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.

Preparations of toxic components from Naja kaouthia venom by selective heat denaturation.

A simple procedure to prepare the toxic components from Naja kaouthia venom for use as immunogens has been studied. The aim was to produce serum rich in antitoxins. By heating the venom (1-6 mg/ml) at 100 degrees C for 10 min at pH 5.0, at least 10 proteins with MW greater than 25,000 daltons were precipitated and removed. The toxic components, i.e., postsynaptic toxins, Direct Lytic Factor (DLF), and phospholipase A2 were relatively stable to this treatment; however, their activities were progressively lost as the heating time was prolonged. The LD50S of the heated (100 degrees C, 10 min) and the unheated venom were 0.37 and 0.325 mg/kg, respectively. As compared to the unheated venom, immunization of rabbits with the heated venom resulted in a 3.38-fold increase in precipitable antibodies against N. kaouthia toxin 3 and a 1.85-fold increase in neutralizing capacity. This toxin preparation should be useful as an immunogen or as a starting material for chemical modification prior to immunization in the production of potent therapeutic antiserum.

Quantitative comparison on the refinement of horse antivenom by salt fractionation and ion-exchange chromatography.

A quantitative comparison was made on the fractionation of pepsin-digested horse antivenoms by ammonium sulfate (AS) fractional precipitation and ion-exchange chromatography on Q-Sepharose. In the precipitation process, pepsin digested horse anti-Naja kaouthia serum was precipitated by 30% saturated AS followed by 50% saturated AS. The recovery of antibody activity [as measured by an enzyme-linked immunosorbent assay (ELISA) against the cobra postsynaptic neurotoxin 3] from the 30-50% saturated AS precipitate was 53% with a 1.93-fold purification. For the chromatographic process, the behavior of the horse antitoxin antibody and its F(ab')2 fragments was first studied. The pepsin digested horse serum was then desalted on a Bio-gel P-2 column followed by chromatography on Q-Sepharose using a linear gradient (20 mM Tris-HCl, pH 8.0 containing 0.0 to 0.5 M NaCl) A peak containing primarily the F(ab')2 antibody could be obtained. This peak constituted 73% of the total antivenom activity with 2.08-fold purification. The total recovery of antibody activity by the chromatographic process was 90%. The yield of antibody activity was about 2-fold higher than that reported previously with other fractionation procedures. The implications of these results for the refining of horse therapeutic antivenoms are discussed.

Production of highly potent horse antivenom against the Thai cobra (Naja kaouthia).

Naja kaouthia (NK) causes the highest fatality due to snake venom poisoning in Thailand. The specific antivenom produced is of low potency and in short supply. The aim of this study was to improve the antivenom potency. Bentonite and complete Freund's adjuvants (CFA) and various immunogens were compared. Six groups of three to five horses were immunized as follows: Group 1, NK venom adsorbed on bentonite; Group 2, NK venom in CFA; Group 3, NK venom in CFA in multi-emulsion formulation; Group 4, NK venom in 25% CFA; Group 5, NK neurotoxin 3 (NK3) conjugated with tetanus toxoid (NK3-TT) in CFA; Group 6, NK3 conjugated with diphtheria toxoid (NK3-DT) in CFA. Horses in Group 2-6 produced antivenom of very high neutralizing activity, up to four times higher than that of horses of Group 1. CFA (100 or 25%) or as a multi-emulsion formulation, induced comparable neutralizing antibody production with all three immunogens. All horses showed normal weight gain during the course of immunization. Group 1 horses exhibited minimal local reactions while horses in the other five groups had mild and comparable local reactions at the injection sites. No significant differences in the reactions caused by CFA in different formulations or different immunogens were observed. The production of highly potent antivenom against N. kaouthia from these horses should help solve problems associated with the currently available antivenom.

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.

Reverse passive hemagglutination tests for rapid diagnosis of snake envenomation.

Reverse passive hemagglutination (RPHA) tests for the detection of six major poisonous snake venoms of Thailand were studied. Three different species of red blood cells i.e., sheep (SRBC), human (HRBC) and chicken (CRBC) were sensitized with protein A-affinity purified rabbit antivenom IgG using chromic chloride as a coupling reagent. The properties of these sensitized erythrocytes with regard to sensitivity, specificity, stability to venom enzymes and storage etc., were studied and compared. The sensitivities of the RPHA tests in venom detection were 2 to 635 ng/ml. Cross-reactions were observed with heterologous venoms at concentrations at least 62 times higher than those observed with homologous venoms. After treatment with glutaraldehyde, the coupled red blood cells showed reduced sensitivity but were stable at 4 degrees C from 1 to 12 months depending upon the antibody and the species of erythrocytes. The entire test required 60 to 120 min. The RPHA using fresh SRBC correctly identified various venoms in 48 of 59 (81.3%) serum samples and 16 of 26 (61.5%) wound swabs. Venom mis-identifications were made in 2 sera (3.4%). In a comparison of 24 paired serum and wound swab samples, more positive identifications were made with serum than with swab samples but the difference was not statistically significant (p > 0.05).

