OBJECTIVETo establish rat models of FSH autoantibody and to investigate the effect of FSH autoantibody on the spermatogenic capability of rat testis.

METHODSThirsty 21-day old SD rats were randomly divided into an experimental and a control group of equal number. A specific polypeptide corresponding to the rat FSHbeta subunit was synthesized and coupled to (keyhole limpet hemocyanin) KLH. The rats in the experimental group were immunized with polypeptide-KLH and these in the control group with KLH. Further immunization was performed every 2 weeks for 7 times. On the 77th, 91st and 105th day of the immunization, 5 rats from the experimental group and another 5 from the control group were killed. Then the structures of the seminiferous tubule and epididymal sperm were observed by light and electron microscope, respectively. Meanwhile, the counts of sperms and the percentage of swelled sperm were calculated. And the level of serum testosterone was detected by enzyme-linked immunospecific assay (ELISA).

RESULTSThe titer of the anti-polypeptide antibody was 1:200 on the 49th day of the immunization, and reached 1:400 on the 63rd. Compared with the control group, the percentage of swelled sperm significantly decreased on the 91st day (60.4 +/- 6.23 vs 50.60 +/- 3.05, P < 0.05), and the number of spermatogenic cells and sperms in seminiferous tubules reduced on the 105th day in the experimental group, the counts of sperms (46.08 +/- 6.56 vs 32.53 +/- 3.41) and the percentage of swelled sperm (60.60 +/- 5.86 vs 48.60 +/- 3.85) significantly lower (P < 0.05), while the level of serum T significantly higher than that in the control group (P < 0.05).

CONCLUSIONFSH autoantibody might cause testis dyszoospermia.

Animals ; Autoantibodies ; physiology ; Follicle Stimulating Hormone ; immunology ; Hemocyanins ; immunology ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sperm Count ; Spermatozoa ; physiology ; Testis ; physiology

To prepare anti-mouse uteroglobin binding protein (mUGBP) polyclonal antibody, two polypeptides were synthesized based on the bioinformatics analysis of mUGBP, and New Zealand white rabbits were immunized separately with each peptide coupled with keyhole limpet hemocyanin (KLH). The data indicate that a 13-amino acid polypeptide (positions 221st-233rd) was able to generate anti-peptide antibodies. The titer of the antisera detected with ELISA was 1:10(8). The antisera were then purified with immuno-affinity chromatography to obtain antibodies. Western blot analysis of mUGBP expressed as a fusion protein with a green fluorescent protein (GFP) was performed on the cell lysates of COS-1 cells with the purified antisera, suggesting that the antisera specifically recognized UGBP. By immunohistochemistry and indirect immunofluorescence analysis, we examined the expression of UGBP in the lung tissues from a patient undergoing surgical lung resection for a tumor and from normal mouse lung tissue, and found for the first time that UGBP protein was widely expressed in both mouse and human lung tissue with the most abundant expression in bronchial epithelial cells. These results suggest that the antigen epitopes of mUGBP are well predicted by using bioinformatics analysis. We have obtained anti-mUGBP polyclonal antibody, which will be useful for further investigation.
Animals ; Antibodies ; chemistry ; COS Cells ; Carrier Proteins ; chemistry ; Cercopithecus aethiops ; Computational Biology ; Enzyme-Linked Immunosorbent Assay ; Hemocyanins ; Humans ; Immune Sera ; Immunohistochemistry ; Mice ; Rabbits ; Recombinant Proteins ; chemistry ; Uteroglobin

OBJECTIVETo study the relationship between the immune response of anti-tetrodotoxin vaccine, including its dose-response, and to select optimal immunization dose so as to enhance antitoxic effect of the anti-tetrodotoxin vaccine.

METHODSTetrodotoxin (TTX) was coupled to Tachypleus tridentatus hemocyanin (TTH) chemically to form artificial antigen (TTX-TTH), and with which Balb/c mice were immunized. Influence of different immunization doses [100 microg as the higher (H) and 25 microg as the lower (L) dose group] on the protective effects of TTX vaccine was compared. The quality of antisera and effects of vaccine in anti-TTX poisoning were observed.

RESULTSThe sera antibody quality increased more quickly in group L than that in group H after immunization. The dose at which the half of immunized mice survived when challenged once with TTX were 16 x LD (1 LD = 13.5 microg/kg, i.p.) in group L and 11 x LD in group H. When TTX was used time and again, the half of immunized mice could tolerate as high as 40 x LD and 22 x LD of accumulated dose, and the maximum tolerable cumulated dose was 104 x LD and 90 x LD for group L and H respectively. The scheme L was better both in antibody quality and effect of protecting against TTX toxicity than that in scheme H.

CONCLUSIONSThe experimental vaccine of TTX could effectively protect animal from TTX intoxication. The lower immunization dose in this study is selected as the optimal immunization scheme.

Animals ; Antibodies ; blood ; Dose-Response Relationship, Immunologic ; Enzyme-Linked Immunosorbent Assay ; Female ; Hemocyanins ; immunology ; Horseshoe Crabs ; Immune Sera ; immunology ; Mice ; Mice, Inbred BALB C ; Tetrodotoxin ; immunology ; toxicity ; Toxicity Tests ; Vaccination ; methods ; Vaccines ; administration & dosage ; immunology

OBJECTIVESTo prepare and purify NogoA vaccination for treatment of spinal cord injury. To study the safety and immune effect of this vaccination.

