OBJECTIVETo establish a two-step pretargeting approach to lymphoma radioimmunoimaging in mice using biotinynaled CD45 monoclonal antibody (McAb) and (188)Re-avidin in a tumor-bearing mouse model.

METHODSSix Nod-Scid mice bearing lymphoma cell xenograft were randomized to receive either an intravenous injection of 50 µg/200 µL biotinyled CD45 McAb followed 24 h later by an intraperitoneal injection of 3.7 MBq (50 µg/100 µL) (188)Re-avidin (two-step pretargeting group), or a single intravenous injection of 3.7 MBq (100 µg/100 µL) (188)Re-CD45 McAb (control group). SPECT was performed at 0.5, 1, 6 and 23 h post-injection to characterize (188)Re isotope biodistribution. At 24 h pos-injection, the mice were sacrificed for measurement of radioactivity uptake in the tumor and normal tissues and calculation of the tumor-to-non-tumor (T/NT) ratios.

RESULTSSPECT showed that the two-step pretargeting method resulted in a low radioactivity in the blood pool during the imaging and a concentrated radioactivity in the liver and spleen. The transplanted tumor began to be displayed at 1 h post-injection and was clearly displayed at 1-6 h; the images were clear even at 23 h. With the two-step pretargeting method, the radioactive uptake at 24 h post-injection were (1.34∓0.52)%, (6.77∓2.32)%, and (2.81∓1.25)% in the tumor, kidney and liver, respectively, with low radioactivity levels in other organs and high tumor/blood and tumor/muscle ratios (4.28∓0.82 and 8.00∓0.88, respectively). In the control group, SPECT revealed intense radioactivity in the liver, spleen, and kidneys with obscure display of the tumor; at 20 h, the radioactivity in the blood pool remained high but that in the tumor was low, and the tumor/blood and tumor/muscle ratios at 24 h were only 0.58∓0.06 and 3.21∓0.24, respectively.

CONCLUSIONCompared with (188)Re-CD45 McAb, the two-step pretargeting approach exhibits a good specificity in targeting lymphoma with an increased T/NT ratio in mice and allows early tumor display at 1 h post-injection.

Animals ; Antibodies, Monoclonal ; Avidin ; Disease Models, Animal ; Lymphoma ; diagnosis ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Radioimmunodetection ; Tissue Distribution ; Tomography, Emission-Computed, Single-Photon

PURPOSE: For periodontal tissue engineering, it is a primary requisite and a challenge to select the optimum types of cells, properties of scaffold, and growth factor combination to reconstruct a specific tissue in its natural form and with the appropriate function. Owing to fundamental disadvantages associated with using a two-dimensional substrate, several methods of seeding cells into three-dimensional scaffolds have been reported and the authors have asserted its usefulness and effectiveness. In this study, we explore the cell attachment of periodontal ligament fibroblasts on nanohydroxyapatite (n-HA) scaffold using avidin biotin binding system (ABBS). METHODS: Human periodontal ligament fibroblasts were isolated from the health tooth extracted for the purpose of orthodontic procedure. HA nanoparticles were prepared and Ca(NO3)2-4H2O and (OC2H5)3P were selected as precursors of HA sol. The final scaffold was 8 mm in diameter and 3 mm in height disk with porosity value of 81.55%. 1x10(5) periodontal ligament fibroblasts were applied to each scaffold. The cells were seeded into scaffolds by static, agitating and ABBS seeding method. RESULTS: The number of periodontal ligament fibroblasts attached was greater for ABBS seeding method than for static or agitating method (P<0.05). No meaningful difference has been observed among seeding methods with scanning electron microscopy images. However, increased strength of cell attachment of ABBS could be deduced from the high affinity between avidin and biotin (Kd=10(-15) M). CONCLUSIONS: The high-affinity ABBS enhances the ability of periodontal ligament fibroblasts to attach to three-dimensionally constructed n-HA scaffolds.
Avidin ; Biotin ; Cell Adhesion ; Dihydroergotamine ; Fibroblasts ; Humans ; Microscopy, Electron, Scanning ; Nanoparticles ; Periodontal Ligament ; Polymethyl Methacrylate ; Porosity ; Seeds ; Tissue Engineering ; Tooth

