Commercial Polyclonal Antisera Research Articles

First Report of Cucumber mosaic virus on Melon in Bosnia and Herzegovina.

During July 2012, field-grown melon plants (Cucumis melo L.) with symptoms of mosaic, chlorotic mottling, and vein banding as well as blistering and leaf malformation were observed in one field in the locality of Kladari (municipality of Doboj, Bosnia and Herzegovina). Disease incidence was estimated at 60%. A total of 20 symptomatic plants were collected and tested with double-antibody sandwich (DAS)-ELISA using commercial polyclonal antisera (Bioreba AG, Reinach, Switzerland) against four the most commonly reported melon viruses: Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), and Papaya ringspot virus (PRSV) (1,3). Commercial positive and negative controls were included in each assay. Only CMV was detected serologically in all screened melon samples. Sap from an ELISA-positive sample (162-12) was mechanically inoculated to test plants using 0.01 M phosphate buffer (pH 7.0). The virus caused necrotic local lesions on Chenopodium amaranticolor 5 days after inoculation, while mild to severe mosaic was observed on Nicotiana rustica, N. glutinosa, N. tabacum 'Samsun,' Cucurbita pepo 'Ezra F1,' and Cucumis melo 'Ananas' 10 to 14 days post-inoculation. All five inoculated plants of each experimental host were DAS-ELISA positive for CMV. The presence of CMV in all naturally and mechanically infected plants was further verified by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and used as template in RT-PCR. RT-PCR was carried out with the One-Step RT-PCR Kit (Qiagen) using primer pair CMVCPfwd and CMVCPrev (4), amplifying the entire coat protein (CP) gene and part of 3'- and 5'-UTRs of CMV RNA 3. Total RNAs obtained from the Serbian CMV isolate from Cucurbita pepo 'Olinka' (GenBank Accession No. HM065510) and healthy melon leaves were used as positive and negative controls, respectively. An amplicon of the correct predicted size (871 bp) was obtained from all naturally and mechanically infected plants as well as from positive control, but not from healthy tissues. The amplified product derived from isolate 162-12 was purified with QIAquick PCR Purification Kit (Qiagen) and sequenced directly using the same primer pair as in RT-PCR (KC559757). Multiple sequence alignment of the 162-12 isolate CP sequence with those available in GenBank, conducted with MEGA5 software, revealed that melon isolate from Bosnia and Herzegovina showed the highest nucleotide identity of 99.7% (100% amino acid identity) with eight CMV isolates originating from various hosts from Serbia (GQ340670), Spain (AJ829770 and 76, AM183119), the United States (U20668, D10538), Australia (U22821), and France (X16386). Despite the fact that CMV is well established in majority of Mediterranean countries and represents an important threat for many agriculture crops, including pepper in Bosnia and Herzegovina (2), to our knowledge, this is the first report of CMV infecting melon in Bosnia and Herzegovina. Melon popularity as well as production value has been rising rapidly and the presence of CMV may have a drastic economic impact on production of this crop in Bosnia and Herzegovina.

First Report of Iris yellow spot virus Infecting Onion in Bosnia and Herzegovina.

In July 2012, a survey was conducted to determine the presence of tospoviruses in Bosnia and Herzegovina, symptoms resembling those caused by Iris yellow spot virus (IYSV; genus Tospovirus, family Bunyaviridae) were observed in an onion (Allium cepa) seed crop in the Gornji Karajzovci locality (Region of Banja Luka). Symptoms included chlorotic to necrotic, straw-colored, spindle- and diamond-shaped lesions, variable in size and randomly distributed on the leaves and particularly on the scapes. Later the lesions enlarged and coalesced, causing scape breakage. Affected plants occurred throughout the field and disease incidence was estimated at 20%. Symptomatic plants were sampled and assayed by double-antibody sandwich (DAS)-ELISA test using commercial polyclonal antisera (Bioreba AG, Reinach, Switzerland) against IYSV and two other tospoviruses, Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV). Commercial positive and negative controls were included in each test. IYSV was detected serologically in 19 of 20 screened samples and none of the samples tested positive for TSWV or INSV. The virus was mechanically transmitted from an ELISA-positive sample (302-12) to five of each Petunia × hybrida and Nicotiana benthamiana using chilled 0.01 M phosphate buffer (pH 7) containing 0.1% sodium sulfite (1). All inoculated P. × hybrida showed local necrotic spots, while N. benthamiana developed mild mosaic 4 and 10 days post-inoculation, respectively. However, difficulties were encountered in reproducing the disease symptoms on mechanically inoculated onion plants corroborating a previous study (2). Serological findings were verified with reverse transcription (RT)-PCR. Total RNAs from all naturally infected onion plants as well as mechanically infected N. benthamiana plants were extracted with the RNease Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with One-Step RT-PCR Kit (Qiagen) using IYSV-specific primers IYSV56U/IYSV917L (3), designed to amplify an 896-bp fragment of the S RNA which includes whole nucleocapsid (N) gene. Total RNAs from Serbian IYSV isolate from onion (GenBank Accession No. EU586203) and from healthy onion plants were used as positive and negative controls, respectively. An amplicon of the expected size was obtained from each of the plants assayed as well as from positive control, but not from the negative control. The amplified products derived from onion isolate 302-12 was purified (QIAquick PCR Purification Kit, Qiagen), sequenced directly (JX861126), and compared with known IYSV isolates. Sequence analysis of the complete N gene, conducted with MEGA5 software (4), revealed the highest nucleotide identity of 99.5% (100% amino acid identity) with IYSV onion isolate (DQ658242) from Texas. To our knowledge, this is the first report of IYSV in Bosnia and Herzegovina. Onion is an important and traditionally grown vegetable crop in Bosnia and Herzegovina and the presence of IYSV could represent an important constraint to onion and other susceptible host production. The discovery of IYSV on onion should prompt more detailed surveys, thorough inspections and subsequent testing to establish the distribution and incidence of IYSV in Bosnia and Herzegovina.

