Quantitative measurement of biotin-binding immunoglobulin G in human sera using F(ab')2 anti-human IgG-coated multi-well plates

Biotin-binding human immunoglobulin G (B-IgG) was quantitatively measured using an F(ab')2anti-human IgG-coated multi-well microplate for the first time. B-IgG was caught by F(ab')2anti-human IgG and was detected by the following detecting reagents: Peroxidase-labeled streptavidin, avidin and peroxidase-biotin, or avidin-biotinylated peroxidase complex with method A, B, or C, respectively. Commercially available B-IgG was detected by all these three methods. However, method A and B could not detect B-IgG in the human sera used in this study, but we quantitatively measured the B-IgG level using method C. The result is probably due to the fact that the sensitivity of method C was higher than that of methods A or B. Properties of B-IgG detected by method C are discussed in the text.     

Biotin-protein ratios and stability of biotinylated immunoglobulins as standards for the quantitation of biotin-binding immunoglobulins

Biotin-binding IgG in human sera was quantitated using F(ab')(2)anti-human IgG-coated multiwell microplates (Muratsugu, M. et al. 2003, Biol. Pharm. Bull., 26, 1605-1608). The biotin-protein ratio of biotinylated IgG, which was used as standard in the assay, was very important to quantitate the level of biotin-binding IgG. We investigated a synthesis method of biotinylated human immunoglobulins, how to determine the biotin-protein ratio of the biotinylated proteins, and their stability to prepare standards for measuring biotin-binding IgG, IgA, and IgM.     

Evaluation of the safety, immunogenicity and pharmacokinetics of equine anti-SARS-CoV F(ab')(2) in macaque

To warrant potential clinical testing, the equine anti-SARS-CoV F(ab')(2) requires evaluation in as many animal models as possible and a safety test in a primate model. In this study, we evaluated the pharmacokinetics, tolerance and immunity of this kind of antibody in macaques and rats. Results showed that the F(ab')(2) fragments had a normal metabolism in injected animals. The general physiological indexes did not differ between animals injected with anti-SARS-CoV F(ab')(2) or saline. However, a mild inflammatory response in local injection site and a moderate immune response against this antibody in the successively injected animals were observed, which however recovered 3 weeks after the last injection. The antibody titring from 1:100 to 400 against the equine anti-SARS-CoV F(ab')(2) in the inoculated hosts could be detected at week 2 during the successive injections of the equine F(ab')(2). The considerable safety of this antibody used in primates and the fact that the immune system of the host can be motivated by post-injection of the F(ab')(2) indicate that this type of anti-SARS-CoV antibody can be used for prevention and treatment of SASR, especially at the early stage of this virus infection. In addition, it can also provide the precious time for the combined use of other anti-SARS-CoV agents such as antiviral drug and vaccine.     

Purification of F(ab')2 anti-snake venom by caprylic acid: a fast method for obtaining IgG fragments with high neutralization activity, purity and yield

Pooled horse plasma containing antibodies against Crotalus durissus terrificus whole venom were digested with pepsin at an enzyme-substrate ratio of 8:1, pH 3.1, for 40 min and the F(ab')2M fragments purified by adding 8.7% caprylic acid (pH 5.0). For comparison, F(ab')2B purified by precipitation with ammonium sulphate and uncleaved IgG purified with caprylic acid were also prepared. Fab' fragments were obtained by reduction and alkylation of F(ab')2B. The anti-whole C.d. terrificus venom titers, determined by Dot-Blot were 12,800 (IgG), 6400 [F(ab')2B], 4800 [F(ab')2M] and 3200 (Fab'B). Immunochemical analysis of these fragments by SDS gel electrophoresis, Western blot and by double immunodiffusion revealed that the solution containing F(ab')2M was free of IgG and of other plasma proteins, whereas that containing F(ab')2B was not. One milligram of either F(ab')2B, F(ab')2M or Fab'B was able to neutralize respectively 20.7 micrograms, 20.2 micrograms and 13.8 micrograms of C.d. terrificus venom.     

