[Transcranial sonographic characteristics of Parkinson's disease with symptoms of restless legs syndrome].

Objective: To evaluate the transcranial sonographic characteristics in patients with Parkinson's disease (PD) with symptoms of restless legs syndrome (RLS). Methods: Patients with diagnosis of definite PD from the Second Affiliated Hospital of Soochow University and 3 other participating hospitals between September 2018 and December 2019 were consecutively enrolled. Concurrent RLS symptoms were determined using Non-motor Symptoms Questionnaire. Transcranial sonography (TCS) and clinical assessments were performed during the same time and the related variables were compared between the two groups using t-test, non-parametric test, Chi-square test and Spearman correlation analysis, respectively. Results: Among 349 patients with PD, the prevalence of RLS symptoms was 22.6%. Compared to patients without RLS symptoms, those with RLS had longer disease duration (43.0 (24.0, 91.0) months vs 37.0 (20.0, 60.0) months, P<0.05) and higher Hoehn-Yahr stage (2.5 (2.0, 3.0) vs 2.0 (1.5, 2.5), P<0.01).TCS revealed that patients with RLS symptoms were more likely to have abnormality in the raphe nucleus (21.50% vs 7.78%, χ²=15.9, P<0.001) and increased third ventricle width ((6.22±1.97) mm vs (5.16±1.90) mm, P<0.001). No significant differences were found regarding parameters of substantia nigra. Conclusions: Concurrent RLS symptoms are common in PD patients. Abnormal echogenicity of raphe nucleus and increased third ventricle width could be characteristics of TCS in PD patients with RLS symptoms.

Depression and anxiety in multiple system atrophy.

BACKGROUND: It has been noticed that the patients with multiple system atrophy (MSA) can accompany with depression and anxiety. This study aimed to establish the incidence and determinants of depression and anxiety symptoms in Chinese MSA patients. METHODS: A total of 237 MSA patients were enrolled in the study. Neuropsychological assessment was performed using Hamilton Depression Rating Scale-24 items and Hamilton Anxiety Rating Scale. RESULTS: We found that 62.0% and 71.7% patients had at least mild depression and anxiety symptoms, respectively. The severity of depression of MSA patients was associated with lower educational years (P=.024), longer disease duration (P<.001), and disease severity (P<.001). The severity of anxiety was associated with increased disease duration (P<.001), disease severity (P=.013), and orthostatic hypotension (P=.005). Binary logistic regression showed the determinants of depression and anxiety were female gender, longer disease duration, and disease severity. CONCLUSION: Depression and anxiety symptoms are common in patients with MSA. Neurologists should pay attention to depression and anxiety in patients with MSA, especially in female patients and those with longer disease duration and severe disease condition.

Non-motor symptoms and quality of life in tremor dominant vs postural instability gait disorder Parkinson's disease patients.

OBJECTIVES: To explore the differences in the features and impact on quality of life (QOL) of non-motor symptoms (NMS) of tremor dominant (TD) and postural instability gait disorder (PIGD) phenotypes early Parkinson's disease (PD), as well as the determinants of poor QOL for TD and PIGD phenotypes. METHODS: This cross-sectional study recruited 301 patients with early PD and 101 healthy controls. Specific assessments used for NMS included NMS scale (NMSS), the Hamilton Rating Scale for Depression (HRSD-24), the Hamilton Anxiety Scale (HAMA), the Mini-Mental state examination (MMSE), and Addenbrooke's Cognitive Exam-Revised (ACE-R). QOL was evaluated with the PD Quality of Life Questionnaire (PDQ-39). RESULTS: Tremor dominant phenotype patients were 117 (38.9%), and PIGD were 155 (51.5%). Compared with TD patients, patients with PIGD had higher frequency of NMS (9.0 ± 5.3 vs 6.7 ± 4.6, P < 0.001), NMSS total scores (39.6 ± 34.5 vs 24.4 ± 22.7, P < 0.001) and more poorly for PDQ-39 summary index (19.2 ± 14.0 vs 13.8 ± 11.5, P = 0.001). There was no difference in the impact of NMS measured with NMSS on QOL between PIGD and TD phenotypes. PIGD phenotype had little impact on poor QOL once the effect of depression was taken into account. Depression was a primary negative predictor for QOL in both TD and PIGD patients (Beta: 0.697 and 0.619, respectively, P < 0.001). CONCLUSIONS: PIGD phenotype had a higher prevalence of NMS and worse QOL than TD phenotype. Depression is related to a dramatic decline in QOL in both TD and PIGD phenotype patients with PD.

