Showing papers in "Biologicals in 1990"
TL;DR: Since the measles, mumps and rubella (MMR) vaccine was introduced into Sweden in 1982, a yearly evaluation of the immunity patterns and sero-conversion rates in 12-year-old children has been carried out and yielded congruent results in early post-vaccination sera from children susceptible to measles prior to vaccination.
Abstract: Since the introduction of the measles, mumps and rubella (MMR) vaccine in Sweden in 1982, a yearly evaluation of the immunity pattern and seroconversion rate of 12-year-old children has been carried out. To be able to realize large-scale, follow-up studies, techniques have to be used which are not too labour-intensive and time-consuming but are sensitive enough to detect past immunity and post-vaccination titres. We report tests with haemolysis-in-gel (HIG) technique and an enzyme-linked, immunosorbent assay (ELISA) compared with neutralization (NT) for the detection of mumps antibodies. This study comprises 226 paired serum samples obtained between 1985 and 1989. One hundred and forty-one of the paired samples had been selected because they had given negative or borderline readings, using HIG technique. The remaining samples were consecutive pre- and post-vaccination sera obtained in 1989 from 85 vaccinees from one area in Sweden. HIG technique gave both false positive and false negative readings, compared with NT as also the false positive sera were detected. Non-inactivated sera could not be used in the NT test against mumps virus, owing to unspecific NT reactions. No differences between non-inactivated and inactivated sera by NT were seen, as against other paramyxoviruses. Cross-reactivity between mumps and parainfluenzae in NT tests was not demonstrated. The ELISA test proved more reliable and specific than HIG and was more sensitive than NT. Some post-vaccination sera from vaccinees who failed to seroconvert by NT contained high levels of mumps antibody detectable by the ELISA. Late post-vaccination sera which were negative by the NT also showed high levels of mumps-specific antibody detectable by the ELISA. Using ELISA test it was demonstrated that sera from children naturally immunized with mumps virus had significantly higher ELISA values than post-vaccination sera from children earlier vaccinated (P
50 citations
TL;DR: The immunocapture method based on the use of a neutralizing monoclonal anti-glycoprotein antibody seems to be a convenient tool for the determination of the in vitro potency of rabies vaccine and of the products corresponding to the different steps of their production process.
Abstract: The replacement of the in vivo potency test (NIH test) for rabies vaccine evaluation by in vitro methods is at present discussed in many reports and also by WHO expert working groups. For this purpose, in vitro glycoprotein titration has been proposed. Among the different glycoprotein assays, we have studied two ELISA methods (immunocapture and direct plate coating with the antigen to be tested) using neutralizing mono- and polyclonal antibodies. In our view, the immunocapture method based on the use of a neutralizing monoclonal anti-glycoprotein antibody seems to be a convenient tool for the determination of the in vitro potency of rabies vaccine and of the products corresponding to the different steps of their production process.
48 citations
TL;DR: In this article, the usefulness of cytopathic indicators for the titration of Cl perfringens beta and epsilon toxins has been investigated and it was concluded that cell culture titration offers a valid in vitro alternative to the use of mouse lethal and guinea-pig dermonecrotic indicators.
Abstract: The usefulness of cytopathic indicators for the titration of Cl perfringens beta and epsilon toxins has been investigated. Neutralization experiments with monoclonal antibodies have shown that the entities responsible for the lethal and dermonecrotic effects of Cl perfringens beta toxin preparations are identical. However, the cytopathic effects of the same preparations are caused by other entities. Therefore, titrations based upon lethal and dermonecrotic indicators of beta toxin are equally valid but those based on cytopathic effects are not. Similar experiments with Cl perfringens epsilon preparations have shown that their lethal, dermonecrotic and cytopathic activities are all caused by the same entity. It follows that all three activities can be valid indicators for toxin neutralization tests. Cell culture titrations of Cl perfringens epsilon antitoxin performed on rabbit sera at the levels of test prescribed by the European Pharmacopoeia have produced consistent results which agree closely with the dermonecrotic test. This test has, in turn, been shown to reflect the results of the mouse lethal test accurately. Titrations of cattle and sheep sera at lower levels of test have also produced results in close agreement with the in vivo test. It is concluded that cell culture titration offers a valid in vitro alternative to the use of mouse lethal and guinea-pig dermonecrotic indicators for the titration of sera generated in the course of potency tests and field trials of Cl perfringens epsilon vaccines.
