Development of a monoclonal antibody sandwich ELISA for the determination of antigen content and quality in diphtheria vaccines.

At present, quality control of diphtheria vaccines by both manufacturers and national control laboratories relies heavily on in vivo assays to confirm potency. As part of the VAC2VAC project we have developed a monoclonal antibody (mAb) enzyme-linked immunosorbent assay (ELISA) to measure the relative amount and quality of diphtheria toxoid (DTxd) in diphtheria-tetanus based vaccines and believe this test has the potential to play a key role in a control strategy no longer including an in vivo potency test. The mAb ELISA is highly specific, has good dilutional linearity, and is suitable for detecting DTxd in a range of different human vaccine products. We demonstrate the ability of the assay to discriminate between batches of different content and quality using vaccine batches that were prepared to contain differing amounts of DTxd or were altered by exposure to heat or oxidative stress. We also demonstrate successful transfer of the method to other laboratories and show that different diphtheria antigen materials may be able to serve as a reference antigen for local standardization of the method. The assay is ideally suited for incorporation into a consistency approach for routine diphtheria vaccine quality control testing and may be suitable to serve as the stability indicating test in replacement of the current in vivo potency test.

Diphtheria vaccines help to protect against diphtheria infection. Currently, animal tests are used to ensure the potency of such vaccines. Since these tests were first introduced, there have been improvements in non-animal technologies that can be used to ensure consistent production of potent vaccine batches. To demonstrate that a new batch of diphtheria vaccine is consistent with a previous batch of known potency, the quality and amount of the component that stimulates the immune response upon vaccination must be assessed in comparison. We have developed an assay that can measure the quality of a range of different diphtheria vaccine product types. The assay is very specific and reliable, and different laboratories obtained comparable results, showing that the assay is suited for routine use. Once validated by manufacturers and recognized by regulators, this assay will greatly reduce the number of animals needed for batch release of diphtheria vaccines.

Investigation of Cysteine Modifications in Recombinant Protein Tetanus Toxoid Heavy Chain Fragment C.

For conjugated HIV-1 fusion peptide vaccine development, recombinant Tetanus toxoid heavy chain fragment C (rTTHC) was applied as a carrier protein to boost peptide immunogenicity. Understanding the characteristics of rTTHC is the first step prior to the peptide conjugation. A comprehensive mass spectrometry (MS) characterization was performed on E. coli expressed rTTHC during its purification process. Intact mass along with peptide mapping analysis discovered the existence of three cysteine modification forms: glutathionylation, trisulfide bond modification, and disulfide bond shuffling, in correlation to a three-peak profile during a hydrophobic interaction chromatography (HIC) purification step. Coexistence of these multiple oxidative forms indicated that the active thiols underwent redox reaction in the rTTHC material. Identity confirmation of the rTTHC carrier protein by MS analysis provided pivotal guidance to assess the purification step and helped ensure that vaccine development could proceed.

Evaluation of a capture antigen ELISA for the characterisation of tetanus vaccines for veterinary use.

We previously developed an ELISA assay for detection of tetanus toxoid antigen in tetanus vaccines for human use. Tetanus vaccines for veterinary use are qualitatively different to those used in humans, often containing a larger number and variety of non-tetanus antigens in the multi-valent products, and adjuvants that are not found in human vaccines. We assessed performance of the capture ELISA with a range of veterinary tetanus vaccines as a first step towards development of an immunoassay as a potential in vivo potency substitute. Nine tetanus vaccines were tested and all produced a good dose response in the ELISA. The shape of the dose response curve for the whole vaccine compared to a matched non-adjuvanted tetanus toxoid antigen was more comparable for vaccines containing a non-aluminium adjuvant than products containing aluminium adjuvants. Elution of the antigen from aluminium adjuvant did not improve the comparability of the dose response curve but did increase the total amount of tetanus antigen available for detection. The ELISA was highly specific for tetanus with no signal obtained for a large number of non-tetanus antigens. These results suggest that a capture ELISA assay can be applied to a control strategy for veterinary tetanus vaccines.