Quantitative meta-analysis of grey matter anomalies in neuropathic pain.

BACKGROUND: Increasing neuroimaging studies have revealed grey matter (GM) anomalies of several brain regions by voxel-based morphometry (VBM) studies in patients with neuropathic pain. The changes have been suggested to be related to central sensitization. Our aim was to investigate concurrence across VBM studies to identify whether different subtypes of neuropathic pain share a common pathophysiological basis revealed by structural abnormalities. METHODS: A systematic search of VBM studies of patients with neuropathic pain and healthy controls published in PubMed and Embase databases from January 2000 to March 2014 was conducted. A quantitative meta-analysis of whole-brain VBM studies in patients with neuropathic pain compared with healthy controls was performed by means of effect-size signed differential mapping. RESULTS: Ten studies comprising 240 patients with neuropathic pain and 263 healthy subjects were systematically included in the present study. Compared to healthy controls, the patients showed consistent decreased GM in bilateral anterior insula and thalamus, right superior frontal gyrus and left postcentral gyrus, and increased GM in right medial frontal gyrus and right posterior insula. The results remained largely unchanged in the following jackknife sensitivity analysis. CONCLUSIONS: This meta-analysis shows strong evidence of brain GM anomalies within the pain matrix in patients with neuropathic pain compared with healthy subjects. Further studies are needed to determine whether the reported changes are specific to neuropathic pain or whether they may be common to other chronic pain.

Gray matter anomalies in anterior cingulate cortex as a correlate of depressive symptoms in drug-naïve idiopathic restless legs syndrome.

BACKGROUND: Depressive symptoms are frequent in idiopathic restless legs syndrome (RLS). However, little is known, so far, about the neurological basis. The present study aimed to explore the neuroanatomical anomalies in depressed drug-naïve RLS patients using voxel-based morphometry (VBM) analysis. METHODS: We recruited 16 drug-naïve idiopathic RLS patients with depressive symptoms (RLS-D), 18 drug-naïve idiopathic RLS patients without depressive symptoms (RLS-ND), and 18 normal controls. All participants underwent structural MRI scans on a 3-T MR system. The differences in regional gray matter (GM) density were determined across groups by VBM8. Additional regression analysis was used to identify any associations between regional GM density and clinical symptoms. RESULTS: GM density of the bilateral anterior cingulate cortex (ACC) was significantly reduced in RLS-D patients when compared to RLS-ND patients or to the healthy controls. However, there were no significant differences of GM density either when the whole RLS group or the RLS-ND group was compared to healthy controls, respectively. Particularly, we found GM density of right ACC was negatively correlated with the severity and duration of depressive symptoms in RLS-D patients. CONCLUSIONS: Depressive symptoms are associated with GM anomalies in ACC in patients with RLS. We propose that ACC is perhaps an important neuroimaging marker for facilitating treatment strategies in patients with RLS when assessing depressive symptoms.

An association analysis of the rs1572931 polymorphism of the RAB7L1 gene in Parkinson's disease, amyotrophic lateral sclerosis and multiple system atrophy in China.

BACKGROUND AND PURPOSE: Recently, the rs1572931 single-nucleotide polymorphism (SNP) of the putative promoter of the member RAS oncogene family-like 1 (RAB7L1) gene was reported to be associated with reduced risk for Parkinson's disease (PD) in the Ashkenazi Jewish population. Ethnic-specific effects are an important consideration in genome-wide association studies. Considering that the clinical manifestations and pathological characteristics overlap between PD, amyotrophic lateral sclerosis (ALS) and multiple system atrophy (MSA), the possible associations between the rs1572931 SNP and these three diseases were studied in the Chinese population. METHODS: A total of 1011 PD patients, 778 sporadic ALS (SALS) patients, 264 MSA patients and 516 healthy controls (HC) were included in this study. All subjects were genotyped for the rs1572931 SNP by using polymerase chain reaction and direct sequencing. RESULTS: Significant differences were observed in the genotype and minor allele frequency (MAF) of rs1572931 between PD and HC (P = 0.0001 and P = 1.08E-04, respectively) and between late-onset PD and matched controls (P = 0.0011 and P = 0.0002, respectively). However, no differences were observed between early-onset PD and HC. The number of minor allele carriers was significantly lower in PD patients than in HC (P = 2.96E-05, odds ratio 0.63, 95% confidence interval 0.51-0.78). No differences were observed between groups with respect to sex, onset symptoms, absence or presence of cognition impairment, anxiety or depression. In addition, no differences were found in the genotype and MAF of rs1572931 between SALS and HC or between MSA and HC. CONCLUSION: Our results suggest that rs1572931 decreases the risk for PD but not for ALS and MSA in the Chinese population. However, the polymorphism is unlikely to be a common cause of SALS and MSA in the Chinese population.

