Horse Antiserum Research Articles

Preparation and Evaluation of a Horse Antiserum against the Venom of Sea Snake Hydrophis curtus from Hainan, China.

Sea snake venom is extremely toxic, and it can induce severe respiratory failure and cause high mortality. The most effective first aid treatment for sea snake bites is to inject antivenom as soon as possible. However, in China, there are only four types of terrestrial snake antivenoms, none of which are effective in the treatment of sea snake bites. In order to develop an antivenom for the dominant species of sea snakes in Chinese seas, Hydrophis curtus venom (HcuV) was chosen as the antigen to immunize horses. From immune plasma, a high-titer Hydrophis curtus antivenom (HcuAV) was prepared. In vitro assessment showed that HcuAV had a cross-neutralizing capacity against HcuV and Hydrophis cyanocinctus venom (HcyV). In vivo assessment indicated that HcuAV injection could significantly improve the survival rates of the HcuV and HcyV envenomated mice (0% to 100% and 87.5%, respectively) when it was injected at a sufficient amount within the shortest possible time. In addition, HcuAV could also effectively alleviate multiple organ injuries caused by HcuV. These results provide experimental support for the future clinical application of HcuAV.

A new protein-coupled antigen of α-conotoxin MI displays high immunogenicity and can produce antiserum with high detoxification activity

α-conotoxin (α-CTX) MI is a small peptide toxin with 14 amino acids and two disulfide bonds. It potently inhibits muscle-type nicotinic acetylcholine receptors (nAChRs), and poses a threat as a toxin to tropical fishermen. However, there are currently no effective drugs for the treatment of MI envenomation due to the toxin's low immunogenicity. In this report, we generated neutralizing antiserum and F(ab')2 to MI by synthesizing a new MI antigen through the coupling of alkynyl-modified MI and azide-modified bovine serum albumin (BSA), followed by immunization into mouse and horse. The new MI-BSA antigen generated high titers of mouse and horse antiserum (1:204,800 and 1:51,200, respectively), and both the antiserum as well as the horse F(ab')2 displayed highly potent neutralization and detoxification efficacy. 12.5 μL of mouse or horse antiserum preincubated with MI could completely neutralize a lethal dose of the MI (0.4 μg, 1.7 × LD50), while 6.25 μL (mouse) or 10.41 μL (horse) of the antiserum could exert complete detoxification of mice injected with 1.7 × LD50 of MI. Moreover, the mouse and horse antiserum exhibited medium cross-reactivity for highly toxic α-CTX GI. These results demonstrate that the integrity of MI's antigen epitope and carrier effect of BSA can improve MI's immunogenicity, and provides an effective detoxification treatment for highly toxic α-conotoxins as well as an effective method for the preparation of antiserum of small peptide toxins.

Antigenic differences between equine influenza virus vaccine strains and Florida sublineage clade 1 strains isolated in Europe in 2019

From late 2018 to 2019, equine influenza virus (EIV) strains of Florida sublineage clade 1 (Fc1), which had until then been circulating mainly in the United States, suddenly spread across Europe causing many outbreaks, and Florida sublineage clade 2 (Fc2) strains, which had been circulating mainly in Europe, have not been detected in Europe since 2018. Since 2010, the World Organisation for Animal Health (OIE) has recommended that EIV vaccines contain an Fc1 strain that is like A/equine/South Africa/4/2003 or A/equine/Ohio/2003. Accordingly, Japanese vaccines contain A/equine/Ibaraki/1/2007 as the Fc1 strain. To evaluate the effectiveness of these vaccines against the Fc1 strains detected in Europe in 2019, we performed virus neutralization tests using horse antisera. Challenge viruses used were Irish strain A/equine/Tipperary/1/2019 and two recombinant viruses generated by reverse genetics. Recombinant viruses possessing hemagglutinin (HA) and neuraminidase (NA) derived from A/equine/Tipperary/1/2019 (rA/equine/Tipperary/1/2019) or British strain A/equine/Essex/1/2019 (rA/equine/Essex/1/2019) were generated. Equine antisera against A/equine/South Africa/2003 and A/equine/Ibaraki/2007 were produced by experimental infection. Antibody titers against A/equine/Tipperary/1/2019, rA/equine/Tipperary/1/2019, and rA/equine/Essex/1/2019 were 2.5- to 6.3-fold lower than those against the homologous vaccine strains A/equine/South Africa/4/2003 or A/equine/Ibaraki/2007. These results suggest that the ongoing evolution of the Fc1 viruses may impact on antigenicity and although antibodies against current vaccine strains neutralize the 2019 strains, ongoing surveillance is essential for optimum choice of candidate vaccine strains.