Improving Analytical Characterization of Glycoconjugate Vaccines through Combined High-Resolution MS and NMR: Application to Neisseria meningitidis Serogroup B Oligosaccharide-Peptide Glycoconjugates.

Conjugate vaccines are highly heterogeneous in terms of glycosylation sites and linked oligosaccharide length. Therefore, the characterization of conjugate vaccines' glycosylation state is challenging. However, improved product characterization can lead to enhancements in product control and product quality. Here, we present a synergistic combination of high-resolution mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) for the analysis of glycoconjugates. We use the power of this strategy to characterize model polysaccharide conjugates and to demonstrate a detailed level of glycoproteomic analysis. These are first steps on model compounds that will help untangle the details of complex product characterization in conjugate vaccines. Ultimately, this strategy can be applied to enhance the characterization of polysaccharide conjugate vaccines. In this study, we lay the groundwork for the analysis of conjugate vaccines. To begin this effort, oligosaccharide-peptide conjugates were synthesized by periodate oxidation of an oligosaccharide of a defined length, α,2-8 sialic acid trimer, followed by a reductive amination, and linking the trimer to an immunogenic peptide from tetanus toxoid. Combined mass spectrometry and nuclear magnetic resonance were used to monitor each reaction and conjugation products. Complete NMR peak assignment and detailed MS information on oxidized oligosialic acid and conjugates are reported. These studies provide a deeper understanding of the conjugation chemistry process and products, which can lead to a better controlled production process.

Hib antibody responses in infants following diphtheria, tetanus, acellular pertussis, and conjugated Haemophilus influenzae type b (Hib) combination vaccines with decreasing amounts of tetanus toxoid.

BACKGROUND: While combination vaccines have contributed to improved vaccine uptake rates in children, studies have documented varied immunogenicity to specific vaccine components. We studied whether varying the amount of tetanus toxoid (TT) in a DTaP and Hib combination vaccine would result in immunogenicity comparable with separate, concurrent administration. METHODS: We evaluated the immunogenicity of Massachusetts Biologic Laboratories (MBL) diphtheria, tetanus, and acellular pertussis (mDTaP) vaccine combined with tetanus-conjugated MBL Haemophilus influenzae type b vaccine (mHib) in a single injection (DTaPH). We compared four DTaPH vaccines containing varying concentrations of TT. We also evaluated the immune response to the DTaP vaccine manufactured by Connaught Laboratories (now known as Sanofi Pasteur) given with mHib and with Wyeth Hib-CRM197 (HbOC) as separate injections. Vaccines were administered to 240 healthy infants at 2, 4, and 6 months of age, and blood specimens for antibody determination were obtained before each immunization and one month after the third immunization. RESULTS: We found no significant differences in immune response to the vaccines between the four DTaPH groups. Hib antibody responses were similar in the mHib and the HbOC groups but significantly lower in the DTaPH groups, as measured by Chinese Hamster Ovary (CHO) cell neutralization titers and filamentous hemagglutinin antigen (FHA) geometric mean concentrations (GMC) of anti-Hib antibodies. There were no significant differences between the groups in pertussis or tetanus toxoid antibody levels. CONCLUSION: Reducing tetanus toxoid amounts did not produce comparable immunogenicity for Hib. The nature of the interaction between immune responses to DTaPH components should be explored further to enable the development of better Hib-containing combination vaccines.

Specific detection of tetanus toxoid using an aptamer-based matrix.

Batch-to-batch variation of therapeutic proteins produced by biological means requires rigorous monitoring at all stages of the production process. A large number of animals are employed for risk assessment of biologicals, which has low ethical and economic acceptability. Research is now focussed on the validation of in vitro and ex vivo tests to replace live challenges. Among in vitro methods, enzyme-linked immunosorbent assay (ELISA) is considered to be the gold standard for estimation of integrity of tetanus toxoid. ELISA utilizes antibodies for detection, which, because of their biological origin and limited modifiability, may have low stability and result in irreproducibility. We have developed a method using highly specific and selective RNA aptamers for detection of tetanus toxoid. Using displacement assay, we first identified aptamers which bind to different aptatopes on the surface of the toxoid. Pairs of these aptamers were employed as capture-detection ligands in a sandwich-ALISA (aptamer-linked immobilized sorbent assay) format. The binding efficiency was confirmed by the fluorescence intensity in each microtire plate well. Using aptamers alone, detection of tetanus toxoid was possible with the same level of sensitivity as antibody. Aptamers were also used in the capture ALISA format. Adjuvanted tetanus toxoid was subjected to accelerated stress testing, including thermal, mechanical and freeze-thawing stress conditions. The loss in antigenicity of the preparation determined by ALISA in each case was found to be similar to that determined by conventional ELISA. Thus, it is possible to replace antibodies with aptamers to develop a more robust detection tool for tetanus toxoid.