Mortality and antibody responses of mice to three successive episodes of experimental scorpion (Centruroides limpidus limpidus) envenomation and immunological rescue.

Mortality rates of mice and their levels of anti-venom and anti-F(ab')2 antibodies were assessed after three episodes of subcutaneous envenomations with or without treatment with horse F(ab')2. Soluble venom from the Mexican scorpion Centruroides limpidus limpidus was used for these experiments. Repetition of episodes did not induce different mortality rates in untreated mice. F(ab')2 rescued about 85% of the mice in the first two episodes and 66% in the third, without distinction of gender or ostensible side-effects: a suggestion of selection of the most resistant mice. Surviving mice produced in vitro neutralizing antibodies to the scorpion venom and also antibodies to F(ab')2, when injected alone but more so if combined: a possible immunological adjuvant or alarm effect of the venom or of the cascading physiopathology of envenomation. In the few surviving mice, both anti-venom and anti-F(ab')2 antibodies increased significantly after the first envenomation but not thereafter, showing no correlation with mortality rates: a suggestion of their clinical irrelevance, the few hard-to kill mice appeared to resist envenomation by mechanisms other than antibody response. Injection of F(ab')2 alone induced production of detectable anti-venom antibodies in a few mice and injection of venom alone induced that of anti-F(ab')2 antibodies, perhaps due to trace amounts of venom in the high affinity fraction of F(ab')2 and to anti-idiotypic antibodies or polyclonal activity in the envenomation episode, respectively.     

Effects of immunoglobulin upon murine myocarditis caused by influenza A virus: superiority of intact type to F(ab')2 type

Influenza viruses play the largest role in the worldwide epidemiology of infectious diseases. Management of some inflammatory disease (eg, Kawasaki disease) with immunoglobulin has been demonstrated to be effective. We examined the effects of intact type and F(ab')2 type of immunoglobulin preparations upon murine influenza A virus myocarditis in mice. In vitro study showed that intact type and F(ab')2 type of immunoglobulin preparations exhibit antiviral activities against many substrains of influenza virus and other cardiotropic viruses. Dose-dependent suppression of an influenza A virus (NWS) was demonstrated by management with both intact immunoglobulin and F(ab')2 fragments of immunoglobulin. The dose inhibiting 50% of plaques was the same between intact type and F(ab')2 type (both 0.0002 mg/dl). Intact immunoglobulin, but not F(ab')2 fragments of immunoglobulin, suppressed serum macrophage inflammatory protein-2 (MIP-2) productions in influenza A virus-infected macrophages in vitro, which is a murine counterpart of interleukin-8. This suppression of MIP-2 production by intact immunoglobulin treatment was blocked by a specific Fc receptor (Fc gamma III/II receptor) antibody pretreatment. Intact immunoglobulin or F(ab')2 fragments of immunoglobulin were administered to virus-inoculated A/J mice intraperitoneally daily, starting simultaneously with virus inoculation (Experiment I) and 2 days after the virus inoculation (Experiment II), until 10 days after virus inoculation. In Experiment I, survival was higher in treated than in control mice; intact type and F(ab')2 type immunoglobulins administration completely suppressed the development of myocarditis. In Experiment II, survival rate was significantly higher and myocarditis was less severe in intact immunoglobulin-treated mice, but not in F(ab')2 fragments-treated mice compared with untreated mice. Serum neutralizing antibody titers in treated mice were significantly higher compared with untreated mice in Experiments I and II. In addition, serum MIP-2 concentrations in intact immunoglobulin-treated mice, but not in F(ab')2 fragments-treated mice, were lower compared with untreated mice in Experiment II. Immunoglobulin therapy suppresses influenza A virus myocarditis by increasing neutralizing antibody titers and the suppression of myocardial virus activities. From the standpoint of suppression of MIP-2 concentrations, intact type is superior to F(ab')2 type. Thus, immunoglobulin treatment may be promising for prevention of influenza virus myocarditis.     

