Estandarización en Colombia de una prueba ELISA para la evaluación de los niveles séricos de anticuerpos IgG contra diez serotipos de Streptococcus pneumoniae / An immunoenzymatic test for IgG antibody levels against 10 serotypes of Streptococcus pneumoniae

Introducción. Streptococcus pneumoniae es causante de gran morbimortalidad en niños pequeños y ancianos. Sin embargo, en Colombia no está disponible una prueba que evalúe la respuesta humoral a la vacunación específica contra este microorganismo Objetivo. Estandarizar en Colombia un ensayo inmunoenzimático para evaluar los niveles séricos de anticuerpos IgG contra diez serotipos de S. pneumoniae en respuesta a la vacunación específica y caracterizar esta respuesta en individuos sanos de nuestra población. Materiales y métodos. Se hizo un ELISA en fase sólida utilizando como antígenos los polisacáridos capsulares 1, 3, 4, 5, 6B, 9V, 14, 18, 19F y 23F de S. pneumoniae. Resultados. Los sueros de referencia y control reaccionaron fuertemente contra los polisacáridos evaluados, especialmente contra 14 y 19F. En los cinco niños sanos evaluados, los polisacáridos 5 y 19F presentaron los mayores títulos antes de la vacunación. Antes de la vacunación en los niños, y antes y después de la vacunación en los adultos, los polisacáridos 14 y 19F reaccionaron fuertemente. Para todos los polisacáridos, excepto para el 5, existe una relación inversa entre títulos altos de anticuerpos IgG antes de la vacunación y la razón de incremento de los títulos después de la misma. Conclusión. Esta prueba ELISA cuantifica de forma confiable los niveles de IgG sérica contra diez serotipos de S. pneumoniae y, de acuerdo con los resultados obtenidos en individuos sanos de nuestra población, en este trabajo se validan los parámetros internacionales para considerar adecuada la respuesta a la vacuna 23-valente contra este microorganismo.

Introduction. Streptococcus pneumoniae is a major cause of morbi-mortality in early childhood and elderly. However, a test to measure the antibody responses after specific vaccination is not available in Colombia. Objective. An immunoenzymatic test was standardized for the measurement of serum IgG levels against 10 serotypes of S. pneumoniae in response to the specific vaccination. Material and methods. Capsular polysaccharides 1, 3, 4, 5, 6B, 9V, 14, 18, 19F, 23F of S. pneumoniae were used as antigens in a solid-phase ELISA. These responses were characterized in a randomized selected healthy individuals from a Colombian population. Results. The reference and control sera showed great reactivity against all the polysaccharides evaluated, especially against polysaccharide 14 and 19F. The lowest reactivity in these two sera was observed against polysaccharide 3 and 4. Among the children evaluated, polysaccharide 5/19F showed the highes pre-vaccination reactivity, and polysaccharide 14/19F showed the highest post-vaccination reactivity. Among the adults, polysaccharides 14 and 19F showed the greatest reactivity pre- and post-vaccination. For all the polysaccharides (excepting polysaccharide 5), an inverse association among high polysaccharide-specific pre-vaccination- and the increase of post-vaccination-IgG levels was observed. Conclusion. This ELISA test reliably quantifies the serum levels of specific IgG against 10 serotypes of S. pneumoniae. According to the responses by healthy individuals, the current study validates parameters used internationally as an adequate the response to the 23-valent pneumococcal vaccine.

Development of immunodetection system for botulinum neurotoxin type B using synthetic gene based recombinant protein.