34 citations
TL;DR: Keywords: Animals ; Cell Line ; Gene Amplification ; Gene Expression Regulation ; Genetic Vectors ; Plasmids/*physiology ; Transfection/genetics; Transfections/genetic Note: Genentech Inc., San Francisco, CA 94080.
Abstract: Keywords: Animals ; Cell Line ; Gene Amplification ; Gene Expression Regulation ; Genetic Vectors/*physiology ; Plasmids/*physiology ; Transfection/genetics Note: Genentech Inc., San Francisco, CA 94080. Reference LBTC-ARTICLE-1990-001doi:10.1016/1045-1056(90)90002-H URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=2257128 Record created on 2007-06-05, modified on 2016-08-08
33 citations
33 citations
TL;DR: The proposed in vitro bioassay for interferon alpha (IFN-alpha) preparations based on their antiproliferative effect in cultured Daudi cells has been developed and had a high sensitivity and precision as well as a good reproducibility compared with antiviral assays.
Abstract: Interferons have, in addition to their antiviral effects, been shown to possess several non-antiviral activities. In this study, an in vitro bioassay for interferon alpha (IFN-alpha) preparations based on their antiproliferative effect in cultured Daudi cells has been developed. Briefly, about 10(5) cells per ml treated with different concentrations of IFN were incubated under standard culture conditions for 3 days. Two different end points, i.e. incorporation of [3H]thymidine and final cell density, were used and responses were evaluated according to established pharmacopoeial principles for quantification of biomolecules. Both methods gave similar results. However, measurement of final cell density yielded the most precise results. The proposed assay, with an effective assay range of 1-10 IU/ml (approximately 0.2-2 x 10(-12)M, had a high sensitivity and precision as well as a good reproducibility. Compared with antiviral assays, it is less resource demanding. In conclusion, the in vitro bioassay described is well suited for potency determinations of IFN-alpha and probably also IFN-beta preparations.
33 citations
TL;DR: Nine measles vaccine preparations, including four different viral strains, provided by eight different manufacturers were analysed by Northern blot for the nature of their nucleocapsid RNAs, and presence or absence of the subgenomic RNAs correlated strictly with the viral strains used.
Abstract: Nine measles vaccine preparations, including four different viral strains, provided by eight different manufacturers were analysed by Northern blot for the nature of their nucleocapsid RNAs. Out of nine preparations, six were shown to contain subgenomic RNAs, along with the full length genomic RNA. Presence or absence of the subgenomic RNAs correlated strictly with the viral strains used. The role of the defective interfering particles in measles virus vaccine attenuation, and in its seroconversion efficacy upon vaccination, as well as the potential hazard of the presence of defective interfering particles in live-virus vaccine preparations, is discussed.
27 citations
TL;DR: A transimmortalisation technique for the isolation of differentiated hepatic cell lines, already engineered to secrete human alpha 1 antitrypsin (alpha 1 AT), a plasma protein which is produced mainly in liver cells, is developed.
Abstract: A number of therapeutic plasma proteins are synthesized by human hepatocytes. Since many of these proteins undergo liver-specific post-translational modifications which are required for full biological activity, it may therefore be necessary to develop hepatocyte-based expression systems for their production. Using transgenic mice we have developed a transimmortalisation technique for the isolation of differentiated hepatic cell lines, already engineered to secrete human α 1 antitrypsin ( α 1 AT), a plasma protein which is produced mainly in liver cells. This was achieved by co-expression of the mouse c- myc proto-oncogene and a genomic copy of the human α 1 AT gene, both under the control of the human α 1 AT promoter. Transgenic mice carrying this construct developed hepatomas producing human α 1 AT. Under defined culture conditions, cell lines secreting active α 1 AT were derived from these tumours. These cells maintain a differentiated hepatic phenotype and continue to secrete human α 1 AT for at least 40 generations.