Initial volume of a drug before it reaches the volume of distribution: pharmacokinetics of F(ab')2 antivenoms and other drugs.

Fast disappearance of F(ab')2 antivenoms from the plasma compartment [Sevcik et al., 2004. Modelling Tityus scorpion venom and antivenom pharmacokinetics. Evidence of active immunoglobulin G's F(ab')2 extrusion mechanism from blood to tissues. Toxicon 44, 731-734; Vazquez et al., 2005. Pharmacokinetics of a F(ab')2 scorpion antivenom in healthy human volunteers. Toxicon 46, 797-805] suggests a quick time course to reach its final distribution volume. An equation was developed to describe how the volume occupied by a drug in the body grows with time. As discussed in the paper this equation is free of some shortcomings of an equation developed for the same purpose by Niazi [1976. Volume of distribution as a function of time. J. Pharm. Sci. 65, 452-454]. Fluorescence microscopy showed that the rapid initial decay in plasmatic F(ab')2 concentration may be related to uptake of F(ab')2 by vascular endothelium which, in combination with accumulation in the vascular wall connective tissue, may produce an intermediate plateau in F(ab')2 V(sl)(t), which reached its final value after 10 h. The V(sl)(t) equation predicts that the plasma concentration half-time of decay has little use to estimate how a drug distributes through the body to exert its action, and predicts that, in some instances, intermediate plateaus in the time course of V(sl)(t) exist. Data from the literature showed that the kinetic considerations for V(sl)(t) also apply to clevidipine, digoxin, digitoxin, lidocaine and thiopentone.     

Toxicokinetic and toxicodynamic analyses of Androctonus australis hector venom in rats: optimization of antivenom therapy

This paper reports the simultaneous determination of toxicokinetic and toxicodynamic properties of Androctonus australis hector venom, in the absence and presence of antivenom (F(ab')(2) and Fab), in envenomed rats. After subcutaneous injection of the venom, toxins showed a complete absorption phase from the site of injection associated with a distribution into a large extravascular compartment. The injection of Fab and F(ab')(2) induced the neutralization of venom antigens in the blood compartment, as well as the redistribution of venom components from the extravascular compartment to the blood compartment. Interestingly, F(ab')(2) and Fab showed distinct efficiencies depending on their route of injection. F(ab')(2) induced a faster venom neutralization and redistribution than Fab when injected intravenously. Fab was more effective than F(ab')(2) by the intramuscular route. The hemodynamic effects of Aah venom were further investigated. Changes in mean arterial pressure and heart rate were observed in parallel with an upper airway obstruction. Fab was more effective than F(ab')(2) for preventing early symptoms of envenomation, whatever their route of administration. Intraperitoneal injection of F(ab')(2) and Fab was similar for the prevention of the delayed symptoms, even after a late administration. Fab was more effective than F(ab')(2) in the inhibition of airway resistance, independent of the route and time of administration. These results show that the treatment for scorpion stings might be improved by the intravascular injection of a mixture of Fab and F(ab')(2). If antivenom cannot be administered intravenously, Fab might be an alternative as they are more effective than F(ab')(2) when injected intramuscularly.     

Inhibition of infection caused by severe acute respiratory syndrome-associated coronavirus by equine neutralizing antibody in aged mice

The high susceptibility of elderly to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) indicates how crucial it is to protect the elderly by various strategies. Aged BALB/c mice displayed a high susceptibility to SARS-CoV and have been a valuable platform for evaluation of strategies against SARS-CoV infection. In this study, we confirmed the validity of this model using various methods, and verified that equine anti-SARS-CoV F(ab')(2) can prevent aged animals from SARS-CoV infection. In a therapeutic setting, treatment with anti-SARS-CoV F(ab')(2) decreased viral load more than several thousand folds in the lungs. Thus, this antibody should be a potential candidate for treatment of elderly patients suffering from SARS.     

