Bovine Αs1-casein Research Articles

Development of a novel polyclonal antibody against bovine αS1-casein IgE epitopes for prediction of potential allergenicity of milk in foods

ABSTRACT Bovine αS1-casein (αS1-CN) is the main allergen in cow's milk, and immunoglobulin E (IgE) epitopes are the material basis to trigger allergic reactions. Thus, IgE epitopes could be as key targets for precise αS1-CN detection and allergenicity assessment. Here, we designed a tandem containing nine IgE linear epitopes of αS1-CN (tαS1-CN), expressed the recombinant tαS1-CN in Escherichia coli, and prepared the rabbit polyclonal antibody against recombinant tαS1-CN (pAb-tαS1-CN). The obtained recombinant tαS1-CN had a purity of 94.43% and retained the antigenicity and potential allergenicity of IgE epitopes. The pAb-tαS1-CN could recognize the nine IgE epitopes of αS1-CN, and also showed high specificity. The ability of pAb-tαS1-CN to evaluate the potential allergenicity of milk in foods was validated by sera from cow's milk allergy patients. These findings indicate that our prepared pAb-tαS1-CN may represents a valuable tool for the evaluation of milk allergenicity in processed foods.

The Anxiolytic-like Properties of a Tryptic Hydrolysate of Bovine αs1 Casein Containing α-Casozepine Rely on GABAA Receptor Benzodiazepine Binding Sites but Not the Vagus Nerve.

(1) Background: A tryptic hydrolysate of bovine αs1-casein (CH) exerts anxiolytic-like properties in many species, including humans. This is mainly related to the presence of α-casozepine (α-CZP), which yields these properties in rodents. This study evaluates, in a rat model, the roles of the vagus nerve and the benzodiazepine binding site of GABAA receptors in the mode of action of CH. (2) Methods: The conditioned defensive burying test was used to evaluate anxiety. (3) Results: Participation of the vagus nerve in the mode of action of CH was excluded, as the global anxiety score in vagotomised rats was not significantly different from that of non-vagotomised animals. The blocking of the binding sites of benzodiazepines with flumazenil antagonised CH anxiolytic-like properties. (4) Conclusions: The vagus nerve does not play a role in the anxiolytic-like properties of CH. On the other hand, this anxiolytic-like activity relies on the benzodiazepine binding site of the GABAA receptors. This result is consistent with previous in vitro studies and, more specifically with the discovery of α-CZP, the peptide responsible for the anxiolytic-like properties of CH.

Concentration, purification and quantification of milk protein residues following cleaning processes using a combination of SPE and RP-HPLC

Detection and quantification of milk protein residues can be of utmost importance for validation of cleaning process efficiency in removing even traces of residues as well as quality assurance and product safety. However, currently available assays cannot provide a combination of high sensitivity and a simultaneous quantification of the individual milk proteins. Furthermore, a low protein-to-protein-variability and high compatibility with other reagents such as residual cleaning agents (e.g. surfactants) cannot be ensured. Therefore, a new method was developed comprised of a pre-concentration of proteins by solid-phase extraction and optimisation of the sensitivity of an existing reversed-phase high performance liquid chromatography method for the separate quantification of bovine milk proteins κ-Casein, αS2-Casein, αS1-Casein, β-Casein, α-Lactalbumin, and β-Lactoglobulin. Hereby, solid-phase extraction enables robust and reproducible purification and concentration of protein residues with a high protein recovery rate and flexible adjustment of concentration factors. The increased sensitivity of the reversed-phase high performance liquid chromatography method was achieved by changes in the measurement wavelength and guanidine buffer concentration. This new method enables reproducible concentration, purification and quantification of protein concentrations below 7 ng mL−1 and thus can be used to detect milk protein residues in highly diluted aqueous systems.•Concentration, purification and quantification of milk protein residues with a high recovery rate of proteins (> 94%) and high reproducibility (coefficient of variation (CV) < 3.0%)•Flexible adjustment of sample volumes allows the utilisation of high concentration factors (≤ 500) without compromising the recovery rate of proteins (recovery rate of proteins decreases by 2.74% per 100 CF)

The Expression of Recombinant Human Serum Albumin in the Mammary Gland of Transgenic Mice

