Chicken IgY Research Articles

Expression of goat RNA binding protein (NANOS2), development and evaluation of anti-NANOS2 IgY antibodies

Available rabbit anti-NANOS2 antibodies failed to recognize the NANOS2 protein of goat origin though they showed better reactivity towards the same protein of human and mice origin. A specific antibody against goat NANOS2 is necessary to explore the biological functions of dairy goat NANOS2 protein. Recombinant NANOS2 protein (dairy goat origin) was expressed in E. coli (BL21). Anti-NANOS2 immunoglobulin Y (IgY; in chicken) was developed and evaluated. NANOS2 protein sequences between mice and dairy goat were compared using bioinformatics tools. The specific IgY titer attained the peak (≥ 1 : 128 000) after the 3 rd booster injection, then it gradually decreased to 1 : 16 000 and remained stable. The specificity and sensitivity of anti- NANOS2 IgY was superior to rabbit anti-NANOS2 IgG. Though, the functional domain 63-116 or 62-115 of NANOS2 protein is highly conserved with 100% identity in dairy goat and mice, the considerable difference in sequence at region 18-61 was identified from the predicted epitopes. According to our survey, this is the first report on preparation of chicken IgY against NANOS2 protein and indicating the prerequisite of species specific NANOS2 antibodies due to epitopes difference in functionally conserved NANOS2 protein. Anti- NANOS2 IgY could be applied as a novel research tool in developmental biology.

Investigating low volume planar surface fluorescent immunoassays with QDs for spatial and spectral multiplexing

The simultaneous detection of two analytes, chicken IgY (IgG) and Staphylococcal enterotoxin B (SEB), in a single spot of a planar surface fluorescent immunoassay was demonstrated using luminescent semiconductor quantum dot nanocrystal (QD) tracers. This spectral multiplexing is made possible by employing one laser to serve as the excitation source for detecting two distinct QD signals from different analytes. Spatial multiplexing – detection of two analytes in different regions of the planar surface – was also demonstrated using fluid handling constructs that allowed for very low solution volumes when performing the assay. The limits of detection for detecting both chicken IgG and SEB within a single spot were 25ng/mL and 1.6ng/mL, respectively. A novel slide reading detection platform involving evanescent wave excitation of a planar surface was created in-house for assay measurement. This platform is potentially more amenable to a point-of-care (POC) environment than conventional slide readers because of its modular design architecture and lack of moving parts. The result is a platform which is simple to repair using primarily off-the-shelf consumer components.

Purification of Immunoglobulins and their Binding to a Bacterial Protein LAG-HRP Conjugate

Objective: To purify IgG molecules from several species by SpA-affinity chromatography and to study the interactions of mammalian IgGs with a peroxidase-labelled SpL, SpA and SpG conjugate (SPLAG-HRP) in an enzyme-linked immunosorbent assay (ELISA). Materials and methods: The periodate method described by Nakane and Kawoi was used to prepare the SPLAG-HRP conjugate. The 10% non-denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of sera and purified immunoglobulins was carried out to characterize molecularly the purified IgGs. The chicken IgY fraction was isolated by the chloroform-polyethylene glycol (PEG) method for its use as a negative control in the ELISA that was used to determine the affinity of different immunoglobulins to a SPLAG-HRP conjugate. Results: The SpA-affinity chromatography and the 10% non-denaturing SDS-PAGE of sera and purified immunoglobulins (IgGs) were useful separation techniques. Most purified IgGs interacted moderately with the SPLAG-HRP including IgGs from horse, dog, skunk, coyote and raccoon. The purified mammalian IgG had a molecular weight (MW) of approximately 150 kDa. Conclusion: The SPLAG-HRP was a versatile heterofunctional reagent useful for the detection of purified immunoglobulins from diverse mammalian species.

