Preparation, characterization and immunogenicity of various polymers and conjugates of elapid postsynaptic neurotoxins.

Seven polymeric forms and conjugates of purified principal postsynaptic neurotoxins of Naja naja siamensis (NNS), Ophiophagus hannah (OH) and Bangarus fasciatus (BF) have been synthesized by controlled polymerization in which only 29-60% of the toxins were allowed to react. A carbodiimide (ECDI) and glutaraldehyde (GA) were used as coupling agents while BSA and tetanus toxoid (TT) were used as carriers. The antigenic mosaic of these immunogens was: NNS-ECDI, NNS-BF-OH-ECDI, NNS-BSA-ECDI, NNS-TT-ECDI, NNS-BF-OH-TT-ECDI, NNS-GA and NNS-BF-OH-GA. By using SDS-PAGE and radioactive toxin, each immunogen preparation was characterized in terms of molecular size and abundance of protein components, percent toxin reacted and toxin density. The relative immunogenicities of the immunogens along with those of NNS venom and pure NNS neurotoxin were evaluated in groups of eight rats. The levels of specific antibody against each of the neurotoxins were determined by ELISAs. Multiple comparisons between antibody responses to these immunogens were made. All the chemically modified immunogens were at least as immunogenic as NNS venom. NNS-TT-ECDI gave the highest antibody response (2.7-6.2-fold higher than that induced by NNS venom). All three multispecific immunogens induced comparable specific antibodies to BF, OH and NNS neurotoxins. The results showed that the presence of TT carrier and the relative degree of toxin density affected the immunogenicities. Some of the immunogens reported here should be useful for the production of potent, polyvalent antivenoms against elapid snakes.

Development of an ELISA to assess the potency of horse therapeutic antivenom against Thai cobra venom.

An ELISA for the quantitation of antibodies against Naja naja siamensis venom proteins has been developed for use as a possible replacement for the in vivo neutralization assay of antivenom potency. Comparison was made with three preparations of venom proteins as antigens of ELISA: these were the crude venom, a toxin fraction and the purified principle postsynaptic neurotoxin of the Thai cobra. Eight batches of horse monovalent therapeutic anti-cobra antivenom, one of which served as positive reference, were assayed by the ELISAs and also by the in vivo neutralization assay using mice. When crude venom, the toxin fraction and the pure neurotoxin were used as antigens in the ELISAs, the correlation coefficients between the ELISA antibody titers and in vivo neutralization of the antivenoms were 0.82 (P less than 0.005), 0.94 (P less than 0.001) and 0.95 (P less than 0.001), respectively. Thus, the ELISA which measures only the antibody against the principle toxin of the snake venom should be most suitable for use as an in vitro assay of antivenom potency. The ELISA should also be useful for potency assessment and standardization of antivenoms against other elapid snake venoms whose lethal components are small, poorly immunogenic peptides.

Development of a latex agglutination inhibition reaction test for amphetamines in urine.

A simple and rapid immunoassay of amphetamines based on latex agglutination inhibition reaction has been developed. N-(3-aminopropyl) amphetamine, a novel amphetamine derivative, and its BSA conjugate were synthesized and characterized. The hapten density in the conjugate was determined spectroscopically to be 62.52 mol/mol of BSA. Two other immunogens, amphetamine-BSA and amphetamino succinamide-BSA, were also synthesized and studied. It was found that N-(3-aminopropyl) amphetamine-BSA exhibits favorable features in terms of immunogenicity and immunochemical specificity when compared to the other two amphetamine immunogens. A latex agglutination inhibition reaction test (LAIRT) using DEAE-cellulose purified rabbit IgG against N-(3-aminopropyl) amphetamine-BSA was found to give a sensitivity of 0.6 microgram/ml and 4.0 microgram/ml of amphetamine and metamphetamine, respectively. Various commonly used drugs and narcotics at concentrations 0.8 mg/ml or less, did not interfere with the test. Interference by normal urine was observed but it could be eliminated by the inclusion of 0.78% normal rabbit serum. The sensitized latex was stable at 4 degrees C for at least 6 months. It was also stable to lyophilization and to at least four cycles of freezing and thawing. The total test time was 35 min. Comparison was made between the LAIRT and EMIT-d.a.u. on 56 urine samples collected from truck drivers. While the EMIT showed 47 positives and nine negatives, the LAIRT gave 38 positives and 18 negatives. The two tests showed no statistical significant difference (P < 0.05).

