A novel and efficient immunoglobulin Y extraction method using poloxamer-polyethylene glycol.

Although many IgY extraction methods (such as polyethylene glycol (PEG) precipitation method, octanoic acid method, water dilution method, etc.) have been established, there is still industrial drive and real need in developing scale-up IgY production methods. Some previous studies have reported that poloxamer degreasing method shows very good result in IgY extraction from egg yolk with high degreasing speed, harmlessness, simpleness in operation and minimal effect on antibody titer. In this study, we developed a new method, poloxamer-PEG method, to obtain functional IgY with high purity and yield. First, the delipidation solution was added into the diluted yolk samples, and then the filtrates were collected from the diluted yolk samples after 3 hr in room temperature. PEG-6000 was added into the collected filtrates and the mixture was centrifuged after shaking on the roller mixer for 45 min at room temperature. Last, the precipitates were resuspended in 1 mL phosphate buffered solution (PBS) buffer and dialyzed overnight. The results showed that the total protein concentrate of extractive could reach at 30 mg/mL and the purity of the IgY could reach at 92.71% with the novel method, which was superior to the PEG precipitation method and water dilution method.

A comparative evaluation of six principal IgY antibody extraction methods.

Egg yolk has been considered a promising source of antibodies. Our study was designed to compare six principal IgY extraction methods (water dilution, polyethylene glycol [PEG] precipitation, caprylic acid extraction, chloroform extraction, phenol extraction, and carrageenan extraction), and to assess their relative extraction efficiencies and the purity of the resulting antibodies. The results showed that the organic solvents (chloroform or phenol) minimised the lipid ratio in the egg yolk. The water dilution, PEG precipitation and caprylic acid extraction methods resulted in high yields, and antibodies purified with PEG and carrageenan exhibited high purity. Our results indicate that phenol extraction would be more suitable for preparing high concentrations of IgY for non-therapeutic usage, while the water dilution and carrageenan extraction methods would be more appropriate for use in the preparation of IgY for oral administration.

Chronobiological studies of chicken IgY: monitoring of infradian, circadian and ultradian rhythms of IgY in blood and yolk of chickens.

IgY is the functional equivalent of mammalian IgG found in birds, reptiles and amphibians. Many of its biological aspects have been explored with different approaches. In order to evaluate the rhythmicity of serum and yolk IgY, four chickens were examined and reared under the same conditions. To monitor biological oscillations of IgY in yolk and serum, the eggs and blood samples were collected over a 60 day period and the rhythm of yolk and serum IgY was determined by direct-ELISA. Results indicated that, there is a significant circaseptan rhythm in yolk IgY and circaquattran rhythm in serum IgY. The serum IgY concentration reached a peak in the morning, decreased to a minimum during the daytime and increased again at night revealing a significant circadian rhythm was superimposed by an ultradian rhythm. These data are suited to address the controversies concerning the IgY concentration in egg yolk and blood of laying hens. In addition, this study raised new questions, if the different rhythms in yolk and serum are concerned.

Stimulation of IgY responses in gene gun immunized laying hens by combined administration of vector DNA coding for the target antigen Botulinum toxin A1 and for avian cytokine adjuvants.

DNA immunization is a convenient and effective way of inducing a specific antibody response. In mammals, co-administration of vectors encoding immunostimulatory cytokines can enhance the humoral response resulting in elevated antibody titers. We therefore set out to investigate the effect using avian interleukin 1ß (IL-1ß) and avian interleukin 6 (IL-6) as genetic adjuvants when immunizing laying hens. A BoNT A1 holotoxoid DNA immunogen carrying two inactivating mutations was evaluated for its ability to induce a specific and sustained IgY antibody response. Both the holotoxoid and the cytokine sequences were codon-optimized. In vitro, the proteins were efficiently expressed in transfected HEK 293T cells and the cytokines were secreted into the culture supernatants. Whereas eggs from hens immunized via gene gun using a prime boost strategy showed no differences in their total IgY content, the specific αBoNT A1 response was slightly elevated up to 1.4× by the IL-1ß adjuvant vector and increased by 3.8× by the IL-6 vector. Finally, although hens receiving the IL-1ß adjuvant had laying capacities above the average, hens receiving the IL-6 adjuvant experienced laying problems.

Development of an IgY-based rocket-immunoelectrophoresis for identity monitoring of Pertussis vaccines.

