The effect of strain differences on the assay of rabies virus glycoprotein by single radial immunodiffusion

Antigenic differences between rabies virus strains used for vaccine manufacture can be demonstrated using monoclonal antibodies. We have shown that these differences are sufficiently large to affect the potency values of vaccines measured in single radial immunodiffusion (SRD) assays if the reference and test vaccines are antigenically heterologous. The production of reagents for use in SRD assays for each strain of rabies virus should be considered.     

Evaluation of the single radial-immunodiffusion assay for measuring the glycoprotein content of rabies vaccines

The glycoprotein content of rabies vaccines containing the Pitman-Moore strain of rabies virus was measured by the single radial immunodiffusion assay and correlated with vaccine potency. The variability of this assay was 6.3% for a single vaccine lot tested over a one-year period. Using sera prepared against rabies virus glycoprotein from different strains of virus, the assay gave different values. These differences could be eliminated by using a homologous vaccine strain as an internal reference. Single radial-immunodiffusion values for Pitman-Moore vaccines correlated with the manufacturers' NIH potency assay, but required a mathematical transformation to convert values from one assay to the other. Single radial-immunodiffusion values for Street Alabama Dufferin and Flury-LEP vaccines did not correlate with NIH values. Modification of the single radial immunodiffusion technique and the feasibility of using this assay for the determination of rabies vaccine potency are discussed.     

Changes in virulence, M protein and IgG Fc receptor activity in a type 12 group A streptococcal strain during mouse passages

A type 12 group A strain (1800) was passaged serially through mice 25 times. The ability to servive in normal human blood dropped from a growth index of 52 after the first passage to 1 after four passages. After 14 passages the growth index increased again and stabilized above 30. The virulence for mice increased from a LD100 of 10(8) colony forming units (CFU) to 10-100 CFU after 7 passages and then remained constant. The Mqw antigen disappeared after 4 passages as tested by immunodiffusion, electroimmunoassay and indirect bactericidal tests. Three antisera, raised in rabbits against strains originally belonging to types M3, M12 and M46 but devoid of type antigens after mouse passages showed high bactericidal indices against the 1800 strain after 14 or more passages on mice. Anti-type M1 serum was also found bactericidal for the passaged strains. The IgG Fc-receptor activity of the strain isolated after each mouse passage was tested in hemagglutination experiments with human red blood cells coated with "incomplete" anti-Rh and hot hydrochloric acid extracts of the strains. The capacity to agglutinate "Ripley"-coated cells increased gradually during the first 12 passages and subsequently the titres of the extracts stabilized between 1:160 and 1:320. The HUN coat, useful for detection of the G3m (5) maraker gave titraes increasing with the number of passages while the titres for IgG1 coats kept at 1:4 or below. On background of these results, the possible role of the IgG Fc-receptor as a virulence factor is discussed.     

Factors affecting the assay of histone H1 and polylysine by binding of Coomassie blue G

Rabbit hepatic microsomal suspensions were bound directly to nitrocellulose sheets using a "Hybridot" apparatus to ensure uniformity. Cytochrome P-450, form 2, was then detected by a modified immunochemical method wherein the nitrocellulose paper was incubated sequentially with antibody to form 2 for 1 h at 25 degrees C, rabbit anti-goat immunoglobulin G (IgG) at a 1:100 dilution for 15 min at 25 degrees C, goat peroxidase-antiperoxidase at a 1:2000 dilution for 15 min at 25 degrees C, and 3,3'-diaminobenzidine at 0.3 mg/ml plus 0.002% hydrogen peroxide for 30 min at 25 degrees C. These conditions, as opposed to those previously published, yielded less background staining. The density of the stain, scanned with a soft laser (Zeineh), increased linearly from 2 to 100 fmol for purified form 2. Cytochrome P-450, form 2, was detected and quantitated in microsomal samples containing 0.1 to 0.5 and 0.02 to 0.05 micrograms protein for preparations from untreated and phenobarbital-treated rabbits, respectively. The results agreed with those obtained by Western blotting and single radial immunodiffusion. This assay is more sensitive than either Western blotting or radial immunodiffusion and has significant advantages such as ease of operation, increased sample numbers, and reduced interference from extraneous proteins.     

