A Multiple-Target Simultaneous Detection Method for Immunosorbent Assay and Immunospot Assay.

Enzyme-linked immunosorbent assay (ELISA) is one of the most common methods in biological studies, and enzyme-linked immunospot (ELISpot) is a method to measure specific cell numbers by detecting protein secretion at a single-cell level. However, these two current methods can only detect one signal at one time and the sensitivity is not high enough to test low-concentration samples, which are major shortcomings in systematically analyzing the samples of interest. Herein, we demonstrated fluorescence-based oligo-linked immunosorbent assay (FOLISA) and fluorescence-based oligo-linked immunospot (FOLISPOT), which utilized DNA-barcoded antibodies to provide a highly multiplexed method with signal amplification. Signal amplification and simultaneous multiple-target detection were achieved by DNA complementary pairing and modular orthogonal DNA concatemers. By comparing FOLISA with traditional ELISA and comparing FOLISPOT with traditional ELISPOT, we found that the detection sensitivities of FOLISA and FOLISPOT are much higher than those of traditional ELISA and ELISPOT. The detection limit of ELISA is around 3 pg/mL, and the detection limit of FOLISA is below 0.06 pg/mL. FOLISPOT can detect more spots than ELISPOT and can detect targets that are undetectable for ELISPOT. Furthermore, FOLISA and FOLISPOT allowed sequential detection of multiple targets by using a single dye or multiple dyes in one round and sequential detection in multiple rounds. Thus, FOLISA and FOLISPOT enabled simultaneous detection of a large number of targets, significantly improved the detection sensitivity, and overcame the shortcomings of ELISA and ELISPOT. Overall, FOLISA and FOLISPOT presented effective and general platforms for rapid and multiplexed detection of antigens or antibodies with high sensitivity, either in laboratory tests or potentially in clinic tests.

Across-cancer specific immune responses induced by nanovaccines or microvaccines to prevent different cancers and cancer metastasis.

Metastatic cancers and recurrent cancers are diverse, different from primary cancers, and organ-dependent. However, how strong are across-cancer immune responses among different types of cancers remain unclear. Herein, vaccines-encapsulated-whole-components-of-tumor-tissue (VEWCOTT) were applied to demonstrate the across-cancer immune responses, thanks to inducing pan-clones T-cell immune responses. Either lung-cancer-tissue- or melanoma-tissue-based VEWCOTT simultaneously prevented melanoma, lung cancer, hepatoma, and metastatic cancer, which showed that strong across-cancer immune responses were induced. Both nanovaccines and microvaccines showed potent across-cancer prevention efficacy. VEWCOTT induced tumor-specific T cells in peripheral immune organs and major organs, and adjusted the immune-microenvironment of cancer-colonized organs. In addition, the allograft of T cells from VEWCOTT immunized mice to allogeneic naive mice efficiently prevent various cancers. Many neoantigens are shared by melanoma cells and lung cancer cells. Across-cancer immune responses exist among different types of cancers, and thus VEWCOTT has the advantage of simultaneously preventing cancer metastasis and cancers in different organs.

Immunotherapy and Prevention of Cancer by Nanovaccines Loaded with Whole-Cell Components of Tumor Tissues or Cells.

Tumor tissues/cells are the best sources of antigens to prepare cancer vaccines. However, due to the difficulty of solubilization and delivery of water-insoluble antigens in tumor tissues/cells, including water-insoluble antigens into cancer vaccines and delivering such vaccines efficiently to antigen-presenting cells (APCs) remain challenging. To solve these problems, herein, water-insoluble components of tumor tissues/cells are solubilized by 8 m urea and thus whole components of micrometer-sized tumor cells are reasssembled into nanosized nanovaccines. To induce maximized immunization efficacy, various antigens are loaded both inside and on the surface of nanovaccines. By encapsulating both water-insoluble and water-soluble components of tumor tissues/cells into nanovaccines, the nanovaccines are efficiently phagocytosed by APCs and showed better therapeutic efficacy than the nanovaccine loaded with only water-soluble components in melanoma and breast cancer. Anti-PD-1 antibody and metformin can improve the efficacy of nanovaccines. In addition, the nanovaccines can prevent lung cancer (100%) and melanoma (70%) efficiently in mice. T cell analysis and tumor microenvironment analysis indicate that tumor-specific T cells are induced by nanovaccines and both adaptive and innate immune responses against cancer cells are activated by nanovaccines. Overall, this study demonstrates a universal method to make tumor-cell-based nanovaccines for cancer immunotherapy and prevention.

A novel thioredoxin reductase inhibitor inhibits cell growth and induces apoptosis in HL-60 and K562 cells.