BACKGROUND: The aim of this study was to assess the feasibility of targeted ultrasound imaging on apoptosis with annexin A5 microbubbles (A5MB) in acute doxorubicin-induced cardiotoxicity. METHODS: Avidinated and octafluoropropan-filled phospholipid microbubbles were conjugated with biotinylated annexin A5. To confirm the specific binding of A5MB, flow cytometry was performed with hydrogen peroxide induced apoptosis in rat aorta smooth muscle cells incubated with fluorescein-5-isothiocyanate (FITC) labeled annexin A5 and A5MB. Adult male rats were injected intraperitoneally with 5 mg/kg doxorubicin weekly for 3 weeks (n = 5). Control rats were injected with normal saline (n = 5). At 24 hours after the final treatment, triggering imaging was performed 15 min after an intravenous bolus injection of A5MB for washout of freely circulating microbubbles. After echocardiography, the heart was isolated for histological detection of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. RESULTS: In the in vitro tests, fluorescence intensity was low for healthy cells and high for apoptotic cells when incubated with FITC-labeled annexin A5 and A5MB. Rats treated with doxorubicin showed significant contrast opacification of the myocardium on contrast echocardiography using A5MB. However, no opacification was observed in control rats. Apoptosis was confirmed by TUNEL assay in doxorubicin treated rats. CONCLUSION: Acute doxorubicin-induced cardiomyopathy based on early apoptosis can be assessed and imaged with targeted ultrasound imaging using A5MB in rats.
Adult ; Animals ; Annexin A5 ; Aorta ; Apoptosis ; Avidin ; Cardiomyopathies ; Doxorubicin ; Echocardiography ; Flow Cytometry ; Fluorescein-5-isothiocyanate ; Fluorescence ; Heart ; Humans ; Hydrogen Peroxide ; In Situ Nick-End Labeling ; Male ; Microbubbles ; Myocardium ; Myocytes, Smooth Muscle ; Rats

OBJECTIVETo detect serum hepatoma-specific datura stramonium lectin-tightly binding gamma-glutamyl transferase (DSA-GGT) in patients with primary hepatic cancer (PHC) by avidin-biotin ELISA method which was established in our laboratory, and carry on a study of its clinical application.

METHODSTo detect serum DSA-GGT in 45 healthy control subjects, 58 PHC patients and 203 non-PHC patients (including 36 patients with other tumors and 167 patients with benign liver diseases) with the method was established; meanwhile, AFP was detected by ELISA method.

RESULTS38 individuals were DSA-GGT positive in 58 PHC patients, the sensitivity was 65.5%. 18 individuals were DSA-GGT positive in 203 patients without PHC, the specificity was 91.1%. The sensitivity and specificity of AFP in diagnosis of PHC patients was 69.0% and 90.6%, respectively. The sensitivity and specificity of combination of DSA-GGT and AFP was 93.1% and 85.7%, respectively. The average intra-CV and inter-CV of DSA-GGT ELISA was 8.9% and 11.5%, respectively.

CONCLUSIONThe sensitivity and specificity of DSA-GGT ELISA method established in our lab is similar with that of AFP assay and the accuracy is good. Combination of DSA-GGT and AFP may improve the diagnostic sensitivity. The method should be potentially as a new way to improve diagnosis of PHC.

Adult ; Avidin ; Biotin ; Carcinoma, Hepatocellular ; blood ; diagnosis ; enzymology ; Enzyme-Linked Immunosorbent Assay ; methods ; Female ; Humans ; Liver Neoplasms ; blood ; diagnosis ; enzymology ; Male ; Middle Aged ; Sensitivity and Specificity ; Young Adult ; alpha-Fetoproteins ; analysis ; gamma-Glutamyltransferase ; blood

PURPOSE: In tissue engineering, it is important that the scaffolds have high affinity with cells for making efficient use of cells. The authors studied the binding affinity of human adipose stem cells(ASCs) to micronized acellular dermal matrix(alloderm) using biotin and avidin linkages. METHODS: Human ASCs were harvested from adipose tissue obtained by abdominoplasty. ASCs(1x10(4), 5x10(4), 1x10(5), 5x10(5), 1x10(6), 5x10(6) cells) were attached to micronized alloderm(1mg) in three groups; 1) control group in which no ASCs and alloderm was treated; 2) serum group in which alloderm was exposed to fetal bovine serum; and 3) biotin group in which biotinylated cells were attached to biotinylated alloderm. The binding affinities were determined 1 day after making ASC-alloderm complexes. The proliferation rates were determined by XTT assays in 4, 7, 14, and 21 days and scanning electron microscopic examination was performed in 7 and 21 days after culture of ASC-alloderm complexes. RESULTS: The binding affinities of the biotin group were significantly increased in all cell concentrations. Maximum binding affinity was observed at 5x10(4)/mg of micronized dermal matrix in biotin group. The viabilities were lowest in biotin group in contrast to binding affinity, but the difference was not significant. SEM showed well attachment of cells to micronized dermal matrix in all groups. CONCLUSION: The use of avidin/biotin facilitated human ASCs attaching to micronized acellular dermal matrix. This attachment would not disturb adipose stem cells viabilities. The present study suggests that avidin/ biotin can be used as making efficient use of cells in adipose tissue engineering.
Abdominoplasty ; Acellular Dermis ; Adipose Tissue ; Avidin ; Biotin ; Collagen ; Electrons ; Humans ; Mesenchymal Stromal Cells ; Stem Cells ; Tissue Engineering