AbstractHuman serum albumin (HSA) is widely used in the clinic for the treatment of several diseases in large amount each year. With the increasing demands of HSA in clinic and limited blood resource, recombinant HSA (rHSA) is becoming an attractive and alternative source for HSA production. In this study, we aimed to express rHSA in the mammary glands of transgenic mice by using a tissue-specific promoter and other regulatory elements. An rHSA expression vector was constructed bearing the cDNA and first intron of HSA under the control of bovine αs1-casein promoter with a 2 × chicken β-globin insulator in the front. Transgenic mice were generated and reverse transcription polymerase chain reaction showed that rHSA was expressed only in the mammary gland, indicating the tissue specificity of the bovine αs1-casein promoter in directing transgene transcription in transgenic mice. Enzyme-linked immunosorbent assay test showed that rHSA was successfully secreted into the milk of transgenic mice with the highest level at 1.98 ± 0.12 g/L. Our results indicate the ability of the bovine αs1-casein promoter to induce successful expression of rHSA in the mammary gland of transgenic mice.

Characterization of a novel antimicrobial peptide from buffalo casein hydrolysate based on live bacteria adsorption

This study aims to isolate the antimicrobial peptide (AMP) from buffalo casein hydrolyzed by Dregea sinensis Hemsl. protease. The AMP was isolated from hydrolysate by live bacteria adsorption, then analyzed using reversed-phase high-performance liquid chromatography, and the fraction with highest antimicrobial activity was identified by liquid chromatography-tandem MS. Further, we characterized the peptide in terms of its peptide sequence, structure, and antimicrobial activity. The results identified the AA sequence of the peptide as YLGYLEQLLRLK, which corresponds to residues 106 to 117 of bovine αS1-casein, and we named it BCp12. BCp12 displays α-helical structure, with high hydrophobic moments and net positive charge. BCp12 can inhibit the growth of indicator bacteria, with minimum inhibitory concentration values ranging from 0.8 to 1.6 mg/mL, and can induce low toxicity in mammalian cells. Antimicrobial activity of the BCp12 peptide remained stable under different salt concentrations but was sensitive to trypsin and high temperatures (121°C and above). The results support further research in the application of our newly generated AMP as an antimicrobial agent in the food industry and in food processing facilities.

Transport of a Peptide from Bovine αs1-Casein across Models of the Intestinal and Blood-Brain Barriers.

The effect of food components on brain growth and development has attracted increasing attention. Milk has been shown to contain peptides that deliver important signals to the brains of neonates and infants. In order to reach the brain, milk peptides have to resist proteolytic degradation in the gastrointestinal tract, cross the gastrointestinal barrier and later cross the highly selective blood–brain barrier (BBB). To investigate this, we purified and characterized endogenous peptides from bovine milk and investigated their apical to basal transport by using human intestinal Caco-2 cells and primary porcine brain endothelial cell monolayer models. Among 192 characterized milk peptides, only the αS1-casein peptide 185PIGSENSEKTTMPLW199, and especially fragments of this peptide processed during the transport, could cross both the intestinal barrier and the BBB cell monolayer models. This peptide was also shown to resist simulated gastrointestinal digestion. This study demonstrates that a milk derived peptide can cross the major biological barriers in vitro and potentially reach the brain, where it may deliver physiological signals.

Expression of a recombinant anti-programed cell death 1 antibody in the mammary gland of transgenic mice

Nivolumab, a fully human IgG4 anti-programed cell death 1（PD-1）antibody, is recently one of the most popular and successful therapeutic monoclonal antibodies in clinical use. With the increasing demands for Nivolumab and other therapeutic monoclonal antibodies, the mammary gland bioreactor has been regarded as another choice for the production of recombinant monoclonal antibodies besides mammalian cell culture. Here, we expressed a recombinant human anti-PD-1 antibody in the mammary glands of transgenic mice. Two expression vectors were constructed bearing the heavy and light chains of anti-PD-1 antibody respectively under the control of bovine αs1-casein promoter. Transgenic mice were then generated by co-microinjection of the two expression cassettes. Three F0 founders with both heavy chain and light chain positive were obtained. Transgenes of both chains were detected to be stably transmitted to the offspring. The recombinant antibody was detected in the milk of transgenic mice with the highest expression level up to 80.52 ± 0.82 mg/L and could specifically binds to the human PD-1 antigen. Therefore, our results suggest the feasibility of anti-PD-1 antibody production in the milk of transgenic animals.