25 citations
TL;DR: The application of an ELISA to measure the response to C. chauvoei vaccines in rabbits is described and the antibody titres of rabbit sera have been compared with the results of guinea-pig challenge potency tests on the same vaccines.
Abstract: Current standards (British Pharmacopeia (Veterinary) 1985) for vaccines containing Clostridium chauvoei require a potency test based on a challenge assay in guinea-pigs. Animal welfare and cost considerations favour the development of alternatives. Most veterinary clostridial vaccines are multi component, requiring assays in rabbits to test the potency of components other than C. chauvoei. We describe the application of an ELISA to measure the response to C. chauvoei vaccines in rabbits. The antigen is a sonicated extract of C. chauvoei strain CH4, intended to include a mixture of cellular and soluble antigens. The rabbit response to more than 70 vaccines containing C. chauvoei has been assessed against a reference serum which has been assigned an arbitrary potency of 100 units ml-1. The antibody titres of rabbit sera have been compared with the results of guinea-pig challenge potency tests on the same vaccines. The pass level in the guinea-pig potency test is equivalent to a rabbit ELISA titre of 50 units ml-1.
25 citations
TL;DR: A double antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) was developed that can simultaneously quantify 146S antigen in many samples, and also indicates when VP1 of 146S particles has disintegrated by the action of proteases.
Abstract: A double antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) was developed to quantify 146S antigen of foot-and-mouth disease virus (FMDV) strain A10 Holland grown in suspension cultures of surviving bovine tongue epithelium When virus harvests were incubated with trypsin--which affects VP1, the most immunogenic structural protein of FMDV--the concentration of 146S antigen as determined by ELISA was reduced by greater than 90% Therefore, the test detected essentially only those virus particles with intact VP1 When the test was compared with the sucrose density gradient method, concentrations of 146S antigen correlated well (r = 087) The rate of variation in both tests was the same In contrast to the sucrose density gradient method, the DAS-ELISA can simultaneously quantify 146S antigen in many samples, and also indicates when VP1 of 146S particles has disintegrated by the action of proteases
23 citations
TL;DR: Methods for enhancing peptide immunogenicity are discussed such as the use of adjuvants and liposomes, and of synthetic branched polypeptides as carriers, and new techniques of automated and multiple peptide synthesis are discussed.
Abstract: Peptides prepared by the solid-phase peptide synthesis (SPPS) approach are used increasingly in biological research, for instance to elicit anti-peptide antibodies that will recognize the intact, cognate protein. Recent advances in SPPS are reviewed, including the use of new coupling reagents, new methods for evaluating peptide purity and new techniques of automated and multiple peptide synthesis. Methods for enhancing peptide immunogenicity are discussed such as the use of adjuvants and liposomes, and of synthetic branched polypeptides as carriers.
TL;DR: There is no difference between levels in different sexes, and no increase with age, but levels in cord blood are raised, and levels in plasma relative to serum are higher.
Abstract: von Willebrand Factor antigen is present in serum, plasma, synovial fluid and semen, and absent from other body fluids including breast milk, urine, saliva, and cerebro-spinal fluid. Levels are higher in plasma relative to serum. There is no difference between levels in different sexes, and no increase with age, but levels in cord blood are raised.
TL;DR: It was shown that flocculation tests using the reference toxoids are very reproducible and reliable and the WHO Expert Committee on Biological Standardization established the toxoid called DIFT as the International Reference Reagent of Diphtheria Toxoid for Flocculation Test with a defined content of 900 Lf-units of diphtherIA toxoid per ampoule.