In this experiment, a novel biotin-avidin conjugation probe was synthesized and employed in the detection of reverse-phase protein microarray. Firstly, the proportion of the biotin-avidin conjugation probe was optimized. Then the rat IgG and goat anti-rat IgG system was served as a model to optimize the fabrication conditions of reverse-phase protein microarray, including the non-specific absorption of streptavidin-Cy3 molecules, spotting buffer as well as protein activities. At last, the biotin-avidin conjugation probe was applied to the detection of the reverse-phase protein microarray. The results show that the protein microarray prepared by using BSA spotting buffer could prevent non-specific absorptions of fluorescent molecules and improve the sensitivity, effectively. In addition, compared with traditional biotin-avidin system, the detection limit could be improved four times using the biotin-avidin conjugation probe. In conclusion, the biotin-avidin conjugation probe has its merits of easy synthesis, low price and could be further conjugated with other signal amplification techniques, which is promising to be used in the detection of protein microarray.
Avidin ; chemistry ; Biotin ; chemistry ; DNA Probes ; Immunoglobulin G ; analysis ; immunology ; Protein Array Analysis ; methods

OBJECTIVETo explore the diagnostic efficiency of circulating antigen using the TM5.28 mAB-biotin-avidin system for the detection of schistosomiasis japonica.

METHODSA mAb-biotin-avidin system was set up using a TM5.28 mAB which was prepared against a gut associated antigen of Schistosoma japonicum. Detection was performed on the sera from 50 acute schistosomiasis patients, 224 chronic patients, 49 advanced patients and 46 schistosomiasis patients who were followed up at 6 months and 12 months post treatment. In addition, 19 cases of clonorchiasis, 31 cases of paragonimiasis, 23 cases of hepatitis B and 100 healthy individuals were also included.

RESULTSThe system showed sensitivity of 83.1% and specificity of 94.0% when applied to detect chronic schistosomiasis and healthy persons respectively, while 94.0% to acute schistosomiasis. The Youden's index of the system was 0.771. The rate of cross-reaction to paragonimiasis, clonorchiasis and hepatitis B was 12.9%, 15.8% and 13.0% respectively. The rates of negative turning were 43.9% and 62.1% respectively in chronic schistosomiasis at the 6 month and 12 month intervals after treatment. Geometric mean of the OD values also decreased from 0.172 before treatment to 0.081 at 6 months and 0.068 at 12 months after treatment with a reduction rate of 60.30%. The detection rate in the heavy infected population reached a maximum of 90.0%. This was similar in moderate and light infected populations, i.e., 83.9% and 82.1%, respectively.

CONCLUSIONThe TM5.28 mAb-biotin-avidin system showed a relatively high efficiency in the diagnosis of schistosomiasis and a high negative turning rate after treatment. It is, therefore, a valuable tool for the estimation of prevalence in endemic populations, as well as individual diagnosis and for assessing the effect of chemotherapy.

Animals ; Antibodies, Helminth ; immunology ; Antibodies, Monoclonal ; immunology ; Avidin ; immunology ; Biotin ; immunology ; Cell Fusion ; Humans ; Mice ; Mice, Inbred BALB C ; Schistosomiasis japonica ; diagnosis ; immunology ; Serologic Tests

BACKGROUND: Numerous cell surface proteins of leukemia cells such as CD33 and CD52 have been identified as diagnostic and therapeutic targets. Thus the profiling of the cell surface proteome and proteins restricted to specific leukemia(s) can provide a way to identify novel targets for leukemia diagnosis and therapy. However, there is a lack of data pertaining to the comprehensive analysis of surface membrane proteins because there are few effective strategies for profiling surface membrane proteomes. METHODS: We report on the application of quantitative proteomic techniques that incorporate affinity-capture and purification on monomeric avidin columns to identify all biotinylated cell surface proteins from leukemia cell lines. RESULTS: An analysis of a subset of biotinylated proteins among the different human leukemia cell lines using matrix-assisted laser desorption ionization and tandem mass spectrometry identified, among others, some widely expressed proteins in leukemia cells, such as CD11a, CD11c, CD18, CD31, CD44, and CD147, as well as a set of proteins identified as chaperone proteins, including HSP90, GRP78, GRP75, HSP70, HSP60 and protein disulfide isomerases. On the basis of their known functional roles, several of these proteins may participate in the progression of leukemogenesis and should be considered as potential markers of leukemia. CONCLUSION: Comprehensive profiling of the leukemia cell surface proteome provides an effective approach for the identification of commonly occurring proteins as well as proteins with restricted expression patterns to a specific cell line.
Avidin ; Cell Line ; Diagnosis ; Humans ; Leukemia* ; Membrane Proteins* ; Membranes ; Protein Disulfide-Isomerases ; Proteome ; Tandem Mass Spectrometry