Background & objectives: Botulinum neurotoxins (A-G) are among most poisonous substances in the world, produced by obligate anaerobic bacteria Clostridum botulinum. Among the seven serotypes A, B, E and F are of human importance. In India, the prevalence of C. botulinum as well as botulism outbreaks have been reported. Due to its extreme toxicity it has been classified in the Category A of biological warfare agent. So far, there is no commercial detection system available in India to detect botulism. The present study aims to develop an immuno detection system for botulinum neurotoxin serotype B using synthetic gene approach. Methods: The truncated fragment of the botulinum neurotoxin type B from amino acid 1-450 was synthesized using PCR overlap primers; the constructed gene was cloned in the pQE30UA vector and transformed to Escherichia coli SG 13009. The recombinant protein expression was optimized using various concentration of isopropylthiogalactoside (IPTG) induction, further the expression was confirmed by Western blot analysis using anti-His antibody. Recombinant protein was purified under denatured condition using Ni-NTA affinity chromatography. Antibody was generated against the recombinant protein using alum adjuvant in BALB/c mice and tested for cross reactivity with other serotypes of C. botulinum as well as closely related clostridia. An ELISA test was developed for the detection of botulinum neurotoxin and the minimum detection limit was also estimated. Results: The recombinant protein was expressed at maximum yield at 4.3 h of post-induction with 0.5 mM IPTG concentration. The recombinant protein was purified using Ni-NTA affinity chromatography up to the homogeneity level. The polyclonal antibodies were raised in mice with a titre of 1:2048000. The developed antibody was highly specific with a sensitivity of detecting approximately 15 ng/ml of recombinant protein and not showing any cross-reactivity with other serotypes. Interpretation & conclusions: There is no commercial immunodetection system available in India to detect botulism. The developed detection system is highly specific. It will be useful for growing food industry to detect botulinum neurotoxin in food samples as well as in clinical samples.

Antigenic competition among different 'O' antigens of Salmonella enterica subspecies enterica serovars during hyperimmunization in pony mares.

The present study on antigenic competition among somatic 'O' antigens of different Salmonella groups (A, B, C1, C2, D and E1) in mares revealed that the immune response to most of the antigens was not (A, B, C2) or little (C1, D) affected by antigenic competition. However, E1 group antigen, which induced high antibody titres (Avg. 12967.3) when given alone, produced almost 3.5 log2 lower antibody titres on giving with other antigens, indicating the antigenic competition among some Salmonella group antigens. The antigenic competition varied for different antigens even of the similar chemical nature. Therefore, antigens belonging to different somatic groups should not be given together for the purpose of raising polyvalent serum or for immunization using multivalent Salmonella vaccines prepared from strains of different 'O' groups revealing antigenic competition.

Production of monoclonal antibodies against Streptococcus mutans antigens

Several studies have been conducted in the last decades aiming to obtain an anti-caries vaccine, however some studies have demonstrated cross reactivity between Streptococcus mutans surface antigens and the human cardiac tissue. In this work, the reactivity of five anti-Streptococcus mutans monoclonal antibodies (MoAb) (24A, 56G, C8, E8 and F6) was tested against oral streptococci, cardiac antigens and skeletal and cardiac myosins, aiming to evaluate the specificity of these MoAb. The hybrid producers of immunoglobulins of the IgG2b class were cloned by limit dilution and expanded in vivo. MoAb were tested by ELISA. The hybrid 24A reacted with S. mutans CCT 1910, S. salivarius CCT 0365 and S. pyogenes T23. No reactivity difference was observed among the tested species. Cross reactivity with heart and cardiac myosin was not confirmed and only reaction with myosin of skeletal muscle was observed (p = 0.0381). The hybrid 56G reacted with all the tested microorganisms and there was statistically significant difference between S. mutans and S. pyogenes T23 (p < 0.001). This hybrid also reacted with myosin of skeletal muscle (p = 0.0095). C8, E8 and F6 presented low reactivity against oral streptococci strains and no reactivity against cardiac antigens. The data of this study showed that the 24A and 56G anti-S. mutans MoAb presented reactivity with S. pyogenes and S. salivarius, reinforcing the occurrence of common antigens between these species. The tested MoAb presented low cross-reactivity with myosin of skeletal muscle, but anti-heart activity could not be confirmed.

Diversos estudos foram realizados nas últimas décadas com o intuito de se obter uma vacina anticárie dentária, mas alguns trabalhos têm demonstrado reatividade cruzada entre antígenos de superfície de Streptococcus mutans e tecido cardíaco humano. Neste trabalho, foi testada a reatividade de cinco anticorpos monoclonais (AcMo) anti-Streptococcus mutans (24A, 56G, C8, E8 e F6) contra estreptococos orais, antígenos cardíacos e miosinas esquelética e cardíaca, no intuito de avaliar a especificidade desses AcMo. Os híbridos produtores de imunoglobulinas da classe IgG2b foram clonados por diluição limite e expandidos in vivo. Os AcMo foram testados por ELISA. O híbrido 24A reagiu com S. mutans CCT 1910, S. salivarius CCT 0365 e S. pyogenes T23. Nenhuma diferença de reatividade foi detectada entre as espécies analisadas. Reatividade cruzada com coração e miosina cardíaca não foi confirmada, existindo somente reação com miosina de músculo esquelético (p = 0,0381). O híbrido 56G reagiu com todos os microrganismos testados e houve diferença estatisticamente significante entre S. mutans e S. pyogenes T23 (p < 0,001). Este híbrido também reagiu com miosina de músculo esquelético (p = 0,0095). C8, E8 e F6 apresentaram baixa reatividade contra cepas de estreptococos orais e nenhuma reatividade com antígenos cardíacos. Os dados deste trabalho demonstraram que os AcMo 24A e 56G anti-S. mutans reagiram com S. pyogenes e S. salivarius, confirmando a existência de antígenos comuns entre essas espécies. Esses AcMo avaliados apresentaram baixa reatividade cruzada com miosina de músculo esquelético, porém a atividade anticoração não foi confirmada.