Abstract: The Lf-unit, which is used in the control of diphtheria and tetanus toxoid production and in some countries also to follow immunization of horses for production of antitoxins, has hitherto been defined by means of antitoxin preparations. A diphtheria toxoid and a tetanus toxoid preparation, both freeze-dried, were examined in an international collaborative study for their suitability to serve as reference reagents in the flocculation tests and for defining the Lf-units. It was shown that flocculation tests using the reference toxoids are very reproducible and reliable and the WHO Expert Committee on Biological Standardization established: the toxoid called DIFT as the International Reference Reagent of Diphtheria Toxoid for Flocculation Test with a defined content of 900 Lf-units of diphtheria toxoid per ampoule; and the toxoid called TEFT as the International Reference Reagent of Tetanus Toxoid for Flocculation Test with a defined content of 1000 Lf-units of diphtheria toxoid per ampoule.
TL;DR: In this paper, an alternative indicator system based on the use of cell cultures is described, and evidence is presented to show that the toxins detected by the in vivo and in vitro indicators are indistinguishable in terms of molecular weight, charge and hydrophobicity.
Abstract: The assay of Clostridium septicum antitoxin currently requires the inoculation of test mixtures intravenously into mice or intradermally into guinea-pig skin. An alternative indicator system based on the use of cell cultures is described. Evidence is presented to show that the toxins detected by the in vivo and in vitro indicators are indistinguishable in terms of molecular weight, charge and hydrophobicity and that there is a close agreement between the two methods of titration. Cell culture indicators are more sensitive than their in vivo counterparts, permitting detection of substantially lower titres than is possible using in vivo indicators. It is suggested that cell culture indicators may prove useful for the titration of Cl septicum antitoxin in sera from vaccine field trials and potency tests. Cell culture methods could also be used for the potency testing of antitoxin preparations.
TL;DR: Fluorogenic and chromogenic substrates were used in direct and trapping enzyme-linked immunosorbent assays (ELISA) for the detection of mouse IgG and foot-and-mouth disease virus and no advantage was found to justify their use.
Abstract: Fluorogenic and chromogenic substrates were used in direct and trapping enzyme-linked immunosorbent assays (ELISA) for the detection of mouse IgG and foot-and-mouth disease virus (FMDV). The detection limits for both antigens were compared using different combinations of enzymes and substrates. Various times and concentrations of chemicals were used to obtain maximum sensitivity for both systems. Similar sensitivities were found using fluorogenic and chromogenic substrates. Tetramethyl benzidine substrate for horse-radish peroxidase enzyme conjugates was found to attain the highest sensitivity levels for chromogenic assays (0·12 ng IgG/ml and 1·0 ng/ml FMDV respectively), after 10 min incubation. Of the two fluorogenic enzyme/substrates studied, B-galactosidase was the most sensitive but required extended incubation times (2–3 h) as compared with chromogenic systems. Special microplates for fluoro-immunoassay (FIA) were compared with conventional microplates and no advantage was found to justify their use. An alkaline phosphatase anti-guinea-pig conjugate was used to confirm the equivalence of fluorogenic and chromogenic substrates in terms of sensitivity. A comparison of the amount of signal generated using various concentrations of enzyme in the absence of antigen was made for two different alkaline phosphatase conjugates to obtain theoretical sensitivity limits. One possible advantage of fluorogenic substrates is that high binding ratio can improve the confidence in discrimination of positive results.
TL;DR: The survival of four strains of Mycoplasma hyorhinis in stock solutions of trypsin was tested and the sensitivity of different cultural procedures for detecting mycoplasmas was compared, suggesting that mycplasmas may be present intrypsin as clumps, which deteriorate very slowly.