Non-perfectly Amphipathic α-Helical Structure Containing the XXYXX Sequence Improves the Biological Activity of Bovine αs2-Casein Antimicrobial Peptides

Non-amphiphilic WIQPKTKVIPYVRYL (WI-6) derived from bovine αs2-casein f (193-207) was modified by a defined mutation method to obtain five engineered peptides with mirror symmetry structures. The five engineered peptide sequences were WF-1 (WFQVKTRVRTKVQFW), FW-2 (FWRRYKKVKKYRRWF), FW-3 (FWQVIKKVKKIVQWF), FK-4 (FKQFYRRVRRYFQKF), and FR-5 (FRQWYRRVRRYWQRF). However, FW-2, FW-3, FK-4, and FR-5 had obvious XXYXX sequences. Among these, FW-3 was demonstrated to have the highest antibacterial activity, which indicates that the non-perfectly amphipathic α-helical structure containing the XXYXX sequence has a better bactericidal effect. Therefore, peptide FW-3 could be widely used as a substitute for antibiotics in food, medicine, and other fields. These findings provide a potential method for designing novel antimicrobial peptides.

Anxiolytic Activity and Brain Modulation Pattern of the α-Casozepine-Derived Pentapeptide YLGYL in Mice.

α-Casozepine (α-CZP) is an anxiolytic-like bioactive decapeptide derived from bovine αs1-casein. The N-terminal peptide YLGYL was previously identified after proteolysis of the original peptide in an in vitro digestion model. Its putative anxiolytic-like properties were evaluated in a Swiss mice model using a light/dark box (LDB) after an intraperitoneal injection (0.5 mg/kg). The effect of YLGYL on c-Fos expression in brain regions linked to anxiety regulation was afterwards evaluated via immunofluorescence and compared to those of α-CZP and diazepam, a reference anxiolytic benzodiazepine. YLGYL elicited some anxiolytic-like properties in the LDB, similar to α-CZP and diazepam. The two peptides displayed some strong differences compared with diazepam in terms of c-Fos expression modulation in the prefontal cortex, the amygdala, the nucleus of the tractus solitarius, the periaqueductal grey, and the raphe magnus nucleus, implying a potentially different mode of action. Additionally, YLGYL modulated c-Fos expression in the amygdala and in one of the raphe nuclei, displaying a somewhat similar pattern of activation as α-CZP. Nevertheless, some differences were also spotted between the two peptides, making it possible to formulate the hypothesis that these peptides could act differently on anxiety regulation. Taken together, these results showed that YLGYL could contribute to the in vivo overall action of α-CZP.

Broad-Spectrum Antimicrobial Activity and Low Cytotoxicity against Human Cells of a Peptide Derived from Bovine αS1-Casein.

The primary objective of this study was to improve our understanding of the antimicrobial mechanism of protein-derived peptides and to provide evidence for protein-derived peptides as food bio-preservatives by examining the antimicrobial activities, low cytotoxicity, stabilities, and mechanism of Cp1 (LRLKKYKVPQL). In this study, the protein-derived peptide Cp1 was synthesized from bovine αS1-casein, and its potential use as a food biopreservative was indicated by the higher cell selectivity shown by 11-residue peptide towards bacterial cells than human RBCs. It also showed broad-spectrum antimicrobial activity, with minimum inhibitory concentrations (MICs) of 64–640 μM against both gram-positive and gram-negative bacteria. The peptide had low hemolytic activity (23.54%, 512 μM) as well as cytotoxicity. The results of fluorescence spectroscopy, flow cytometry, and electron microscopy experiments indicated that Cp1 exerted its activity by permeabilizing the microbial membrane and destroying cell membrane integrity. We found that Cp1 had broad-spectrum antimicrobial activity, low hemolytic activity, and cytotoxicity. The results also revealed that Cp1 could cause cell death by permeabilizing the cell membrane and disrupting membrane integrity. Overall, the findings presented in this study improve our understanding of the antimicrobial potency of Cp1 and provided evidence of the antimicrobial mechanisms of Cp1. The peptide Cp1 could have potential applications as a food biopreservative.