Evaluation of the humoral immune response in BALB/c mice immunized with a naked DNA vaccine anti-methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is the major pathogen involved in nosocomial infections, leading to high rates of morbidity and mortality in hospitals worldwide. The methicillin resistance occurs due to the presence of an additional penicillin-binding protein, PBP2a, which has low affinity for b-lactam antibiotics. In the past few years, vancomycin has been the only antibiotic option for treatment of infections caused by multiresistant MRSA; however, reports of vancomycin-resistant strains have generated great concerns regarding the treatment to overcome these infections. In the present study, we report preliminary results regarding the humoral immune response generated in BALB/c mice by two different doses of naked DNA vaccine containing an internal region, comprising the serine-protease domain, of the PBP2a of MRSA. The immunization procedure consisted of four immunizations given intramuscularly within 15-day intervals. Blood was collect weekly and anti-PBP2a-specific antibodies were screened by ELISA. BALB/c mice immunized with DNA vaccine anti-PBP2a have shown higher antibody titers mainly after the fourth immunization, and intriguingly, no correlation between the humoral immune response and DNA dose was observed. Our results suggest that the DNA vaccine anti-PBP2a induced an immune response by production of specific antibodies anti-MRSA in a non-dose-dependent manner, and it could represent a new and valuable approach to produce specific antibodies for passive immunization to overcome MRSA infections.

Effectivity of crude versus purified mycobacterial secretory proteins as immunogen for optimum antibody production.

Monospecific antibodies have been successfully utilized in antigen detection, which is better indicator of active infection. Mycobacterium tuberculosis excretory secretory (M tb ES) antigens such as ES 31, ES 41 and ES 43 (31 kDa, 41 kDa and 43 kDa protein, respectively) have been shown to be present in Mycobacterium tuberculosis H37Ra culture filtrate and are of diagnostic interest. To study the immunogenic potential of crude versus purified antigen, goat was immunized with M tb detergent soluble sonicate (DSS) antigen as well as purified antigen fraction (ESAS 7) containing ES 31 antigen. Both anti-DSS IgG antibody and anti ESAS 7 IgG antibody were found to be reactive with ES 31 antigen upto 1 ng concentration of antibody by ELISA. Crude DSS antigen was found to be quite effective in producing high titre antibodies and showed further high reactivity with other ES antigens (ES 41 and ES 43) of diagnostic interest.

Production of monoclonal antibody to subtype 9 of Neisseria meningitidis and the distribution of this subtype in Brazil

A new monoclonal antibody (5F81A4P1.9), which is specific for subtype 9 antigen of meningococci, was studied. The antibodies were raised against a previously non-typable (NT) serogroup B strain from Brazilian patients and were found to react with the subtype antigen of prototype reference strains for subtype 9 (M982), as well as with those of homologous strains. The subtype 9 epitope was found in 6.8 percent of serogroup B strains among 602 strains of Neisseria meningitidis case isolates, including representative isolates from Brazilian states. Subtype P1.9 was predominantly related to serogroup B in Brazil among the isolates collected during the N. meningitidis epidemic in 1992. No significant differences were observed in the occurrence of subtype P1.9 among strains isolated from several Brazilian states. Fluorescence-activated cell-sorter analysis showed that 5F81A4 MAb recognized a 46 kDa protein on the surface of a homologous strain of N. meningitidis (B:4:P1.9). These results, in association with a bactericidal activity assay for 5F81A4, and with experimental passive protection in mice, demonstrated the importance of subtype 9 class 1 proteins of N meningitidis in Brazil. Serotyping is essential for the development of vaccination strategies.

