Animals ; Enzyme Inhibitors ; pharmacology ; Hepatitis ; immunology ; Immune Sera ; pharmacology ; Mice ; Sodium-Potassium-Exchanging ATPase ; antagonists & inhibitors ; metabolism

Due to the serovar diversity in Haemophilus (H.) parasuis, it is difficult to develop a universal serological method for detection of this pathogen. Here, we report a universal plate-agglutination test for detecting H. parasuis. Diagnostic antisera were prepared by mixing antisera of serovars 4, 5, 12, 13 and 14 in the optimized ratio. The results of the plate-agglutination test showed that the diagnostic antisera could agglutinate with all 15 reference strains of H. parasuis and 74/75 clinical isolates. Further, the specificity of the method was validated with 22 bacterial strains from 12 related species.
Agglutination Tests/*methods ; Animals ; Cross Reactions ; Haemophilus parasuis/isolation & purification/*physiology ; Immune Sera/*metabolism ; Reproducibility of Results ; Sensitivity and Specificity

Background: Triiodothyronine(T3) is a hormone secreted from thyroid gland which exerts a stimulating effect on metabolism. The disorder of thyroid system brings about several serious diseases like hypothymidism or hyperthyroidism. Therefore, the determination of T in blood is very important on monitoring thyroid function. Methods: Rabbit anti-T3 antibody was generated by immunization of T-BSA as an immunogen and purified hom antisera using Affi-gel protein A kit. The titer and specificity of purified antibody were characterized. To detect T3, competitive ELISA was performed using anti-T3 antibody and T3-HRP conjugate which was synthesized by glutaraldehyde method. The sensitivity and precision assay wer~e deterrnined and compared with that of RIA. Results: The titer of purified anti-T3 antibody was about 1:100 and the optimal dilution of T3- HRP conjugate was 1:1000. When the standard curve was constructed by ELISA, its sensitivity was about 0.5ng/ml. The eoefficient variations of intra- and inter-assay were 4.9~9.3% and 7.5~13.8%, respectively. The results obtained by ELISA and RIA correlated well with each other(n =50, r= 0.97), The linear regression equation was y= 1.09*0.08(P<0.01). Conclusion: We successfully developed a method for the measurement of T3 on ELISA which was based on competitive reaction between antigen(T3) and enzyme labeled antigen(T3-HRP). These results demonstrated that competitive ELISA is a convenient, fast, reproducible and aecurate method for the determinstion of T in serum and can be used as practical alternative to RIA.
Enzyme-Linked Immunosorbent Assay ; Glutaral ; Hyperthyroidism ; Immune Sera ; Immunization ; Linear Models ; Metabolism ; Methods ; Sensitivity and Specificity ; Staphylococcal Protein A ; Thyroid Gland ; Triiodothyronine

OBJECTIVETo express human HZF1 fusion protein in E. coli and to obtain an anti-HZF1 antibody.

METHODSA DNA fragment encoding non-zinc finger region of HZF1 protein was inserted into pET30a vector to get the recombination expression plasmid pET30a-HZF1. E. coli was transformed with pET30a-HZF1 and the selected clones were cultured with isopropy-beta-D-thiogalactoside induction. The proteins were prepared from the culture and the fusion protein was purified by Ni column. Rabbits were immunized and reinforced three times with the purified fusion protein. The antiserum was collected and the titer and the specificity of the antibody were checked by ELISA and Western blot.

RESULTSAntibody against HZF1 was obtained and its titer was more than 1:100 000, as proven by ELISA. Western blot analysis showed specific reaction between this antibody and HZF1 fusion protein or the endogenetic HZF1 protein in hemin-induced K562 cells.

CONCLUSIONSThe specific antibody against HZF1 is obtained. The antibody may have potential application in farther HZF1 function study and HZF1 determination in tissues and cells.

DNA-Binding Proteins ; genetics ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Transfer Techniques ; Immune Sera ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Transformation, Bacterial

OBJECTIVETo evaluate the effect of sensitized sera on engraftment of hematopoietic stem cells (HSCs) and its mechanism.