Human thioredoxin reductase (TrxR) system is associated with cancer cell growth and anti-apoptosis process. Effects of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]ethane (BBSKE), a novel TrxR inhibitor, were investigated on human leukemia cell lines HL-60 and K562. BBSKE treatment induced cell growth inhibition and apoptosis in both cell lines. Apoptosis induced by BBSKE is through Bcl-2/Bax and caspase-3 pathways. Ehrlich's ascites carcinoma-bearing mice were used to investigate the anti-tumor effect of BBSKE in vivo. Tumor-bearing mice treated with BBSKE showed an increase of life span with a comparable effect to cyclophosphamide (CTX). These results suggest a potential usage of BBSKE as a therapeutic agent against non-solid tumors.

[Realgar nano-particles induce apoptosis and necrosis in leukemia cell lines K562 and HL-60].

OBJECTIVE: To examine the growth-inhibitory, apoptosis- and necrosis-inducing effects of realgar nano-particles (RNP) in human chronic myelogenous leukemia cell line K562 and acute myeloid leukemia cell line HL-60, and to find out the chemical species with efficacy. METHOD: A "solvent-relay" strategy was used for the preparation of RNP suspension. Cell viability was determined by MTT assay. Cell apoptosis and necrosis were characterized with Annexin V-PI double staining in association with flow cytometry and with morphological examination with Hoechst 33258 staining. Parallel experiments with arsenous acid (H3AsO3), the dominant form of arsenic trioxide in the solution, were conducted for comparison. RESULT: The mean diameter of RNP was 159.0 nm. RNP showed growth-inhibitory effect on both cell lines. The double staining test indicated that RNP induced both apoptosis and necrosis, and this was further confirmed by morphological examination. CONCLUSION: RNP induced both apoptosis and necrosis in leukemia cell lines K562 and HL-60. Thioarsenite species with both As-O and As-S bonds may be the active intermediates in the RNP.

Preparation of tri-block copolymer micelles loading novel organoselenium anticancer drug BBSKE and study of tissue distribution of copolymer micelles by imaging in vivo method.

BBSKE (1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)] ethane, PCT: CN02/00412) is a novel organoselenium anticancer drug that plays a role in anticancer through inhibiting TrxR (thioredoxin reductase). In this study, we prepared a tri-block copolymer micelles loading BBSKE utilizing the amphiphilic tri-block copolymers (PEG6000-PLA6000) which we synthesized. And then the characters of the copolymer micelles were investigated. When packaged in polymeric micelles, the water solubility of BBSKE was improved to 0.21 mg/ml. The IC(50) were 7.14 microM, 5.05 microM and 4.23 microM when MCF-7 breast cancer cells were treated with BBSKE after 24h, 48h and 72h. The inhibition effect of polymeric micelles to MCF-7 tumor cells was bettered when folate, whose receptor was highly expressed in various tumors, was coated on the surface of these nanoparticles. Finally, by adopting a new way of imaging in vivo, we studied the distribution of micelles in nude mice with and without MCF-7 tumor. The results demonstrated that this copolymer micelles loading BBSKE can accumulate into tumor efficiently.

[Pilot application of optical in vivo imaging on acupuncture research].

OBJECTIVE: As a new technique of biomedical imaging, the optical in vivo imaging has been used in many research fields of biological processes, such as analyzing gene expression pattern, observing action of drug target. For extending its application field, this technique is initially used to investigate the effect of acupuncture in this study. METHODS: Seven SPF mice were randomly divided into an acupuncture group (4 cases) and a control group (3 cases). Fluorescent labeling Cy7 (Tf-Cy7) and Doxorubicin (Dox) were injected into the caudal vein of the nude mice, respectively. With the.stabilized distribution of Tf-Cy7 and Dox in vivo, "Yaoyangguan" (GV 3) point was acupunctured in the acupuncture group. The animals in the control group were not acupunctured. Then, the fluorescent irradiance of Tf-Cy7 and Dox was displayed by the optical in vivo imaging, the effect of acupuncture was analyzed. RESULTS: (1) After acupuncture, the fluorescent intensity of Tf-Cy7 started to decrease in the blood-brain barrier and heart, but tended to increase in the liver and spleen, in which the highest intensity of Tf-Cy7 was demonstrated at 50 min during the acupuncture. It was obviously different from that of the control group. (2) The fluorescent intensity of Dox tended to increase in the brain and lung after 20 min of acupuncture, and reached the highest concentration at 30 min. CONCLUSION: Acupuncture can influence the action of Tf-Cy7 targeting and distribution of Dox, which is clearly showed with the optical in vivo imaging. Thus, this technique provides a new approach for the acupuncture and meridian research.