Immunogenicity and reactogenicity of two regimens of diphtheria-tetanus-acellular pertussis-hepatitis B-inactivated polio and Haemophilus influenzae type b vaccines administered to infants primed at birth with hepatitis B vaccine.

An open, randomized study evaluated the immune response and safety of two different regimens of diphtheria-tetanus-acellular pertussis-hepatitis B-inactivated poliovirus-Haemophilus influenzae type b (DTPa-HBV-IPV-Hib) immunization in infants primed at birth with hepatitis B vaccine. One-half of the 150 healthy, full-term infants received a DTPa HBV-IPV-Hib vaccine at 1 1/2, 3 and 5 months of age; the other received a DTPa-IPV-Hib vaccine at 1 1/2, 3 and 5 months of age with separate HBV vaccine at 1 and 5 months of age. Immune response was similar following the two regimens with 100% of the vaccinees seroprotected for HBV, diphtheria, tetanus, Hib and poliovirus types 2 and 3 diseases after the full vaccination course. One vaccinee in the DTPa HBV-HPV- Hib group failed to respond to the poliovirus type 1 antigen. Response to the three pertussis antigens ranged from 92-97% in the DTPa-IPV-Hib plus separate HBV group and 100% in the DTPa HBV-IPV-Hib group. The most frequently reported post-vaccination symptoms were irritability in the DTPa-IPV-Hib plus separate HBV group (49% of vaccinees) and fever, defined as axillary temperature > or =37.5 degrees C, in the DTPa HBV- IPV-Hib group (50% of vaccinees).

Group A streptococcal vaccine delivery by immunization with a self-adjuvanting M protein-based lipid core peptide construct.

BACKGROUND & OBJECTIVES: To develop a broad strain coverage GAS vaccine, several strategies have been investigated which included multi-epitope approaches as well as targeting the M protein conserved Cregion. These approaches, however, have relied on the use of adjuvants that are toxic for human application. The development of safe and effective adjuvants for human use is a key issue in the development of effective vaccines. In this study, we investigated the lipid polylysine core peptide (LCP) system as a self-adjuvanting GAS vaccine delivery approach. METHODS: An LCP-GAS construct was synthesised incorporating multiple copies of a protective peptide epitope (J8) from the conserved carboxy terminal C-repeat region of the M protein. B10.BR mice were immunized parenterally with the LCP-J8 construct, with or without conventional adjuvant, prior to the assessment of immunogenicity and the induction of serum opsonic antibodies. RESULTS: Our data demonstrated immunogenicity of LCP-J8 when coadministered in complete Freund's adjuvant (CFA), or administered in the absence of conventional adjuvant. In both cases, immunization led to the induction of high-titre J8 peptide-specific serum IgG antibody responses, and the induction of heterologous opsonic antibodies that did not cross-react with human heart tissue proteins. INTERPRETATION & CONCLUSION: These data indicated the potential of a novel self-adjuvanting LCP vaccine delivery system incorporating a synthetic GAS M protein C-region peptide immunogen in the induction of broadly protective immune responses, and pointed to the potential application of this system in human vaccine development against infectious diseases.

Prospecting for new group A streptococcal vaccine candidates.

BACKGROUND & OBJECTIVES: Most group A streptococcal (GAS) vaccine strategies focused on the surface M protein of the GAS. However, vaccine based on M protein have some drawbacks. In the present study, we used two approaches to identify new proteins and peptides that may have utility as vaccine candidates. METHODS: A whole gel elution procedure was used to separate GAS surface antigens into 9 size fractionated pools. Mice were vaccinated with each pool and antibody titre, opsonic ability and protective capacity measured. In an alternative approach BioInformatics was used to identify putative GAS surface proteins. Peptides from within these proteins were then selected on the basis of predicted antigenicity or location. These peptides were conjugated to keyhole lymphocyanin (KLH) and immunogenicity measured in a mouse model. RESULTS: One pool of GAS surface proteins (approximately 29kDa) induced antibodies that were both opsonic and potentially protective. Immunoflourescent microscopy demonstrated that these antibodies bound to the surface of M1 GAS. Amino acid sequencing subsequently identified superoxide dismutase as the major antigen in this pool. A BioInformatic search of the M1 GAS genome and subsequent analysis identified several peptides that fulfilled criteria as potential vaccine candidates. Each peptide when conjugated to KLH was able to induce a strong antibody response. INTERPRETATION & CONCLUSION: Several new antigens were identified that may have potential as vaccine targets. A future GAS vaccine may have multiple peptide epitopes, providing protection against multiple GAS strains.