Botulinum type A toxin neutralisation by specific IgG and its fragments: a comparison of mouse systemic toxicity and local flaccid paralysis assays.

In this study, we have compared two in vivo assay methods to measure the type A botulinum toxin neutralising activity of specific immunoglobulin G (IgG) and its fragments (F(ab')(2), Fab', Fab) purified from pentavalent botulinum antisera raised in goats. Each assay method was repeated on three separate occasions in mice and relative potencies calculated with respect to a type A equine reference antitoxin. The conventional assay, which measures the number of mice surviving typically after 72 or 96 h following the intraperitoneal administration of a mixture of toxin and antitoxin, gave the following order of potency IgG>F(ab')(2)>Fab'>Fab (6.8>4.7>3.5>2.6 IU/mg). Differences in potency are likely to be due to differences in the pharmacokinetics of the antitoxins, which are related to their molecular weight. The alternative local flaccid paralysis assay, where toxin and antitoxin are injected subcutaneously into the left inguinocrural region, gave results with a narrower range of activities: IgG>Fab'>F(ab')(2)>Fab (6.0>5.9>5.5>4.6 IU/mg). Comparison of the two assay methods showed no significant differences for IgG, F(ab')(2) or Fab', although the Fab fragment was significantly more potent in the non-lethal assay probably because of the reduced influence of antitoxin pharmacokinetics in this localised assay. These findings show that a local flaccid paralysis assay provides a less time consuming and more humane alternative to the lethal assay for the potency testing of botulinum IgG and F(ab')(2) antitoxins.     

Preparation and development of equine hyperimmune globulin F（ab^1）2 against severe acute respiratory syndrome coronavirus

AIM: The resurgence of severe acute respiratory syndrome (SARS) is still a threat because the causative agent remaining in animal reservoirs is not fully understood, and sporadic cases continue to be reported. Developing high titers of anti-SARS hyperimmune globulin to provide an alternative pathway for emergent future prevention and treatment of SARS. METHODS: SARS coronavirus (CoV)F69 (AY313906) and Z2-Y3 (AY394989) were isolated and identified from 2 different Cantonese onset SARS patients. Immunogen was prepared from SARS-CoV F69 strain. Six health horses were immunized 4 times and serum was collected periodically to measure the profile of specific IgG and neutralizing antibodies using indirect enzyme-linked immunosorbent assay and a microneutralization test. Sera were collected in large amounts at the peak, where IgG was precipitated using ammonium sulphate and subsequently digested with pepsin. The product was then purified using anion-exchange chromatography to obtain F(ab')2 fragments. RESULTS: The specific IgG and neutralizing antibody titers peaked at approximately week 7 after the first immunization, with a maximum value of 1:14210. The sera collected at the peak were then purified. Fragment of approximately 15 g F(ab')2 was obtained from 1litre antiserum and the purity was above 90% with the titer of 1:5120, which could neutralize the other strain (SARS-CoV Z2-Y3) as well. CONCLUSION: This research provides a viable strategy for the prevention and treatment of SARS coronavirus infection with equine hyperimmune globulin, with the purpose of combating any resurgence of SARS.     

Preparation,characterization and application of anti-human OX40 ligand (OX40L) monoclonal antibodies and establishment of a sandwich ELISA for autoimmune diseases detection

OX40L (CD252, TNFSF4), a type II transmembrane protein which like other tumor necrosis factor ligands, involved in the costimulation and differentiation of T cells, functions as a positive signal in immune response. To investigate the biological function of soluble OX40L (sOX40L), three functional anti-OX40L monoclonal antibodies (mAbs) 3D2, 3F7 and 2H3 were obtained by hybridoma technology. Besides, specificity of the mAbs was further demonstrated by ELISA, Western blot and Immunofluorescence experiments. We also developed a novel enzyme-linked immunosorbent assay (ELISA) based on two anti-human OX40L antibodies 3D2 and 3F7 with different epitopes. Using the ELISA system, we found that sOX40L in the sera of healthy donors increases in an age-dependent manner and that enhanced sOX40L expression in some autoimmune diseases especially in rheumatoid arthritis (RA) patients, suggesting the potential diagnostic significance of sOX40L in the autoimmune diseases. Together, these data demonstrate that the existence of circulating sOX40L in human sera might play an important role in immunoregulation.     