Venom and Antivenom of the Redback Spider (Latrodectus hasseltii) in Japan. Part I. Venom Extraction, Preparation, and Laboratory Testing.

The redback spider (Latrodectus hasseltii Thorell) reportedly invaded Japan in September 1995. To date, 84 redback spider bite cases have been reported; 7 of these cases employed the antivenom. Antivenom has been imported from Australia in the past, but because of restrictions on exportation it was evident that nearly all of the antivenom present in Japan would expire during 2014. In 2014, a plan was proposed to experimentally manufacture and stockpile a horse antiserum for ourselves, using redback spiders indigenous to Japan. A total of 11,403 female spiders were captured alive: 1,217 from the vicinity of Nishinomiya City, Hyogo prefecture, and 10,186 from Osaka prefecture. Of these, 10,007 females were dissected, and the venom was extracted from the venom glands of individuals and subjected to crude purification to yield 4 lots, of which the majority was α-latrotoxin. Among them, a large amount of single lots with an estimated protein content of 236 mg is subsequently scheduled to be used for immunizing horses. We also determined lethal toxicity of the venom (LD50: 9.17 μg per mouse), and established the assay for the determination of an anti-lethal titer of antivenom in mice.

Development of a recombinant camelid specific diabody against the heminecrolysin fraction of Hemiscorpius lepturus scorpion

Scorpion sting is one of the most important problems in public health practice in many countries. Khuzestan yellow scorpion, scientifically known as Hemiscorpius lepturus, belongs to the family Hemiscorpidae, and is one of the most common and dangerous scorpions in Iran. Hemiscorpius lepturus scorpion has potentially cytotoxic venom. The first treatment for scorpion envenoming is horse antisera. Due to their unique characteristics, camels have been considered as an important source for antiserum and monoclonal antibody (Nanobody) production. In the previous study, we reported a specific nanobody against the hemolytic fraction (heminecrolysin) of Hemiscorpius lepturus venom. In the present study, a recombinant diabody was constructed and produced by fusion of two nanobodies. Results from in vitro and in vivo assays showed that the diabody has a promising ability to neutralize venom activity.

Assessment of antigenic difference of equine influenza virus strains by challenge study in horses.

We previously reported that horse antiserum against the Japanese equine influenza vaccine virus, A/equine/La Plata/1993 (LP93) exhibited reduced cross‐neutralization against some Florida sublineage Clade (Fc) 2 viruses, for example, A/equine/Carlow/2011 (CL11). As a result, Japanese vaccine manufacturers will replace LP93 with A/equine/Yokohama/aq13/2010 (Y10, Fc2). To assess the benefit of updating the vaccine, five horses vaccinated with inactivated Y10 vaccine and five vaccinated with inactivated LP93 were challenged by exposure to a nebulized aerosol of CL11. The durations of pyrexia (≥38.5°C) and other adverse clinical symptoms experienced by the Y10 group were significantly shorter than those of the LP93 group.

A 2015 outbreak of Getah virus infection occurring among Japanese racehorses sequentially to an outbreak in 2014 at the same site.