First Report of Tomato spotted wilt virus on Gloxinia in Bosnia and Herzegovina.

In June and July 2012, symptoms resembling those caused by a tospovirus infection were observed on the greenhouse-grown gloxinia (Sinningia speciosa Benth. and Hook.) in the Lijevče polje, in the vicinity of Banja Luka (Bosnia and Herzegovina). Infected plants exhibited chlorotic ring spots and chlorotic and necrotic patterns followed by necrosis and distortion of leaves. Disease symptom incidence was estimated at 30% out of 400 inspected plants. Symptomatic leaves were collected and tested by double-antibody sandwich (DAS)-ELISA test using commercial polyclonal antisera (Bioreba AG, Reinach, Switzerland) for two of the most important tospoviruses in the greenhouse production of ornamentals: Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) (2). TSWV was detected serologically in 27 out of 30 tested gloxinia samples, and all were negative for INSV. Symptomatic leaves of five selected ELISA-positive gloxinia plants were separately ground in chilled 0.01 M phosphate buffer (pH 7) containing 0.1% w/v sodium sulphite and were mechanically inoculated on five plants of Petunia × hybrida. All inoculated plants produced typical symptoms of TSWV (1), necrotic spots on inoculated leaves in 2 to 5 days post-inoculation. For further confirmation of TSWV infection, total RNAs were extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from all 27 infected gloxinia plants and tested by reverse transcription (RT)-PCR assay. A 738-bp fragment of TSWV nucleocapsid (N) gene was amplified with One-Step RT-PCR Kit (Qiagen) using primer pairs TSWV CP-f and TSWV CP-r (4). Total RNAs from Serbian tobacco TSWV isolate (GenBank Accession No. GQ373173) and RNA extract from healthy gloxinia plants were used as positive and negative controls, respectively. Amplicons of the expected size were obtained from all 27 naturally infected gloxinia plants, while no amplification products were obtained from the healthy control. After the purification with QIAquick PCR Purification Kit (Qiagen), the RT-PCR product obtained from one selected isolate 160-12 was sequenced directly in both directions and submitted to GenBank (JX468079). Sequence analysis of the partial N gene, conducted by MEGA5 software (3), from isolate 160-12 showed the highest nucleotide identity of 99.7% (100% amino acid identity) with eight pepper isolates of TSWV from Spain (FR693229, FR693231, FR693152-153, FR693078, FR693081, FR693089, and FR693092). To our knowledge, this is the first report on the occurrence of TSWV in Bosnia and Herzegovina. The presence of this harmful pathogen into a new area could have a serious threat to intensive and increasing production of ornamentals and numerous other TSWV susceptible species in Bosnia and Herzegovina. The discovery of TSWV on gloxinia should prompt more surveys, thorough inspections, and subsequent testing of other TSWV susceptible plants cultivated in Bosnia and Herzegovina.

