Genetic Loci of Major Antigenic Protein Genes of Edwardsiella tarda

ABSTRACT Seven antigenic proteins of Edwardsiella tarda were identified by using a rabbit polyclonal antiserum. Four of these proteins also reacted with a Japanese flounder antiserum. The amino acid sequences had identity to lipoproteins, periplasmic proteins, and exported and secreted proteins with roles in transport of metabolites across the cell membrane, stress response, and motility. These genes and their products are useful for developing DNA or recombinant subunit vaccines to control edwardsiellosis.

An Immunoaffinity Column for the Determination of Peanut Protein in Chocolate

An immunoaffinity column was prepared from rabbit polyclonal antiserum for the determination of peanut protein from food matrixes. The anti-peanut immunoglobulin G was isolated from antiserum by affinity chromatography on a column coupled with peanut protein and then attached to an AminoLink gel. The column was applied to the determination of peanut protein in chocolate after extraction, immunoaffinity chromatography, and enzyme-linked immunosorbent assay (ELISA). Overall recoveries from chocolate spiked with 0.2–3.2 μg/g of peanut protein averaged 77% (range, 72–84%), and the minimum detection limit was 0.1 μg/g. Chromatography of extracts with the column improved detection limit and eliminated the matrix effect experienced with direct ELISA of chocolate extracts.

First Report of Banana Streak Badnavirus in Plantain Landraces in Southern Cameroon, Central Africa

Banana streak badnavirus (BSV) has been reported from Musa spp. in many parts of West Africa, including Benin, Côte d'Ivoire, Ghana, and Nigeria (1). Symptoms of BSV infection in Musa spp. are sometimes similar to and confused with those caused by cucumber mosaic virus (CMV). BSV is prevalent in areas of southern Nigeria bordering Cameroon, and the disease may also be present in other Central African countries. In June 1996, six leaf samples with viruslike yellow/chlorotic streak symptoms were collected from plantain in the four villages, Awae, M'Balmayo, Nkolfep, and Nkolfoulou, within a 60-km radius of Yaoundé, Cameroon's capital. The samples were indexed for BSV and CMV by both triple antibody sandwich indirect enzyme-linked immunosorbent assay (TAS-ELISA) and immunosorbent electron microscopy (ISEM) to ascertain the presence of these two viruses. The TAS-ELISA was performed with rabbit polyclonal antiserum (obtained from B. E. L. Lockhart, University of Minnesota) for trapping and mouse polyclonal antiserum (obtained from G. Thottappilly, IITA) for detection. Out of the six samples, one tested strongly positive (>×2 A405 of the healthy control) and four were weakly positive (<×2 but >×1.5 A405 of the healthy control) for BSV by TAS-ELISA. However, all six samples contained BSV particles when examined by ISEM with rabbit polyclonal antiserum (from B. E. L. Lockhart) for trapping. None of the samples tested positive for CMV. These results confirm that BSV is present in Cameroon and that BSV is likely to be the causal agent of the symptoms. Reference: (1) C. Pasberg-Gauhl et al. Plant Dis. 80:224, 1996.

High molecular weight forms of IGF-II ('big-IGF-II') released by Wilms' tumor cells

Insulin-like growth factor-II (IGF-II) is thought to play a critical role in the development of embryonic tumors such as Wilms' tumor. However, despite highly elevated IGF-II mRNA levels in tumors, IGF-II is not elevated in the serum of patients with Wilms' tumors. Recently high molecular weight forms of IGF-II ('big'- or pro-IGF-II) have been found to be produced by some tumors. In order to prove whether or not high molecular weight forms of IGF-II are produced by Wilms' tumor cells and secreted into the culture medium, we established Wilms' tumor cell lines. After column chromatography of the culture medium, IGF-II and pro-IGF-II concentrations were measured. For pro-IGF-II measurement we established a pro-IGF-II RIA using a rabbit polyclonal antiserum directed against amino acids 7-21 (E7-21) of the E-domain of pro-IGF-II. Gel electrophoresis and Western blotting with anti-IGF-II antibodies revealed a band at 7.5 kDa corresponding to fully processed IGF-II and bands between 10 and 20 kDa. Using pro-IGF-II antiserum, bands between 10 and 25 kDa were detected. We conclude that in vitro cultured Wilms' tumor cells produce and release various forms of 'big IGF-II' with molecular masses between 10 and 25 kDa. It remains uncertain whether these high molecular weight forms of IGF-II represent normal precursors of IGF-II or incorrectly processed IGF-II.