Human erythrocyte band 3 functions as a receptor for the sialic acid-independent invasion of Plasmodium falciparum. Role of the RhopH3–MSP1 complex

Plasmodium falciparum takes advantage of two broadly defined alternate invasion pathways when infecting human erythrocytes: one that depends on and the other that is independent of host sialic acid residues on the erythrocyte surface. Within the sialic acid-dependent (SAD) and sialic acid-independent (SAID) invasion pathways, several alternate host receptors are used by P. falciparum based on its particular invasion phenotype. Earlier, we reported that two putative extracellular regions of human erythrocyte band 3 termed 5C and 6A function as host invasion receptor segments binding parasite proteins MSP1 and MSP9 via a SAID mechanism. In this study, we developed two mono-specific anti-peptide chicken IgY antibodies to demonstrate that the 5C and 6A regions of band 3 are exposed on the surface of human erythrocytes. These antibodies inhibited erythrocyte invasion by the P. falciparum 3D7 and 7G8 strains (SAID invasion phenotype), and the blocking effect was enhanced in sialic acid-depleted erythrocytes. In contrast, the IgY antibodies had only a marginal inhibitory effect on FCR3 and Dd2 strains (SAD invasion phenotype). A direct biochemical interaction between erythrocyte band 3 epitopes and parasite RhopH3, identified by the yeast two-hybrid screen, was established. RhopH3 formed a complex with MSP119 and the 5ABC region of band 3, and a recombinant segment of RhopH3 inhibited parasite invasion in human erythrocytes. Together, these findings provide evidence that erythrocyte band 3 functions as a major host invasion receptor in the SAID invasion pathway by assembling a multi-protein complex composed of parasite ligands RhopH3 and MSP1.

IgY pharmacokinetics in rabbits: Implications for IgY use as antivenoms

This paper presents the first study of chicken IgY pharmacokinetics (PK) in rabbits. We measured IgY blood serum concentrations using a specific high sensitivity ELISA method. The fast initial component observed when studying horse Fab, F(ab′)2 or IgG was absent from IgY PK. During the first 80 min of observation there was only a single slow exponential decay, which sped up afterward to the point that IgY became undetectable after 216 h of observation; due to this time course, PK parameters were determined with trapezoidal integration. The most significant IgY pharmacokinetic parameters determined were (all presented as medians and their 95% confidence interval): Area Under the Curve = 183.8 (135.2, 221.5) mg·h·L−1; Distribution volume of the central compartment·[Body Weight (BW)]−1 = 46.0 (21.7, 70.3) mL·kg−1; Distribution volume in steady state·BW−1 = 56.8 (44.4, 68.5) mLkg−1; Mean Residence Time = 40.1 (33.6, 48.5) h; Total plasma clearance·BW−1 = 1.44 (1.15, 1.66) mL·h−1·kg−1. Anti IgY IgG titers determined by ELISA increased steadily after 72 h, and reached 2560 (1920, 5760) dilution−1 at 264 h; anti-chicken IgG concentrations rose up to 3.19 (2.31, 6.17) μg/mL in 264 h. Our results show that IgY PK lacks the fast initial decay observed in other PK studies using horse IgG, F(ab′)2 or Fab, remains in the body 39.0 (28.7, 47.2) % much as IgG and is ≈3 times more immunogenic that horse IgG in rabbits.

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.

Effect of chicken egg yolk antibodies (IgY) against diarrhea in domesticated animals: a systematic review and meta-analysis.

BackgroundIgY antibodies are serum immunoglobulin in birds, reptiles and amphibians, and are transferred from serum to egg yolk to confer passive immunity to their embryos and offspring. Currently, the oral passive immunization using chicken IgY has been focused as an alternative to antibiotics for the treatment and control of diarrhea in animals and humans. This systematic review was focused to determine the effect of IgY in controlling and preventing diarrhea in domesticated animals including Piglets, Mice, Poultry and Calves.Methods and ResultsPrevious research reports focused on treatment effect of Chicken IgY against diarrhea were retrieved from different electronic data bases (MEDLINE, EMBASE, SPRINGER-LINK, WILEY, AGRICOLA, MEDWELL Journals, Scientific Publish, Chinese articles from Core periodicals in 2012). A total of 61 studies in 4 different animal classes met the inclusion criteria. Data on study characteristics and outcome measures were extracted. The pooled relative risk (RR) of 49 studies of different animals [Piglets – 22; Mice – 14; Poultry – 7 and Calves – 6] in meta-analyses revealed that, IgY significantly reduced the risk of diarrhea in treatment group when compare to the placebo. However, the 95% confidence intervals of the majority of studies in animal class piglets and calves embrace RR of one. The same results were obtained in sub group analyses (treatment regiment – prophylactic or therapeutic; pathogen type – bacterial or viral). Perhaps, this inconsistency in the effect of IgY at the individual study level and overall effect measures could be influenced by the methodological heterogeneity.ConclusionThe present systematic review (SR) and meta-analysis demonstrated the beneficial effect of IgY. This supports the opinion that IgY is useful for prophylaxis and treatment. However, more intensive studies using the gold standard animal experiments with the focus to use IgY alone or in combination with other alternative strategies are indispensable.