Huntingtin gene CAG repeat numbers in Chinese patients with Huntington's disease and controls.

BACKGROUND AND PURPOSE: Huntington's disease is due to a CAG triplet repeat elongation in the huntingtin gene. Boundaries in CAG numbers have been found between healthy people with and without risk to pass the disorder to the next generation, and between people without, with a mild, or with a fully penetrant phenotype. These data have been generated in western populations and it is not clear whether they are also valid amongst Chinese. METHODS: In order to establish normative data in the huntingtin gene for Chinese people, 966 chromosomes from normal controls were tested. Further, the range of CAG repeats was examined in a cohort from six centres and a total of 368 patients with the disease were included. RESULTS: The CAG triplet repeat range in normal controls was between 9 and 35 (mean 18.9, SD 2.57). Triplets in the range between 26 and 35 were found in 2.5%. In the patient cohort, triplet repeats in the shorter allele were between 8 and 37 (mean 17.7, SD 1.6). In the longer allele, a range between 36 and 120 was found. There was a negative correlation (-0.65, r = 0.42) between age at onset and the number of triplet repeats in the larger allele. The mean age at onset was 38 years, with a range between 2 and 70 years. In 23 patients (6%) a childhood or juvenile onset was noted. CONCLUSION: These data show comparable ranges of huntingtin gene CAG triplet repeats in normal people and in patients with Huntington's disease as in western populations.

PRRT2 mutation screening in patients with paroxysmal kinesigenic dyskinesia from Southwest China.

BACKGROUND AND PURPOSE: Proline-rich transmembrane protein 2 (PRRT2) has recently been identified as a causative gene of paroxysmal kinesigenic dyskinesia (PKD). However, the frequencies of its mutations and their correlation with the clinical features of PKD remain largely unknown. METHODS: Four exons of PRRT2 in 33 patients with PKD from Southwest China were screened by direct sequencing in this study. RESULTS: The mean onset age of the patients was 12.50 ± 2.70 years. Sixteen patients (48.48%) had sensory aura before their attacks. In total, 66.67% of the patients were running when the attacks occurred. c.649_650insC (p.P217fsX7), the most commonly reported insertion mutation, was identified in nine patients (27.27%). CONCLUSIONS: Other genes are involved in the development of PKD, but PRRT2 is a common causative gene for patients with PKD from Southwest China.

Expression of Toll-like receptors 2 and 4 and the OPG-RANKL-RANK system in inflammatory external root resorption and external cervical resorption.

AIM: To investigate and compare the expression of the pathogen recognition receptors Toll-like receptor (TLR) 2 and TLR4, and the hard tissue resorption triad osteoprotegerin (OPG)-receptor activator of nuclear factor kappa-B ligand (RANKL)-receptor activator of nuclear factor kappa-B (RANK) in external inflammatory root resorption of endodontic origin (ER) and external cervical root resorption (ECR) by immunohistochemistry. METHODOLOGY: Formalin-fixed, paraffin-embedded archival specimens collected from teeth that were diagnosed clinically, radiographically and histopathologically with either ER (n = 9) or ECR (n = 9) were processed for immunohistochemistry to investigate and compare levels of TLR2, TLR4, OPG, RANKL, RANK, CD3, CD19 and CD83 expression. The histological features were evaluated via haematoxylin and eosin stain. Taylor's modification of the Brown and Brenn Gram stain was used for examining the presence and distribution of bacteria. All stained slides were digitally photographed and qualitatively analysed, and F test and unpaired Student's t-test were used for statistical analysis. RESULTS: Both ER and ECR showed similar immuno-histopathology characteristics of a fibrovascular connective tissue with varying degrees of inflammatory infiltrate consisting of T and B lymphocytes, dendritic cells, polymorphonuclear lymphocytes and plasma cells. Colonies of bacteria were identified in the majority of lesions, and this correlated with the cellular expression of TLR2 and TLR4 in all lesions. Similarly, all lesions showed a significantly higher (P < 0.05) level of cells expressing RANKL than OPG, indicating hard tissue resorption processes where active in the lesions. CONCLUSION: The immunohistopathology patterns of ECR samples were consistent with the bacteria-driven ER specimens, suggesting bacteria-induced inflammation may be involved in ECR.