Immunohistochemical Detection of Haemophilus Parasuis Serovar 5 in Formalin-Fixed, Paraffin-Embedded Tissues of Experimentally Infected Swine

An avidin-biotin complex immunohistochemistry technique was developed to detect Haemophilus parasuis serovar 5 in experimentally infected 18-21-day-old conventional pigs, using a rabbit polyclonal antiserum. Seven of 10 intratracheally inoculated animals developed a low to medium degree of fibrinous polyserositis; meninges and pleura were the most severely affected areas. Haemophilus parasuis was recovered from 9 of 10 pigs; in 2 of them H. parasuis was isolated from tracheal swabs only. Positive immunohistochemistry results, mainly observed as free bacteria or bacteria within inflammatory cell cytoplasm in the fibrinopurulent exudate, were observed in 8 of 10 animals. Cross-reactivity with Actinobacillus pleuropneumoniae was detected but not with other gram-positive and gram-negative bacteria tested. This immunohistochemistry technique seemed to be at least as sensitive as microbiologic cultures and could be useful in studies of pathogenesis and retrospective diagnosis. However, cross-reactivity with A. pleuropneumoniae means that positive immunohistochemistry results in lung tissue from field cases would be dubious.

A glycoprotein specific to the amphids of Meloidogyne species

SUMMARYIndirect immunofluorescence studies using a rabbit polyclonal antiserum have been used to localize the presence of a 32 kDa glycoprotein in the region of the amphids of 2nd-stage juveniles of the root-knot nematode, Meloidogyne incognita. Similar immunoreactivity was also demonstrated in 5 other Meloidogyne species but was not found in representatives of 8 other nematode genera including the closely related cyst nematodes (Globodera and Heterodera). Immunoelectron microscopical studies have shown that the immunoreactivity in M. incognita is associated with the secretory material filling the amphidial channel and probably with the sheath cell.

The antigenicity of the carbohydrate moiety of an insect glycoprotein, honey-bee (Apis mellifera) venom phospholipase A2. The role of α1,3-fucosylation of the asparagine-bound N-acetylglucosamine

A rabbit polyclonal antiserum raised against honey-bee (Apis mellifera) venom phospholipase A2 (PLA2) contains antibodies that react exclusively with its glycosylated variants and cross-react with plant glycoproteins. The interaction of anti-(horseradish peroxidase) antiserum with PLA2 suggests the existence of a carbohydrate determinant common to both glycoproteins. E.l.i.s.a. binding and inhibition experiments, employing glycoproteins and glycopeptides of plant and animal origin with known N-glycan structures, in combination with chemical and enzymic deglycosylation, identified alpha 1,3-fucosylation of the asparagine-bound N-acetylglucosamine as the antigenic determinant. This fucose residue is present in the N-glycan of PLA2 and is frequently found in plant glycoproteins, whereas mammalian glycoproteins lack this modification.

Immunological studies on the rat peripheral-type benzodiazepine acceptor

Photoaffinity labelling of rat adrenal mitochondrial preparations with [3H]PK 14105 resulted in a single 3H-labelled band on SDS/PAGE gels with an apparent-molecular-mass peak of 18 kDa. This represents a polypeptide associated with the peripheral-type benzodiazepine-binding site. Solubilization of photoaffinity-labelled membranes with 6 M-guanidine hydrochloride, followed by gel filtration and reversed-phase h.p.l.c. of the solubilized material, resulted in the purification to homogeneity of the [3H]PK 14105-labelled polypeptide. This purified polypeptide was used to raise a rabbit polyclonal antiserum which recognized the immunogen in pure form and exclusively recognized it in a crude preparation of rat adrenal mitochondria as judged by immunoblotting. By the same analysis the antiserum identified the corresponding polypeptide from rat kidney and salivary gland, demonstrating its cross-reactivity. Subsequent immunocytochemical studies localized the polypeptide to the cortex of the adrenal gland, the distal tubules of kidney, the interstitial cells of testis, the biliary epithelium of liver and the choroid plexus and ependyma cells within the brain. This selective localization within organs may provide an insight into the physiological role of the peripheral-type benzodiazepine acceptor.

Enzyme-linked Immunosorbent Assay of Versicolorin A and Related Aflatoxin Biosynthetic Precursors

An immunochemical approach is described for the detection of versicolorin (VA) and other aflatoxin precursors in Aspergillus parasiticus cultures. VA was purified from A. parasiticus ATCC 36537 cultures by semipreparative high performance liquid chromatography and confirmed by mass spectrometry and ultraviolet (UV) absorption. To be rendered immunogenic, VA was converted to a hemiacetal and conjugated to bovine serum albumin (BSA) by reductive alkylation. Rabbit polyclonal antiserum prepared against the VA hemiacetal-BSA conjugate was employed in a competitive ELISA using VA hemiacetal-horseradish peroxidase conjugate as the marker ligand. Based on the amount of VA analogue required to inhibit binding by 50% in competitive ELISA, cross-reactivity relative to VA for VA hemiacetal, averufanin, averantin, norsolorinic acid, averufin, sterigmatocystin, and aflatoxin B1 were 106, 85, 7, 6, 2, &lt;1, and &lt;1%, respectively. The ELISA was used to monitor enhanced production of VA equivalents by A. parasiticus ATCC 36537 in a modified culture procedure. The VA antibody should be extremely useful in the biochemical and genetic investigation of aflatoxin biosynthesis.