Agar Gel Double Diffusion Research Articles

Evaluation of Enzyme-Linked Immunosorbent Assay of Narrow- and Broad-Spectrum Anti-Adult Southern Pine Beetle Serum1

An enzyme-linked immunosorbent assay (ELISA) was conducted with broad-spectrum and specific anti-adult southern pine beetle sera against two-fold dilutions of 7.0 mg/ml antigens prepared from adult southern pine beetles, Dendroctonus frontalis Zimmerman; black turpentine beetles, Dendroctonus terebrans (oliver), and the engraver beetles, Ips avulsus (Eichhoff), Ips calligraphus (Germar), and Ips grandicollis (Eichhoff). Analysis of variance of absorbance values at all dilutions shows that broad-spectrum and specific sera can differentiate these genera and species. ELISA was markedly more sensitive than agar-gel double diffusion or counter-immunolectrophoresis with low-titer anti-insect sera.

Purification and partial characterization of an antigen from Fusarium oxysporum f. sp. vasinfectum that cross-reacts with antiserum to cotton ( Gossypium hirsutum) root antigens

Conidia of Fusarium oxysporum f. sp. vasinfectum contained an antigen that cross-reacted with antiserum to cotton root tissue antigens. In agar-gel double diffusion tests, one precipitin band was formed when antiserum to cotton antigens was reacted with crude fungal antigens, or when antiserum to crude fungal antigens was reacted with cotton antigens. The cross-reactive antigen from fungal conidia (CRA) was isolated, purified, and partially characterized. The CRA migrated as a single band in polyacrylamide- or agar-gel electrophoresis, and sedimented as a single band during analytical ultracentrifugation. It was antigenic in rabbits, and was a protein-carbohydrate complex. The possible significance of the CRA in host-parasite compatibility is discussed.

The tissue and cellular location of a major cross-reactive antigen shared by cotton and soil-borne fungal parasites

A major cross-reactive antigenic substance (CRA), common to cotton ( Gossypium hirsutum ) and certain fungal parasites of cotton roots, was isolated and purified to homogeneity from conidial cultures of Fusarium oxysporum f.sp. vasiufectum (F.o.v,). Agar gel double-diffusion tests indicated the prcscncc of CRA not only in F.o.v. and in cotton roots and seed but also in Thielaviopsis bosicola . Indirect staisung of antibodies using fluorescein isothiocyanate (FITC) indicated that in cross-sections of roots, cut near or just below the root hair zone, the CPA was concentrated mainiy around xylem elements, the endodernais and epidermal cells and was present throughout the cortex tissue. Protoplasts prepared from cross-sections of young cotton roots also contained the CPA which was concentrated in the region of the plasmalemma. Treatment of conidia and mycelia of F.o.v. with antiserum to cotton and using indirect staining with FITC indicated that the CRA was mainly present in hyphal tips and in patch-like aseas on conidia. The possible role of CRA in basic compatibility of host and parasite is discussed.

Serotypes among Neisseria meningitidis serogroups B and C strains isolated in Canada.

Antisera made to prototype serogroup B strains of Neisseria meningitidis were used to serotype, by agar gel double diffusion, 262 meningococcal serogroups B and C strains isolated in Canada. The strains included 93 from patients and 169 from carriers. Serotype 2 was associated with 39 of 75 (52%) of group B strains and 14 of 18 (77.8%) of group C strains isolated from patients. The group B strains were mainly (87.2%) serotype 2b, while the majority (92.2%) of group C strains was serotype 2a. Other serotypes (including a new provisional serotype) represented 25.3 and 5.5% of groups B and C strains, respectively. The new serotype accounted for 13% of the group B strains. Approximately 23% of the strains isolated from patients were nontypable. The distribution of serotype 2, nontype 2 (other serotypes), and nontypable strains isolated from carriers was 2.1, 36.6, and 61.3%, respectively, for group B meningococci and 22.2, 29.6, and 48.25, respectively, for group C meningococci. Serotype 11 was the most prominent of the strains isolated from carriers. Approximately 7% of all the strains were multiple serotypes. Serotype 2 is an important virulence marker associated with meningococcal groups B and C disease in Canada, with serotypes 2a and 2b being markedly associated with groups C and B meningococcal disease, respectively.

Grouping of beta-haemolytic streptococci by enzyme-linked immunosorbent assay.

A method of grouping beta-haemolytic streptococci serologically by enzyme-linked immunosorbent assay is described. A comparison of this method with double diffusion in agar gel showed complete correlation of results when highly absorbed grouping sera were used.