Mechanisms of Antigen Adsorption Onto an Aluminum-Hydroxide Adjuvant Evaluated by High-Throughput Screening.

The adsorption mechanism of antigen on aluminum adjuvant can affect antigen elution at the injection site and hence the immune response. Our aim was to evaluate adsorption onto aluminum hydroxide (AH) by ligand exchange and electrostatic interactions of model proteins and antigens, bovine serum albumin (BSA), ß-casein, ovalbumin (OVA), hepatitis B surface antigen, and tetanus toxin (TT). A high-throughput screening platform was developed to measure adsorption isotherms in the presence of electrolytes and ligand exchange by a fluorescence-spectroscopy method that detects the catalysis of 6,8-difluoro-4-methylumbelliferyl phosphate by free hydroxyl groups on AH. BSA adsorption depended on predominant electrostatic interactions. Ligand exchange contributes to the adsorption of ß-casein, OVA, hepatitis B surface antigen, and TT onto AH. Based on relative surface phosphophilicity and adsorption isotherms in the presence of phosphate and fluoride, the capacities of the proteins to interact with AH by ligand exchange followed the trend: OVA < ß-casein < BSA < TT. This could be explained by both the content of ligands available in the protein structure for ligand exchange and the antigen's molecular weight. The high-throughput screening platform can be used to better understand the contributions of ligand exchange and electrostatic attractions governing the interactions between an antigen adsorbed onto aluminum-containing adjuvant.

Use of a tetanus toxoid marker to allow differentiation of infected from vaccinated poultry without affecting the efficacy of a H5N1 avian influenza virus vaccine.

Tetanus toxoid (TT) was assessed as a positive marker for avian influenza (AI) virus vaccination in chickens, in a vaccination and challenge study. Chickens were vaccinated twice with inactivated AI H5N2 virus vaccine, and then challenged three weeks later with highly pathogenic AI H5N1 virus. Vaccinated chickens were compared with other groups that were either sham-vaccinated or vaccinated with virus with the TT marker. All sham-vaccinated chickens died by 36 hours postinfection, whereas all vaccinated chickens, with or without the TT marker, were protected from morbidity and mortality following exposure to the challenge virus. Serological testing for H5-specific antibodies identified anamnestic responses to H5 in some of the vaccinated birds, indicating active virus infection.

Calibration of replacement international standards of diphtheria and tetanus toxoids for use in flocculation test.

The 1st International Reference Reagents (IRR) of Diphtheria and Tetanus Toxoids for Flocculation Test (DIFT and TEFT) were established by the WHO in 1988. These reagents are essential for the standardization of assays used to calculate Lf units of toxoids. Candidate replacement materials were provided by several European vaccine manufacturers and were formulated and freeze-dried at NIBSC. This paper provides a summary of the results of an international collaborative study including 18 laboratories from 16 countries, which examined the candidate replacement materials in a variety of methods. Materials 02/176 and 04/150 were proposed and adopted by the Expert Committee on Biological Standardization of WHO in October 2007 as 2nd WHO International Standards of Diphtheria and Tetanus Toxoid for use in Flocculation Test. The replacement standards were assigned the value of 1100 and 690Lf/ampoule, respectively, based on results of flocculation tests carried out using provided reagents. Material coded 02/176 fully complied with the WHO specifications for stability, residual moisture content, precision of fill and sterility. Stability of material coded 04/150 was slightly lower than expected but predictions were based only on 2-year data and were to be further monitored, post-adoption.