Salmonella collected from nest run cart shelves in commercial shell egg processing facilities

Salmonella, a member of the bacterial family Enterobacteriaceae, may be recovered from foods and processing facilities. High levels of Enterobacteriaceae in the processing plant environment can be an indication of inadequate sanitation. This experiment was designed to determine if nest run egg carts serve as reservoirs for Salmonella. Eggs that are produced by hens not housed in buildings connected to the processing plant are referred to as nest run. Many of these eggs are transported to a central processing facility before they are washed, graded, and packed. Two plants in the Southeastern United States were sampled; one was a mixed operation and the other was an off-line operation. On each of 3 visits, 5 shelves on each of 5 carts were sampled (n = 25/visit). A 12 × 12 cm area on each shelf was swabbed with a sterile gauze pad moistened with PBS and transported on ice back to the laboratory. Each swab was preenriched in buffered peptone at 37°C for 24 h, selectively enriched using TT and Rappaport-Vassiliadis broth at 42°C overnight, then plated onto brilliant green sulfa and XLT-4 incubated at 37°C for 24 h. Presumptive colonies were transferred to lysine iron agar and triple sugar iron slants for 24 h at 37°C. Isolates with presumptive reactions were confirmed using commercial polyclonal antisera. After initial confirmation, serogrouping was performed using commercial antisera. Mixed-operation swab samples were 12% positive for Salmonella, whereas off-line samples were 36% positive for Salmonella; isolates were confirmed as serogroups B, C1, and C2. Kauffman-White serotyping was performed by a contract laboratory. Serotypes (n = 30) recovered were Anatum, Heidelberg, Infantis, Kentucky, Mbandanka, and Typhimurium. This work demonstrated that nest run egg carts may serve as reservoirs for Salmonella in the shell egg processing environment.

Identification of Maize dwarf mosaic virus in Maize in Poland.

From 2005 to 2007 in Southern Wielkopolska, Lower Silesia, and Malopolska regions, maize (Zea mays) plants showing leaf mosaic and stunting symptoms were found. ELISA tests using commercial polyclonal antisera against Maize dwarf mosaic virus (MDMV) obtained from Bioreba (Basel, Switzerland) and Loewe (Munich, Germany) gave positive results in 71 samples. However, the ELISA response for symptomatic plants, in most cases, was low, with OD values ranging from 0.05 to 0.18. Therefore, only eight plants with relatively high virus concentration were chosen for further identification assays. Examination of leaf extracts with an electron microscope revealed the presence of potyvirus-like particles. Symptomatic leaves were positive for MDMV by using immunosorbent electron microscopy (ISEM) with antiserum raised against the Spanish isolate of MDMV (supplied as positive MDMV control from A. Achon, Centre Vdl-Irta, Lleida, Spain). A set of test plants, including sweet corn, dent corn, sorghum (Sorghum vulgare), and true millet (Panicum miliaceum), were mechanically inoculated with extracts from symptomatic plants in 0.05 M phosphate buffer plus 1% β-mercaptoethanol. Inoculated plants developed symptoms typical of MDMV in 2 to 5 weeks (1,2). For further investigations, three virus isolates were chosen. To confirm the identification of MDMV, reverse transcription (RT)-PCR was performed with total RNA isolated from infected plants with primers 3MDF (5' GAT GAG TTR AAY GTY TAT GCA CGA C 3'), a forward primer in the 3' region of NIb gene and either 1MDR (5' RTG CAT RAT TTG TCT GAA AGT TGG 3') or 3MDR (5' ACC AVA CCA TYA TWC CAC TC 3'), reverse primers in the 3' region of the coat protein gene (A. Zare, Shiraz University, personal communication). 3MDF corresponds to nucleotides 8306 to 8330, 3MDR is complementary to nucleotides 8791 to 8813, and 1MDR is complementary to nucleotides 8917 to 8939 of the MDMV genome (GenBank Accession No. AJ001691). The RT-PCR products obtained were analyzed by agarose gel electrophoresis. Amplicons of the expected sizes (635 and 560 bp) were obtained with RNA from symptomatic plants, but not from asymptomatic plants. The sequence of the 576-bp PCR product was deposited in GenBank (Accession No. EU240460). In alignments done with BlastN ( www.ncbi.nlm.nih.gov/blast ), the highest nucleotide sequence identities were 99% with Spanish MDMV isolates ("SP" AM110758, "SP" AJ416645, and "S1" AJ416635), 91% with the Hungarian isolate "Sc/H, sweet corn" AJ542536, 90% with "MDMV-A" U07216, and 87% with an Israeli MDMV (AF395135). On the basis of these findings, the virus isolated from diseased maize plants was identified as MDMV. The significance of MDMV detection is noteworthy because maize has become an important crop in Poland in recent years and acreage is increasing systematically.