Antibodies specific to a deoxyribotrinucleotide sequence

Bovine serum albumin conjugates of two trinucleotides, dpTpTpA and dTpTpAp, were prepared by linking the trinucleotides through their end phosphates by the ‘carbodiimide method’. Antibodies were raised in rabbits by injecting the trinucleotide-bovine serum albumin conjugates. Analysis by double diffusion in agar gel, quantitative precipitin reaction and its inhibition by haptens showed clearly the presence of antibodies specific to the whole trinucleotide molecule. The titre of antibodies obtained by the trinucleotide-rabbit serum albumin conjugates with their respective antisera was approximately the same, indicating that linking the trinucleotide through either 5′ or 3′ phosphate does not have an appreciable effect on the titre of antibodies. The results also demonstrate that the nucleotide(s) away from the carrier protein is more immunodominant than the one linked directly to the protein.

The Trapping of Tymovirus Particles on Electron Microscope Grids by Adsorption and Serological Binding

Summary Many purified tymovirus particles are trapped on buffer-treated grids due to non-specific adsorption. Pre-treatment of grids with serum or addition of crude plant sap to virus preparations greatly inhibited non-specific adsorption. Specific serological binding on grids coated with homologous antiserum produced particle counts comparable to those achieved by non-specific adsorption on buffer-treated grids in the absence of plant sap. Specific serological binding was not influenced by crude sap of Nicotiana clevelandii. For both adsorption and serological binding there was a linear relationship between the log virus concentration and log particle counts at virus concentrations below approx. 10 µg/ml; at higher concentrations the grids were saturated. In the presence of excess virus, antisera at dilutions of less than 10-3 yielded lower particle counts than more dilute antisera. Heterologous reactions were detected only with viruses for which very close serological relationships had been found in the agar gel double-diffusion test. Heterologously bound virus was apparently not replaced by subsequently applied homologous virus. However, adsorbed bottom component virus particles could be replaced by top component particles of the same virus and vice versa.

Serological Studies on the Relationships of Some Isometric Viruses of Gramineae

SUMMARY Particles of 17 isolates of viruses from Gramineae were purified and the virus relationships assessed by different serological techniques. From the reaction end-points of antisera in agar gel double diffusion tests the serological differentiation indices of reciprocal tests were calculated. These indices were used to construct a scheme of serological interrelationship. Spur formation tests were used to distinguish between closely related, non-identical viruses. The isolates were shown to belong to four groups, one of which (the cocksfoot mild mosaicpanicum mosaic virus group) contains most of the isolates and can be subdivided into two main clusters (typified by cocksfoot mild mosaic and panicum mosaic viruses, respectively). Additional information was obtained by immunoelectrophoresis of the viruses with their homologous antisera and by the determination of the mol. wt. of the coat protein subunits using SDS-polyacrylamide gel electrophoresis. The data obtained by both methods agree with the above mentioned results, although some minor differences were found between the immunoelectrophoretic patterns of panicum mosaic virus and its relatives and also the three isolates of Cynosurus mottle virus. Reversed rocket electrophoresis did not provide any information of use in virus grouping. Brome mosaic virus did not react with the antisera to the 17 viruses, but tobacco mosaic virus reacted weakly with all the antisera and also with one of the antisera to brome mosaic virus. These and other recently published results are discussed.

Preparatory Immunodiffusion for Production of Specific Anti-Adult Southern Pine Beetle Serum

Preparatory immunodiffusion was used to produce an antiserum specific for the southern pine beetle, Dendroctonus frontalis (Zimm.), and the black turpentine beetle, D. terebrans (Olivier). Precipitin bands were excised from two-dimensional electroimmunodiffusion agar gel plates of crude antigen and antiserum, extracted in saline, emulsified with Freund's incomplete adjuvant, and injected subcutaneously near the inguinal lymph nodes of rabbits. The specific antiserum did not cross react with Ips avulsus Eichhoff, I. calligraphus Germar, or I. grandicollis (Eichhoff) when tested at high antigen concentrations by agar gel double diffusion, Crowle's template micro-double diffusion, two-dimensional electroimmunodiffusion, counter immunoelectrophoresis, or cellulose acetate membrane electrophoresis with immunofixation. The antiserum is genus specific in all tests, and species specific when run in the agar gel double diffusion test against a standard Dendroctonus antigen (7.0 mg protein/ ml).