Expression and Purification of Hydrophobic PEP‐Inhibiting Peptides from Bovine αS1 Casein

According to previous studies, there is a prolyl endopeptidase‐inhibiting sequence (LNENLLRFFVAPFPEVFG) within a relatively hydrophobic region of αS1 casein (Juillerat‐Jeanneret et al., 2011). Recently, sixteen recombinant peptides were designed in assorted lengths, each containing the inhibitory sequence at varied positions within the αS1 casein sequence. The corresponding cDNA sequences were originally ligated into the pET15b vector, which provides an N‐terminal poly‐histidine tag for purification. The vector was in turn transformed into a BL21(DE3)pLysS E. coli expression host (Vishram et al., 2016). However, the system proved unsuccessful in adequate expression of the hydrophobic peptide. The intention of this study was to assemble a new pET28a expression system, kindly provided by Dr. X. Cheng (MD Anderson in Houston, TX) that utilizes a SUMO (small ubiquitin‐like modifier) fusion tag. The SUMO tag is known for enhancing expression levels of potentially toxic or insoluble recombinant peptides (Bommarius et al., 2010). The expressed peptides were purified via Ni‐NTA column chromatography, with subsequent UlpI cleavage. This was followed by reversed‐phase high performance liquid chromatography (RP‐HPLC) to further purify the peptides. Tricine‐SDS‐PAGE, a useful screening tool for hydrophobic peptides smaller than 30kDa, was used to analyze expression and purification. A comparison was made between SUMO‐fused expression and non‐fused expression of the hydrophobic casein peptides.Support or Funding InformationOffice of Research and Sponsored Projects, SFASU and Ed &amp; Gwen ColeThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Milk authenticity by ion-trap proteomics following multi-enzyme digestion

The practice of adding adulterating substances in milk in order to raise profits is unfortunately worldwide. In addition, higher priced milk, coming from minor dairy species, is often illegally integrated with the lower priced cow milk. The presence of species-specific proteins, different from those declared in label, may be a serious problem for people with allergies. The development of proper analytical methods is therefore essential to protect consumer benefits and product authenticity.In this study, a proteomic approach for the detection of adulteration processes of specific milks in mixtures is proposed. Few microliters of milk samples have been digested with trypsin and chymotrypsin and analyzed by nanoLC-ESI-IT-MS/MS. A post-database processing was performed to obtain confident peptide sequence assignments, allowing the detection of milk adulteration at a level lower than 1%. Species-specific peptides from bovine β-lactoglobulin and αS1 casein were identified as suitable peptide markers of milk authenticity.

Mapping in mice the brain regions involved in the anxiolytic-like properties of α-casozepine, a tryptic peptide derived from bovine αs1-casein

α-Casozepine, a bioactive peptide from milk casein, displays an anxiolytic-like activity in many species. Since its mode of action is still not elucidated, a study was conducted in Swiss mice to investigate c-Fos expression, a marker of neuronal activity, in different brain areas. After an intraperitoneal injection of α-casozepine (1mg/kg), animals were placed either in a non-stressful or in an anxiety-inducing situation triggered with a light/dark box. No effect of α-casozepine on c-Fos expression was observed in the non-stressful situation. In the stressful situation, modulation of neuronal activity by α-casozepine was observed in different brain regions compared to that of vehicle. However, while diazepam, a benzodiazepine, modulated neuronal activity the same way in hippocampus, accumbens nucleus and hypothalamus, differences were observed in c-Fos expression in amygdala and prefrontal cortex compared to α-casozepine. These results strengthen the assumption that the anxiolytic mechanisms of α-casozepine differ partly of those of diazepam.

A tryptic hydrolysate from bovine milk αs1-casein enhances pentobarbital-induced sleep in mice via the GABAA receptor

Studies have shown that enzymatic hydrolysis of casein, the primary protein component of cow’s milk, produces peptides with various biological activities, and some of these peptides may have sleep-promoting effects. In the present study, we evaluated the sedative and sleep-promoting effects of bovine αS1-casein tryptic hydrolysate (CH), containing a decapeptide αS1-casein known as alpha-casozepine. CH was orally administered to ICR mice at various concentrations (75, 150, 300, or 500mg/kg). An hour after administration, assessment of its sedative (open-field and rota-rod tests) and sleep-potentiating effects (pentobarbital-induced sleeping test and EEG monitoring) were conducted. Although a trend can be observed, CH treatment did not significantly alter the spontaneous locomotor activity and motor function of mice in the open-field and rota-rod tests. On the other hand, CH (150mg/kg, respectively) enhanced the sleep induced by pentobarbital sodium in mice. It also promoted slow-wave (delta) EEG activity in rats; a pattern indicative of sleep or relaxation. These behavioral results indicate that CH has sleep-promoting effects, but no or has minimal sedative effects. To elucidate the probable mechanism behind the effects of CH, we examined its action on intracellular chloride ion influx in cultured human neuroblastoma cells. CH dose-dependently increased chloride ion influx, which was blocked by co-administration of bicuculline, a competitive GABAA receptor antagonist. Taken together, the results of the present study suggest that CH has sleep-promoting properties which are probably mediated through the GABAA receptor–chloride ion channel complex.