Assessment of D216H DYT1 polymorphism in a Chinese primary dystonia patient cohort.

BACKGROUND: The D216H single-nucleotide polymorphism (SNP) (rs1801968) in DYT1 exon 4 has been suggested to be a genetic modifier in primary dystonia. METHODS: To further explore this question, we assessed rs1801968 variations in a cohort of 210 Chinese patients with primary dystonia devoid of DYT1 mutations. RESULTS: We found that focal dystonia, specifically cervical dystonia, was the most common form of dystonia, with 8.1% of all the patients having a positive family history of dystonia. No association of the D216H SNP with primary dystonia was identified. In a subsequent subgroup analysis, the 216H allele was found to occur more frequently in patients with writer's cramp, but no correlation was found between the allele and other forms of dystonia or age of onset. CONCLUSIONS: Our findings do not confirm that the allele contributes to the risk of D216H SNP primary dystonia.

Voxel-wise meta-analysis of gray matter abnormalities in idiopathic Parkinson's disease.

Structural neuroimaging studies on idiopathic Parkinson's disease (IPD) with voxel-based morphometry (VBM) yielded variable and conflicting findings. A systematic review of VBM studies of patients with IPD and healthy control (HC) subjects published in PubMed, ISI Web of Science, Embase, and Medline databases from 1995 to 25 October 2010 was conducted. Coordinates were extracted from clusters of significant gray matter (GM) difference between patients with IPD and HC subjects. Meta-analysis was performed using signed differential mapping. A total of 17 VBM studies involving 498 patients with IPD and 375 HC subjects met the inclusion criteria. A significant regional GM volume decrease was detected in the left inferior frontal gyrus (BA47) extending to the left superior temporal gyrus (BA38) and the left insula (BA13) of patients with IPD compared with HC subjects. The findings of this study remain largely unchanged in quartile and jackknife sensitivity analyses and in subgroup analyses. Robust GM reductions in the inferior frontal/orbitofrontal gyrus (BA47) are implicated in IPD, and the reductions may be related to the mediation of the non-motor IPD symptoms, such as cognitive, emotional, and autonomic functions. Further studies must be conducted to determine whether the findings are specific to all IPD subtypes or different from the atypical Parkinsonism.

Clinical feature and DYT1 mutation screening in primary dystonia patients from South-West China.

BACKGROUND: Clinical presentation and DYT1 status amongst Chinese patients with primary dystonia have not been well studied. METHODS: One hundred and twenty patients with primary dystonia from South-West China were studied in a prospective survey for 3.5 years. Severity and the resulting disability were assessed using the Burke-Fahn-Marsden dystonia rating scale (BFMDRS). Health related quality of life (HRQL) was measured through the 36-item short-form (SF-36). The Hospital Anxiety and Depression Scale (HADS) was utilized to identify and quantify depression and anxiety. Mutations in the DYT1 exon 5 were screened by direct sequencing. RESULTS: Cervical dystonia was found to be the most frequent form of focal dystonia and was discovered to occur at an early age. Pain and tremor were the common associated symptoms. Family history was positive in 19.5% of the cases, with a trend of earlier onset. Depression (14.5%) and anxiety (6.6%) were the main HRQL impairments. Multiple linear regression analysis suggested that gender, depression, anxiety and functional disability were amongst the principal determinants of lower HRQL. Only one instance of DYT1 GAG deletion (1.4%) was detected in 71 patients. CONCLUSION: Our data on a cohort of Chinese patients show some difference from descriptions in other ethnic groups. This includes an earlier age of onset, a lower incidence of depression and female serving as a predictor factor of a HRQL. Similar to other cohorts, DYT1 gene mutations are rare.

Antitumor and immunostimulating effects of Anoectochilus formosanus Hayata.