Comparison between IgG and F(ab')(2) polyvalent antivenoms: neutralization of systemic effects induced by Bothrops asper venom in mice, extravasation to muscle tissue, and potential for induction of adverse reactions.

Whole IgG and F(ab')(2) equine-derived polyvalent (Crotalinae) antivenoms, prepared from the same batch of hyperimmune plasma, were compared in terms of neutralization of the lethal and defibrinating activities induced by Bothrops asper venom, their ability to reach the muscle tissue compartment in envenomated mice, and their potential for the induction of adverse reactions. Both preparations were adjusted to the same potency against the lethal effect of B. asper venom in experiments involving preincubation of venom and antivenom. Then, "rescue" experiments were performed, i.e. antivenom was administered either intravenously or intramuscularly at various times after envenomation. IgG and F(ab')(2) antivenoms were equally effective in the neutralization of lethality, both being more effective when administered i.v. than after i.m. injection. Neutralization decreased as the time lapse between envenomation and treatment increased. No significant differences were observed in the ability of antivenoms to neutralize defibrinating activity of B. asper venom in experiments involving independent injection of venom and antivenoms. There was a much higher accumulation of equine antibodies in muscle tissue that had been injected with B. asper venom than in non-envenomated tissue, indicating that venom-induced microvessel damage probably favors a prominent and similar extravasation of both IgG and F(ab')(2) antibodies. This may explain the similar effectiveness of both types of antivenom in previously reported studies on the neutralization of venom-induced local tissue damage. Both IgG and F(ab')(2) antivenoms activate human complement in vitro and induce an anti-equine immunoglobulin response in mice, indicating that Fc removal per se does not eliminate the potential for inducing adverse reactions. However, IgG antivenom had higher anticomplementary activity and induced a stronger anti-immunoglobulin response than F(ab')(2) antivenom.     

Increased plasma paraquat levels in intoxicated mice following antiparaquat F(ab')2 treatment

Death most often results from human acute poisonings due to paraquat, a widely used herbicide. It causes a quick and insidious accumulation in lungs. It was proposed to study the effects of the administration of antiparaquat F(ab')2 fragments in mice intoxicated with paraquat. Antisera against a paraquat acid derivative coupled to bovine serum albumin were prepared in rabbits, then purified using immunoaffinity chromatography columns and fragmented by pepsin. Antiparaquat F(ab')2 antibodies obtained were preventively injected to mice. After intravenous paraquat injection of 8 mg/kg, plasma paraquat levels were measured from 0.25 to 48 hours. Plasma from antiparaquat F(ab')2 pretreated mice as compared with non-specific immunoglobulin pretreated control mice showed a significant increase (p less than 0.001) of the paraquat concentrations from the 4th (1.17 +/- 0.06 versus 0.20 +/- 0.01 microgram/ml) to the 48th hour (0.47 +/- 0.08 versus 0.02 +/- 0.01 microgram/ml). Although pulmonary paraquat concentrations presented no modification, it could be considered that these preliminary results would have to be studied thoroughly with a view to finding an efficient treatment in human acute poisoning with paraquat.     