Proteolytic Activities of Kiwifruit Actinidin (Actinidia deliciosa cv. Hayward) on Different Fibrous and Globular Proteins: A Comparative Study of Actinidin with Papain

Actinidin, a member of the papain-like family of cysteine proteases, is abundant in kiwifruit. To date, a few studies have been provided to investigate the proteolytic activity and substrate specificity of actinidin on native proteins. Herein, the proteolytic activity of actinidin was compared to papain on several different fibrous and globular proteins under neutral, acidic and basic conditions. The digested samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry to assess the proteolytic effect. Furthermore, the levels of free amino nitrogen (FAN) of the treated samples were determined using the ninhydrin colorimetric method. The findings showed that actinidin has no or limited proteolytic effect on globular proteins such as immunoglobulins including sheep IgG, rabbit IgG, chicken IgY and fish IgM, bovine serum albumin (BSA), lipid transfer protein (LTP), and whey proteins (α-lactalbumin and β-lactoglobulin) compared to papain. In contrast to globular proteins, actinidin could hydrolyze collagen and fibrinogen perfectly at neutral and mild basic pHs. Moreover, this enzyme could digest pure α-casein and major subunits of micellar casein especially in acidic pHs. Taken together, the data indicated that actinidin has narrow substrate specificity with the highest enzymatic activity for the collagen and fibrinogen substrates. The results describe the actinidin as a mild plant protease useful for many special applications such as cell isolation from different tissues and some food industries as a mixture formula with other relevant proteases.

Egg yolk antibodies (IgY) against Bovine Leukemia Virus

Bovine Leukemia Virus (BLV) is distributed worldwide and causes important economic losses on dairy farms. Currently, there are no effective vaccines or antivirals against BLV. Egg yolk antibodies (IgY) has many advantages over mammalian IgG. Despite the higher yields, they are non-invasively extracted from egg yolk, do not cross react against mammalian antigens or activate the mammalian complement system. In this work we evaluate the reactivity of Igy antibodies against Bovine Leukemia Virus p24 core protein and against the whole virus particle. Hens were immunized by intramuscular inoculation with purified p24 or the virus particle until the development of high antibody-titers. Total IgY was purified from egg yolks by ammonium sulfate precipitation. The purified egg yolk antibodies strongly reacted with BLV particles from a persistently infected cell line, with supernatants from ex vivo cultures of PBMCs from natural infected animals and also with purified p24 by both ELISA and Western blot. These data suggest that chicken IgY may be a suitable platform to produce large amounts of anti-BLV antibodies for diagnostic systems. Furthermore, the use of IgY for passive immunization against BLV infection should also be explored in order to develop new strategies to control the disease in cattle.

Characterization of the optimized C2 domain of protein G: finding its additional chicken IgY-binding ability

The C2 domain of streptococcal protein G is a small (55 residue) peptide with immunoglobulin-binding activity. Following codon optimization, the gene was divided into four oligonucleotide fragments and amplified by overlap PCR. The recombinant plasmid pET30a-C2 was transformed into Escherichia coli Rosetta (DE3) PLysS for expression. After purification by Ni-NTA, the fusion protein was identified by western-blotting, Dot-ELISA and ELISA. His-tagged C2 bound to human, rabbit, cattle, pig, goat, mouse or guinea pig IgG had no affinity for goose, duck, wild duck, wild turkey and red-crowned crane IgY. Its affinity for chicken IgY, however, was comparable to that of guinea pig IgG. The C2 domain may therefore provide an ideal material for the purification and detection of immunoglobulin G from various mammals.

Effect of processing on the detectability of peanut protein by ELISA

Chicken IgY was used for the detection and quantification of peanut proteins by indirect competitive ELISA. The method was optimized by using a checker board approach to determine the optimal concentration of coating antigen, primary antibody and secondary antibody. Peanut protein could be detected in foods down to levels of 10ppm. The effect of physical (heat treatment at 80°C and 100°C) and chemical (acid, alkali and reducing sugar) treatments on the IgY binding of peanut proteins was investigated. The optimized assay was relatively sensitive for the roasted peanut proteins. However, the binding ability of chicken IgYs to peanut proteins was found to be significantly altered by denaturation and hydrolysis of proteins. It was also observed that the effect of Millard chemistry on the detectability of peanut protein was less pronounced at high temperatures than at low temperatures.