Immunological studies of the anticryptococcal factor of normal human serum

Preliminary studies have shown a very high inhibitory activity in the alpha₂ and gamma zone of human serum towards the growth of Cryptococcus neoformans. These findings are now corroborated by single radial immunodiffusion tests, which showed the some loss of IgA and IgM globulins and of the other three globulin fractions (ceruloplasmin, alpha₂ macroglobulin and alpha₂ HS glycoprotein) which migrate in the alpha₂ zone. The data was obtained by single radial immunodiffusion tests. The losses were not statistically significant however. No change in the immunoglobulin content of the sera kept for 6 days in contact with a heat-killed suspension of C. neoformans was noted. These findings suggest, that the inhibitory activity of the normal human serum on the in-vitro growth of C. neoformans is due to the above mentioned globulin fractions and not to a single specific factor.     

Toxoplasma gondii: growth in ovine fetal kidney cell cultures

Serial, in vitro passage of Toxoplasma gondii (Rh strain) was successfully performed in a cell line derived from ovine fetal kidney cells. Invasion of this parasite into the kidney cells was easily discernible 1 hr after inoculation. The subsequent proliferation of the parasite was followed in the cytoplasm of the kidney cells. Very active endodyogeny and rosette formations, as many as 13 in a cell, were observed in the cytoplasm of the kidney cells 48 hr postinoculation. After 96 hr of incubation, the parasite population had increased about 132-fold. The virulence of T. gondii against mice was not attenuated after 2 years of in vitro growth which represented 100 serial passages through the kidney cell cultures. Although no "exotoxin" was produced by T. gondii grown in vitro, a Toxoplasma sp. agar gel immunodiffusion test antigen was isolated from the cell-free supernatant fluid of the kidney cell cultures which was identical to an antigen isolated from "toxogenic" organisms harvested from infected mice.     

Genetic and biochemical characterization of D-arabinose dehydrogenase from Neurospora crassa.

D-Arabinose dehydrogenase has been purified to homogeneity from wild-type Neurospora crassa 74-A (FGSC 262) and from two colonial mutants, col-15a (FGSC 1391) and col-16a (FGSC 1349), found to contain more of the enzyme. The enzymes were characterized by measurement of several kinetic and physicochemical parameters. The enzymes were the same in all characteristics studied thus far. Immunological studied performed with enzyme preparations from the three strains showed antigenic identity and indicated that those colonial strains contain more normal enzyme, rather than the usual amount of an altered "improved" enzyme. Quantitation of the enzyme in crude extracts, performed by single radial immunodiffusion, showed that the colonial strains have twice the level of enzyme as the wild-type strain. Genetic characterization, performed by analysis of meiotic products, heterokaryosis, and reversions, indicated that the difference in D-arabinose dehydrogenase activity detected among the three strains is probably determined by one gene. The genetic control, structural or regulatory of this enzyme activity is different from that determining the morphological alterations exhibited by mutant strains carrying the col-15 or col-16 gene.     

Quantitative Measurement of Precipitating Antibodies in Streptococcal Grouping Antisera by the Single Radial Immunodiffusion Technique

A comparative study was made of the single radial immunodiffusion test and the classical quantitative precipitin test for determining the amount of precipitable antibodies present in streptococcal groups A and C antisera. The potency of 21 group A and 54 group C antisera was determined by both methods; purified group-specific carbohydrates were used as antigens. The coefficient of correlation between the results from the two methods was 0.976 for group A antisera and 0.946 for group C antisera. When the concentration of antigen, the volume of antiserum used, and the depth of the antigen-agar mixture are kept constant, the diameter of the precipitin disc is directly related to the concentration of precipitable antibodies present in the antiserum. The use of the radial immunodiffusion test for evaluating and standardizing streptococcal grouping antisera is discussed as well as the advantages and disadvantages of using a concentrated vaccine for producing these antisera.     

Immunochemical quantitation of dextran (α-1,6-glucan) traces in sucrose solutions by a rapid and sensitive automated nephelometric procedure

Dextran was quantitated in sucrose preparations using a polymer-enhanced immunonephelometric technique. The procedure is rapid, automated, and specific and has high sensitivity. The method shows excellent precision and good correlation with a reversed single radial immunodiffusion method. The dextran content of different saccharide preparations has been analyzed and the results are reported.     

Purification and immunochemical properties of human low molecular weight kininogen.