CpG DNA, liposome and refined antigen oral cholera vaccine.

An oral cholera vaccine made up of three Vibrio cholerae antigens, i.e. lipopolysaccharide (LPS), recombinant toxin co-regulated pili (rTcpA) and heat-treated cholera toxin (H-CT) has been developed in six different formulations. Eight-week-old Wistar rats were divided into nine groups and immunized as follows: the first group received the oral vaccine 1 consisting of the three antigens (LPS, rTcpA and H-CT) associated with a liposome (L) and bacterial CpG-DNA (ODN#1826). The rats of groups 2 and 3 received oral vaccines 2 and 3 consisting of the liposome-associated three antigens with and without non-bacterial CpG-DNA (ODN#1982), respectively. Rats of groups 4 received oral vaccine 4 consisting of the three antigens mixed with the ODN#1826, similar to vaccine 1, but without liposome. Rats of groups 5 and 6 received oral vaccines 5 and 6 consisting of the three antigens with and without ODN#1982, respectively, similar to vaccines 2 and 3, but without liposome. Rats of groups 7, 8 and 9 received oral placebos, namely liposomes (L), ODN#1826 (CpG), and vaccine diluent, i.e. 5% NaHCO3 solution, respectively. All vaccines were given in three doses at 14-day intervals. It was found that the combination of liposome and ODN#1826 in vaccine 1 evoked the highest immune response to V. cholerae antigen compared to other vaccine formulations and placebos, as measured by the appearance of antigen-specific antibody-producing cells in the intestinal lamina propria. The immunogenicity according to the magnitude of the immune response was: V1>V2=V3>V4>V5=V6>V7=V8=V9. The results of this study indicate that CpG-DNA and liposome are effective mucosal adjuvants for an oral cholera vaccine prepared from refined V. cholerae antigens and their combination seems to be synergistic. The potential role of liposome as a vaccine delivery vehicle has been confirmed.

A single strain of Mycobacterium bovis bacillus Calmette-Guérin (BCG) grown in two different media evokes distinct humoral immune responses in mice

Two attenuated bacillus Calmette-Guérin (BCG) preparations derived from the same Moreau strain, Copenhagen but grown in Sauton medium containing starch and bacto-peptone (onco BCG, O-BCG), or asparagine (intradermal BCG, ID-BCG), exhibited indistinguishable DNA sequences and bacterial morphology. The number of viable bacilli recovered from spleen, liver and lungs was approximately the same in mice inoculated with the vaccines and was similarly reduced (over 90 percent) in mice previously immunized with either BCG vaccine. The humoral immune response evoked by the vaccines was, however, distinct. Spleen cell proliferation accompanying the growth of bacilli in tissue was significantly higher in mice inoculated with O-BCG. These cells proliferated in vitro upon challenge with the corresponding BCG extract. Previous cell treatment with mAb anti-CD4 T cells abolished this effect. Anti-BCG antibodies, as assayed either in serum by ELISA or by determining the number of antibody-producing spleen cells by the spot-ELISA method, were significantly higher in mice inoculated with ID-BCG. Anti-BCG antibodies were detected in all immunoglobulin classes, but they were more prevalent in IgG with the following distribution among its isotypes: IgG1>(IgG2a = IgG2b)>IgG3. When some well-characterized Mycobacterium tuberculosis antigens were used as substitutes for BCG extracts in ELISA, although antibodies against the 65-kDa and 96-kDa proteins were detected significantly, antibodies against the 71-kDa, 38-kDa proteins and lipoarabinomannan were only barely detected or even absent. These results indicate that BCG bacilli cultured in Sauton-asparagine medium permitted the multiplication of bacilli, tending to induce a stronger humoral immune response as compared with bacilli grown in Sauton-starch/bacto-peptone-enriched medium

Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice

The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins

Low cost production and purification of polyclonal antibodies to staphylococcal enterotoxin A

An immunization scheme for production of antiserum to staphylococcal enterotoxin A (SEA) is proposed. The reference method of Robbins and Bergdoll was modified to reduce the number of doses and the amount of toxin used per animal. The best immunization scheme used injections in days 0,8,24,59,62 and 67. The amount of toxin at each injection was 5,6,20,50,100 and 200ug, respectively. The total amount of toxin was 381ug, which corresponded to a reduction of 107ug in the amount of toxin for each animal when compared to the reference method. The average antiserum titer using the Optimum Sensitivity Plate - OSP was 1:60 and using ELISA the titer was 1:100.000. The lack of cross-reactivity with other staphylococcal enterotoxins indicated high specificity of the antibody to SEA. The proposed immunization scheme was adequate to produce specific SEA antisera, with high titers and the aditional advantage of reducing the amount of purified SEA required for immunization.

