgG. The antigen binding portion of the antibody recognizes a specific epitope of a specific protein. Antibodies can recognize epitopes on folded proteins that may not exist when the protein is denatured and vice versa. Therefore, the availability of the epitope depends on protein folding - identifying an important factor to consider when choosing antibodies and conditions for IP.
Both prokaryotic and eukaryotic systems have antibody-binding proteins. In eukaryotic systems, the purpose is immune protection from bacteria while in prokaryotic systems, the purpose is protection from the immune system. Antibody-binding proteins affect IP methodology in two ways. First, there is a necessary pre-clearing step (Figure 1) to rid the lysate of proteins that bind antibodies - thereby reducing non-specific binding in the final product. This step uses an isotype antibody that has the same constant domain as but a different antibody binding domain than your protein-specific antibody. Bacterial antibody-binding proteins are the second key component of this method. After the protein-specific antibody binds the target protein, the antibody: protein complex must be pulled down (Figure 1). Proteins A, G, and L are bacterial proteins that bind the constant domain of antibodies. While bacteria use this to subvert the immune system, researchers have co-opted this system for easy antibody purification, and it is used during both the pre-clearing and pull-down steps. These proteins have different binding affinities for different species and different constant domain subtypes - another factor to consider when choosing conditions for IP. Many companies sell Protein A/G labeled agarose beads (Figure 1), pre-made spin columns, or resins to make columns. In general, beads and spin columns are used for smaller sample sizes while resins are used for bulk purification.
In this lab exercise, we demonstrate how to purify the endogenous protein c-myc, from primary murine thymocytes, using Protein A/G Plus agarose beads based basic immunoprecipitation technique. The protocol starts from cell lysate preparation and ends with the with verification of successful protein pull down using Western blot analysis.

Immunoprecipitation Techniques: Purification of Proteins

Source: Susannah C. Shissler1, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation ...

A Technique to Purify Monoclonal Antibodies Produced by Chinese Hamster Ovary Cells

Source: Velugula-Yellela, S. R., et al. <a href="https://app.jove.com/v/58947">Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System.</a> J. Vis. Exp. (2019).
This video demonstrates an assay for purifying monoclonal antibodies from cell culture fluid harvested from Chinese Hamster Ovarian cells. It employs a porous glass resin with immobilized protein A, which binds to the Fc region of the antibodies in the sample, effectively separating them from contaminants. The use of a low-pH elution buffer weakens the interaction, facilitating the retrieval of purified antibodies.

1. Purification of antibody
NOTE: The equilibration buffer for the in-house antibody is 25 mM Tris, 100 mM NaCl, pH 7.5. The elution buffer used is 0.1 M ...