Abstract: The survival of four strains of Mycoplasma hyorhinis in stock solutions of trypsin was tested at 22, 4 and −15°C. Low (10 4 –10 5 cfu/ml) and high (10 6 –10 7 cfu/ml) initial concentrations of each strain were used, each was tested three times. A regular decrease of low and high concentrations (1 log in 10 and 20 min, respectively) was seen at 22°C. At 4°C the low concentrations showed a reduction of about 1 log/h, while apart from one strain high concentrations hardly decreased during the first 6 h and the survival time ranged from 24 to more than 30 h at the end of which there was a reduction of 4 logs. At −15°C low concentrations survived up to 1 week in only one of the three tests, high concentrations survived for more than 12 weeks (reduction 3 logs). These latter results suggest that mycoplasmas may be present in trypsin as clumps, which deteriorate very slowly. A study was also performed to compare the sensitivity of different cultural procedures for detecting mycoplasmas.
TL;DR: This article describes a new application of high performance liquid chromatography (HPLC), based on molecular sieving, for such an evaluation of bacterial capsular polysaccharides, which is rapid, accurate, reproducible, requires only very low amounts of product and presents good correlation with conventional gel permeation chromatography.
Abstract: Bacterial capsular polysaccharides are major virulence factors and some are used as vaccinal antigens. Their molecular size is an important physicochemical criterion which correlates with immunogenicity. This article describes a new application of high performance liquid chromatography (HPLC), based on molecular sieving, for such an evaluation. This HPLC method is rapid, accurate, reproducible, requires only very low amounts of product and presents good correlation with conventional gel permeation chromatography.
TL;DR: A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure antibody to bovine herpesvirus type 1 (BHV-1) in cattle sera and showed a good positive correlation between the two tests.
Abstract: A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure antibody to bovine herpesvirus type 1 (BHV-1) in cattle sera. The optical density produced from a single dilution of test serum was compared with a standard curve and the results were read and printed out from a computer interfaced to a multichannel ELISA reader. The printed results were expressed in ELISA units. The ELISA results obtained on 370 cattle sera were compared with those of the serum neutralisation test (SNT). An agreement of 90·5% was obtained when reciprocal SNT titres equal to or greater than 4 and IgG ELISA units equal to or greater than 50 were taken as indicative of a specific reaction. Of the 370 sera, 35 gave discrepant results of which 21 were SNT positive/IgG ELISA negative and 14 were SNT negative/IgG ELISA positive. When the SNT positive sera negative in the IgG ELISA were tested in an IgM ELISA, 19 were found to be positive. Thus, when the IgG and IgM ELISA results were combined the overall agreement between the ELISA and SNT increased to 95·7%. The IgG ELISA had a sensitivity of 82·4% and specificity of 94·4% relative to the SNT, whereas the combined IgG and IgM ELISA results gave a sensitivity and specificity of 98·3% and 94·4% respectively. There was a good positive correlation between the two tests ( r = 0·86).
TL;DR: The potency of individual lots of recombinant (r)TPA could only be estimated in International Units (IU) of TPA activity with the automated clot-lysis assay and the potency values obtained were in remarkably close agreement with the manufacturers' values.
Abstract: An assessment was made of two methods for determining the potency of tissue-type plasminogen activator (TPA). A chromogenic microtitre plate assay was established which contained TPA, plasminogen, a synthetic plasmin substrate (H-D-valyl-L-leucyl-L-lysyl-p-nitroaniline dihydrochloride, S2251) and any one of the following stimulators: native fibrinogen, enzymatic and chemical digests of fibrinogen, poly-D-lysine (PDL) and chemical derivatives of the latter. The chromogen assay was compared with an automated clot-lysis (turbidimetric) assay for sensitivity, reproducibility and validity for potency determination. Reference preparations of TPA were titrated in both assays: in the chromogen assay the dose-response curves were non-parallel, whereas parallelism was observed in the clot-lysis assay. Thus, the chromogen assay was restricted in its applicability and disqualified from any routine regulatory use. The potency of individual lots of recombinant (r)TPA could only be estimated in International Units (IU) of TPA activity with the automated clot-lysis assay and the potency values obtained (IU/vial) were in remarkably close agreement with the manufacturers' values.