Short communication: Measuring the angiotensin-converting enzyme inhibitory activity of an 8-amino acid (8mer) fragment of the C12 antihypertensive peptide

An 8-AA (8mer) fragment (PFPEVFGK) of a known antihypertensive peptide derived from bovine αS1-casein (C12 antihypertensive peptide) was synthesized by microwave-assisted solid-phase peptide synthesis and purified by reverse phase HPLC. Its ability to inhibit angiotensin-converting enzyme (ACE) was assessed and compared with that of the parent 12mer peptide (FFVAPFPEVFGK) to determine the effect of truncating the sequence on overall hypotensive activity. The activity of the truncated 8mer peptide was found to be almost 1.5 times less active than that of the 12mer, with ACE-inhibiting IC50 (half-maximal inhibitory concentration) values of 108 and 69μM, for the 8mer and 12mer, respectively. Although the 8mer peptide is less active than the original 12mer peptide, its overall activity is comparable to activities reported for other small proteins that elicit physiological responses within humans. These results suggest that microbial degradation of the 12mer peptide would not result in a complete loss of antihypertensive activity if used to supplement fermented foods and that the stable 8mer peptide could have potential as a blood pressure–lowering agent for use in functional foods.

Alpha S1-casein polymorphisms in camel (Camelus dromedarius) and descriptions of biological active peptides and allergenic epitopes.

Milk samples of 193 camels (Camelus dromedarius) from different regions of Sudan were screened for casein variability by isoelectric focusing. Kappa-casein and beta-casein were monomorphic, whereas three protein patterns named αs1-casein A, C, and D were identified. The major allele A revealed frequencies of 0.79 (Lahaoi), 0.75 (Shanbali), 0.90 (Arabi Khali), and 0.88 (Arabi Gharbawi) in the different ecotypes. CSN1S1*C shows a single G > T nucleotide substitution in the exon 5, leading to a non-synonymous amino acid exchange (p.Glu30 > Asp30) in comparison to CSN1S1*A and D. At cDNA level, no further single nucleotide polymorphisms could be identified in CSN1S1* A, C, and D, whereas the variants CSN1S1*A and CSN1S1*C are characterized by missing of exon 18 compared to the already described CSN1S1*B, as consequence of DNA insertion of 11bp at intron 17 which alter the pre-mRNA spliceosome machinery. A polymerase chain-restriction fragment length polymorphism method (PCR-RFLP) was established to type for G > T nucleotide substitution at genomic DNA level. The occurrence and differences of IgE-binding epitopes and bioactive peptides between αs1-casein A, C, and D after digestion were analyzed in silico. The amino acid substitutions and deletion affected the arising peptide pattern and thus modifications between IgE-binding epitopes and bioactive peptides of the variants were found. The allergenic potential of these different peptides will be investigated by microarray immunoassay using sera from milk-sensitized individuals, as it was already demonstrated for bovine αs1-casein variants.

In silico Designing of Protein Rich in Large Neutral Amino Acids Using Bovine αs1 Casein for Treatment of Phenylketonuria

Phenylketonuria (PKU) is a genetically inherited disease where the body fails to convert Phenylalanine (Phe) to Tyrosine (Tyr) due to the defective Phenylalanine Hydroxylase (PAH) enzyme, resulting in the elevated blood Phe level causing neurological damage. Of all therapies, Large Neutral Amino Acid (LNAA) supplementation has become a promising approach to the dietary treatment of PKU, where the LNAA compete with Phe for the same L-Type Large Neutral Amino Acid Transporter (LAT1, SLC7A5) across the blood-brain barrier, decreasing brain Phe level. In this study we have designed an easily digestible protein enriched with LNAA (except Phe), using bovine αs1 casein as template by homology modeling. Our challenge was to maximize the LNAAs (except Phe) in the protein model by finding a suitable scaffold for hosting LNAAs, thereby turning the usual concept of homology modeling. Bioinformatics tools like SWISS-MODEL, EXPASY, PROFUNC, I-TASSER, RaptorX, and SAVeS Server were used for the structure prediction, and validation of the designed protein. Out of 63 different models designed, Protein Model-54 was selected based on sequence similarity to template (61.4%), compact 3D structure containing only α-helices and coils without β-sheets (QMEAN score of 0.567), and good in silico digestibility. The molecular weight of protein was 12,094.6 Da. Ramachandran plot revealed that the designed protein contained 89.9% amino acid residues in the favored region with ERRAT score of 88.04. Based on these evaluations, the Protein Model-54 was found to be the best, stable and reliable model which may be of high nutritional significance for PKU patients..