Purification and characterization of potato leafroll virus

Potato leafroll virus (PLRV) was purified from infected potato ( Solanum tuberosum L.) with yields of 0.4–0.6 mg/kg of foliage. The virus sedimented as a single component of 127 S. An antiserum prepared against purified virus had a maximum titer of 1:1024 in agar gel double diffusion tests. PLRV had a buoyant density of 1.39 g/ml and an estimated nucleic acid content of 28%. The nucleic acid had a molecular weight of 2.0 × 10 6 and was degraded by RNase but not by DNase, indicating that the PLRV nucleic acid is RNA. The sedimentation coefficient of the RNA molecule was 34.5 S and after treatment with formaldehyde, 20.7 S. Dissociated coat protein migrated as a single band in polyacrylamide gel electrophoresis and the average subunit molecular weight was 26,300. PLRV should be considered a member of the luteovirus group.

Rapid serogroup identification of Neisseria meningitidis by using antiserum agar: Prevalence of serotypes in a disease-free military population.

Nasopharyngeal cultures from 414 Marines were plated directly onto antiserum agar containing the antibiotics vancomycin, colistin, and nystatin for meningococcal isolation and serogroup identification. Meningococci were isolated from 267 Marines, giving a carrier prevalence of 64.5%. A total of 58% of the isolates could be placed into serogroups; of these 22.3% were group B, 4.7% were group C, 25.7% were group Y, 24.3% were group W135, and 23.0% were group 29E. No serogroup A organisms were recovered. Serotyping by agar gel double diffusion was performed on 148 strains. More than 70% of these strains were nontypable, and the disease-associated serotype 2 was present only in two group Y isolates. The same 148 isolates were also classified by major outer membrane protein patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis pattern IV was most common among all serogroups. The data demonstrate the effective use of antiserum agar for meningococcal surveillance and document the frequency of specific serotypes and polyacrylamide gel electrophoresis types among carrier isolates obtained from a nonrecruit military population.

Evaluation of Immunodiffusion and Immunoelectrophoretic Tests Using a Broad Spectrum Anti-Adult Southern Pine Beetle Serum

A broad spectrum anti-adult southern pine beetle rabbit serum was evaluated in the following test systems: agar gel double diffusion, rocket immunoelectrophoresis, electroimmunodiffusion, crossed electroimmunodiffusion, tandem-crossed electroimmunodiffusion and counter immunoelectrophoresis. Counter immunoelectrophoresis was the most sensitive and rapid procedure, but crossed electroimmunodiffusion separated and quantified individual antigen- antibody systems. Possible laboratory and field applications of each procedure are discussed.

Immunological cross-reactivity of pathogenic Nocardia and related organisms

Aerobic actinomycetes associated with various clinical conditions in man and animals were studied for their antigenic relationships along with related organisms. Culture filtrate antigens obtained from 16 species were tested by antigen-antibody crossed-immunoelectrophoresis and by agar gel double diffusion against rabbit antibodies to Nocardia asteroides, N. brasiliensis and N. caviae. Weak cross-reactivity was detected among the three, Nocardia species and Micropolyspora brevicatena, but no cross-reactivity was noted with antigens from any of the other species studied.

Antigenic relationships among thermophilic actinomycetes.

Antigens from Micropolyspora faeni, Saccharomonospora viridis, Thermoactinomyces candidus, T. sacchari and T. vulgaris were prepared by growing them on dialysate of trypticase soy broth. Sera from rabbits immunized with these antigens were used to study cross-reactivity between thermophilic actinomycetes by antigen-antibody crossed immunoelectrophoresis and by agar gel double diffusion. Mi. faeni and S. viridis showed some degree of cross-reaction, but both failed to show any cross reactivity with Thermoactinomyces species. Antigens from Thermoactinomyces cross-reacted with members within the genus, but no reactivity against S. viridis or M. faeni antisera was detected. Hence, the presence of antibodies to several thermophilic actinomycetes in the sera of patients may be attributed to the exposure of the individual to different thermophilic actinomycetes rather than to the antigenic cross-reactivity between the organisms.

Attempt to detect bovine antibody against Fusobacterium necrophorum by the agar gel double diffusion test.

In order to detect bovine serum antibody against Fusobacterium necrophorum, serological investigation was made on bovine isolates of this species by the agar gel double diffusion test. Precipitin lines were observed between most of the HC1-heat extracts prepared from these strains and four anti-F. necrophorum sera. Precipitin was developed in rabbits inoculated experimentally with the organism, in the late stage of infection. Precipitin lines appeared between concentrated antigen prepared from the VPI 2891 strain and 16 of 23 sera from cattle affected with hepatic abscess, and between this antigen and 4 of 88 sera from healthy cattle.