An important step in vaccine production and quality control is the analysis of identity of different lots. For that purpose chicken was immunized with acellular Pertussis components (Pertussis toxoid, Filamenteous haemagglutinin, Pertactin, Fimbriae 2/3 antigen). The resulting antibodies (IgY) were non-invasive extracted from egg yolk and used for rocket immunoelectrophoresis (RIE). We demonstrated that the Ab reacted with characteristic peaks ("rockets") with the corresponding antigen. The shape of the peaks varied depending on the manufacturer and the nature of antigen (adsorbed or non-adsorbed). The coefficients of variation was about 20% during a year period. In summary, our data illustrate that an IgY-based RIE is not only a cost-effective method but also proficient for monitoring Pertussis vaccines.

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°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).

Simultaneous quantification of five bacterial and plant toxins from complex matrices using a multiplexed fluorescent magnetic suspension assay.

Proteotoxins such as ricin, abrin, botulinum neurotoxins type A and B (BoNT/A, BoNT/B) and staphylococcal enterotoxin B (SEB) are regarded as potential biological warfare agents which could be used for bioterrorism attacks on the food chain. In this study we used a novel immunisation strategy to generate high-affinity monoclonal and polyclonal antibodies against native ricin, BoNT/A, and BoNT/B. The antibodies were used along with antibodies against SEB and abrin to establish a highly sensitive magnetic and fluorescent multiplex bead array with excellent sensitivities between 2 ng/L and 546 ng/L from a minimal sample volume of 50 microL. The assay was validated using 20 different related analytes and the assay precision was determined. Advancing the existing bead array technology, the novel magnetic and fluorescent microbeads proved amenable to enrichment procedures, by further increasing sensitivity to 0.3-85 ng/L, starting from a sample volume of 500 microL. Furthermore, the method was successfully applied for the simultaneous identification of the target toxins spiked into complex food matrices like milk, baby food and yoghurt. On the basis of our results, the assay appears to be a good tool for large-scale screening of samples from the food supply chain.

Gene gun-supported DNA immunisation of chicken for straightforward production of poxvirus-specific IgY antibodies.

Orthopoxviruses code for numerous immunomodulatory proteins, the structure and function of which are clarified inadequately. Antibodies constitute a potent tool to study such proteins, enabling conclusions on protein location and time course of expression. However, common antibody production in mice or rabbits requires tedious protein expression and injection, as well as blood collection at regular intervals. To simplify this procedure, IgY antibodies specific for poxviral proteins (F1L and p28) were generated by immunisation of chickens, because antibody retrieval from eggs allows the non-invasive generation of huge amounts of antibodies. The main intentions were (i) to decrease invasiveness, (ii) to immunise with native forms of proteins and (iii) to circumvent previous protein expression and purification. Therefore, chicken were immunised with DNA expression vectors coding for conserved domains of the selected proteins delivered for the first time by a gene gun. Four weeks after initial immunisation specific antibodies were found in the egg yolk as proven by immunofluorescence staining of poxvirus-infected cells. The specific IgY titre rose to 1:80,000 and was stable for more than 120 days. With this investigation we present an universal procedure for IgY design and production that can be applied for various issues in the future.

Thymulin gene therapy prevents the reduction in circulating gonadotropins induced by thymulin deficiency in mice.

Integrity of the thymus during perinatal life is necessary for a proper maturation of the pituitary-gonadal axis in mice and other mammalian species. Thus congenitally athymic (nude) female mice show significantly reduced levels of circulating gonadotropins, a fact that seems to be causally related to a number of reproductive derangements described in these mutants. Interestingly, a number of in vitro studies suggest that the thymic peptide thymulin may be involved in thymus-pituitary communication. To determine the consequences of low serum thymulin in otherwise normal animals, we induced short (8 days)- and long (33 days)-term thymulin deficiency in C57BL/6 mice by neonatally injecting (intraperitoneally) an anti-thymulin serum and assessed their circulating gonadotropin levels at puberty and thereafter. Control mice received an irrelevant antiserum. Gonadotropins were measured by radioimmunoassay and thymulin by bioassay. Both long- and short-term serum thymulin immunoneutralization resulted in a significant reduction in the serum levels of gonadotropins at 33 and 45 days of age. Subsequently, we injected (intramuscularly) an adenoviral vector harboring a synthetic DNA sequence (5'-ATGCAAGCCAAATCTCAAGGTGGATCCAACTAGTAG-3') encoding a biologically active analog of thymulin, methionine-FTS, in newborn nude mice (which are thymulin deficient) and measured circulating gonadotropin levels when the animals reached 52 days of age. It was observed that neonatal thymulin gene therapy in the athymic mice restored their serum thymulin levels and prevented the reduction in circulating gonadotropin levels that typically emerges in these mutants after puberty. Our results indicate that thymulin plays a relevant physiological role in the thymus-pituitary-gonadal axis.