BackgroundAs we reported previously, Getah virus infection occurred in horses at the Miho training center of the Japan Racing Association in 2014. This was the first outbreak after a 31-year absence in Japan. Here, we report a recurrent outbreak of Getah virus infection in 2015, sequential to the 2014 one at the same site, and we summarize its epizootiological aspects to estimate the risk of further outbreaks in upcoming years.ResultsThe outbreak occurred from mid-August to late October 2015, affecting 30 racehorses with a prevalence of 1.5 % of the whole population (1992 horses). Twenty-seven (90.0 %) of the 30 affected horses were 2-year-olds, and the prevalence in 2-year-olds (27/613 [4.4 %]) was significantly higher than that in horses aged 3 years or older (3/1379 [0.2 %], P < 0.01). Therefore, the horses newly introduced from other areas at this age were susceptible, whereas most horses aged 3 years or older, which had experienced the previous outbreak in 2014, were resistant. Among the 2-year-olds, the prevalence in horses that had been vaccinated once (10/45 [22.2 %]) was significantly higher than that in horses vaccinated twice or more (17/568 [3.0 %], P < 0.01). Horse anti-sera raised against an isolate in 2014 neutralized both the homologous strain and a 2015 isolate at almost the same titers (256 to 512), suggesting that these viruses were antigenically similar. Among horses entering the training center from private surrounding farms in 2015, the seropositivity rate to Getah virus increased gradually (11.8 % in August, 21.7 % in September, and 34.9 % in October). Thus, increased virus exposure due to the regional epizootic probably allowed the virus to spread in the center, similarly to the outbreak in 2014.ConclusionsThe 2015 outbreak was caused by a virus which was antigenically close to the 2014 isolate, affecting mostly 2-year-old susceptible horses under epizootiological circumstances similar to those in 2014. The existence of 2-year-olds introduced from regions free from Getah virus could continue to pose a potential risk of additional outbreaks in upcoming years. Vaccination on private farms and breeding farms would help to minimize the risk of outbreaks.

A Simple and Novel Strategy for the Production of a Pan-specific Antiserum against Elapid Snakes of Asia

Snakebite envenomation is a serious medical problem in many tropical developing countries and was considered by WHO as a neglected tropical disease. Antivenom (AV), the rational and most effective treatment modality, is either unaffordable and/or unavailable in many affected countries. Moreover, each AV is specific to only one (monospecific) or a few (polyspecific) snake venoms. This demands that each country to prepare AV against its local snake venoms, which is often not feasible. Preparation of a ‘pan-specific’ AV against many snakes over a wide geographical area in some countries/regions has not been possible. If a ‘pan-specific’ AV effective against a variety of snakes from many countries could be prepared, it could be produced economically in large volume for use in many countries and save many lives. The aim of this study was to produce a pan-specific antiserum effective against major medically important elapids in Asia. The strategy was to use toxin fractions (TFs) of the venoms in place of crude venoms in order to reduce the number of antigens the horses were exposed to. This enabled inclusion of a greater variety of elapid venoms in the immunogen mix, thus exposing the horse immune system to a diverse repertoire of toxin epitopes, and gave rise to antiserum with wide paraspecificity against elapid venoms. Twelve venom samples from six medically important elapid snakes (4 Naja spp. and 2 Bungarus spp.) were collected from 12 regions/countries in Asia. Nine of these 12 venoms were ultra-filtered to remove high molecular weight, non-toxic and highly immunogenic proteins. The remaining 3 venoms were not ultra-filtered due to limited amounts available. The 9 toxin fractions (TFs) together with the 3 crude venoms were emulsified in complete Freund’s adjuvant and used to immunize 3 horses using a low dose, low volume, multisite immunization protocol. The horse antisera were assayed by ELISA and by in vivo lethality neutralization in mice. The findings were: a) The 9 TFs were shown to contain all of the venom toxins but were devoid of high MW proteins. When these TFs, together with the 3 crude venoms, were used as the immunogen, satisfactory ELISA antibody titers against homologous/heterologous venoms were obtained. b) The horse antiserum immunologically reacted with and neutralized the lethal effects of both the homologous and the 16 heterologous Asian/African elapid venoms tested. Thus, the use of TFs in place of crude venoms and the inclusion of a variety of elapid venoms in the immunogen mix resulted in antiserum with wide paraspecificity against elapid venoms from distant geographic areas. The antivenom prepared from this antiserum would be expected to be pan-specific and effective in treating envenomations by most elapids in many Asian countries. Due to economies of scale, the antivenom could be produced inexpensively and save many lives. This simple strategy and procedure could be readily adapted for the production of pan-specific antisera against elapids of other continents.