TL;DR: In 9457 dairy farms located in an area with low prevalence of bovine leukemia virus (BLV) infection, bulk tank milk was examined to detect for the presence of antibodies using an ELISA test, used to establish decision rules in the event of a positive or doubtful result during mass screening.
Abstract: In 9457 dairy farms located in an area with low prevalence of bovine leukemia virus (BLV) infection, bulk tank milk was examined to detect for the presence of antibodies using an ELISA test. If the result was positive or doubtful, serum of all animals in the farm was tested and bulk tank milk was tested again five times every 8–12 days. The results were used to establish decision rules in the event of a positive or doubtful result during mass screening.
TL;DR: Gamma radiation is safer than chemical inactivation procedures and is a reliable and effective replacement for BPL in preparing diagnostic reagents.
Abstract: The objective of this study was to apply the pertinent findings from gamma inactivation of virus infectivity to the production of high quality diagnostic reagents. A Gammacell 220 (Atomic Energy of Canada, Ltd., Ottawa, Canada§) was used to subject 38 viruses grown in either susceptible tissue cultures or embryonated chicken eggs to various doses of gamma radiation from a cobalt-60 source. The radiation required to reduce viral infectivity was 0·42 to 3·7 megarads (Mrad). The effect of gamma treatment on the antigenic reactivity of reagents for the complement fixation (CF), hemagglutination (HA) and neuraminadase assays was determined. Influenza antigens inactivated with 1·7 Mrad displayed comparable potency, sensitivity, specificity and stability to those inactivated by standard procedures with beta-propiolactone (BPL). Significant inactivation of influenza N1 and B neuraminidase occurred with >2·4 Mrad radiation at temperatures above 4°C. All 38 viruses were inactivated, and CF or HA antigens were prepared successfully. Antigenic potency remained stable with all antigens for 3 years and with 83% after 5 years storage. Influenza HA antigens evaluated after 9 years of storage demonstrated 86% stability. Gamma radiation is safer than chemical inactivation procedures and is a reliable and effective replacement for BPL in preparing diagnostic reagents.
TL;DR: The authors have developed a simplified activity test for Tetanus Toxoids Adsorbed based on the comparison of antitoxin levels in mice 4 weeks after injection of a reference toxoid and of the vaccine to be tested and found close correlation in immunized mice between the titre of circulating antibodies and the survival/death response after challenge by tetanus toxin.
Abstract: The authors have developed a simplified activity test for Tetanus Toxoids Adsorbed, based on the comparison of antitoxin levels in mice 4 weeks after injection of a reference toxoid and of the vaccine to be tested. Titration of tetanus antitoxin is achieved by passive agglutination of turkey RBC sensitized by means of glutaraldehyde. After preliminary experiments establishing feasibility of this method, the authors have obtained reproducible and quantitative results. They observed an increase of the immune response by a booster and an immunostimulation when pertussis component is present. They have found close correlation in immunized mice between the titre of circulating antibodies and the survival/death response after challenge by tetanus toxin as done in the official control. This simplified method using a reduced number of animals, yields, nevertheless, quantified results with confidence limits. Thus it is suitable for laying down a norm and can in many cases take the place of the official potency test which is tedious, expensive and often criticized.
TL;DR: Two enzyme-linked antiglobulin tests (ELAT) for assessing anti-D IgG preparations are described; one is performed in tubes and the other in microtitre plates, and both give good agreement with results obtained in automated haemagglutination.
Abstract: Two enzyme-linked antiglobulin tests (ELAT) for assessing anti-D IgG preparations are described; one is performed in tubes and the other in microtitre plates. An anti-human IgG alkaline phosphatase conjugate and the substrate p-nitrophenyl phosphate are used. Both methods were sensitive and reproducible, with variations coefficients of 7·8 and 8·6% for enzyme immunoassay in tubes and microplates, respectively. The linear relationship between the amount of red cell-bound anti-D and the optical density shows that the method is suitable for quantitative studies. Results obtained by the two methods show a very good correlation (r = 0·99) in 12 of the 14 samples assayed, and both give good agreement with results obtained in automated haemagglutination. Since microtitre plate ELAT has numerous advantages over the tube method, it could provide an alternative method for assessing anti-D activity of specific IgG preparations in control laboratories.