Some parameters of affinity chromatography in the purification of antibody against Naja naja siamensis toxin 3.

Some parameters in the purification of antibodies against Naja naja siamensis toxin 3 by affinity chromatography were studied on toxin Sepharose, toxin-succinylaminoethyl Sepharose, toxin-albumin Sepharose and toxin succinylaminoethyl Biogel adsorbents. Immunologically pure antibody with 10-12-fold increase in potency was obtained by chromatography of horse refined globulin on all these adsorbents. The maximum antibody binding capacities were higher for adsorbents containing linear spacers but represented only 7-16% of the theoretical binding capacity. The operational half-lives of the adsorbents ranged from 19 to 108 days with toxin-albumin Sepharose showing the highest stability. The recovery of neutralizing capacity was about 30-356% for any of the 4 adsorbents. It is concluded that improvements regarding the antibody binding capacity and the recovery of neutralizing capacity should be made before large scale purification of antibody for therapeutic purposes can be attempted.

A volatile, heat-stable anesthetic is not present in the skin of Bufo asper (Gravenhorst).

A study was made concerning the possible existance of a volatile, heat-stable anesthetic popularly believed by the Thai people to be in the skin of Bufo asper. The skin together with the paratoid glands of two toads were dried and pyrolyzed. Mice and rats inhaling a continuous stream of the smoke or injected with a high dose of the volatile material showed signs of extreme irritation with no loss of consciousness. It is concluded that the skin of this toad does not contain a volatile, heat-stable anesthetic.

A comparative study of three vehicles on antibody responses against elapid snake neurotoxin immunogens.

A study was undertaken to compare the effects of three vehicles (incomplete Freund's adjuvant, IFA; bentonite and squalene/Arlacel A) on the antibody responses to various elapid neurotoxin immunogens. Three neurotoxin immunogens were prepared using purified principal postsynaptic neurotoxin(s) of Naja naja siamensis (NNS), Ophiophagus hannah (OH) and Bungarus fasciatus (BF). Glutaraldehyde (GA) or a carbodiimide (ECDI) was used as a coupling agent while diphtheria toxoid (DT) was used as a protein carrier. The immunogens (NNS-OH-BF-GA, NNS-DT-ECDI and NNS-OH-BF-DT-ECDI) were characterized in terms of percentage of toxin polymerized, mol. wt and abundance and toxin density by SDS-PAGE using 125I-NNS neurotoxin 3. The immunogens, with crude NNS venom as control, were immunized in groups of eight rats with either of the three vehicles given above. Serum titres of specific antibodies against NNS, OH and BF neurotoxins were determined by ELISAs. Multiple comparisons of the results showed IFA to enhance significantly higher antibody titres than the other two vehicles for all immunogens. All the chemically modified immunogens stimulated higher antibody titres against NNS neurotoxin 3 than the crude NNS venom in most vehicles. The two multispecific immunogens also stimulated high antibody titres against OH and BF neurotoxins. These results should lead to the preparation of potent, polyvalent antivenoms against these elapid snakes.

Production of potent polyvalent antivenom against three elapid venoms using a low dose, low volume, multi-site immunization protocol.