Production and establishment of a new candidate horse antiserum (Common OIE International Standard/ European Pharmacopoieia Biological Reference Preparation) to the Florida Clade 2 equine influenza virus A/eq/Richmond/1/07

Production and establishment of a new candidate horse antiserum (Common OIE International Standard/ European Pharmacopoieia Biological Reference Preparation) to the Florida Clade 2 equine influenza virus A/eq/Richmond/1/07

A Rational Design for the Nanoencapsulation of Poisonous Animal Venoms in Liposomes Prepared with Natural Phospholipids

Liposomes have been used since the 1970's to encapsulate drugs envisaging enhancement in efficacy and therapeutic index, avoidance of side effects and increase in the encapsulated agent stability. The major problem when encapsulating snake venoms is the liposomal membrane instability caused by venom phospholipases. Here the results obtained encapsulating Crotalus durissimus terrificus and a pool of Bothropic venoms within liposomes (LC and LB, respectively) used to produce anti-venom sera are presented. The strategy was to modify the immunization protocol to enhance antibody production and to minimize toxic effects by encapsulating inactivated venoms within stabilized liposomes. Chemically modified venoms were solubilized in a buffer containing an inhibitor and a chelating agent. The structures of the venoms were analyzed by UV, CD spectroscopy and ELISA. In spite of the differences in the helical content between natural and modified venoms, they were recognized by horse anti-sera. To maintain long-term stability, mannitol was used as a cryoprotectant. The encapsulation efficiencies were 59 % (LB) and 99 % (LC), as followed by filtration on Sephacryl S1000. Light scattering measurements led us to conclude that both, LB (119 ±47 nm) and LC (147±56 nm) were stable for 22 days at 4 °C, even after lyophilization. Genetically selected mice and mixed breed horses were immunized with these formulations. The animals did not show clinical symptoms of venom toxicity. Both, LB and LC enhanced by at least 30 % the antibody titers 25 days after injection and total IgG titers remained high 91 days after immunization. The liposomal formulation clearly exhibited adjuvant properties.

Seroprevalence of Equine Rhinitis Virus in Louisiana Horses

Equine influenza caused byequine influenza virus (H3N8) is considered themost important respiratory disease of horses due to its rapid transmission. Inactivated vaccines are widely used for limiting the spread of disease and reducing the clinical severity in individual level. However, many outbreaks have been reported among vaccinated horses primarily because of the antigenic differences between the vaccine strains and epidemic strains. It is therefore necessary to periodically review the strain composition of vaccines. Thehemagglutination inhibition (HI) test has been widely used for assessing the cross-reactive antibody responses induced by vaccines to epidemic strains. Though there is general agreement between results of HI tests and vaccine efficacies, it remains unclear whether cross-antibody titer measured by the HI test correlates with crossneutralizing reactivity. Here, we assessed the cross-reactivity of the antibodies elicited by the Japanese local vaccine strains to the current epidemic strains, by virus neutralization (VN) test.Wemade the horse antisera by inhalations of the Japanese vaccine strains [A/equine/La Plata/1993 (American) and A/equine/Ibaraki/1/2007 (Fc1)] [108.3 50% egg infectious dose (EID50)/horse each] to horses. Two weeks after the inhalations, each serum was taken from each horse. All the antisera were treated with trypsin-heatpotassiummetaperiodate to remove non specific inhibitors before VN tests. Then the required final dilution of treated antiserum (1:8) was prepared and absorbed with packed chicken red blood cells. Two-fold serial dilutions of the antiserumwere prepared and added to the equal volume of each virus (approx. 104.0 EID50/200 ml). After incubation for 60 min at 34 C, 200 ml of the mixture was injected into an embryonated hen’s egg (5 eggs per each serum dilution). After three overnights incubation at 34 C, the allantoic fluids were harvested and examined hemagglutination activities. VN titers were expressed as the log (2) of the reciprocals of the dilution of antisera which reduced infectivity to1 EID50/200ml.Whereas thehorse antiserumraised to A/equine/Ibaraki/1/2007 (Fc1) showed significantly lower VN titer (6.2) to A/equine/Yokohama/aq13/2010 (Fc2) than that to thehomologous virus (9.0), thehorse antiserum raised to A/equine/La Plata/93 neutralized A/equine/Yokohama/aq13/2010 well at the similar VN titer (9.3) to the homologous VN titer (9.2). The World Organization for Animal Health (OIE) annually reports the antigenic traits of circulating viruses in the world. It is recommended that vaccines for the international market should contain both Fc1 and Fc2 viruses. Our results however showed that the inoculation of A/equine/La Plata/93, which is one of the Japanese local vaccine strains and not genetically classified into Fc2, can elicit the antibody cross-neutralizing the Fc2 viruses well in horse serum.