METHODSBone marrow cells (BMCs) from C57BL/6 mice were incubated with sensitized sera (group I) or normal sera (group II), and then washed and transplanted into irradiated BALB/c mice. The survival analysis and engraftment evaluation of the recipients were observed. The incubated BMCs were bound with goat anti mouse IgG for and labeled with Annexin V for apoptosis detection.

RESULTSSeven out of ten recipient mice in group I died of bone marrow failure at day 10 after transplantation, while all of those in group II were long-term survived. Engraftment assay showed recipients blood count and BMCs progressively decreased along with time passing in group I; in addition, the chimeric percentage of donor cells progressively decreased as well. The percentage of BMCs binding with goat anti mouse IgG in group I or group II were (90.3 +/- 5.1)% and (5.2 +/- 2.4)%, respectively, and the difference was statistically significant (P < 0.01). However, no significant difference was found in the apoptosis detection between the two groups (P > 0.05).

CONCLUSIONThe engraftment capacity of HSCs is significantly impaired by sensitized sera, the antibodies in sensitized sera may bind to antigens expressed on HSCs but do not induce apoptosis.

Animals ; Annexin A5 ; metabolism ; Apoptosis ; Bone Marrow Cells ; metabolism ; pathology ; Cells, Cultured ; Hematopoietic Stem Cell Transplantation ; Immune Sera ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL

AIMTo investigate the role of delayed rectifier K+ channel (Kv), Ca2+ -activated K+ channel (K(Ca)) and ATP-sensitive K+ channel (K(ATP)) in the regulation of the resting and contracting tone of human control and passively sensitized bronchial smooth muscle (BSM).

METHODSThe regulating effects of the three K+ channels on the tone of human BSM (HBSM) were observed by measuring the isometric tone of bronchial rings in vitro.

RESULTS(1) The contraction of passively sensitized bronchial ring was significantly increased by histamine. (2) Kv blocker 4-aminopyridine (4-AP) caused concentration dependent contraction in resting bronchial rings of two groups, and the contraction sensitivity of the sensitized group rings was significantly stronger than that of control, that is, the negative logarithm of the drug concentration causing 50% of maximal effect (pD2) of the sensitized group rings were significantly larger than that of control rings, but there was no difference in the maximal effect (Emax) of two groups; Kca blocker tetraethylammonium (TEA) and K(ATP) blocker glibenclamide (Glib) had no such effects as those of 4-AP. (3) After pretreatment with 4-AP, the contraction of the control rings could significantly increased by histamine. After 4-AP treatment the Emax was significantly larger than that before 4-AP treatment. But the sensitized group rings had no such change, there was no significant difference in Emax before and after 4-AP treatment.

CONCLUSION(1) Not K(Ca) and K(ATP) but Kv participated in regulation of the resting tone of HBSM. (2) The activity of Kv decreased in bronchial smooth muscle passively sensitized by asthmatic serum compared with that of nonsensitized group. This change might be involved in the mechanism of asthma.

Asthma ; metabolism ; physiopathology ; Bronchi ; physiology ; Delayed Rectifier Potassium Channels ; physiology ; Female ; Humans ; Immune Sera ; Immunization, Passive ; In Vitro Techniques ; Male ; Middle Aged ; Muscle Tonus ; Muscle, Smooth ; physiology

For studying the expression and distribution of angiotensinogen (AGT), the C-teminus of rat AGT gene was expressed in E.coli. Rabbits were immunized with expressed AGT protein and sera from different rabbits were raised. ELISA showed a high titre (1:25600) of the antiserum. With the antiserum, Western blotting recognized not only the prokaryotic expressed AGT, but also the endogenous AGT protein in liver tissue of both rats and humans. Using this antiserum, immunohistochemistry showed the expression of AGT protein in islet cells of human pancreas as well as in epithelium of human bile duct. These results suggest that the prokaryotic expressed AGT protein is an effective immunogen for the preparation of anti-AGT antiserum. Our present work provides an important tool for study of the pathophysiological role of AGT as well as local renin-angiotensin system.
Angiotensinogen ; genetics ; immunology ; Animals ; Antibodies, Monoclonal ; biosynthesis ; Escherichia coli ; genetics ; metabolism ; Humans ; Immune Sera ; biosynthesis ; immunology ; Immunization ; Rabbits ; Rats ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Renin-Angiotensin System ; physiology