The purpose of this study was to prepare a potent polyvalent antivenom against three elapids namely, the Thai cobra (Naja kaouthia, NK), the King cobra (Ophiophagus hannah, OH) and the banded krait (Bungarus fasciatus, BF). Two groups of horses were immunized. Group 1, comprising five horses, was immunized twice with a mixture of postsynaptic neurotoxins followed by an additional six immunizations with a mixture of crude venoms of the three elapids. Group 2, comprising four horses, was immunized with a mixture of crude venoms throughout the course. For the first immunization, the immunogens were emulsified in Complete Freund's adjuvant and injected using a low dose, low volume multi-site immunization protocol previously developed in this laboratory (Pratanaphon, R., Akesowan, S., Khow, O., Sriprapat, S. and Ratanabanangkoon, K. (1997) Production of highly potent horse antivenom against the Thai cobra (Naja kaouthia). Vaccine 15, 1523-1528). The second immunization was carried out with the immunogens in Incomplete Freund's adjuvant. Blood was drawn to assay the antibody titer by ELISA. Sera at the peak of ELISA titers were pooled and assayed for the median effective dose (ED(50)). The ED(50)'s of antivenom from Group 1 horses against NK, OH and BF venoms were 1.44, 0.22 and 0.23 ml serum/mg venom, respectively, while those from Group 2 horse sera were 0.88, 0.20 and 0.49 ml serum/mg venom, respectively. The potency of sera from Group 2 against BF venom was significantly higher, while the potencies against NK and OH venoms were comparable to those of the corresponding monovalent antivenoms produced under the same protocol. This potent, truly polyvalent antivenom should be useful in saving lives of victims envenomed by these elapids and the immunization protocol should be useful in the production of potent polyvalent antivenoms against other medically important elapids.

Antigenic relationships and relative immunogenicities of venom proteins from six poisonous snakes of Thailand.

Venoms from Naja naja siamensis, Ophiophagus hannah, Bungarus fasciatus, Vipera russelli, Calloselasma rhodostoma and Trimeresurus albolabris have been studied by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting. The immunoblots were stained with rabbit homologous and heterologous antibodies. In general, the higher the mol. wt the protein the higher the immunogenicity although two proteins with mol. wts of 23,000 and 25,000 from O. hannah venom are extraordinarily immunogenic. Cross reacting and species specific venom proteins were readily identified by the immunoblot techniques. Only a small number of venom proteins were cross-reactive among the snake species tested while the remaining appeared to be species specific.

Inhibition of mitochondrial ATPase by 2,4,3',5'-tetrahydroxystilbene.

2,4,3',5'-tetrahydroxystilbene (THS) wa s found to inhibit rat liver mitochondrial adenosine triphosphatase (ATPase) activity induced by various concentrations of 2,4-dinitrophenol (DNP). The I50 was found to be 17 nmoles/mg mitochondrial protein. The maximum inhibitory effects of oligomycin and atractyloside on the DNP-activated mitochondrial ATPase activity can be enhanced by adding THS. The atractyloside-insensitive ATPase activity of Lubrol-treated rat liver mitochondria was also inhibited by low concentration of THS. The tetramethoxyderivative of THS was much less effective than the parent compound in depressing the ATPase activity of both intact and Lubrol-treated mitochondria. These observations suggest that the phenolic groups are essential for the mitochondrial actions of THS, and this compound most probably acts by a mechanism different from oligomycin on the mitochondrial ATPase complex.

Studies on the cobra neurotoxin inhibiting activity in an extract of Curcuma sp. (Zingiberaceae) rhizome.