A low molecular weight (LMW) kininogen was isolated from pooled human serum by chromatography on DEAE-Sephadex A-50, CM-Sephadex C-50, Sephadex G-150, and Sephadex G-100. It was shown to be homogeneous by ultracentrifugation, polyacrylamide gel electrophoresis, and immunoelectrophoresis. The sedimentation coefficient, S020,W, of purified LMW kininogen was 3.85 s, and its molecular weight was determined to be 78,000 by Sephadex G-100 gel-filtration. The LMW kininogen contained 79.3% protein, 8.0% hexose, 3.9% hexosamine, and 4.9% sialic acid. In order to determine the immunochemical properties of LMW kininogen, specific antiserum was prepared in rabbits. The antigenic determinant of LMW kininogen was not related to the sialic acid and kinin moieties in the kininogen molecule, but could not be distinguished from that of high molecular weight (HMW) kininogen. In the quantitative single radial immunodiffusion test, a sialic acid-free LMW kininogen reacted to a greater extent with the antiserum than the native LMW kininogen. The kininogen level in human serum was estimated by single radial immunodiffusion. The antiserum cross-reacted with monkey serum, but not with sera from dogs, rats, and mice, horses, pigs, guinea pigs, oxen, and rabbits.     

Purification of a mannose/glucose-specific hemagglutinin/lectin from a Vibrio cholerae O1 strain

A cell-associated mannose/glucose-specific hemagglutinin (MSHA) has been purified from a strain of Vibrio cholerae O1 by chromatography on a chitin column followed by affinity purification on Sephadex G100. The purified protein gave a single stained band of 40 kDa by SDS-PAGE, exhibited high affinity towards D-mannose and D-glucose but was resistant to L-fucose and N-acetyl-D-glucosamine. The purified MSHA was revealed as a globular form of protein under electron microscope. In immunodiffusion tests the purified MSHA produced a single precipitin band against homologous antisera and antisera raised against the whole cell bacteria without any reactivity towards antisera raised against the purified N-acetyl-D-glucosamine-specific lectin of the same bacterial strain. Immunogold labelling confirmed the location of hemagglutinin on the surface of the bacteria. Purified MSHA reacted strongly with sera from convalescent cholera patients in immunodiffusion tests.     

Diagnosis of Corynebacterium pyogenes infection in pigs by immunodiffusion test with protease antigen.

A protease antigen was prepared from the culture supernatant of Corynebacterium pyogenes by concentrating with a flash evaporator and ultrafiltration. It was adjusted to the concentration of 32 units by the single radial immunodiffusion with a tentative standard serum. In the immunodiffusion test, the antigen of 4 units reacted enough with sera having an antibody titer ranging from 1 to 128. As a result, it was decided that the antigen of 4 units should be used in the immunodiffusion test for the detection of protease antibody. By the immunodiffusion test, protease antibody was demonstrated in about 35% of 443 sera from pigs collected at random. The antibody titer showed the distribution of 2 peaks. The summits of the two peaks were seen at 4 and 32 of antibody titer, respectively. The valley between the two peaks was seen at 16 of titer. From the result, a diagnostic criterion of the immunodiffusion test was decided provisionally as follows: above 16 of antibody titer is positive, 1 to 8 suspect, and less than 1 negative. On the other hand, protease antibody was demonstrated in sera from 13 of 14 pigs carrying abscesses from which C. pyogenes had been isolated. Its titer was 8 (in 2 pigs), 16 (in 1), 32 (in 3), 64 (in 6), and 128 (in 1). From these results, it was proposed that the immunodiffusion test with protease antigen be used for the diagnosis of C. pyogenes infection in pigs.     

Serum IgG, IgM, and C3 levels in Schistosoma japonicum infected rabbits

New Zealand white rabbits were infected with 250 or 500 Schistosoma japonicum cercariae of Philippine-Leyte strain. Following the initial pre-infection bleed, blood was collected 2, 4, 6, 8, 11, 14, 17, 20, 23, 26, 29, and 32 weeks post-infection, and IgG, IgM, and C3 levels were assayed in the serum samples by single radial immunodiffusion. IgG and IgM achieved peak levels at 14 weeks post-infection and began to decrease in concentration by 26 weeks. At 32 weeks, some of the infected rabbits exhibited significantly reduced IgG and IgM levels which approached the concentrations assayed in the control animals. The infected animals demonstrated significantly elevated C3 levels (p less than or equal to 0.001) during the 6th to 26th weeks post-infection, with the exception of the 20th week.     

The nature of mutation in Krabbe disease.

Galactosylceramide beta-galactosidase cross reacting material was demonstrated in brain, liver, and skin fibroblasts from patients with Krabbe disease. The mutant enzyme was antigenically identical to the normal enzyme and exhibited similar electrophoretic mobility. Normal quantities of the catalytically deficient enzyme were measured in the patients' tissues by a sensitive single radial immunodiffusion assay, indicating that the mutation is in structural gene for the enzyme protein.     