The Joubert Syndrome-associated Missense Mutation (V443D) in the Abelson-helper Integration Site 1 (AHI1) Protein Alters Its Localization and Protein-Protein Interactions

Missense mutations in AHI1 result in the neurodevelopmental ciliopathy called Joubert syndrome. Mutations in AHI1 decrease cilia formation, alter its localization and stability, and change its binding to HAP1 and NPHP1. Mutations in AHI1 affect ciliogenesis, AHI1 protein localization, and AHI1-protein interactions. This study begins to describe how missense mutations in AHI1 can cause Joubert syndrome. Mutations in AHI1 cause Joubert syndrome (JBTS), a neurodevelopmental ciliopathy, characterized by midbrain-hindbrain malformations and motor/cognitive deficits. Here, we show that primary cilia (PC) formation is decreased in fibroblasts from individuals with JBTS and AHI1 mutations. Most missense mutations in AHI1, causing JBTS, occur in known protein domains, however, a common V443D mutation in AHI1 is found in a region with no known protein motifs. We show that cells transfected with AHI1-V443D, or a new JBTS-causing mutation, AHI1-R351L, have aberrant localization of AHI1 at the basal bodies of PC and at cell-cell junctions, likely through decreased binding of mutant AHI1 to NPHP1 (another JBTS-causing protein). The AHI1-V443D mutation causes decreased AHI1 stability because there is a 50% reduction in AHI1-V443D protein levels compared with wild type AHI1. Huntingtin-associated protein-1 (Hap1) is a regulatory protein that binds Ahi1, and Hap1 knock-out mice have been reported to have JBTS-like phenotypes, suggesting a role for Hap1 in ciliogenesis. Fibroblasts and neurons with Hap1 deficiency form PC with normal growth factor-induced ciliary signaling, indicating that the Hap1 JBTS phenotype is likely not through effects at PC. These results also suggest that the binding of Ahi1 and Hap1 may not be critical for ciliary function. However, we show that HAP1 has decreased binding to AHI1-V443D indicating that this altered binding could be responsible for the JBTS-like phenotype through an unknown pathway. Thus, these JBTS-associated missense mutations alter their subcellular distribution and protein interactions, compromising functions of AHI1 in cell polarity and cilium-mediated signaling, thereby contributing to JBTS.

Application of IgY to sandwich enzyme-linked immunosorbent assays, lateral flow devices, and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products

Staphylococcal enterotoxins (SEs), produced by Staphylococcus aureus, are a major cause of staphylococcal food poisoning. Traditionally, sandwich enzyme-linked immunosorbent assay (ELISA) and reverse passive latex agglutination with rabbit antibody IgG have been used to detect SEs. However, most of these kits require a long processing time and there is a risk of false-positive results since IgG reacts nonspecifically with protein A produced by S. aureus. In this study, we prepared antienterotoxin chicken IgY antibodies specific for each SE (SEA to SEE) without reaction to protein A, which enabled a drastic reduction in nonspecific reactions. ELISAs, lateral flow device (LFDs), and IgY-based immunopillar chips were developed for SE detection. All the ELISAs developed were as sensitive as commercially available kits. The SEs in milk were successfully detected by the ELISAs, LFDs, and immunopillar chips without any sample pretreatment. The LFD could detect SEA even at the low concentration of 0.2ng/ml within 15min in milk. The detection limit of the immunopillar chips for the SEs ranged from 0.01 to 0.1ng/ml in milk; the SEs were detected within 12min and specialized skills were not required. The ELISA and LFD detected SEA in dairy products artificially contaminated with S. aureus, including ice cream, yogurt, and café au lait, in a dose-dependent manner. In conclusion, IgY allows highly specific detection of SEs, and ELISAs, LFDs, and immunopillar chips should be useful tools for screening SEs in milk and dairy products.

The impact of PEGylation on protein immunogenicity

Covalent attachment of PEG (PEGylation) is widely used to improve the pharmaceutical properties of therapeutic proteins. The applicability and safety of this method have been proven by the use of various PEGylated pharmaceutical proteins approved by the Food and Drug Administration (FDA). One of the properties attributed to PEGylation is immunogenicity reduction of the PEGylated protein. In this study, the impact of PEGylation on immunogenicity was tested and compared for two proteins (chicken IgY and horse IgG) in two strains of mice (Balb/c and C57BL/6) for two routes of administration (i.v. and i.m.) and two sizes of PEG (5kD and 20kD). The influence of PEG was shown to be inconsistent between the mouse strains and routes of administration, even with the same tested protein. Consequently, immunogenicity reduction by PEGylation cannot be predicted or assumed; it must be tested on an individual case basis.