Chicken egg yolk antibodies (IgY-technology): a review of progress in production and use in research and human and veterinary medicine.

The production of antibodies (Abs) in chickens and the extraction of specific Abs from egg yolk (IgY Abs) are increasingly attracting the interest of the scientific community, as demonstrated by the significant growth of the IgY literature. This review offers detailed and comprehensive information about IgY-technology, including: a) possibilities for hen keeping in accordance with the Three Rs principles; b) new insights into the IgY transfer mechanism from blood to yolk as a biological basis for the technology; c) the comparative characteristics of IgY Abs and IgG Abs; d) the high efficacy of the technique, in view of the extraordinary amount of IgY Ab produced by one hen in one year (between 20 g and 40 g IgY in total); e) comparisons between the efficacies of IgY Abs and IgG Abs (rabbit, sheep, mouse) in several immunological assays; f) immunisation protocols, as well as the most commonly used IgY-extraction procedures; g) new possibilities for application in human and veterinary medicine, including strategies for the treatment of Helicobacter pylori infection or fatal intestinal diseases in children, particularly in poor countries, for reducing the use of antibiotics, and, in Asia and South America, for producing Abs against snake, spider and scorpion venoms; and h) the use of IgY Abs in various fields of research, also taking into consideration recent developments in South America (particularly Argentina and Cuba) and in Asia.

The zinc finger of the CSN-associated deubiquitinating enzyme USP15 is essential to rescue the E3 ligase Rbx1.

The COP9 signalosome (CSN) is a conserved protein complex found in all eukaryotic cells and involved in the regulation of the ubiquitin (Ub)/26S proteasome system. It binds numerous proteins, including the Ub E3 ligases and the deubiquitinating enzyme Ubp12p, the S. pombe ortholog of human USP15. We found that USP15 copurified with the human CSN complex. Isolated CSN complex exhibited protease activity that deubiquitinated poly-Ub substrates and was completely inhibited by o-phenanthroline (OPT), a metal-chelating agent. Surprisingly, the recombinant USP15 was also not able to cleave isopeptide bonds of poly-Ub chains in presence of OPT. Detailed analysis of USP sequences led to the discovery of a novel zinc (Zn) finger in USP15 and related USPs. Mutation of a single conserved cysteine residue in the predicted Zn binding motif resulted in the loss of USP15 capability to degrade poly-Ub substrates, indicating that the Zn finger is essential for the cleavage of poly-Ub chains. Moreover, pulldown experiments demonstrated diminished binding of tetra-Ub to mutated USP15. Cotransfection of USP15 and the Ub ligase Rbx1 revealed that the wild-type deubiquitinating enzyme, but not the USP15 mutant with a defective Zn finger, stabilized Rbx1 toward the Ub system, most likely by reversing poly/autoubiquitination. In summary, a functional Zn finger of USP15 is needed to maintain a conformation essential for disassembling poly-Ub chains, a prerequisite for rescuing the E3 ligase Rbx1.

Characterization of the alternative sigma factor sigma54 and the transcriptional regulator FleQ of Legionella pneumophila, which are both involved in the regulation cascade of flagellar gene expression.

We cloned and analyzed Legionella pneumophila Corby homologs of rpoN (encoding sigma(54)) and fleQ (encoding sigma(54) activator protein). Two other genes (fleR and pilR) whose products have a sigma(54) interaction domain were identified in the genome sequence of L. pneumophila. An rpoN mutant strain was nonflagellated and expressed very small amounts of the FlaA (flagellin) protein. Like the rpoN mutant, the fleQ mutant strain of L. pneumophila was also nonflagellated and expressed only small amounts of FlaA protein compared to the amounts expressed by the wild type. In this paper we show that the sigma(54) factor and the FleQ protein are involved in regulation of flagellar gene operons in L. pneumophila. RpoN and FleQ positively regulate the transcription of FliM and FleN, both of which have a sigma(54)-dependent promoter consensus sequence. However, they seemed to be dispensable for transcription of flaA, fliA, or icmR. Our results confirmed a recently described model of the flagellar gene regulation cascade in L. pneumophila (K. Heuner and M. Steinert, Int. J. Med. Microbiol. 293:133-145, 2003). Flagellar gene regulation was found to be different from that of Enterobacteriaceae but seems to be comparable to that described for Pseudomonas or Vibrio spp.