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)

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).

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.

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.

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.

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.

Diagnosis of snake venoms by a reverse latex agglutination test.

A reverse latex agglutination test using protein A column purified rabbit antivenom IgG-sensitized latex particles was developed for the detection of the six medically important snake venoms of Thailand. The detection limit of the reverse latex agglutination test was 0.16 to 1.2 micrograms/mL of crude venoms. Cross-reactions with heterologous venoms were observed at concentrations 460 to 16000 times that of homologous venoms. Detection of various snake venoms in clinical specimens was carried out by the reverse latex agglutination test. The sensitivity was 52.5% of the 59 serum samples. There was one (1.69%) false positive sample. The positive detection of venom in wound swabs (26 cases) was 38.5% and was not statistically different from that observed in paired serum samples. Interference from human plasma, serum and urine on the reverse latex agglutination test could be eliminated by adsorption with normal rabbit IgG-coated latex suspension or by heat inactivation at 56 degrees C for 30 min. Prozone effect observed in some sera was eliminated by heat inactivation at 56 degrees C for 30 min. The sensitized latex particles were stable at 4 degrees C and -20 degrees C for at least 3 months. Cycles of freezing-thawing and lyophilization did not change their reactivities. The total test time was about 40 min.

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.

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.

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.

Phosphorylation of human choriogonadotropin. Stoichiometry and sites of phosphate incorporation.

Human chorionic gonadotropin (hCG) and its subunits were studied as substrates for cAMP-dependent protein kinase. Phosphorylated residues were identified in peptide fragments by sequence analysis following appropriate hydrolysis and purification. Only the free beta-subunit was phosphorylated. Intact beta subunit incorporated 1 mol of phosphate/mol of peptide. This was localized to Thr 97, within the sequence -Arg-Arg-Ser-Thr-, which resembles one type of acceptor site for cAMP-dependent phosphorylation. Since beta Thr 97 did not phosphorylate in native hCG, this residue may be masked by the alpha subunit. Reduced and carboxymethylated hCG beta phosphorylated to 2.8 mol of phosphate/mol of peptide. Besides Thr 97, phosphate was also found at Ser 66 and Ser 96. Ser 66 occurs within a segment recognized to be favored for phoshorylation with the general sequence -Arg-X-X-Arg-X-X-Ser-. The appearance of this site in the linearized peptide suggests that Ser 66 is "buried" in the native conformation of the beta subunit. Two synthetic peptides representing residues 93-102 of hCG beta were also examined. The disulfide form of the peptide phosphorylated at Thr 97 while the linear peptide phosphorylated at Ser 96. Conformation as well as primary structure can thus influence the site of phosphate incorporation.

Properties of the phosphorylated beta subunit of human choriogonadotropin.

The beta subunit of human choriogonadotropin (hCG) was previously shown to be phosphorylated by beef skeletal muscle cAMP-dependent protein kinase (Keutmann, H. T., Ratanabanangkoon, K., Pierce, M. W., Kitzmann, K., and Ryan, R. J. (1983) J. Biol. Chem. 258, 14522-14527). The phosphorylation site was primarily at Thr 97, located within the "determinant loop" region proposed by Ward and Moore (Ward, D. N., and Moore, W. T. (1979) Animal Models for Research in Fertility and Contraception (Alexander, N. J., ed) pp. 151-164, Harper and Row, Baltimore, MD) to be important for hormonal activity and specificity. Biological and immunological studies were carried out to determine the effect of this modification on the activity of hCG. The phosphorylated hCG beta recombined with hCG alpha to form phosphorylated hCG (*hCG). The recombined *hCG retained full immunological activity in a radioimmunoassay using anti-hCG serum and 125I-hCG. The biological activities of the *hCG on appropriate rat gonadal cells, as studied by hCG radioreceptor assay, follitropin radioreceptor assay, and adenylate cyclase assay, were 0.29 +/- 0.04, 0.29 +/- 0.07, and 0.69 +/- 0.13 times as potent as hCG, respectively. The reduced receptor binding activity of *hCG was not due to dissociation of the hormone into its subunits. The far ultraviolet CD spectrum of the *hCG showed no gross conformational change induced by phosphorylation. We conclude the following: 1) Thr 97 is not involved in subunit-subunit interaction and is not part of the hCG antigenic site recognized by this particular antiserum; 2) Thr 97 does appear to participate in the hCG-receptor interactions; 3) modification of Thr 97 does not result in an enhancement of follitropin-like activity.