TL;DR: An informal meeting was held at the National Institute for Biological Standards and Control (NIBSC) on 24 August 1989 between representatives from industry, the World Health Organisation and NIBSC to discuss issues related to the use of human diploid cells in virus vaccine production.
Abstract: An informal meeting was held at the National Institute for Biological Standards and Control (NIBSC), Potters Bar, U.K. on 24 August 1989 between representatives from industry, the World Health Organisation (WHO) and NIBSC to discuss issues related to the use of human diploid cells (HDC) in virus vaccine production. This record of the discussions and concensus views which emerged are provided to stimulate consideration by a wider audience. A list of participants is given in the Appendix.
TL;DR: The human CD4 positive cell lines JM, CCRF, CEM, U937, HL60 and THP-1 have been cleared of mycoplasma contamination and defined by DNA fingerprinting and cell surface phenotype marker analysis and are now available as a source of standardized cell lines for HIV related research.
Abstract: The human CD4 positive cell lines JM, CCRF, CEM, U937, HL60 and THP-1 have been cleared of mycoplasma contamination and defined by DNA fingerprinting and cell surface phenotype marker analysis. These cells have been banked and are now available as a source of standardized cell lines for HIV related research.
TL;DR: The genetic linkage results with GSS raise the possibility that sporadic CJD arises from a somatic mutation, and recent genetic linkage studies provide the best evidence, to date, that GSS is a genetic disorder in which infectious prions are produced.
Abstract: Over the last 40 years, an impressive array of data has been accumulated indicating that many features of scrapie, kuru, CreutzfeldMakob disease (CJD) and Gerstmann-Straussler syndrome (GSS) are not typical of infectious diseases. Futhermore, the last two decades have witnessed the collection of numerous experimental results which argue strongly that the scrapie agent is a novel pathogen. To distinguish the scrapie and CJD agents from viroids and viruses, the term ‘prion’ was introduced.’ Although scrapie can be transmitted to animals by inoculation with extracts of diseased tissue, whether infection features in the spread of natural scrapie is unclear.2-g Some investigators proposed that natural scrapie is a genetic disease and that its transmissibility is incidenta12s3 while others have argued vehemently that natural scrapie is an infectious disease.a*’ The tripartite manifestations of prion diseasesinfectious, sporadic and genetic-are illustrated by kuru, CJD and GSS, respective1y.l’ Consistent with some of the observations on natural scrapie noted above, GSS and familial CJD are the only known human diseases which appear to be both inherited and transmissible.11-15 Recent genetic linkage studies provide the best evidence, to date, that GSS is a genetic disorder in which infectious prions are produced.” An alternative hypothesis, for which there is little supporting data, is that prions are ubiquitous infectious pathogens causing CJD sporadically across the earth at a rate of one case per million population and GSS in all individuals carrying a mutation in one allele of their prion protein (PrP) gene.10-12,1618 Our genetic linkage results with GSS raise the possibility that sporadic CJD arises from a somatic mutation.5*1’T17 Over the past 5 years, a large amount of experimental data about the particles causing scrapie has accumulated. Most of the information has been confirmed, and much of it is widely accepted.lg At times, this confirmed body of information has been overshadowed by what appears to be controversy due to the diverse terminology used by different laboratories.
TL;DR: Anti-PT antibodies seem to play an important role in protection when animals are challenged intracerebrally, as is the case in the standard potency test for pertussis vaccine.