A novel DNA vaccine targeting macrophage migration inhibitory factor protects joints from inflammation and destruction in murine models of arthritis.

OBJECTIVE: Previous studies have demonstrated that neutralization of macrophage migration inhibitory factor (MIF) by anti-MIF antibodies decreases joint inflammation and destruction in a type II collagen-induced arthritis model in mice. The aim of this study was to develop and describe a simple and effective method of active immunization that induces anti-MIF autoantibodies, which may neutralize MIF bioactivity. METHODS: We developed a MIF DNA vaccine by introducing oligonucleotides encoding a tetanus toxoid (TTX) Th cell epitope into the complementary DNA sequence of murine MIF. Mice were injected with this construct in conjunction with electroporation. The ability of this immunization to inhibit the development of collagen antibody-induced arthritis (CAIA) in BALB/c mice and spontaneous autoimmune arthritis in interleukin-1 receptor antagonist (IL-1Ra)-deficient mice was then evaluated. RESULTS: Mice that received the MIF/TTX DNA vaccine developed high titers of autoantibodies that reacted to native MIF. Compared with unvaccinated mice, vaccinated mice also produced less serum tumor necrosis factor alpha after receiving an intravenous injection of lipopolysaccharide. In addition, vaccination with MIF/TTX DNA resulted in significant amelioration of both CAIA in BALB/c mice and symptoms of autoimmune arthritis in IL-1Ra-knockout mice. CONCLUSION: These results suggest that MIF/TTX DNA vaccination may be useful for ameliorating the symptoms of rheumatoid arthritis.

Toxoid flocculation assay by laser light-scattering.

Internationally accepted designations of antigen content for toxoid vaccines are provided by the WHO in Lf (limes flocculationis) units, based on the formation of antigen-antibody complexes. The current assay method for Lf determination involves observation of the complexes by eye, making the development of a more objective system highly desirable. Here we report a novel detection system using a laser light-scattering platelet aggregometer. The system was highly reproducible and more objective than the current method. Only three sets of duplicate data were sufficient for statistically significant determination of toxoid Lf by parabolic regression.

Development of rocket electrophoresis technique as an analytical tool in preformulation study of tetanus vaccine formulation.

Rocket Electrophoresis (RE) technique relies on the difference in charges of the antigen and antibodies at the selected pH. The present study involves optimization of RE run conditions for Tetanus Toxoid (TT). Agarose gel (1% w/v, 20 ml, pH 8.6), anti-TT IgG - 1 IU/ml, temperature 4-8 degrees C and run duration of 18 h was found to be optimum. Height of the rocket-shaped precipitate was proportional to TT concentration. The RE method was found to be linear in the concentration range of 2.5 to 30 Lf/mL. The method was validated and found to be accurate, precise, and reproducible when analyzed statistically using student's t-test. RE was used as an analytical method for analyzing TT content in plain and marketed formulations as well as for the preformulation study of vaccine formulation where formulation additives were tested for compatibility with TT. The optimized RE method has several advantages: it uses safe materials, is inexpensive, and easy to perform. RE results are less prone to operator's bias as compared to flocculation test and can be documented by taking photographs and scanned by densitometer; RE can be easily standardized for the required antigen concentration by changing antitoxin concentration. It can be used as a very effective tool for qualitative and quantitative analysis and in preformulation studies of antigens.

Pertusis en lactantes, niños y adolescentes: diagnóstico, tratamiento y prevención / Whooping Cough in infants, children, and adolescents: diagnosis, treatment, and prevention

La vacuna celular contra pertusis, asociada a toxoide tetánico y diftérico (DPT) ha disminuido sustancialmente la enfermedad. El alto porcentajede reacciones adversas: reacción local, fiebre y síntomas sistémicos, han dado origen al desarrollo de una vacuna menos reactogénica llamada vacuna antipertúsica acelular (DaPT), cuya eficacia es comparable a la celular, aunque la duración de la protección después de la inmunización, no ha sido establecida definitivamente. La DaPT se aplica a los 2, 4 y 6 meses, un refuerzo entre los 15 y 18 meses y uno último entre 4 y 6 años de edad. A partir del año 2005 se licenció la vacuna Tdap para adultos y adolescentes entre 11 y 18 años.