A study was carried on the mode of action and some properties of a cobra neurotoxin inhibitor found in the extract of Curcuma sp. (Zingiberaceae). When the principal postsynaptic neurotoxin (STX) of the Thai cobra (Naja naja siamensis) was mixed with an aqueous extract of Curcuma sp. rhizome, the STX was inactivated as tested in mice or in vitro using a rat hemidiaphragm preparation. The 'neurotoxin inhibitor' ('NTxI') was found only in the water insoluble fraction of the rhizome extract. Using radioactively labeled neurotoxins, 125I-STX and 3H-STX, it was demonstrated that the neurotoxin did not form a stable complex with the 'NTxI'; the inactivated neurotoxin remained in the supernatant of the reaction mixture. After inactivation by 'NTxI', the STX exhibited an unchanged molecular weight as judged by SDS-polyacrylamide gel electrophoresis and an unchanged isoelectric point in isoelectric focusing. Extraction of the Curcuma sp. rhizome with at least 0.2% Triton X-100 resulted in solubilization of a component capable of forming a soluble and stable complex with 3H-STX. By column chromatography on Sephadex G-200 in the presence of 0.1% Triton X-100, the toxin-binding compound was shown to have a molecular weight of about 150 kDa. This 150 kDa component was obtained by Triton extraction of the water-insoluble fraction, and much less from the water soluble fraction, of Curcuma sp. rhizome. It did not possess any carbohydrate side-chain capable of binding the lectin Concanavalin A. The time course of the 150 kDa-3H-STX complex formation was extremely slow (approx 22 hours).(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the 52 kDa antigen of Salmonella typhi: physicochemical stability, purification by affinity chromatography and immunochemical specificity.

The 52 kDa specific protein antigen of Salmonella typhi, as identified by monoclonal antibodies (Ekpo et al. 1990) has been studied with respect to its physicochemical stability, purification by affinity chromatography and immunochemical specificity. It was found that the 52 kDa protein was degraded into smaller antigenic fragments of MW 30-51 kDa when treated with acetone, ethanol, sodium thiocyanate, 0.3M sodium chloride and Veronal and Tris buffers. The exact chemical nature of the degradation of the protein under these conditions is not known but digestion by conventional proteases and dissociation of the non-covalent subunit type have been ruled out. It is proposed that the degradation may be the result of yet unidentified enzyme(s) which become activated by various physical or chemical treatments. Affinity chromatography using a specific monoclonal antibody has been carried out in an attempt to purify the 52 kDa protein. The binding of S. typhi protein to the column was saturable at 65.6 microgram protein/ml gel. The amount of S. typhi protein adsorbed on the column was 0.51% of the total sonicated cell protein. SDS-PAGE of the immunoadsorbent purified protein revealed bands at Mr 15-58 kDa, indicating that the protein obtained had been severely degraded. However, Western blot of the purified protein stained with a specific monoclonal antibody and with rabbit polyclonal antibody against S. typhi showed striking similarity, indicating that the protein obtained was close to immunochemical purity. The 52 kDa protein purified by affinity adsorbent was used as an antigen for the detection of specific IgM in sera of patients. It was shown that sera of patients infected with S. typhi as well as those infected with other bacteria, contained specific IgM against the 52 kDa protein. Thus, it appears that the 52 kDa protein contains species specific as well as cross-reacting epitopes. The possible development of specific diagnosis of S. typhi based on the present experimental results in discussed.

The absence of antagonism between extracts of Clinacanthus nutans Burm. and Naja naja siamensis venom.

Clinacanthus nutans Burm, a herb reputed in Thailand and Malaysia to be "snakebite antidote" has been tested in vitro and in vivo for antivenin activity. The aqueous extract of C. nutans leaves has been found to have no effect on the inhibition of neuromuscular transmission produced by purified Naja naja siamensis neurotoxin in isolated rat phrenic-nerve diaphragm preparations. The extract of C. nutans, when given orally or intraperitoneally, are ineffective in prolonging the survival time of experimental mice receiving lethal doses of N.n. siamensis crude venom. Oral administrations of the herb extracts pretreated with alpha-amylase or beta-amylase also fail to protect the animal. It is concluded that the extract of C. nutans can not antagonize the action of cobra venom.

Juvenile rheumatic fever and rheumatic heart disease at Ramathibodi Hospital, Thailand.