Antiserum to myotoxin from prairie rattlesnake ( Crotalus viridis viridis) venom

C. L. Ownby, W. M. Woods and G. V. Odell. Antiserum to myotoxin from prairie rattlesnake ( Crotalus viridis viridis) venom. Toxicon 17, 373–380. 1979.—A myotoxic component was isolated from rattlesnake ( Crotalus viridis viridis) venom and an antibody to it was produced in rabbits. Gel-filtration and cation exchange chromatography were used to fractionate the crude venom and two components were shown to be locally myotoxic using an in vivo assay. One of these components, fraction 3 of the Sephadex cation exchange column, was shown to be homogeneous by electrophoresis. This purified myotoxin was injected into rabbits, and when the resulting antiserum was reacted with the myotoxin in agar-gel double-diffusion plates, one precipitin line was formed. Anti-myotoxin serum reacted with only 2 of 14 crude venoms forming one precipitin line to both C. v. viridis and C. durissus terrificus. The line which formed against these two crude venoms was identical to the one that formed against myotoxin indicating the presence in C. d. terrificus venom of a component immunologically very similar to myotoxin from C. v. viridis venom. It is postulated that this component might be crotamine, a known myotoxic component in C. d. terrificus venom. Antibodies to myotoxin could not be detected in Wyeth's polyvalent Crotalidae antivenin although at least one antibody to crude C. v. viridis venom was present. It is possible that antiserum to pure myotoxin could prevent the local myonecrosis induced by the pure myotoxin, crude C. v. viridis venom and perhaps by other crotalid venoms.

A Comparison Between four Serological Methods Used in the Identification of Streptococcus Agalactiae

Four serological methods used for the identification of group B streptococci were compared; the Lancefield precipitin method using double diffusion in agar gel, co-agglutination employing commercial reagents, immunofluorescence staining and counterimmunoelectrophoresis. In two experiments using 177 and 673 streptococcal isolates the results indicated that the Lancefield technique is not as sensitive as the other three tests. The counterimmunoelectrophoresis test is very sensitive to minimal quantities of antigen and/or antibody, but tends to detect cross reactions with group C antigens. The other two methods showed the same number of positive reactions, but fewer false positives. The reasons for preferring either the immunofluorescence or the co-agglutination technique for the routine identification of group B streptococci are discussed, together with the comments from other authors who have made similar comparisons.

The occurrence of an antigen associated with pregnancy in the ewe.

Precipitating antisera raised in calves against whole 14-16 day ovine conceptuses were rendered specific to antigens associated with ovine pregnancy by absorption with liver and kidney tissue from nonpregnant ewes. Using agar gel double diffusion, the pregnancy associated antigen was detected in 100% of samples of trophoblast tissue (n=30) and uterine tissue (n=31) and 94% of samples of uterine flushings (n�89) from pregnant ewes. Antigen was also detected in 27% of samples of uterine tissue (n=148), but not in uterine flushings (n=128) from nonpregnant ewes. The incidence of detection of antigen in uterine tissue from nonpregnant ewes was higher in ewes which were exhibiting cyclic ovarian activity (43%, n=83) than in nonpregnant ewes which were anestrous or prepubertal (7%, n=60). The results suggest that the incidence of detection of antigen in uterine tissue varies depending on the reproductive state of the animal.

Molecular distribution of allotypic determinants Lpm7 and Lpm8 of mink very high density α 2-lipoprotein

Agar gel double-diffusion experiments demonstrated that the allodeterminants Lpm7 and Lpm8, very high density lipoproteins of mink, reside on different molecules. Molecules possessing Lpm7 and Lpm8 have no other known Lpm-allodeterminants. These results extend our earlier observations on the distribution of Lpm1, Lpm2, Lpm3, Lpm4 and Lpm5 on lipoprotein molecules. It is concluded that Lpm-lipoproteins from mink sera are represented by molecules of several types. identical in their species-specific antigenic properties and each having a unique allodeterminant. The distribution patterns of Lpm7 and Lpm8 among molecules support the suggestion that monogenically inherited allogroups are controlled by closely-linked structural genes.

Evaluation of Methods to Detect Antibodies against Aspergillus fumigatus

Seventy-five sera, including sera from 23 patients with aspergillosis, 17 with tuberculosis, asthma or carcinoma, and 35 normal controls, were studied for antibody activity against Aspergillus fumigatus by agar gel double diffusion (DD), counterimmunoelectrophoresis (CIE), indirect hemagglutination (IHA) and indirect immunofluorescence (IF). Metabolic antigens produced from a selected strain of A. fumigatus were used for DD, CIE and IHA, while slide cultures of the same organism were used for IF. The results indicated that sera from the patients with aspergillosis had high IHA and IF titers, while only 91 and 87% were positive for CIE and DD methods, respectively. IHA was more sensitive than DD and CIE, and IF was equally sensitive but had false-positive reactions. CIE was simple and fast, but had false-negative and occasionally false-positive results.