The water extract of Anoectochilus formosanus Hayata showed a potent tumor inhibitory activity in BALB/c mice after subcutaneous transplantation of CT-26 murine colon cancer cells. The tumor-inhibition ratios of mice pre-administered with A. formosanus for 2 days before tumor transplantation, and treated further for 12 consecutive days, were 55.4% and 58.9% at the oral dose of 50 and 10 mg/mouse per day, respectively. Even for the tumor-bearing mice, after oral administration of the water extract of A. formosanus for 12 consecutive days, the tumor inhibition ratios were still 23.8% and 40.5% at doses of 50 and 10 mg/mouse, respectively. Because the low-concentration water extract of A. formosanus does not show direct cytotoxicity in CT-26 tumor cells, we observed further that oral administration of the water extract of A. formosanus may activate murine immune responses, such as stimulating the proliferation of lymphoid tissues and activating the phagocytosis of peritoneal macrophages against Staphylococcus aureus. This study suggests that the antitumor activity of A. formosanus may be associated with its potent immunostimulating effect. It is worth further analyzing the immunomodulating component purified from A. formosanus, and evaluating its potential value for the treatment of human cancers.

Recombinant protein composed of Pseudomonas exotoxin A, outer membrane proteins I and F as vaccine against P. aeruginosa infection.

We have constructed a chimeric protein composed of the receptor binding and membrane translocation domains of Pseudomonas exotoxin A (PE) with the outer membrane proteins I and F, together designated as PEIF. The potential of PEIF as a vaccine against Pseudomonas infection was evaluated in BALB/c mice and New Zealand white rabbits. We examined titers of anti-PE and anti-OprF antibodies, and the ability both to neutralize PE cytotoxicity and to increase opsonophagocytic uptake of Pseudomonas aeruginosa strain PAO1, serogroups 2 and 6. The results showed that PEIF can induce antibodies not only to neutralize the PE cytotoxicity but also to promote the uptake of various strains of P. aeruginosa by murine peritoneal macrophages. In a burned mouse model, PEIF afforded significant protection against infection by the homologous P. aeruginosa strain PAO1, heterologous serogroup 2, and the PE hyperproducing strain PA103. These observations thus indicate that PEIF may be used as a novel vaccine against P. aeruginosa infection.

A nontoxic Pseudomonas exotoxin A induces active immunity and passive protective antibody against Pseudomonas exotoxin A intoxication.

Pseudomonas exotoxin A (PE) is one of the most potent cytotoxic agents produced by Pseudomonas aeruginosa. In this study, we examined the possibility of using PE with a deletion of 38 carboxyl-terminal amino acid residues, designated PE(Delta576-613), for active immunization against PE-mediated disease. We first examined the toxic effects of PE and PE(Delta576-613) on 5- and 9-week-old ICR mice. The results show that the subcutaneous administration of PE(Delta576-613) at a dose of 250 microg was still nontoxic to 5- and 9-week-old ICR mice, while native PE was lethal at a dose of 0.5 and 1 microg, respectively. PE(Delta576-613) was then used to immunize ICR mice. The minimum dose of PE(Delta576-613) that could effectively induce anti-PE antibodies in 5- and 9-week-old ICR mice was found to be 250 ng. However, immunization with 250 ng PE(Delta576-613) failed to protect the immunized mice from a lethal dose of PE. The effective immunization dose of PE(Delta576-613) that could protect mice against a 2 microg PE challenge was found to be 15 microg. In addition, sera obtained from PE(Delta576-613)-immunized ICR mice were able to neutralize PE intoxication and effectively protect mice from PE. Thus, PE(Delta576-613) may be used as an alternative route to new PE vaccine development.

Characterization of monoclonal antibody B7, which neutralizes the cytotoxicity of Pseudomonas aeruginosa exotoxin A.

A nontoxic Pseudomonas aeruginosa exotoxin A (PE), which has the carboxyl-terminal 38 amino acid residues of native PE deleted, was used as an antigen to immunize BALB/c mice, which were then challenged with native PE in order to raise monoclonal antibodies (MAbs) that can neutralize PE cytotoxicity. A murine MAb against PE, designated MAb B7, was established. MAb B7 was characterized in terms of its ability to neutralize PE cytotoxicity, epitope mapping, inhibition of PE receptor binding, and influence on cellular processing of PE and ADP-ribosylation activities. We found that MAb B7 could neutralize PE cytotoxicity in cell culture and in BALB/c mice. The epitope recognized by MAb B7 was mapped to the carboxyl-terminal amino acid residues 575 to 595 of PE. Consistent with the results of epitope mapping, MAb B7 did not block PE receptor-binding activity or the cellular processing of PE but strongly inhibited the ADP-ribosylating activity of PE. In addition, MAb B7 retained strong binding to PE even at pH 4.0, indicating that the complex of MAb B7 and PE is stable in the phagolysosome. On the basis of these observations, the neutralization of PE cytotoxicity by MAb B7 could be due to its binding to the carboxyl terminus of PE. As a result, MAb B7 may interfere with the interaction of the carboxyl-end amino acid residues REDLK of PE with cellular factors. However, we could not rule out the possibility that MAb B7 directly blocks the ADP-ribosylation activity of PE in the cytosol.