Replacement of the in vivo neutralisation test for efficacy demonstration of tetanus vaccines ad us. vet.

The bacterium Clostridium (C.) tetani is an ubiquitous pathogen. This anaerobic, gram-positive bacterium can form spores and can be found in the whole environment. It enters the body via injuries of the skin and wounds where it releases the neurotoxin "tetanospasmin" (= tetanus toxin). The animals most susceptible to tetanus infection are horses and sheep. Only active immunisation by tetanus vaccine provides effective protection against tetanus intoxication. The marketing authorisation requirements stipulate that efficacy of tetanus vaccines ad us. vet. must be demonstrated in all target animal species via determination of neutralising tetanus serum antitoxin concentrations. The standard method used for this purpose is still the toxin neutralisation test (TNT), as it quantifies the tetanus toxin-neutralising effect of tetanus serum antibodies in vivo. In this test, tetanus toxin is added to dilutions of serum from vaccinated horse and sheep. The serum dilutions are then administered to mice or guinea pigs, which are observed for toxic symptoms. Against the background of animal protection, the goal of one project of the Paul-Ehrlich-Institut (Bundesministerium fuer Bildung und Forschung (Federal Ministry for Education and Research), 0312636) was to establish an alternative to the toxin neutralisation test, enabling the testing of efficacy of tetanus vaccines with serological in vitro methods. For this purpose, a so-called double antigen ELISA (DAE) was established which enables the testing of sera of different species in one assay. In addition, the sera were tested in an indirect ELISA for horses and sheep separately. Altogether, ten groups of horses and eight groups of sheep were immunised with ten animals per group each. The tetanus vaccines comprised almost all products authorised for the German market at the start of the project. 564 horse sera and 257 sheep sera were tested using the two ELISA methods. Some sera were also tested in vivo. The kinetics of antibody responses were recorded. The in vitro DAE method seems to be suitable to replace the mouse neutralisation test used for the detection of tetanus antitoxin in sera of target animal species. The comparison of some sera in the ELISA and the TNT showed good equivalence of results. Nevertheless, before an ELISA titre in horse and sheep sera indicating unambiguous protection against tetanus can be fixed, further comparative assays of low titre sera in the TNT and the DAE will have to be performed.

Diagnosis of tetanus immunization status: multicenter assessment of a rapid biological test.

Diagnosis of tetanus immunization status by medical interview of patients with wounds is poor. Many protected patients receive unnecessary vaccine or immunoglobulin, and unprotected patients may receive nothing. The aim of this study is to evaluate the feasibility and accuracy of the Tetanos Quick Stick (TQS) rapid finger prick stick test in the emergency department for determining immunization status. We designed a prospective multicenter study for blinded comparison of TQS with an enzyme-linked immunosorbent assay (ELISA). Adults referred for open wounds in 37 French hospital emergency departments had the TQS after receiving standard care (emergency-TQS). TQS was also performed in the hospital laboratory on total blood (blood/lab-TQS) and serum (serum/lab-TQS). ELISA was performed with the same blood sample at a central laboratory. We assessed concordance between emergency-TQS and blood/lab-TQS by the kappa test and the diagnostic accuracy (likelihood ratios) of medical interview, emergency-TQS, and lab-TQS. ELISA was positive in 94.6% of the 988 patients included. Concordance between blood/emergency-TQS and blood/lab-TQS results was moderate (kappa=0.6), with a high proportion of inconclusive blood/emergency-TQS tests (9.8%). Likelihood ratios for immunization were 3.0 (95% confidence interval [CI], 1.8 to 5.1), 36.6 (95% CI, 5.3 to 255.3), 89.1 (95% CI, 5.6 to 1,405.0), and 92.7 (95% CI, 5.9 to 1,462.0) for medical interview, blood/emergency-TQS, blood/lab-TQS, and serum/lab-TQS, respectively. The sensitivity of the blood/emergency-TQS was 76.7%, and the specificity was 98% by reference to the ELISA. TQS use in the emergency room could make tetanus prevention more accurate if its technical feasibility were improved, and our assessment will be supplemented by a cost effectiveness study.