Production, purification, and characterization of rat pro-CCK from serum-free adapted Drosophila cells.

The precursor of cholecystokinin (pro-CCK) was expressed and purified from media of stably transfected D.Mel-2 cell as an V5-His tagged fusion protein. Its identity was confirmed using SDS-PAGE, immunoblotting, gel filtration chromatography, HPLC, and Mass Spectroscopy. Two major forms of pro-CCK were found with a molecular weight of about 14.4 and 11.3 kDa. The smaller form represents the V5-His tagged pro-CCK after cleavage at a single arginine residue at CCK-58. This cleavage is probably being performed by endogenous proteases in these cells. Purification of the desired larger form of pro-CCK is possible using a nickel column with a recovery of about 20%, yielding 500 microg/L media. The purified protein is stable for several months and can be used for further functional studies of pro-CCK.

Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome.

The COP9 signalosome (CSN) purified from human erythrocytes possesses kinase activity that phosphoryl ates proteins such as c-Jun and p53 with consequence for their ubiquitin (Ub)-dependent degradation. Here we show that protein kinase CK2 (CK2) and protein kinase D (PKD) co-purify with CSN. Immunoprecipitation and far-western blots reveal that CK2 and PKD are in fact associated with CSN. As indicated by electron microscopy with gold-labeled ATP, at least 10% of CSN particles are associated with kinases. Kinase activity, most likely due to CK2 and PKD, co-immuno precipitates with CSN from HeLa cells. CK2 binds to DeltaCSN3(111-403) and CSN7, whereas PKD interacts with full-length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modifies CSN7. Both CK2 and PKD phosphorylate c-Jun as well as p53. CK2 phosphoryl ates Thr155, which targets p53 to degradation by the Ub system. Curcumin, emodin, DRB and resveratrol block CSN-associated kinases and induce degradation of c-Jun in HeLa cells. Curcumin treatment results in elevated amounts of c-Jun-Ub conjugates. We conclude that CK2 and PKD are recruited by CSN in order to regulate Ub conjugate formation.

Extraction of a monospecific Coombs-reagent from chicken eggs.

During the last ten years the extraction of specific antibodies (ab) from the yolk of eggs of immunised chickens is more and more accepted as an useful alternative to the immunisation of mammals. The subject of this work is the immunisation of chickens with human IgG and the extraction of specific anti human IgG ab from egg yolk in order to obtain monospecific Coombs reagent. 12 Leghorn hens (25 weeks old) were immunised with intact human IgG (INTACGLOBIN). The chickens were immunised with 100 microg IgG/animal once per week for a period of seven weeks. The highest titre was observed after the 5th immunisation, the following immunisations achieved no further titre increase. The IgY purification was performed according to the method of Akita and Nakai (1993). The resulting IgY preparation was tested for the presence of hetero-agglutinine by means of direct agglutination using human erythrocytes of all blood groups. Thereafter 58 blood donors were tested by means of direct or indirect Coombs-test using a reference reagent (DAKO) and a Coombs reagent isolated from chicken eggs (IgY antibodies). No differences have been found between the results obtained using both Coombs reagents. Presented results show that there is a possibility to produce Coombs-reagent in chickens. Advantages of this method are: 1) non invasive antibody sampling by egg collection instead of bleeding the animal (refinement of antibody production); 2) decreasing amount of animals necessary to produce high amounts of reagent; 3) IgY-preparation contains no hetero-agglutinine in contrast to serum ab from mammals, therefore additional step in reagent production e.g. the absorption of hetero-agglutinins is not necessary.

Stable biocompatible adjuvants--a new type of adjuvant based on solid lipid nanoparticles: a study on cytotoxicity, compatibility and efficacy in chicken.