Tetanus antitoxin binds to intracellular tetanus toxin in permeabilized chromaffin cells without restoring Ca2(+)-induced exocytosis

Tetanus toxin blocks Ca2(+)-evoked catecholamine release from permeabilized bovine adrenal chromaffin cells preloaded with gangliosides. Tetanus toxin preincubated with its specific antibodies F(ab')2 is without any effect on exocytosis. Specific antitetanus F(ab')2 presented to chromaffin cells which are pretreated with tetanus toxin and permeabilized by digitonin cannot restore exocytosis. Under the same conditions, however, 125I-labeled F(ab')2 accumulates in chromaffin cells. The accumulation depends on the presence and concentration of tetanus toxin and can be prevented by an excess of unlabeled F(ab')2. Once tetanus toxin has initiated block of exocytosis, it cannot be neutralized by binding to its specific antibody.     

Effect of conjugation on the biodistribution of 111In-labelled anti-PAP and anti-PSA monoclonal antibodies examined in nude mice with PC-82 human tumor xenografts.

The effect of conjugation on the biodistribution of 111In-labelled antibodies was studied in nude mice carrying human prostatic cancer xenografts (PC-82). Two monoclonal antibodies and their fragments raised against human prostate-specific acid phosphatase (PAP) and prostate-specific antigen (PSA) were used. We used the cyclic anhydride of DTPA (CA-DTPA) as a chelating agent, or, alternatively, 1-(p-aminobenzyl)diethylenetriaminepentaacetic acid (NH2-Bz-DTPA) was attached as a linker to the carbohydrate components of the parent molecules. The conjugation method, the amount of circulating antigen and the size of the antibody component affected the blood clearance of the labelled derivatives. F(ab')2 fragments displayed a faster blood clearance than the corresponding derivatives of intact IgG1s. Aminobenzyl derivatives of anti-PAP-IgG1 showed a faster blood clearance than the corresponding CA-DTPA derivatives, but, in the case of derivatives of anti-PSA-IgG1, this was less clear, possibly due to the high PSA concentrations in the mouse sera. All the derivatives studied accumulated in the liver independently of the size of the antibody derivative, most probably due to the formation of antigen-antibody complexes. All CA-DTPA derivatives showed a higher kidney accumulation than the corresponding aminobenzyl derivatives. CA-DTPA-anti-PAP-F(ab')2 fragments showed a higher kidney uptake than the corresponding anti-PSA-F(ab')2 derivatives, since a large fraction of the latter are complexed with circulating antigen, thereby slowing down its reabsorption by the kidney. In addition, the lower kidney accumulation for anti-PSA-F(ab')2 fragments might be, at least partly, due to the electronegative charge of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulating pharmacokinetics of an anti-interleukin-8 F(ab')(2) by amine-specific PEGylation with preserved bioactivity

By covalently attaching biocompatible polyethylene-glycol (PEG) groups to epsilon-amino groups of the F(ab')(2) form of a humanized anti-interleukin-8 (anti-IL-8) antibody, we sought to decrease the in vivo clearance rate to give a potentially more clinically acceptable therapeutic. The in vivo clearance was modulated by changing the hydrodynamic size of the PEGylated antibody fragments. To achieve significant increases in the hydrodynamic size with minimal loss in bioactivity, high molecular weight linear or branched PEG molecules were used. Modification involved N-hydroxy-succinamide reaction of the PEGs with primary amines (lysines and/or the N-terminus) of the anti-IL-8 F(ab')(2). The process of adding up to four linear 20 kDa PEG, or up to two branched 40 kDa PEG, gave reproducible distribution of products. The components with uniform size (as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were purified by a single-step ion-exchange high-performance liquid chromatography and showed no significant loss of biological activity in ligand binding and cell-based assays. Addition of a single branched 40 kDa PEG to a F(ab')(2) (molecular weight (MW)=1.6 million Da) or up to two 40 kDa branched PEG (MW=1.9 million Da) increased the serum half-life to 48 h as compared with the unPEGylated F(ab')(2) with a half-life of 8.5 h. This study shows that by attaching high molecular weight PEGs at a one or two sites, bioactive antibody fragments can be made reproducibly with sizes tailored to achieve the desired pharmacokinetics.     