Use of monoclonal antibodies against horse immunoglobulin in an enzyme immunoassay of bacterial toxins and toxoids

Immunization of BALB/c mice by horse antiserum against diphtheria made it possible to obtain IgG1 monoclonal antibodies (MoAbs) 2B7E4 specific for light chains of horse immunoglobulin (Ig). Unlike commercial preparations of anti-horse immunoglobulin antibodies, which are specific for the whole Ig molecule or its Fc-fragment, the peroxidase (HRP) conjugate of the MoAb, 2B7E4-HRP did not interact with human, mouse, rabbit, and sheep Igs, or horse albumin. The conjugate obtained was used with MoAbs against bacterial toxins and commercial horse anatoxins, as a universal reagent in sandwich enzyme immunoassay (ELISA) for bacterial toxins and anatoxins. The detection sensitivity of diphtheria toxin/anatoxin equaled 0.0005 Lf/ml; tetanus toxin and anatoxin were detected with sensitivities of 20 LD50/ml and 0.005 UI/ml, respectively. A similar sandwich ELISA for botulinum anatoxins (group measurement) allowed types A, B, and E to be detected at 0.02, 0.002, and 0.001 UI/ml, respectively; selective measurement was only possible in the case of type E anatoxin (0.001 UI/ml).

The Hooke Folio

Few readers of Notes and Records of the Royal Society will be unaware of the purchase by the Society in March of this year of the Hooke folio. This is an important bundle of long-lost seventeenth-century documents in the hand of Robert Hooke relating to the early years of the Society when he was

Structural Basis of Citrate-dependent and Heparan Sulfate-mediated Cell Surface Retention of Cobra Cardiotoxin A3

Anionic citrate is a major component of venom, but the role of venom citrate in toxicity other than its inhibitory effect on the cation-dependent action of venom toxins is poorly understood. By immobilizing Chinese hamster ovary cells in microcapillary tubes and heparin on sensor chips, we demonstrated that heparan sulfate-mediated cell retention of the major cardiotoxin (CTX) from the Taiwan cobra, CTX A3, near membrane surfaces is citrate-dependent. X-ray determination of a CTX A3-heparin hexasaccharide complex structure at 2.4 A resolution revealed a molecular mechanism for toxin retention in which heparin-induced conformational changes of CTX A3 lead to citrate-mediated dimerization. A citrate ion bound to Lys-23 and Lys-31 near the tip of loop II stabilizes hydrophobic contact of the CTX A3 homodimer at the functionally important loop I and II regions. Additionally, the heparin hexasaccharide interacts with five CTX A3 molecules in the crystal structure, providing another mechanism whereby the toxin establishes a complex network of interactions that result in a strong interaction with cell surfaces presenting heparan sulfate. Our results suggest a novel role for venom citrate in biological activity and reveal a structural model that explains cell retention of cobra CTX A3 through heparan sulfate-CTX interactions.

Mapping of the antibody-binding regions on the HN-domain (residues 449-859) of botulinum neurotoxin A with antitoxin antibodies from four host species. Full profile of the continuous antigenic regions of the H-chain of botulinum neurotoxin A.