An antimicrobial peptide screened from casein hydrolyzate by Saccharomyces cerevisiae cell membrane affinity method

A Saccharomyces cerevisiae cell membrane affinity screening method was developed to separate targeted antimicrobial peptides from the pepsin hydrolyzate of bovine casein. S. cerevisiae cell membranes were first immobilized on the surface of the silica gel to construct an affinity binding medium. A membrane-binding fraction was successfully screened by comparing the RP-HPLC fingerprint chromatograms of the hydrolyzate before and after adsorption with the adsorption medium. The amino acid sequence of the peptide was identified as LRLKKYKVPQL with the use of a matrix-assisted laser desorption/ionisation quadrupole time-of-flight tandem mass spectrometer. The sequence corresponded to amino acid residues 99–109 of bovine αS1-casein. The results indicated that it is feasible to target screen antimicrobial peptides from protein hydrolyzate using S. cerevisiae cell membranes. The influences of thermal treatment, pH, ions, and enzymes on the activity of the purified peptide were also determined. The activity of the peptide was relatively thermally stable and was pH dependent. It retained more than 90% of its activity in the presence of 15% Na+, K+ and pepsin. Trypsin, proteinase K, divalent cation Mg2+ and Ca2+ reduced the activity to different extents. The peptide also showed antibacterial effectiveness in fresh pear juice. These observations provide further information on the application of protein-derived antimicrobial peptides in food systems.

Relative quantitation analysis of the substrate specificity of glutamyl endopeptidase with bovine α-caseins

A bovine α-caseins’ preparation digested with glutamyl endopeptidase (GE) at 37 and 50°C was quantitatively analysed with the isobaric tag for relative and absolute quantification (iTRAQ) technique using nano-LC-ESI-QTOF-MS/MS. Incubation temperature was shown to affect protein digestion. MS analysis of the digestion products indicated that phosphorylated peptides were less sensitive than non-phosphorylated peptides according to the MS intensities. GE hydrolysed Glu(51)-Tyr(52) and Glu(50)-Glu(51) in Glu(49)-Glu(50)-Glu(51)-Tyr(52) of bovine αs1-casein. The results herein helped to confirm the precise process of α-caseins’ hydrolysis with GE, which is significant for quantifying the release of bio- and techno-functional peptides.

Development of a surface display ELISA to detect anti-IgG antibodies against bovine αS1-casein in human sera

The aim of the present study was to develop a surface display ELISA (SD-ELISA) for IgG-serum reaction against bovine casein αS1 (CSN1S1). In a SD-ELISA, the antigen is displayed on the surface of Escherichia coli using the autodisplay technology and whole cells of E. coli are used to coat the microplates for serum testing.After establishing the setup of the SD-ELISA with polyclonal rabbit antiserum against bovine CSN1S1, the SD-ELISA was validated with 20 human sera, of which 10 sera were proven to have an IgG-mediated reaction against bovine CSN1S1 and 10 sera were shown to be negative for this reaction. Receiver operating characteristics (ROC) analysis revealed sensitivity of 100% and a specificity of 100% at a cut-off value of 0.133. Furthermore, human serum of 48 patients with known reactivity against human CSN1S1 (31 positive and 17 negative) was examined by the newly developed SD-ELISA to exclude cross-reactivity. Twenty human sera showed an IgG-mediated reaction against bovine CSN1S1. Eleven of these sera were positive for the reactivity against human CSN1S1, and nine were negative.In conclusion it was demonstrated that the performance of SD-ELISA is comparable to established ELISA without loss in sensitivity or specificity. Based on the advantages of this method – in particular no need for time-consuming and expensive antigen production and purification – the SD-ELISA is a potent alternative to convenient methods for identification and especially high-throughput screening of new antigens in the field of food allergies.