Site-specific conjugation of bifunctional chelator BAT to mouse IgG1 Fab' fragment

AIM: To perform a site-specific conjugation of Fab' fragments of a mouse monoclonal antibody(MoAb) B43(of IgG1 subtype) to a bifunctional chelator 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N',N' 'N' ' '-tetraacetic acid (BAT) via the thiol groups in the hinge distal to the antigen-binding site of the Fab'. METHODS: B43 was cleaved using a simple 2-step method. First, stable F(ab')(2) was produced by pepsin treatment. Fab' with free thiol in the hinge region was then obtained by cysteine reduction of F(ab')2. Second, a site-specific conjugation of Fab' to thiol-specific BAT was performed in a one-step reaction. RESULTS: The Fab' fragment had approximately 1.8 free thiol groups per molecule after cysteine reduction. The conjugation efficiency and the chemical yield were approximately 1.28 moles chelator/Fab' and 74% of the initial concentration of Fab', respectively. The F(ab')2, Fab' and Fab'-BAT all maintained reasonable antigen-binding properties. (67)Cu labeling of the conjugate under standard conditions did not impair the immunoreactivity of Fab'-BAT. CONCLUSION: This is a simple and efficient method for producing immunoreactive conjugates of Fab'-BAT, which can be used to make radiometal-labeled conjugates for further diagnostic and therapeutic applications.     

A new ELISA for determination of potency in snake antivenoms.

A competitive ELISA for potency determination of bothropic equine antivenom was developed and compared to the conventional in vivo ED(50) assay, with the aim of partially substituting the in vivo assay in the monitoring of antivenom immunoglobulin levels. On this purpose, blood samples were taken at different times during and after the immunization protocol of the lot of horses used for production of snake antivenom at the Instituto de Higiene, Uruguay. Both the competitive ELISA and the ED(50) assay were performed on those samples. In addition, a group of five commercial pepsin-digested antivenoms were tested by both methods. A significant (P<0.001) correlation (Pearson's r=0.957) was found between the ELISA titres and the corresponding ED(50) values, indicating that the in vitro test can estimate the neutralizing antibody capacity of the sera as well as the in vivo assay. By means of this new ELISA, it was found that the immunized animals maintained good venom antibody titres, in the order of 20-50% of the maximum achieved, even 10 month after the end of the immunization schedule. The main advantage of our ELISA design is its ability to correctly estimate the neutralization capacity of crude hyperimmune plasma and antivenom sera independently of their antibody composition in terms of whole IgG or F(ab')(2) fragment.     

Neutralisation of venom-induced haemorrhage by IgG from camels and llamas immunised with viper venom and also by endogenous, non-IgG components in camelid sera.

Envenoming by snakes results in severe systemic and local pathology. Intravenous administration of antivenom, prepared from IgG of venom immunised horses or sheep, is the only effective treatment of systemic envenoming. Conventional antivenoms, formulated as intact IgG, papain-cleaved (Fab) or pepsin-cleaved F(ab')2 fragments, are however ineffective against the local venom effects because of their inability to penetrate the blood/tissue barrier. We have embarked on a new research program to examine (i) whether the unusually small (15 kDa) antigen-binding fragment of camelid heavy chain IgG (V(H)H) can be exploited to neutralise the local effects of envenoming and (ii) whether a novel antivenom to treat both the systemic and local effects of envenoming can be formulated by combining anti-snake venom V(H)H and conventional F(ab')2. In this preliminary study, we demonstrate that camels and llamas respond to immunisation with Echis ocellatus venom with high antibody titres and broad antigen specificity. These encouraging immunological results were matched by the successful elimination of venom-induced haemorrhage by IgG from the venom-immunised camels and llamas. Unexpectedly, we report for the first time that camelid serum contains a non-IgG, highly potent inhibitor of venom-induced haemorrhage.