One hundred consecutive cases of rheumatic fever and rheumatic heart disease who were seen at Department of Pediatrics. Ramathibodi Hospital were reviewed. Particular attention was given to the pattern and the outcome of the cardiac status of the patients. The high incidence of severe carditis and tight mitral stenosis was similar to most reports from other developing countries. There was a poor prognosis for the cardiac status of those who came late, had more than valvular lesions, were in congestive heart failure, or had preexisting heart disease and atrial fibrillation. In spite of this, 6 patients had no evidence of heart disease after being followed up for less than 5 years.

Vitamin B12 and vitamin B12 binding proteins in iron deficiency anaemia.

Vitamin B12 and vitamin B12 binding proteins were determined in 20 patients with iron deficiency anaemia who showed low haemoglobin, haematocrit, serum iron levels and hypochromic microcytic red blood cells. The serum vitamin B12 levels in these patients were significantly lower than that of the normal subjects. Nine of 20 patients had serum vitamin B12 less than 350 pg/ml. There was a significant increase in serum UBBC and TBBC levels in patient group and 9 of 20 patients had higher UBBC values than those of the normal subjects. The absolute values of TCI and TCII increased significantly while TCIII was within the normal limit even though the percentage of UBBC were not different from that of the normal subjects.

Serum folate and folic acid binding proteins in iron deficiency anaemia.

Serum and red cell folate and folic acid binding protein (FABP) concentrations were determined on 20 iron deficiency anaemic children of both sexes aged 6--12 years. All cases had haemoglobin level less than 12 gm% or haematocrit less than 36% with low serum iron and elevated unsaturated iron binding capacity. Serum folate levels in the anaemic group was not significantly different from that of normal subjects while red cell folate level was significantly lower in the anaemic group. However, all cases had red cell folate levels higher than 100 ng/ml. There was a direct relationship between the haemoglobin concentration and serum folate level. Serum FABP level in the anaemic group was found to be significantly higher than that of normal subjects and showed no correlation with haemoglobin, haematocrit, serum or red cell folate levels. The significance of elevated serum FABP was discussed.

Immunoassays of amphetamines: immunogen structure vs antibody specificity.

Various immunoassays have been developed for the detection of amphetamines. These have varying degrees of cross-reactivity to other drug and food components. Information on the immunogen structures used, and the specificities of the antibodies obtained, have allowed formulation of a "structure-specificity" pattern delineated on the basis of immunochemistry and stereochemistry. The 'structure-specificity' relationship should be useful to future developments of these immunoassays. Specifically, immunoassays intended to detect either amphetamine or methamphetamine with minimal cross-reaction, should employ immunogens with amphetamine (or methamphetamine) derivatized via the para position of the phenyl ring. Such assays should show minimal cross-reaction with other secondary (or tertiary) amines but should strongly cross-react with phenyl ring substituted analogs. On the other hand, assays intended for detection of both amphetamine and methamphetamine should employ amphetamine (rather than methamphetamine) derivatized via its amino group as an immunogen. Such assays should show minimal cross-reaction with other tertiary amines and phenyl-substituted amphetamine/methamphetamine.

Immunodiagnosis of snake venom poisoning.

Uncertainty as to the species diagnosis remains a serious problem in the management of snake venom poisoning. This is particularly so in areas inhabited by numerous species, the venoms of which elicit similar pharmacological effects and clinical symptoms and against which para-specific cross-neutralizing antivenom is not available. Attempts have been made to eliminate some of this ambiguity through the development of various immunodiagnostic tests. Tests based on ELISA are sensitive, specific and even quantitative and adaptable to field application. In the development of diagnostic tests for use in developing countries, however, practical consideration must be given to speed, cost, simplicity in terms of equipment and expertise, and stability to the climate and storage conditions. This may dictate further modification or selection of more suitable alternative methodologies. Furthermore, the test may have to allow more flexibility in accommodating local species distributions and to address probable complications of heterophile antibodies in test samples from rural people.