High-throughput simultaneous genotyping of human platelet antigen-1 to -16 by using suspension array.

BACKGROUND: Comprehensive and accurate detection of human platelet antigens (HPAs) plays a significant role in diagnosis and prevention of the platelet (PLT) alloimmune syndromes and ensuring clinical safety of patients undergoing PLT transfusion. The majority of the available methods are incapable of performing high-throughput simultaneous detection of HPA-1 to -16, and the accuracy of many methods needs to be further enhanced. STUDY DESIGN AND METHODS: We have developed a new HPA-genotyping method for simultaneous detection of HPA-1 to -16 based on suspension array technology. A total of 216 samples from Chinese Han donors in Xi'an were genotyped using the developed method, and all the samples again were genotyped using polymerase chain reaction (PCR) sequence-based typing (PCR-SBT), which is considered the gold standard. RESULTS: All 216 samples were successfully genotyped for HPA-1 to -16 using both our method and PCR-SBT. Results showed that the genotype and allele frequencies obtained using our method were fully consistent with those obtained using PCR-SBT. CONCLUSION: Our method provides accurate, high-throughput, and simultaneous genotyping of HPA-1 to -16 and will serve as the foundation for large-scale clinical genotyping of HPAs and for the establishment of an HPA-typed PLT donor registry.

[Application of suspension array assay to detect marker genes expression of circulating tumor cells for early prediction of breast cancer metastasis].

OBJECTIVE: To develop a suspension array assay to detect the expression of multiple genes in the circulating breast cancer cells simultaneously so as to identify the marker genes for human breast cancer metastasis. METHODS: Peripheral blood samples were obtained from 73 breast cancer patients, including 31 breast cancer metastasis patients, 30 patients with benign breast diseases, and 40 healthy women, and peripheral blood mononuclear cells (PBMCs) were isolated. Total RNA was extracted and cDNA was synthesized. PCR was used to amplify 8 breast cancer-related genes: hMAM, HER2, CK19, SBEM, EPG2, hTERT, beta-HGG, and B305D. Suspension array of the PCR products was developed and underwent Luminex 100 laser Flow-type analysis to read the fluorescence signal. COX proportional hazard model was used to find the independent prognostic predictors of breast cancer metastasis. RESULTS: hMAM expression was detected in 57.5%, HER2 in 57.5%, CK19 in 53.4%, SBEM in 52.1%, EPG2 in 31.5%, hTERT in 26.1%, beta-HCG in 21.9%, and B305D in 15.1% of the blood samples respectively. Compared with serum CA15-3 detection, the multigene detection has higher sensitivity (P < 0.05). The expression of SBEM-mRNA in the peripheral blood was correlated with the stage of breast cancer (P < 0.05); and hMAM, SBEM, HER2, and ER could be considered as the independent prognostic predictors of breast cancer metastasis (all P < 0.05). CONCLUSION: The suspension array assay thus developed is practical in diagnosis of the prognosis of breast cancer. The expression of hMAM, SBEM, and HER2 in peripheral blood can be considered as the independent prognostic predictors of breast cancer metastasis.

[Screening and identification of egg mimotope of Schistosoma japonicum].

OBJECTIVE: To explore egg mimotope of Schistosoma japonicum that can be used in the development of diagnostic reagents for schistosomiasis. METHODS: By performing three rounds of biopanning in the affinity selection before picking out single clones for identification, target specific phages were effectively enriched from a random 15-peptides phage library with an immobilized mAb 6B12 which is specific to egg antigen of S. japonicum as a bait. By using ELISA, competitive ELISA with natural egg antigen as competitor, and Western blotting, 13 phage clones with high affinity and specificity to 6B12 were obtained and amplified from 400 single clones. RESULTS: DNA sequencing revealed that all the 13 selected single clones were identical in displaying gene sequence. Serum samples were tested by ELISA for the presence of IgG antibodies to the phages with mimic epitope, and showed that there was a significant difference between healthy volunteers and schistosomiasis patients. CONCLUSION: It is possible that the phage display peptide antigen, egg mimotope of S. japonicum may be a replacement of natural egg antigen for the diagnosis of schistosomiasis.