Detection of Methicillin-Resistant Staphylococcus aureus Using a Specific Anti-PBP2a Chicken IgY Antibody

Immunodetection of methicillin-resistant Staphylococcus aureus (MRSA) by conventional methods employing mammalian immunoglobulins has unknown detection limits, and often yields false-positive results because of the presence of S. aureus protein A, which binds the Fc region of mammalian IgG. In this study, a new PBP2a-specific chicken IgY antibody was developed in inbred and conventional chickens, and used for the detection of MRSA using whole cell lysate samples. Our results showed that this chicken IgY antibody minimized the side effects of protein A. Moreover, enzyme-linked immunosorbent assay and immunochromatography systems were used with a monoclonal and polyclonal anti-PBP2a IgY antibody, clearly differentiating MRSA from methicillin-sensitive S. aureus and other methicillin-sensitive Staphylococcus spp. The detection limit of the immunochromatography was 10(8) colony-forming units; therefore, 1 colony on an agar plate was adequate to distinguish MRSA from non-MRSA. The specificity and sensitivity of this assay were almost similar to that of a commercially available latex agglutination test; however, the procedure used in this study was less complicated. The entire detection procedure, including sample preparation, takes only 20 min and does not require special equipment. Therefore, the use of this IgY antibody as a new tool for the detection of MRSA is highly recommended.

Development of Chicken Immunoglobulin Y for Rapid Detection of Cronobacter muytjensii in Infant Formula Powder

The present study was aimed to produce a chicken polyclonal antibody against Cronobacter muytjensii and to develop an immunoassay for its detection. Purification of anti-C. muytjensii IgY from egg yolk was accomplished using various methods such as water dilution and salt precipitation. As a result, sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced two bands around 30 and 66 kDa, corresponding to a light and a heavy chain, respectively. Indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was performed to determine the effectiveness of the chicken IgY against C. muytjensii. The optimum conditions for detecting C. muytjensii by indirect ELISA and checkerboard titration of the antigen revealed an optimum average absorbance at the concentration of 18 <TEX>${\mu}g/mL$</TEX>, having ca. <TEX>$10^8$</TEX> coated cells per well. The anti-C. muytjensii IgY antibody had high specificity for C. muytjensii and low cross-reactivity with other tested pathogens. In this assay, no cross-reactivity was observed with the other genera of pathogenic bacteria including Escherichia coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus, Bacillus cereus, Enterobacter aerogenes, Salmonella Enteritidis and Listeria monocytogenes. In addition, detection of C. muytjensii in infant formula powder showed a low matrix effect on the detection curve of IC-ELISA for C. muytjensii, with similar detection limit of <TEX>$10^5$</TEX> CFU/mL as shown in standard curve. These findings demonstrate that the developed method is able to detect C. muytjensii in infant formula powder. Due to the stable antibody supply without sacrificing animals, this IgY can have wide applications for the rapid and accurate detection of C. muytjensii in dairy foods samples.

Avian IgY is selectively incorporated into the egg yolks of oocytes by discriminating Fc amino acid residues located on the Cυ3/Cυ4 interface

In avian species, maternal IgY is selectively incorporated into the egg yolks of maturing oocytes, but the relevance of receptor-mediated uptake is unclear. Here we investigated the critical amino acid residues of IgY required for egg yolk transport by conducting mutational analyses of selected residues located along the Cυ3 and Cυ4 domains of chicken IgY. Recombinant wild-type IgY-Fc (WT) and its mutants were synthesized, and their uptakes into the egg yolks of quail were determined. Among the 17 amino acid residues located on the Cυ3/Cυ4 interface, the substitution of Y363 at the Cυ3 domain to alanine abolished the IgY-Fc uptake into egg yolks. The comprehensive substitution of Y363 with other amino acids revealed that the residue at 363 needs to be allocated with aromatic amino acids to maintain the high transport ability. The deglycosylation of the N-linked carbohydrate chain by substituting N407 at the Cυ3 domain with alanine also caused a marked reduction of IgY-Fc uptake. The microscopic detection of the injected WT and Y363A mutant in ovarian follicles showed that the WT was concentrically accumulated in yolk granules, whereas the Y363A mutant was hardly accumulated in yolk granules, but it had infiltrated into the granulosa cell layer, suggesting that a major hurdle disturbing the infiltration of the Y363A mutant lies on the inside of the granulosa cell layer. The identification of important amino acid residues required for efficient IgY transport enhances our understanding of the molecular mechanisms underlying IgY transport through a specific IgY receptor in ovarian follicles.