Protective immunity induced by a Yersinia enterocolitica serovar 0: 8 cellular extract

The purpose of this study was to investigate the protective power of a cellular extract (CE) from Y. enterocolitica 0:8 grown in condition of expression of chromosomal antigens. Mice were immunized by s.c. route and challenged with: 0 LD50 (1 x 10(4) CFU/ml). Immunoblotting showed that CE-specific serum reacted with several CE antigens. Prominent bands, of molecular weights 60 and 35.5, were present in cytoplasmic and membrane fraction, respectively. The lipopolysaccharide (LPS) was detected in CE. These findings suggest that chromosomally-encoded antigens present in CE may induce protection against Y. enterocolitica infection. Both humoral and cellular immune response contribute to protection in mice.

Effect of onion extract on immune response in rabbits.

A total of 40 NZW rabbits were selected for this study to see the effect of onion extract on immune response following antigenic challenge. These animals were randomly divided into four groups, each composed of ten rabbits. Group I and II were challenged with typhoid H (TH) antigen and groups III and IV with sheep red blood cells (SRBC). Groups I and III were considered as control and II and IV as treated groups. The latter two groups were treated with onion extract orally. The immunosuppressive effect of onion extract was evaluated by estimating antibody levels by Widal test and hemolysin titer. It was found that mean antibody titers were significantly lower in the treated groups than in controls. The weights of thymus and lymph nodes were higher and of adrenal glands were lower in the control groups than in the treated groups. It appeared from the current study that onion extract has an inhibitory effect on immune response.

Evaluation of potency of inactivated cholera vaccine by mouse protection assay & antibody induction method.

Twenty one batches of whole cell inactivated cholera vaccine manufactured at Central Research Institute, Kasauli were evaluated for potency by mouse protection assay (MPA) and antibody induction method. In the antibody induction method the sera of immunized mice were screened for the presence of antibodies against Vibrio cholerae by microagglutination (MA) test and IgG ELISA. The number of organisms estimated by MPA were correlated with agglutinating and neutralizing antibodies against individual serotypes by MA and ELISA respectively. Correlation coefficient(r) of 0.692 and 0.815 were observed for the titres evaluated by MA and ELISA when compared with standard MPA method for the serotype Ogawa. Similarly r values of 0.925 and 0.849 were observed for titres evaluated by MA and ELISA when compared with standard MPA method for the serotype Inaba. Antibody induction method can be as an alternative method for determining the potency of inactivated cholera vaccine.

Production of monoclonal antibodies to Vi polysaccharide antigen of Salmonella typhi.

Twenty-four Vi antigen-specific monoclonal antibodies were produced in this study. The MAbs were found to be highly specific to Vi possessing bacteria. Selected MAbs were used in a direct agglutination assay for rapid identification of S. typhi in primary bacterial culture and also used to develop an assay to detect Vi antigen in clinical specimens. The result showed that they could not detect the antigen in urine and serum from acute patients even they could detect as low as 0.02 micrograms/ml of Vi antigen added in normal urine. The study has shown that these MAbs are very useful for rapid identification of S. typhi in primary bacterial culture and they can replace polyclonal anti-Vi antibodies which have been used routinely in bacteriological laboratories.

Effects of transcutaneous needle stimulation on the immune response in rabbits.

Studies in the field of neuroimmunomodulation have shown that endorphins such as methionine-encephalin may have an immunomodulatory role. This study was undertaken to test whether transcutaneous needle stimulation, which is known to release such peptides from the central neurones, has an effect on immune response in rabbits. Twelve healthy rabbits, in three similar subgroups, were exposed to pathogenic Escherichia coli and their immune response was studied under different experimental conditions. Our results show that needle stimulation causes a reversal of stress induced suppression of antibody production. Further studies are necessary to evaluate any therapeutic uses these observations may have in humans.