Dataset Information

Development of a Microsphere-Based Immunoassay Authenticating A2 Milk and Species Purity in the Milk Production Chain.

ABSTRACT: Processed milk and milk products produced from bovine milk, commonly contain β-casein A1 (βCA1) and β-casein A2 (βCA2). Since the presence of βCA1 is linked to milk intolerance and digestion problems, A2A2 milk, which only contains βCA2, is proposed as a healthier alternative. To support this health claim, the purity of A2A2-milk has to be guaranteed. In the presented study, a multiplex immunoassay, able to distinguish between βCA2 and βCA1, was developed and real-life applicability was shown on raw milk samples from genotyped A1A1, A1A2 and A2A2 cows. Because of its ability to discriminate between βCA2 and βCA1, this newly developed method was able to detect the addition of common bovine A1A2 milk to A2A2 milk, as low as 1%. Besides the detection of A2A2 milk purity, the developed assay can also be implemented as a rapid phenotyping method at dairy farms to replace the more invasive DNA-based screening. Additionally, the developed method was capable of detecting the addition of common bovine milk up to 1% in sheep, goat, buffalo, horse and donkey milk, which conforms to EU recommendations. In conclusion, a newly developed multiplex method capable of reliably detecting the dilution of A2A2 milk of multiple species, with common bovine milk up to 1%, is presented.

SUBMITTER: Elferink AJW

PROVIDER: S-EPMC9144198 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Development of a Microsphere-Based Immunoassay Authenticating A2 Milk and Species Purity in the Milk Production Chain.

Elferink Alexander J W AJW Entiriwaa Deborah D Bulgarelli Paolo P Smits Nathalie G E NGE Peters Jeroen J

Molecules (Basel, Switzerland) 20220517 10

Processed milk and milk products produced from bovine milk, commonly contain β-casein A1 (βCA1) and β-casein A2 (βCA2). Since the presence of βCA1 is linked to milk intolerance and digestion problems, A2A2 milk, which only contains βCA2, is proposed as a healthier alternative. To support this health claim, the purity of A2A2-milk has to be guaranteed. In the presented study, a multiplex immunoassay, able to distinguish between βCA2 and βCA1, was developed and real-life applicability was shown on ...[more]

PMID: 35630686

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Project description:BackgroundZoonotic schistosomiasis, caused by Schistosoma japonicum, is prevalent in China, the Philippines and Indonesia. Rapid point-of-care (POC) diagnostics are attractive and promising tools for evaluating the efficacy of intervention strategies for schistosomiasis control.MethodologyThe diagnostic potential of five recombinant antigens was tested by enzyme-linked immunosorbent assay (ELISA) using sera from individuals with positive Kato-Katz (KK) results for S. japonicum (n = 28) and non-endemic controls (n = 12). A latex microsphere (LM)-based lateral flow immunoassay (LFIA) incorporating the recombinant SjSAP4 (rSjSAP4) was developed for the diagnosis of schistosomiasis japonica. The test conditions including diluent, dilution factor and reaction time, were optimised for the developed LFIA. Under the optimised conditions, serum samples from individuals living in a barangay endemic for S. japonicum (n = 549) and non-endemic controls (n = 50) were tested with the established LFIA cassettes. The results were imaged by a smartphone and analysed by the ImageJ program. The intensity ratio of the test line to the control line (T/C ratio) was calculated for each cassette.Main findingsELISA confirmed that rSjSAP4 was the optimal candidate for serological diagnosis of schistosomiasis japonica. Under optimal testing conditions, the developed LFIA strips had a sensitivity of 80.6% and a specificity of 98.0% at a cut-off T/C ratio of 0.1031. Moreover, the results of the LM-based LFIA was positively correlated with those obtained from the rSjSAP4-ELISA (r = 0.8270, 95% CI, 0.7990-0.8514; p < 0.0001). The schistosomiasis prevalence determined by the LFIA strips was about 1.8 times greater than that obtained with the 6-slide KK procedure performed on three stool samples.Conclusions/significanceThe developed LFIA represents a POC diagnostic tool that is suitable for onsite screening of human S. japonicum infection with minimal equipment needed. The established immunochromatographic assay complies with most of the WHO's ASSURED criteria for POC diagnostics.

Project description:Surveillance of highly pathogenic viruses circulating in both human and animal populations is crucial to unveil endemic infections and potential zoonotic reservoirs. Monitoring the burden of disease by serological assay could be used as an early warning system for imminent outbreaks as an increased seroprevalance often precedes larger outbreaks. However, the multitude of highly pathogenic viruses necessitates the need to identify specific antibodies against several targets from both humans as well as from potential reservoir animals such as bats. In order to address this, we have developed a broadly reactive multiplex microsphere immunoassay (MMIA) for the detection of antibodies against several highly pathogenic viruses from both humans and animals. To this aim, nucleoproteins (NP) of Ebola virus (EBOV), Marburg virus (MARV) and nucleocapsid proteins (NP) of Crimean-Congo haemorrhagic fever virus, Rift Valley fever virus and Dobrava-Belgrade hantavirus were employed in a 5-plex assay for IgG detection. After optimisation, specific binding to each respective NP was shown by testing sera from humans and non-human primates with known infection status. The usefulness of our assay for serosurveillance was shown by determining the immune response against the NP antigens in a panel of 129 human serum samples collected in Guinea between 2011 and 2012 in comparison to a panel of 88 sera from the German blood bank. We found good agreement between our MMIA and commercial or in-house reference methods by ELISA or IIFT with statistically significant higher binding to both EBOV NP and MARV NP coupled microspheres in the Guinea panel. Finally, the MMIA was successfully adapted to detect antibodies from bats that had been inoculated with EBOV- and MARV- virus-like particles, highlighting the versatility of this technique and potentially enabling the monitoring of wildlife as well as human populations with this assay. We were thus able to develop and validate a sensitive and broadly reactive high-throughput serological assay which could be used as a screening tool to detect antibodies against several highly pathogenic viruses.

Dataset Information

Development of a Microsphere-Based Immunoassay Authenticating A2 Milk and Species Purity in the Milk Production Chain.

Publications

Development of a Microsphere-Based Immunoassay Authenticating A2 Milk and Species Purity in the Milk Production Chain.

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