A new type of adjuvant was tested for its ability to initiate antibody production in chickens, and its cellular and tissue compatibility were assessed. The stable biocompatible adjuvants tested are based on surface-modified solid lipid nanoparticles (SLNs), made from paraffin or biodegradable glycerides, and are simply admixed to the antigens before administration. The tissue-damaging potency of four formulations of the new adjuvants (H1, H2, H3 and H4) were first tested in vitro by using human foreskin fibroblasts and RAW 264.7 macrophages. The adjuvants were well tolerated by both cell types. Immunisation studies in chickens were performed by using a Mycoplasma bovis antigen and mouse immunoglobulin G (IgG). The resulting antibodies were non-invasively extracted from egg yolk. The use of the various adjuvant formulations resulted in a significant production of specific antibodies after the first and second booster immunisations. Freund's complete adjuvant (FCA), considered until now to be the "gold standard" among the adjuvants, revealed the highest antibody titre against mouse IgG. SLNs with a particle size of more than 100 nm exhibited a clear adjuvant activity, whereas SLNs with a particle size below 100 nm, in various concentrations, revealed a lower adjuvant activity. Immunisation of chickens with the mouse IgG alone, dissolved in phosphate-buffered saline, resulted in a slow antibody titre development. At the end of the experiment, the chickens were examined for vaccination-associated tissue damage. In contrast to FCA, the SLN formulations caused only minor tissue irritation at the injection sites. In conclusion, SLNs seem to be a promising alternative to FCA for antibody production in chickens, and potentially in other animals.

A flagellar gene cluster from the oral spirochaete Treponema maltophilum.

A flagellar gene cluster from the oral spirochaete Treponema maltophilum ATCC 51939T was cloned. Sequence analysis revealed six putative ORFs, two of which encode the flagellar subunit proteins FlaB2 (286 aa) and FlaB3 (285 aa). Northern blot analysis revealed two flagellin transcripts with the expected size of monocistronic mRNAs. Sequence analysis and primer extension experiments indicated that the transcription of the flaB2 gene is directed by a sigma28-like FliA factor. Using fliA and fliA+ Escherichia coli K-12 strains, it was shown that flaB2 expression in E. coli required the sigma28 factor using an initiation site identical to that in Treponema maltophilum. Primer extension analysis revealed two transcriptional start sites 5' of the flaB3 gene, a strong promoter with a sigma28-like -10 promoter element and a weak promoter with a putative sigma54 promoter consensus sequence. Downstream of flaB3, a putative fliD homologue was found, probably encoding the flagellar cap protein of Treponema maltophilum. Flagellin-gene-specific DNA probes hybridized to all 13 Treponema strains investigated, whereas a fliD-specific DNA probe only hybridized to Treponema maltophilum, other treponemal group IV isolates and Treponema brennaborense.

Egg Yolk Antibodies, State of the Art and Future Prospects.

Immunization of chickens and extraction of antibodies from egg yolk belongs to the alternative methods since the animals suffering is reduced by non-invasive antibody-sampling. Also, the number of animals needed to produce a certain amount of antibody can be reduced since chickens produce a significant higher antibody quantity than rabbits. Despite its several advantages this technology (IgY-technology) is rather scarcely used. Traditional behavior as well as limited or no information at all may hamper a broader acceptance at present. However, significant arguments exist in chicken housing, the choice of appropriate IgY-extraction methods and a lack of information regarding the use of IgY-antibodies. This paper intends to give a short introduction in the IgY-technology, to briefly discuss the state of the art and to inform on recent developments and discussions in this field. The suitability of IgY for special fields of application (as a result of the structural differences between IgY and IgG) is emphasized (e.g. assays combining IgG and IgY, immunization of chickens against highly conserved anti-genes). In addition, it is stressed that the IgY-technology as an alternative method can particularly integrate requirements of animal protection (reduce, replace, refine), science (characteristics of avian immune system and resulting properties of IgY) and economy (amount of IgY produced from one chicken).

Assay Development of a Serum Marker for Myocardial Injury: Experiences with an Avian anti-Troponin I Antibody.

The diagnosis of acute myocardial infarction is still difficult in certain cases. The measurement of human cardiac Troponin I, a new marker for heart muscle cell necrosis, can help to support current procedures of diagnosis. The detection is usually done with antibodies in a sandwich immunoassay. For development of such a test for human cardiac Troponin I on the automated analyser Technicon Immuno 1 avian egg yolk antibodies were tested. Antibody preparations from two immunised chickens were tested for content of specific anti-Troponin I antibodies. Following affinity purification against a peptide from the Troponin I sequence the antibodies were conjugated with alkaline phosphatase and used as detector antibodies. The combination with different monoclonal antibodies resulted in standard curves for Troponin I, which were in terms of sensitivity inferior to a commercial polyclonal antibody from goat.