A dual chicken IgY against rotavirus and norovirus

Rotavirus (RV) and norovirus (NoV) are the two most important causes of viral gastroenteritis. While vaccine remains an effective prophylactic strategy, development of other approaches, such as passive immunization to control and treat clinical infection and illness of these two pathogens, is necessary. Previously we demonstrated that high titers of NoV-specific IgY were readily developed by immunization of chickens with the NoV P particles. In this study, we developed a dual IgY against both RV and NoV through immunization of chickens with a divalent vaccine comprising neutralizing antigens of both RV and NoV. This divalent vaccine, named P-VP8∗ particle, is made of the NoV P particle as a carrier with the RV spike protein VP8∗ as a surface insertion. Approximately 45mg of IgY were readily obtained from each yolk with high titers of anti-P particle and anti-VP8∗ antibodies detected by ELISA, Western blot, HBGA blocking (NoV and RV) and neutralization (RV) assays. Reductions of RV replication were observed with viruses treated with the IgY before and after inoculation into cells, suggesting an application of the IgY as both prophylactic and therapeutic treatment. Collectively, our data suggested that the P-VP8∗ based IgY could serve as a practical approach against both NoV and RV.

Employing Immunomarkers to Track Dispersal and Trophic Relationships of a Piercing-Sucking Predator, &lt;I&gt;Podisus maculiventris&lt;/I&gt; (Hemiptera: Pentatomidae)

Immunoproteins are markers that are useful for monitoring dispersal and/or pest consumption, but current application techniques are less effective for the large guild of piercing-sucking predators important in biocontrol. We quantified the use of protein immunomarks in tracking emigration of spined soldier bug, Podisus maculiventris Say (Hemiptera: Pentatomidae) and predation on the hornworm caterpillar, Manduca sexta L. (Lepidoptera: Sphingidae). An external protein mark was topically applied to adult P. maculiventris to assess persistence under field conditions for >2 wk. Internal marks were incorporated into the artificial diet of M. sexta to test retention of the internal mark in the prey and uptake of the mark by predators. External marks remained detectable in 100% of individuals after 3 d and >50% still tested positive at 12 d after application in the field. Internal diet-based marking was also effective in tracking feeding by P. maculiventris on M. sexta, especially using rabbit IgG that was far more persistent than chicken IgY. Nearly 90% of stink bugs fed caterpillars previously reared on protein-enriched diet retained their mark for 24 h. Surprisingly, diet concentration and time reared on diet had comparatively little impact on mark retention. Development on unmarked tomato leaves clearly diluted the initial diet mark, but plant-reared individuals that were marked were still successfully detected in 35 and 20% of the predators.

Development and immunochemical evaluation of a novel chicken IgY antibody specific for KLK6

BackgroundHuman kallikrein-related peptidase 6 (KLK6) has been implicated in various types of cancer and in neurodegenerative and demyelinating diseases including multiple sclerosis. Further, anti-KLK6 antibodies attenuated disease manifestations in the mouse model of multiple sclerosis. Availability of specific antibodies against KLK6 is fundamental to the development of improved diagnostic and/or immunotherapeutic applications. Here, we exploited the enhanced immunogenicity of mammalian proteins in avian species to generate a polyclonal antibody against KLK6.ResultsChicken were immunized with recombinant KLK6 and antibodies Y (IgYs) were purified from egg yolk with a simple procedure and evaluated for KLK6 detection by ELISA and Western blot using recombinant proteins and human cell lysates and supernatants. The anti-KLK6 Y polyclonal exhibited high affinity for KLK6 with a detection limit of 30 fmol. On the other hand, the widely used rabbit polyclonal antibody that was raised against the same recombinant KLK6 had a detection limit of 300 fmol. Moreover, the IgYs did not display any crossreactivity with recombinant KLKs or endogenous KLKs and other cellular proteins.ConclusionsBased on its high specificity and sensitivity the developed anti-KLK6 IgY is expected to aid the development of improved diagnostic tools for the detection of KLK6 in biological and clinical samples.