Characterization of human DNA topoisomerase II as an autoantigen recognized by patients with IDDM.

Autoantibodies against several cytoplasmic autoantigens such as glutamic acid decarboxylase, heat shock protein 65, insulin, and carboxypeptidase H have been identified in the sera of patients with IDDM. To investigate whether type II DNA topoisomerase (TopII) is an autoantigen in IDDM patients, we have constructed a series of overlapping DNA TopII fragments that covered the entire length of this enzyme. These fragments were used as antigens to screen sera of IDDM patients. We have examined 195 Chinese IDDM patients (mean age 14.2 +/- 7.5 years, age at onset 9.2 +/- 6.4 years, duration of diabetes 4.6 +/- 3.4 years) and 51 nondiabetic individuals. The results showed that DNA TopII autoantibodies were detected in 49.2 and 47.2% of IDDM patients using purified TopII fragments and full-length TopII as antigens, respectively. The frequency of anti-TopII positivity was relatively stable irrespective of sex and disease duration. The patients were slightly older at onset and the prevalence of anti-thyroglobulin/anti-microsomal autoantibodies was twice that in the IDDM subgroup positive for anti-TopII than in IDDM patients who were negative for anti-TopII. We also characterized the epitopes of DNA TopII that were recognized by IDDM sera. Those epitopes resided mostly in three distinct domains. One resided in amino acid residues 1-147, another in amino acid residues 286-472, and the third in the COOH-terminal one-third of DNA TopII. Intriguingly, we found that these epitopes shared similarity (up to 36% identity and 63.6% homology) to previously identified epitopes of IDDM autoantigens.

Increased synthesis and degradation of DNA topoisomerase I during the initial phase of human T lymphocyte proliferation.

The regulation of DNA topoisomerase I was studied in human T lymphocytes following phytohemagglutinin (PHA) stimulation. As T lymphocytes began to enter the S phase 24 h after stimulation, there was a rapid increase in DNA topoisomerase I mRNA. The level of DNA topoisomerase I mRNA increased continuously over the next 18 h and peaked (> 50-fold increase) 42 h after stimulation with PHA. A concomitant increase in DNA topoisomerase I protein was also observed. However, the maximal increase in DNA topoisomerase I protein was only 6-fold. To explain the quantitative difference between the mRNA and protein levels, we investigated the change in the rates of DNA topoisomerase I protein synthesis versus degradation in human T lymphocytes following PHA stimulation. The increase in the mRNA parallels the increase in protein synthesis. However, the half-life of the enzyme protein was reduced to 9 h in proliferating T lymphocytes compared to a half-life of 36 h in resting lymphocytes. These results indicate that, in addition to the growth-regulated increase in the expression of DNA topoisomerase I, there was also a concomitant increase in the degradation of DNA topoisomerase I protein.

Protective murine monoclonal antibodies to tetanus toxin.

Monoclonal antibodies against tetanus toxin were generated by fusion of mouse NS-1 myeloma cells with spleen cells from BALB/C mice immunized with tetanus toxoid. Twenty seven hybridomas against tetanus toxin were obtained. Six hybridoma clones, designated as 1A6B12, 1H7D9, 3A8G9, 3A9F2, 3F9H9, 4A6D11 were selected for further studies. All of them were IgG1, k chain and bound specifically to tetanus toxin and toxoid. All six clones were injected intraperitoneally into pristane-primed BALB/C mice. Antibodies with titer up to 10(6) were obtained in the ascites. Results obtained from in vivo neutralization test showed that 1A6B12, 3A8G9, 3F9H9, 4A6D11 mAbs did have neutralizing activities against tetanus toxin. Monoclonal antibody 4A6D11 had the strongest neutralizing activity. 4A6D11 were purified from ascites by DEAE-52 ion exchange chromatography. Comparing to U.S.A. standard antitetanus toxin antiserum, 50 micrograms purified 4A6D11 mAb had 1 international unit neutralizing activity. The purified 4A6D11 mAb was also coupled to cyanogen bromide-activated sepharose to make an affinity column. Pure tetanus toxin can be obtained by passing crude tetanus toxin through this column and eluting the adsorbed toxin with 4M urea. Large scale purified tetanus toxin could be obtained by this method.