BACKGROUND: Catenins are the associated protein with E-cadherin in the formation of adhesion complexes in normal and tumor cells related with epithelial differentiation and development of organ formation as well as in the tumor spread. The present study was aimed to find the distribution of alpha- and gamma-catenins in fetal skin development. OBJECTIVE: The purpose of this study was to observe the distribution of above two adhesion related proteins in the fetal skin during development, and to find its relationship by expression and their distribution pattern. METHODS: Skin was obtained from the scalp, chest, and sole of 21 human fetuses, ranging from 13 to 37 weeks of gestational age. Immunohistochemical staining was performed by the avidin biotin peroxidase complex method on paraffin embedded tissue using the anti-human monoclonal antibody against the human alpha- and gamma-catenins. RESULTS: alpha- and gamma-catenins were expressed strongly in basal cells of the epidermis and germ cells of skin adnexa, such as hair and eccrine glands at 13th week, followed by decreased basal cell expression. Increase in the suprabasal epithelium and differentiated adnexal epithelium, such as outer root sheath cells and eccrine ducts and glands at 18th week, and adult pattern in 23th week of gestation. Both showed similar distribution pattern in skin though gamma-catenin appeared two or three weeks later. alpha- and gamma-catenins are expressed not only in the epithelium of the skin, but also in the mesenchymal cells such as endothelial cells and fibroblasts. Though both catenins are more strongly expressed in the membrane portion, cytoplasmic expression is also noted. CONCLUSION: Both alpha- and gamma-catenin showed basically the same expression distribution pattern in the fetal skin developmental stage, suggesting that both adhesion molecules are highly related to each other in function and development of epidermis and adnexae of the skin in fetal stage.
Adult ; Avidin ; Biotin ; Cadherins ; Catenins ; Cytoplasm ; Eccrine Glands ; Endothelial Cells ; Epidermis ; Epithelium ; Fetus ; Fibroblasts ; gamma Catenin* ; Germ Cells ; Gestational Age ; Hair ; Humans ; Membranes ; Paraffin ; Peroxidase ; Pregnancy ; Scalp ; Skin* ; Thorax

BACKGROUND: Glutathione S-transferases(GST) are a family of multi-functional enzymes involved in cellular detoxification and excretion of a variety of exogenous and endogenous toxic or carcinogenic compounds. The GST family has been divided into three classes, alpha, mu, and pi, based on substrate specificity and sequence homology. GST-pi is an acidic type and predominant in skin, small intestine, breast, lung and prostate. The overexpression of GST-pi associated with skin tumor and tumor-like lesion suggests that GST-pi is a major detoxifying enzyme in skin tumors. OBJECTIVE: The purpose of this study was to observe the expression and the distribution pattern of GST-pi in the human fetal skin. METHODS: Skin was obtained from the scalp, chest, and sole of 49 human fetuses, ranging from 8th to 40th weeks of gestational age. Immunohistochemical staining was performed using avidin biotin peroxidase complex method on paraffin embedded tissue using antirabbit polyclonal antibody against the human GST-pi. RESULTS: GST-pi was expressed in intermediated layer of epidermis at 8th week, and gradually increased in strength of expression stronger in suprabasal layer. In hair unit, GST-pi was expressed in sebaceous gland, bulge, hair matrix cell and outer root sheath cell from 15th week. In eccrine gland, also GST-pi was expressed in central differentiated cells of intradermal eccrine duct from 18th week, and in terminal duct and acini from 26th week of fetal age. CONCLUSION: GST-pi was expressed from the 8th week of gestation suggesting that GST-pi plays an important role in detoxification for the protection of the skin in fetal stage from the various toxic agent.
Avidin ; Biotin ; Breast ; Eccrine Glands ; Epidermis ; Fetus ; Gestational Age ; Glutathione Transferase* ; Glutathione* ; Hair ; Humans ; Intestine, Small ; Lung ; Paraffin ; Peroxidase ; Pregnancy ; Prostate ; Scalp ; Sebaceous Glands ; Sequence Homology ; Skin* ; Substrate Specificity ; Thorax