Previously, we mapped the antibody (Ab) and T-cell recognition regions on the HC domain (residues 855-1296) of the 848-residue heavy (H) chain of botulinum neurotoxin A (BoNT/A). We have mapped here the HN-domain (residues 449-859) regions that bind protective anti-BoNT/A Abs raised in four different species. We synthesized, purified, and characterized 29 19-residue peptides that spanned the entire HN and overlapped consecutively by 5 residues, and also region L218-231 around the L-chain's substrate-binding site. Human, horse, mouse, and chicken anti-BoNT/A Abs did not bind to the L-peptide but recognized similar HN regions within peptides 519-537/533-551/547-565/561-579 (with slight left- or right-shifts), 743-761, 785-803, and 813-831/827-845 overlap. Recognition of other peptides that bound lower Ab levels showed similarities and also some differences. Peptide 463-481, strongly immunodominant with horse antisera, did not bind human, mouse, and chicken Abs. However, peptide 449-467 bound Abs in these three antisera, and the region may have shifted to the right (peptide 463-481) with horse Abs. The overlap 659-677/673-691 reacted strongly with human Abs whereas with mouse and chicken antisera, only peptide 673-691 showed low reactivity. Horse antisera had no detectable Ab binding to region(s) 659-691. The Ab-recognition regions on the H chain occupy surface locations in BoNT/A three-dimensional structure, but the great part of the surface is not immunogenic. Regions recognized by the protective antisera of the four different species are prime candidates for inclusion in synthetic vaccine designs.

Passive immunization of pigs against experimental infection with Streptococcus suis serotype 2

The safety and protective efficacy of a horse antiserum raised against inactivated whole cell preparations of Streptococcus suis serotype 2 was investigated in pigs by experimental challenge. The antiserum was evaluated in two similar experiments each comprising 12 4-week-old pigs treated with 6 ml of antiserum the day before challenge and four pigs used as challenge controls. Pigs were infected by subcutaneous injection with approximately 10 11 colony forming units of S. suis serotype 2. Clinical disease in the pigs that could be attributed to infection with S. suis was reduced from 88 to 35% ( P=0.015). The percentage of pigs with lesions that could be associated with S. suis was reduced from 88 to 22% ( P=0.002) and isolation of S. suis serotype 2 was reduced from five (63%) out of eight pigs in the combined challenge control groups to 3 (13%) out of 23 pigs in the combined treatment groups. These results indicate that passive immunization of pigs may be a way to reduce or control S. suis serotype 2 infections in pigs.

Antigen-Antibody Interaction: Application of the Ouchterlony Method for Determining Evolutionary Relationships

The purpose of this experiment is to illustrate how antigen-antibody interactions can be applied to the study of evolution. Antigens are substances that stimulate an immune response from the body. There are two general categories of antigens: pathogens and allergens (Karp 1996). Pathogens include viruses, bacteria, fungi, protozoan parasites and toxins. Allergens include pollen, spores, and food additives. Antibodies are globular proteins produced by B lymphocytes that bind to an antigen and target the antigen for destruction. Antibodies are located in interstitial fluid and plasma and are mostly effective against pathogens that have not invaded host cells. The Ouchterlony test is a double diffusion method on an agar plate that clearly shows any reaction between antigens and antibody (Ouchterlony & Nilsson 1973). Antigens and antibody are inoculated onto an agar plate into separate wells where they diffuse through the medium. As long as the pore sizes within the agar gel substance are large enough, an assumption is made that diffusion of randomly moving particles can freely occur. The agar must be an inert material that does not react with the testing materials. If the antibody recognizes and binds to an antigen on the plate, a white precipitate will form. The precipitate itself is too large to continue to diffuse-once a precipitate is formed, it is stationary. The antigens for this experiment are albumins from various ungulates: horses, pigs, cows and sheep. Albumins are small globular proteins isolated from blood. Albumin is produced by the liver and participates in homeostasis by influencing the osmotic balance of plasma. Antiserum is a liquid containing antibodies that have been exposed to a specific antigen. For example, antihorse serum includes antibodies that will react against antigens isolated from a horse. Antisera were purchased from Sigma, a chemical supply company (PO Box 14508, St. Louis, MO 63178, USA; 800-325-3010). Students learn in lecture and lab that an outstanding characteristic of antibodies is their property of specificity. The general hypothesis being tested is: a specific antigen will react with a specific antibody that has been formed against it, and form a precipitate. Therefore, horse albumin should react only with anti-horse serum, pig albumin with anti-pig, and so on. The results can be used to support the current classification of the ungulates at the order and suborder level. To accomplish this, students must become informed about the following subjects:

Iron Loading into Ferritin by an Intracellular Ferroxidase

An intracellular, membrane-bound enzyme exhibiting bothp-phenylenediamine oxidase activity and ferrous iron oxidase activity was isolated with the plasma membrane fraction of horse heart and studied for its ability to load iron into ferritin. The ferroxidase activity of the tissue oxidase was stimulated approximately twofold by horse spleen apoferritin, and the iron was loaded into ferritin. The loading of iron into ferritin by the tissue oxidase was inhibited by anti-horse serum ceruloplasmin antibody. The stoichiometry of iron oxidation and oxygen consumption during iron loading into ferritin by the tissue-derived oxidase and serum ceruloplasmin were 3.6 ± 0.2 and 3.9 ± 0.2, respectively. These data provide evidence that an enzyme analogous to ceruloplasmin is present on the plasma membrane of horse heart and that this ferroxidase is capable of catalyzing the loading of iron into ferritin. The implications of these data on the present models for the uptake and storage of iron by cells are discussed.

Neutralization Determinants of Laboratory Strains and Field Isolates of Equine Arteritis Virus: Identification of Four Neutralization Sites in the Amino-Terminal Ectodomain of the GLEnvelope Glycoprotein

The N-terminal hydrophilic ectodomain of the GLenvelope glycoprotein of equine arteritis virus (EAV) contains neutralization determinants of the virus. We developed a panel of 17 neutralizing murine monoclonal antibodies (MAbs) to further characterize the neutralization determinants of EAV. Included were 6 MAbs previously raised against a laboratory strain (EAVUCD) of the original Bucyrus strain of EAV, as well as 11 additional MAbs that were raised against a neutralization-resistant variant [escape mutant (EM)] virus (EM6D10) that was derived from EAVUCD. All MAbs raised against EAVUCD and 4 of the MAbs raised against EM6D10 (2B3, 5F8, 8D4, and 10B4) reacted with the corresponding GLenvelope glycoprotein in a Western immunoblotting assay, whereas the remaining 7 MAbs raised against EM6D10 did not react with any viral protein in the immunoblotting assay but competitively inhibited the binding of MAbs 2B3, 5F8, 8D4, and 10B4, indicating that they also recognize epitopes on the GLprotein. A panel of 18 EM viruses raised to the MAb panel, 19 field isolates of EAV from North America and Europe, the modified-live virus vaccine (ARVAC), and 3 other laboratory strains of EAV were characterized by microneutralization assay with the panel of neutralizing MAbs and polyclonal rabbit and horse antisera. Comparative analysis of the nucleotide sequences of ORF5 and the deduced amino acid sequences of the GLprotein of individual EM viruses and field isolates of EAV identified four distinct neutralization sites. These sites include amino acids 49 (site A), 61 (site B), 67 through 90 (site C), and 99 through 106 (site D). With the notable exception of site A, the sites were all located in the V1variable region (amino acids 61–121) within the second half of the N-terminal hydrophilic ectodomain of the GLprotein. Site D includes several overlapping linear epitopes which appear to interact with amino acids in the other three sites to form conformationally dependent epitopes. Amino acid substitutions within any of these four sites can alter the neutralization phenotype of individual strains of EAV.

Detección de antígenos de Neisseria meningitidis del grupo B con la prueba MB-Dot-ELISA en pacientes con meningitis

Infection with Neisseria meningitidis group B has been difficult to detect, partly because this bacterial group's polysaccharide is a weak immunogen. This article describes work carried out to test a new procedure (MB-Dot-ELISA) employing a high-titered horse antiserum for detection of N. meningitidis group B antigens. The study assayed cerebrospinal fluid samples from 585 subjects, 574 with suspected meningitis cases and 11 with neurologic disorders. The results of the assay indicated a sensitivity of 0.991 and a specificity of 0.826. These results were superior to those obtained with latex agglutination and in substantial agreement with the results of counterimmunoelectrophoresis and bacteriologic methods. Overall, the MB-Dot-ELISA was found to be sensitive, inexpensive, and suitable for public health laboratory investigations.