The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na+ and H+ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca2+. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.
Animals ; Cell Line ; Immune Sera/genetics/immunology/*metabolism ; Male ; Mice ; Protozoan Proteins/genetics/*metabolism ; Rabbits ; Recombinant Proteins/immunology ; Sheep ; Sodium-Hydrogen Antiporter/genetics/immunology/*metabolism ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/parasitology/prevention & control

Human metapneumovirus (hMPV) is a recently identified respiratory virus more like human respiratory syncytial virus in clinical symptoms. Matrix protein (M) is one of the most important structural proteins. For further studying of hMPV, the full length of M genes from the recombinant plasmid pUCm-M1816 and pUCmM1817 were cloned by PCR and sub-cloned into the pET30a(+) vector, which is a prokaryotic expression vector, after dual-enzyme digestion with Bam HI and Xho I. The positive recombinated plasmids were transformed into E. coli BL21 (DE3) and expressed under the inducing of IPTG. Target proteins were characterized by SDS-PAGE and Western blotting. In this article, we' ve successfully constructed the recombinated plasmids pET30a-M1816 and pET30a-M1817 which have correct open reading frames confirmed by dual-enzyme digestion analysis and sequencing. The fusion proteins with 6 x His-N were highly produced after inducing by 1mmol/ L IPTG at 37 degrees C. A unique protein band with approximate 27.6 kD was characterized by SDS-PAGE. Most of the target protein existed in inclusion body. Western blot analysis showed that the target protein has specific binding reaction to rabbit antiserum against polypeptides of the matrix protein of hMPV. So the M genes were highly expressed in the prokaryotic system and the expressed M proteins have specific antigenic activities. It can be used for further studying of hMPV infections in Beijing.
Animals ; Antigens, Viral ; genetics ; immunology ; metabolism ; Blotting, Western ; China ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; Immune Sera ; immunology ; Metapneumovirus ; genetics ; immunology ; metabolism ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; Rabbits ; Species Specificity ; Viral Structural Proteins ; genetics ; immunology ; metabolism

Prior to fertilization sperm has to undergo an activation process known as capaciation, leading to the acrosome reaction. Till now, little is known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources have been thought to be involved. In this study, we report that NYD-SP27, an isoform of phospholipase C Zeta 1 (PLCZ1), is localized to the sperm acrosome in mouse and human spermatozoa by immunofluorescence using a specific antibody. Western blot and double staining analyses show NYD-SP27 becomes detached from sperm, as they undergo capacitation and acrosome reaction. The absence of HCO3-, a key factor in activating capacitation, from the capacitation-inducing medium prevents the loss of NYD-SP27 from sperm. The anti-NYD-SP27 antibody also prevents the loss of NYD-SP27 from sperm, reduced the number of capacitated sperm, inhibited the acrosome reaction induced by ATP and progesterone, and inhibited agonist-induced PLC-coupled Ca2+ mobilization in sperm, which can be mimicked by the PLC inhibitor, U73122. These data strongly suggest that NYD-SP27 is a physiological inhibitor of PLC that acts as an intrinsic decapacitation factor in sperm to prevent premature capacitation and acrosome reaction.
Acrosome ; drug effects ; metabolism ; Acrosome Reaction ; physiology ; Adult ; Animals ; Calcium ; metabolism ; Fluorescent Antibody Technique, Indirect ; Humans ; Immune Sera ; pharmacology ; Male ; Mice ; Middle Aged ; Phosphoinositide Phospholipase C ; immunology ; metabolism ; Sperm Capacitation ; drug effects ; physiology ; Spermatozoa ; drug effects ; metabolism