METHODSArtificial NogoA-13 polypeptide was coupled with KLH to improve the immunogenicity of vaccination. Sixty three-week-old Wistar female rats were divided into 3 groups randomly. Group A was immunized with NogoA vaccination, group B with incomplete freund's adjuvant + complete freund's adjuvant; group C with KLH. Rats received abdominal cavity immunization. The level of antibody and the binding capability were detected with ELISA. The safety of vaccination was evaluated by the incidence and severity of experimental autoimmune encephalomyelitis (EAE).

RESULTSThe IgG antibody against the NogoA-13 polypeptide had been detected with ELISA in group A. A value of serum presented regular gradient during multiple proportion dilution. In group B and C, no antibodies were detected. The statistical significant difference in A value was revealed between group A and B, C group. No statistical significant difference was found in A value between group B and group C and non-immunized negative control serum. The features of EAE were not found in the immunized rats.

CONCLUSIONSNogoA polypeptide vaccination can stimulate the antibody against the polypeptide. The immune effect of this vaccination is confirmed by binding reaction revealed in the ex vivo experiment. The good safety of vaccination is revealed by no features of EAE found in the immunized rats.

Animals ; Encephalomyelitis, Autoimmune, Experimental ; chemically induced ; Female ; Hemocyanins ; adverse effects ; immunology ; Immunoglobulin G ; immunology ; Myelin Proteins ; adverse effects ; immunology ; Random Allocation ; Rats ; Rats, Wistar ; Safety ; Spinal Cord Injuries ; immunology ; Vaccination

OBJECTIVETo prepare a single-epitope polyclonal antibody against mouse long peptidoglycan recognition protein (mPGRP-L).

METHODSB cell dominant epitopes of mPGRP-L predicted by bioinformatics were synthesized, and the immunogen was prepared by conjugation of the synthetic peptide and the carrier protein key-hole limpet hemocyanin (KLH) by MBS method. The single-epitope polyclonal antibody was obtained by immunizing rabbits with the KLH-peptide conjugate, purified by SPG affinity columns or antigenic peptide affinity columns, and identified by ELISA and Western blotting.

RESULTS AND CONCLUSIONA dominant epitope in N-terminal region of mPGRP-L, with amino acid sequence of NH(2)-(C)DPHSLSPELQALISEVAQHD-COOH, was chosen and synthesized. The titer of anti-serum of the rabbits immunized with the KLH-peptide conjugate was 1:256,000. The polyclonal antibody purified with SPG affinity columns and antigenic peptide affinity columns were named as mPGRP-Ln1 and mPGRP-Ln2, respectively. Western blotting demonstrated that the antibody mPGRP-Ln1 could recognize the recombined N-terminal fragment of mPGRP-Ln with a clear band at relative molecular mass of about 29,000, suggesting the successful preparation of the single-epitope polyclonal antibody against the N-terminal region of mPGRP-L.

Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; chemistry ; immunology ; isolation & purification ; Antibody Specificity ; immunology ; Blotting, Western ; Carrier Proteins ; chemistry ; immunology ; Enzyme-Linked Immunosorbent Assay ; Epitope Mapping ; Epitopes ; chemistry ; immunology ; Hemocyanins ; chemistry ; Immune Sera ; immunology ; Mice ; Molecular Sequence Data ; Protein Binding ; Rabbits

BACKGROUNDNR2B containing N-methyl-D-aspartate (NMDA) receptor plays an important role in the facilitation and maintenance of neuropathic pain. The discrete distribution of NR2B subunit in the central nervous system (CNS) may support reduced side effects of agents that act selectively at this site. Therefore, we investigated the hypothesis that a humoral autoimmune response targeting the NR2B subunit of NMDA receptor relieves pain like behaviours produced by peripheral injury.

METHODSRats were immunized subcutaneously with NR2B-Keyhole Limpet Hemocyanin (NR2B-KLH) three times at two-week intervals. NR2B specific IgG titres in sera and cerebrospinal fluid were determined by indirect ELISA. Seven days after the third immunization, 2 of the 3 terminal branches of the sciatic nerve (tibial and common peroneal nerves) were tightly ligated. Behavioural testing was carried out on every other day after surgery, until 7 days after surgery. The lumbar spinal cord (L4-6) was removed on day 7 after ligation. The expression of NR2B protein in the lumbar spinal cord was determined using Western blotting.

RESULTSAfter the second vaccination, NR2B specific IgG in sera was detected to be > 0.5 microg/ml in six of nine rats. After the third vaccination, all the immunized rats had > 2.2 microg/ml. Titres of NR2B specific IgG in sera peaked 42 days post initial immunization and persisted for over 70 days. No NR2B specific IgG was detected in sera from PBS or KLH group. The behavioural thresholds in NR2B group were significantly higher than those in PBS and KLH groups on day 7 after ligation. NR2B specific IgG in CSF in NR2B group could not be detected on day 1 before spinal dissection; but could be detected on day 7 after surgery. The expression of NR2B protein in group NR2B was significantly lower than in PBS and KLH groups on day 7 after surgery.

CONCLUSIONThe NR2B peptide could be used as a vaccine against neuropathic pain, which could be associated with reduction of NR2B protein in the lumbar spinal cord.

Adjuvants, Immunologic ; Animals ; Blotting, Western ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Female ; Hemocyanins ; immunology ; Immunoglobulin G ; immunology ; Neuralgia ; immunology ; physiopathology ; prevention & control ; Pain Measurement ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; immunology ; metabolism ; Recombinant Fusion Proteins ; administration & dosage ; immunology ; Spinal Cord ; drug effects ; metabolism ; physiopathology ; Time Factors ; Vaccines ; administration & dosage ; immunology