Abstract: The antibody response to filamentous haemagglutinin and pertussis toxin was studied in N:NIH mice vaccinated according to the WHO recommendations for potency test with the International Standard for Pertussis Vaccine (ISPV). Some of the vaccinated animals were challenged intracerebrally on day 14. All animals, whether challenged or not, were bled on days 7, 14, 21, 28 and 35 after immunization. The relationship between anti-PT and anti-FHA antibodies measured by ELISA and protection from intracerebral challenge was examined. All those mice with anti-PT titres on day 14 higher than 43 EU/ml survived challenge. No relationship was found between anti-FHA antibodies and survival. Anti-PT titres on day 14 below 43 EU/ml were related to the days of survival after challenge; a linear regression curve of y = 13 + 2·4 x , with a correlation coefficient r = 0·61 was found. Anti-PT antibodies seem to play an important role in protection when animals are challenged intracerebrally, as is the case in the standard potency test for pertussis vaccine.
TL;DR: Protection to intracerebral challenge with B. pertussis is T-cell dependent as is the humoral response to PT, FHA, OMP and pili, and the T-independent B-cell activation by the whole cell preparation is due to the presence of LPS.
Abstract: Athymic (nu/nu) and euthymic (+/nu) BALB/c mice were immunized with a whole cell pertussis vaccine or with an acellular vaccine which contained detoxified pertussis toxin (PT) and filamentous hemagglutinin (FHA). Only the euthymic mice were protected against intracerebral challenge with virulent Bordetella pertussis which implies involvement of T-cells. As a cell transfer from mice immunized with whole cell or acellular vaccine prior to the challenge did not protect naive euthymic recipients, cellular immunity seems to be non-protective as an effector mechanism. Mice could be protected passively against a challenge by administration of immune sera. Therefore, T-cell dependent humoral immune responses to B. pertussis appear to be crucial for protection. The humoral response was further studied with athymic and euthymic mice. In euthymic mice the whole cell vaccine induced antibodies to FHA, pililipopolysaccharides (LPS) and an outer membrane protein (OMP) preparation, whereas the acellular vaccine induced antibodies to PT, FHA and OMP. Both IgM and IgG could be detected. From the nude mice only those immunized with the whole cell vaccine showed an antibody response which consisted of low titres of IgM directed to LPS. Sera from both +/nu and nu/nu mice immunized with the whole cell vaccine were bactericidal in vitro. These data demonstrate that in the mouse model protection to intracerebral challenge with B. pertussis is T-cell dependent as is the humoral response to PT, FHA, OMP and pili. The T-independent B-cell activation by the whole cell preparation is due to the presence of LPS.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: A simple but specific, sensitive and reproducible latex agglutination inhibition assay for the determination of foetal calf sera in viral vaccines has been developed and standardized and the detection limit was at nanogram level.
Abstract: A simple but specific, sensitive and reproducible latex agglutination inhibition assay for the determination of foetal calf sera in viral vaccines has been developed and standardized. The detection limit was at nanogram level. The assay procedure requires two pipetting steps, a short centrifugation stage and the use of a spectrophotometer.
TL;DR: Extraction of palmitic acid from Tice BCG vaccine was not appreciably affected by lyophilization and the calculated dry cell mass values of freeze-dried vaccine samples correlated well with particle number, so this method may be useful in measuring BCG cell mass during all stages of vaccine manufacture and storage.
Abstract: The presence of additives and large cellular aggregates in freeze-dried BCG vaccines precludes accurate measurement of total cell content by traditional methods. The possibility that extraction and quantitation of a cell membrane fatty acid may provide a suitable means of cell mass determination was tested. The palmitic acid methyl ester peak area determined by gas chromatography was directly proportional to the wet weight of freshly grown Tice-, Pasteur-, and Glaxo-substrain BCG, as well as the dry weight of the ampoule contents after removal of soluble material. Extraction of palmitic acid from Tice BCG vaccine was not appreciably affected by lyophilization and the calculated dry cell mass values of freeze-dried vaccine samples correlated well with particle number. This method, therefore, may be useful in measuring BCG cell mass during all stages of vaccine manufacture and storage.