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

64.2K Views.  Robert Koch-Institute. Questo protocollo descrive in particolare l'estrazione di IgY totale di tuorlo d'uovo per mezzo di precipitazione polietilene glicole e fornisce informazioni generali sulla tecnologia IgY.

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

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall 1,2. The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients 3,4.
In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients 3. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate.

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell transplant recipients1. Detection of IPA represents a formidable diagnostic challenge and, in the absence of a 'gold standard', relies on a combination of clinical data and microbiology and histopathology where feasible. Diagnosis of IPA must conform to the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycology Study Group (EORTC/MSG) consensus defining "proven", "probable", and "possible" invasive fungal diseases2. Currently, no nucleic acid-based tests have been externally validated for IPA detection and so polymerase chain reaction (PCR) is not included in current EORTC/MSG diagnostic criteria.
Identification of Aspergillus in histological sections is problematic because of similarities in hyphal morphologies with other invasive fungal pathogens3, and proven identification requires isolation of the etiologic agent in pure culture. Culture-based approaches rely on the availability of biopsy samples, but these are not always accessible in sick patients, and do not always yield viable propagules for culture when obtained.
An important feature in the pathogenesis of Aspergillus is angio-invasion, a trait that provides opportunities to track the fungus immunologically using tests that detect characteristic antigenic signatures molecules in serum and bronchoalveolar lavage (BAL) fluids. This has led to the development of the Platelia enzyme immunoassay (GM-EIA) that detects Aspergillus galactomannan and a 'pan-fungal' assay (Fungitell test) that detects the conserved fungal cell wall component (1 &rarr;3)-&beta;-D-glucan, but not in the mucorales that lack this component in their cell walls1,4. Issues surrounding the accuracy of these tests1,4-6 has led to the recent development of next-generation monoclonal antibody (MAb)-based assays that detect surrogate markers of infection1,5.
Thornton5 recently described the generation of an Aspergillus-specific MAb (JF5) using hybridoma technology and its use to develop an immuno-chromatographic lateral-flow device (LFD) for the point-of-care (POC) diagnosis of IPA. A major advantage of the LFD is its ability to detect activity since MAb JF5 binds to an extracellular glycoprotein antigen that is secreted during active growth of the fungus only5. This is an important consideration when using fluids such as lung BAL for diagnosing IPA since Aspergillus spores are a common component of inhaled air. The utility of the device in diagnosing IPA has been demonstrated using an animal model of infection, where the LFD displayed improved sensitivity and specificity compared to the Platelia GM and Fungitell (1 &rarr; 3)-&beta;-D-glucan assays7.
Here, we present a simple LFD procedure to detect Aspergillus antigen in human serum and BAL fluids. Its speed and accuracy provides a novel adjunct point-of-care test for diagnosis of IPA in haematological malignancy patients.

A rapid and accurate point-of-care test for invasive pulmonary aspergillosis is presented. It takes advantage of lateral-flow technology using a specific monoclonal antibody that binds to an Aspergillus antigen secreted during pulmonary infections. The assay is compatible with serum and brochoalveolar lavage and represents a novel adjunct test for disease diagnosis.

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

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 ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

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 ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...