Weed competition in drilled summer cabbage

A single immunodiffusion drop test has been developed for the serological detection of lily symptomless virus in lilies. The presence of virus is demonstrated by intra-gel precipitation in drops of an antiserum-agar mixture which are contiguous with droplets of pyrrolidine-treated leaf extracts covered with paraffin oil. Single immunodiffusion drop tests require c. twelve times less antiserum than radial diffusion plate tests. The results of virus detection by this technique agree closely with those obtained by electron-microscopical screening of negatively stained leaf extracts.     

The identification of diphtheria, tetanus and pertussis vaccines by single radial and double immunodiffusion techniques

The identification tests for adsorbed diphtheria, tetanus and pertussis vaccines, which are required by the European Pharmacopoeia to be undertaken in animals, may be replaced by precipitation tests, for instance in agaros gels. Such in vitro tests eliminate the use of animals and are less expensive and time-consuming. The single radial immunodiffusion technique is a suitable semiquantitative test, while the double diffusion test is necessary for the investigation of complete or partial identity. The precipitates obtained in the single radial diffusion tests and in double diffusion tests with diphtheria toxoid were visible without staining; those obtained in the double diffusion tests with tetanus toxoid were weaker and staining was sometimes needed.     

Affinity chromatography of α-amylase from Bacillus licheniformis

An affinity chromatographic method with a novel eluant from Bacillus licheniformis is described. α-amylase was bound to starch, starch-celite, starch-Sepharose columns and the bound α-amylase was rapidly eluted with 2% (w/v) white dextrin. The binding capacity of α-amylase to starch column is 380 μmol/g of starch. The purified enzyme showed a single polypeptide on SDS-polyacrylamide gel electrophoresis with a molecular weight of 58 kD. The specificity of purified enzyme was confirmed by immunodiffusion, immunoelectrophoresis. Single radial immunodiffusion and western blotting studies analyzed the synthesis of enzyme at different time points.     

Inactivation of SAM-Methyltransferase is the Mechanism of Attenuation of a Historic Louse Borne Typhus Vaccine Strain

Louse borne typhus (also called epidemic typhus) was one of man''s major scourges, and epidemics of the disease can be reignited when social, economic, or political systems are disrupted. The fear of a bioterrorist attack using the etiologic agent of typhus, Rickettsia prowazekii, was a reality. An attenuated typhus vaccine, R. prowazekii Madrid E strain, was observed to revert to virulence as demonstrated by isolation of the virulent revertant Evir strain from animals which were inoculated with Madrid E strain. The mechanism of the mutation in R. prowazekii that affects the virulence of the vaccine was not known. We sequenced the genome of the virulent revertant Evir strain and compared its genome sequence with the genome sequences of its parental strain, Madrid E. We found that only a single nucleotide in the entire genome was different between the vaccine strain Madrid E and its virulent revertant strain Evir. The mutation is a single nucleotide insertion in the methyltransferase gene (also known as PR028) in the vaccine strain that inactivated the gene. We also confirmed that the vaccine strain E did not cause fever in guinea pigs and the virulent revertant strain Evir caused fever in guinea pigs. We concluded that a single nucleotide insertion in the methyltransferase gene of R. prowazekii attenuated the R. prowazekii vaccine strain E. This suggested that an irreversible insertion or deletion mutation in the methyl transferase gene of R. prowazekii is required for Madrid E to be considered a safe vaccine.     

The enhancin gene: One of the genetic determinants of population variation in baculoviral virulence

It was established that the virulence of the North American baculovirus strain LdMNPV-45 is almost two orders of magnitude higher than the virulence of the Asian strain LdMNPV-27 and does not depend on the test host population (gypsy moth). The Asian strain carries deletions in bro-p and vef-1 genes (82 and 91%, respectively). In accordance with the published data, the product of the latter can greatly increase the virulence of the virus. This result indicates that the population polymorphism of the virulence of baculoviruses can be explained by the vef-1 gene deletion.     

用免疫单扩散法测定低pH静注丙球lgG含量及其影响因素初探

本文报道了低 pH静注丙球IgG含量测定免疫单扩散法的建立 ,并对其影响因素进行了初步试验。结果表明 ,该试验方法较为稳定 ,简单易行。在低pH静注丙球IgG含量检测时 ,进口Sigma琼脂糖效果优于国产琼脂糖(P <0 .0 1) ,国产 (东海 )琼脂糖不适于IgG含量的定量检测 ;应先将静注丙球pH调至中性后再进行IgG含量测定(P <0 .0 5 ) ;样品中含有的 10 %麦芽糖对试验结果无影响 (P >0 .0 5 )。