[Determination of tetanus toxin and toxoid by ELISA using monoclonal antibodies]. / Immunofermentnoe opredelenie stolbniachnogo toksina i anatoksina s ispol'zovaniem monoklonal'nykh antitel.

The procedure of obtaining monoclonal antibodies TT-1, TT-2, and TT-3 against tetanus toxin/toxoid is described. It is shown that both commercial DTP vaccine and tetanus toxoid conjugated with a low-molecular-weight hapten can be used an immunogens. Monoclonal antibodies TT-1 and TT-2 neutralized tetanus toxin in vivo. The monoclonal antibodies obtained were used to design and compare several schemes of quantitative determination of tetanus toxoid and toxin by ELISA. A more sensitive competitive ELISA allowed detecting as much as 0.01 EC/ml toxoid and 50 LD50/ml toxin.

Development of sandwich enzyme-linked immunosorbent assay for determination of tetanus toxoid concentration.

According to the recommendation of the World Health Organization (WHO), the use of an in vivo test for measuring of the potency of tetanus toxoid vaccine (TTdV) is still unavoidable, but the establishment of a convenient in vitro test would significantly improve the work in this field. A sandwich enzyme-linked immunosorbent assay (sELISA) was developed for a rapid and sensitive quantification of tetanus toxoid (TTd). We produced four monoclonal antibodies (MAbs) designated 41, 51, 62, and 71 that reacted with TTd and recognized different antigenic determinants on TTd. We also used two of these antibodies for developing a sELISA, with MoAb 71 as an immobilized and MoAb 51 as a capture antibody. The measurement range of this assay was from 31-1000 ng/mL and the minimum detection limit for TTd was 31 ng/mL. This high sensitivity of this sELISA and its good reproducibility suggest that the developed method could be reliably used to estimate the concentration of TTd, which could be easily extrapolated to the estimation of vaccine potency.

Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines-part 1.

A collaborative study on the evaluation of an alternative functional assay, the Vero cell method, to the Ph. Eur. in vivo challenge procedures for potency determination of diphtheria toxoid in 6 different combined vaccines was initiated in January 2001. The study was an extension of a previous study for the validation of serological methods for potency testing of tetanus toxoid vaccines for human use. To allow interim evaluation of test results and to monitor study progress, the project was divided into three consecutive phases. The results of Phase I and II studies are presented in this report. Pre-validation (Phase I) study, performed in two laboratories, indicated that comparable diphtheria potency estimates were obtained in the Ph. Eur. direct intradermal challenge assay in guinea pigs, in Vero cell assay and in indirect ELISA for five vaccines of different potencies (range of estimates: ca. 20-200 IU/ml). The correlation coefficients between the challenge assay and the Vero cell assay corresponded to those between the challenge assay and ELISA, confirming that the antibodies play an important role in protection and that predominantly protective/neutralising antibodies are present in guinea pigs, at the time point investigated. It was observed, for Vero cell assays, that about 16-35 (9-28 in Phase II study) fold lower titre of individual serum samples were obtained when using equine, rather than guinea pig reference serum. The study also provided preliminary information that sera from the same guinea pigs may be used for potency determination of both diphtheria and tetanus toxoid components of vaccines. In Phase II, another five laboratories analysed a subset of the vaccines included in Phase I study plus an additional vaccine. Four laboratories performed the lethal challenge assay and one laboratory carried out the intradermal challenge assay. All laboratories also performed the Vero cell assay and both ELISA for diphtheria antitoxin and ELISA for tetanus antitoxin. One laboratory also performed the tetanus ToBI assay. The correlation coefficient (r) between Vero cell assay and ELISA for diphtheria antitoxin ranged from 0.76 to 0.91 in the different laboratories. The correlation between diphtheria serological assays and challenge assays were confirmed satisfactory as ca. 90 per cent of serum-estimates lead to correct prediction of mortality. All laboratories had identical rankings of the vaccines in all serological assays and in the valid challenge assays. The ranking order was identical to assumed/provided potency for the highest and the lowest vaccine. Two of the vaccines had an inversion in some assays and laboratories. As these two vaccines have almost identical potencies in all assays, these inversions are not significant. As the vaccine doses were optimised for the diphtheria component, serum anti-tetanus toxoid/toxin activities varied widely between the vaccines, making it questionable to apply a parallel line model to calculate exact potencies. However, the dose levels used showed a clear regression and good linearity in general. DTaP vaccines containing the IPV component did not always meet the present Ph. Eur. requirements in the serological assays. It should be further investigated in the Phase III study if this is a general feature of such combined vaccines. Preliminary investigations on samples from two laboratories indicate that the neutralising activity of type 1, 2 and 3 polioviruses can also be detected, in a dose-dependent way. Further studies are in progress with serum samples from other laboratories. In the light of results obtained in the first two phases, it is recommended to proceed with Phase III study to investigate reliability of the in vitro assays. In Phase III it will also be further investigated whether the serological assays for D and T components are suitable for the control of the multi-component vaccines currently marketed in Europe.

Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines - extended study: correlation of serology with in vivo toxin neutralisation.

Phase I of BSP034 collaborative study was extended in two laboratories to include correlation of serology with in vivo toxin neutralisation test (TNT) using 2 separate sets of 20 serum pools, produced in-house. The study investigated the extent to which the in vitro methods for diphtheria antibodies, Vero cell assay and diphtheria enzyme-linked immunosorbent assay for diphtheria antitoxin (D-ELISA), can detect neutralising antibodies by comparison with TNT in guinea pigs. The study was also performed to compare the antibody neutralising potency obtained in relation to guinea pig (GP) or equine (DI) antitoxin standard. In addition, the study provided an opportunity to compare ELISA for tetanus antitoxin (T-ELISA) and TNT assay for detection of anti-tetanus antibodies, from the same set of serum pools. The data obtained show that antitoxin potency obtained by Vero cell assay, D-ELISA and T-ELISA using the same GP standard, highly correlated with neutralising potency as determined in respective TNT assays. Vero cell assay with DI provided estimates that also correlated with neutralising potency, but were of significantly lower titre. Since reference to DI standard is widely used in serodiagnosis, as well as in clinical studies where diphtheria antitoxin titres obtained in the Vero cell method are taken as surrogate markers for vaccine efficacy, it should be investigated if a similar difference is also observed for human serology.

Vacunas adsorbidas: ¡no congelar! / Vaccines inactived: ¡not to freeze!

Las vacunas inactivadas son vacunas a base de gérmenes muertos, enteros o fraccionados, que generalmente requieren la presencia de un agente adyuvante que adsorbe los antígenos y los libera lentamente para garantizar una respuesta inmunológica adecuada. Los adyuvantes más utilizados son las sales de aluminio como el hidróxido y el fosfato de aluminio, de los cuales se conoce su eficacia y seguridad. Sin embargo, su presencia es indicativo de que la vacuna deberá ser conservada a una temperatura de entre 2 y 8° C y evitar su congelación, ya que pierden su estructura coloidal, cristalizando, lo cual ocasiona además de la pérdida de potencia de la vacuna, severas reacciones locales. En este trabajo se evaluó la relación que existe entre el tiempo de congelación, el aspecto macro y microscópico de diferentes tipos de vacunas adsorbidas y la posibilidad de determinar a simple vista si la vacuna sufrió congelación. Los resultados señalan que no se puede establecer relación entre la velocidad de congelación, el tipo de vacuna, envase en que se encuentra y la aparición de cristales y partículas. Todas las muestras desarrollaron cristalización en apenas 1 hora de estar sometidas a temperatura de -20°C, aun cuando la mayoría se encontraba en estado líquido y sin presencia de partículas visibles a simple vista. La cristalización fue fácilmente observada en microscopio óptico, siendo una forma rápida y confiable para determinar si una vacuna adsorbida sufrió congelación y evitar así su administración