Direct Immunodiffusion Research Articles

Verification of immunofluorescence colony‐staining of Erwinia carotovora subsp. atroseptica by reisolation and immunodiffusion or PCR

In this study, the reliability and efficiency of three procedures for verification of IFC‐positive colonies of Erwinia carotovora subsp. atroseptica were compared: (1) PCR amplification, (2) reisolation on a non‐selective medium (trypticase soy agar) followed by direct immunodiffusion (TSA‐DID), developed for isolation of target and cross‐reacting bacteria, and (3) reisolation on a selective medium (crystal‐violet pectate) and characterization of selected isolates with Ouchterlony double diffusion (DLCVP‐ODD), developed for isolation of pectinolytic Erwinia spp. The reliability of a PCR amplification procedure for characterization of E.c. atroseptica was evaluated. Specific amplification products could be produced from DNA of all 187 European strains of the bacterium, while no amplification products were obtained from DNA of four distinctive serological groups of bacteria cross‐reacting with antibodies against E.c. atroseptica, nor from DNA of randomly selected saprophytic bacteria isolated from potato peel extracts. All 60 immunofluorescent‐positive target colonies from a potato peel extract with added E.c. atroseptica tested were positive by PCR compared with 68 and 72% successful determinations by TSA‐DID and DLCVP‐ODD, respectively. PCR enabled verification of fluorescent colonies from IFC preparations of naturally infected seed lots with an efficiency of 93%, compared with 48 and 71% successful determinations by TSA‐DID and DLCVP‐ODD, respectively. It is concluded that PCR is useful for routine confirmation of the identity of fluorescent colonies in IFC.

New approach in detecting phytopathogenic bacteria by combined immunoisolation and immunoidentification assays1

Antibodies coated onto a suitable solid phase, e. g. polystyrene beads, rods for inoculating agar plates, or petri dishes, enable selective trapping of homologous bacteria and of immunologically related bacteria by immunoaffinity. After washing away unbound organisms, the bound organisms are desorbed and plated on a suitable medium. Selective immunoisolation has been demonstrated for various phytopathogenic bacteria including Corynebacterium michiganense ssp. michiganense, Erwinia chrysanthemi, E. carotovora ssp. atroseptica, Xanthomonas campestris pv. begoniae and Pseudomonas syringae pv. phaseolicola. Three serological methods are described for additional rapid immunoidentification of colonies directly on agar plates and for screening for cross‐reacting microorganisms: (a) direct immunodiffusion on dilution plates, (b) agar mixed‐antibody assay, (c) fluorescent antibody colony staining. Schemes are presented for increasing the reliability and sensitivity of sample screening for quality indexing of plant material, and for efficient screening and isolation of possible cross‐reacting microorganisms to enable production of more specific antisera.

Identification of serotypes of Haemophilus pleuropneumoniae by counterimmunoelectrophoresis.

Counterimmunoelectrophoresis, direct immunofluorescence and immunodiffusion procedures were used to serotype 15 strains of Haemophilus pleuropneumoniae isolated from the respiratory tract of pigs in southern Brazil. Antigens were prepared by extracting cultures with a saline solution or by the phenol-water method. Antisera were prepared in rabbits against serotypes 1, 2, 3 and 5. Thirteen of the isolates were type 5 and two were type 3. No differences were observed between the results obtained in serotyping with counter immunoelectrophoresis and direct immunodiffusion, but both procedures were significantly better than immunodiffusion except with the saline extracted antigen. Counterimmunoelectrophoresis was quicker, more sensitive and more easily performed than the other techniques.

Amyloid P-component(C1t) and complement: Lack of physical or functional relationship

Studies were carried out to determine if Clt, which is identical to the P-component of amyloid (AP), plays a role in Cl structure or function, or modulates the activity of Cl or its sub-units. Analyses of Cl in serum by a direct immunodiffusion method showed conclusively that AP was not a constituent of Cl. Further, AP did not interact physically with Cl or with Clq, Clr or Cls individually or in any combination, nor did it activate precursor Cl. Although some preparations contained Cl r-like hemolytic activity, this was due to the variable presence of a contaminating serine esterase, with properties suggesting that the contaminant was Clr. AP was also completely devoid of ability to provide Clr-like activity to sera genetically deficient in Clr. Immunodepletion studies showed that AP was not essential for hemolytic activity. As reported by others, Ap formed a calcium-dependent bond with agarose. In addition, AP interacted with IgG and aggregated in the presence of calcium. These studies cumulatively demonstrate that AP is not a sub-component of Cl and further show that AP does not interact with, or modulate the activity of Cl or any of its sub-units. The present experiments also furnish alternative explanations for earlier studies which led to the conclusion that AP was a fourth Cl subcomponent.

Immunological Comparison of Various Human Pregnancy-Associated Plasma Proteins

Direct immunodiffusion comparison with specific antisera demonstrated that all of the four pregnancy-associated plasma proteins (PAPPs) described in our laboratory are distinct from the pregnancy zone protein (von Schoultz); alpha2-pregnoglobulin (Berne); pregnancy-associated alpha2-glycoprotein (SP3) (Bohn); new serum alpha2-macroglobulin (Stimson); PAG (Horne); pregnancy-associated alpha2-globulin (Kasukawa); Pal (McLaren), and Xh protein (Dunston). All the latter proved to be immunologically idnetical to each other, and apparently represent the same protein described under different names. It was confirmed that none of the PAPPs is immunochemically related to placental alkaline phosphatase. PAPP-A, PAPP-C, and the pregnancy zone protein, but not HPL (PAPP-D), showed a decreased anodic mobility when treated with neuraminidase; their reactivities with antibody were essentially unaffected by the ezyme, however. Certain detergents had no effect on the immunological reactivities of the PAPPs and the pregnancy zone protein in whole plasma, while butanol and desoxycholate partially, and urea, SDS and cetylpyridinium largely inactivated them.

Direct immunodiffusion technique for Ig quantitation

The present study established a direct immunodiffusion precipitin method for the determination of immunoglobulin levels. Agar gel was solidified on a glass slide and the desired number of wells were made. Five μl of test samples were placed in each well of agar gel and the serum proteins were allowed to diffuse for a period of adequate time. An aliquot size of Whatman No.1 filter paper which has been impregnated with antiserum was placed on the agar gel and kept in a moist chamber for 1 to 3 h depending on the immune reaction. The precipitin ring was clearly observed in an agar plate and the diameter of the ring could be measured before and after staining. The present method is economical, using a small quantity of antiserum when compared with the previously described methods and the results can be obtained rapidly with a sharply edged ring.