Identification of Proteomic Signatures of Exposure to Marine Pollutants in Mussels (Mytilus edulis)

Bivalves and especially mussels are very good indicators of marine and estuarine pollution, and so they have been widely used in biomonitoring programs all around the world. However, traditional single parameter biomarkers face the problem of high sensitivity to biotic and abiotic factors. In our study, digestive gland peroxisome-enriched fractions of Mytilus edulis (L., 1758) were analyzed by DIGE and MS. We identified several proteomic signatures associated with the exposure to several marine pollutants (diallyl phthalate, PBDE-47, and bisphenol-A). Animals collected from North Atlantic Sea were exposed to the contaminants independently under controlled laboratory conditions. One hundred and eleven spots showed a significant increase or decrease in protein abundance in the two-dimensional electrophoresis maps from the groups exposed to pollutants. We obtained a unique protein expression signature of exposure to each of those chemical compounds. Moreover a set of proteins composed a proteomic signature in common to the three independent exposures. It is remarkable that the principal component analysis of these spots showed a discernible separation between groups, and so did the hierarchical clustering into four classes. The 14 proteins identified by MS participate in alpha- and beta-oxidation pathways, xenobiotic and amino acid metabolism, cell signaling, oxyradical metabolism, peroxisomal assembly, respiration, and the cytoskeleton. Our results suggest that proteomic signatures could become a valuable tool to monitor the presence of pollutants in field experiments where a mixture of pollutants is often present. Further studies on the identified proteins could provide crucial information to understand possible mechanisms of toxicity of single xenobiotics or mixtures of them in marine ecosystems.

Nontoxigenic Strains of Pseudomonas syringae pv. phaseolicola Are a Main Cause of Halo Blight of Beans in Spain and Escape Current Detection Methods

ABSTRACT From a collection of 152 pseudomonads isolated from diseased beans in Spain, 138 (91%) of the strains were identified as Pseudomonas syringae pv. phaseolicola and the rest as P. syringae pv. syringae. The P. syringae pv. phaseolicola strains produced typical water-soaked lesions on bean pods, although 95 of them did not produce phaseolotoxin in vitro. Ninety-four of these isolates did not produce the expected 0.5-kb product after polymerase chain reaction (PCR) amplification using primers specific for open reading frame (ORF) 6 of the phaseolotoxin (tox) gene cluster and did not contain DNA homologous to ORF 6 in Southern hybridization experiments. To our knowledge, this is the first report of the widespread occurrence in the field of strains of P. syringae pv. phaseolicola lacking the tox cluster, which contrasts sharply with the general belief that Tox(+) isolates are the only ones with epidemiological importance. Additionally, the tox(-) isolates were not specifically detected by a commercial polyclonal antisera in an enzyme-linked immunosorbent assay. Accordingly, it is possible that the certification of seed lots as free of the pathogen cannot be reliably done in Spain, or in any other country where tox(-) strains might occur frequently, using current PCR or serological protocols. The amplification of three avirulence genes by PCR allowed us to make predictions of the P. syringae pv. phaseolicola race structure, as confirmed by plant assays. Six races (races 1, 2, 5, 6, 7, and 9) were identified, with race 7 being the most prevalent (46.1%) followed by races 6 (21.3%) and 1 (9.0%). All the tox(-) isolates contained gene avrPphF, typical of races 1, 5, 7, and 9.

Correction: Corrigendum: IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA

Nature 415, 92–96 (2002). In this Letter, the commercial polyclonal antiserum used to detect endogenous XBP-1 was sc-7160 (Santa Cruz Biotechnology) and not sc-8015, as erroneously stated. The antibody sc-8015 is a mouse monoclonal and, in our hands, is not useful in detecting the endogenous proteinby immunoblotting.

Immunolocalisation of oestrogen receptor beta in human tissues.

Oestrogens exert their actions via specific nuclear protein receptors that are members of the steroid/thyroid receptor superfamily of transcription factors. Recently, a second oestrogen receptor (ERbeta) has been cloned, and using reverse transcription-PCR and immunohistochemistry it has been shown to have a wide tissue distribution in the rat that is distinct from the classical oestrogen receptor, ERalpha. Using commercial polyclonal antisera against peptides specific to human ERbeta, we have determined the sites of ERbeta expression in archival and formalin-fixed human tissue and compared its expression with that of ERalpha. ERbeta was localised to the cell nuclei of a wide range of normal adult human tissues including ovary, Fallopian tube, uterus, lung, kidney, brain, heart, prostate and testis. In the ovary, ERbeta was present in multiple cell types including granulosa cells in small, medium and large follicles, theca and corpora lutea, whereas ERalpha was weakly expressed in the nuclei of granulosa cells, but not in the theca nor in the copora lutea. In the endometrium, both ERalpha and ERbeta were observed in luminal epithelial cells and in the nuclei of stromal cells but, significantly, ERbeta was weak or absent from endometrial glandular epithelia. Epithelial cells in most male tissues including the prostate, the urothelium and muscle layers of the bladder, and Sertoli cells in the testis, were also immunopositive for ERbeta. Significant ERbeta immunoreactivity was detected in most areas of the brain, with the exception of the hippocampus - a tissue that stained positively for ERalpha. In conclusion, the almost ubiquitous immunohistochemical localisation of ERbeta indicates that ERbeta may play a major role in the mediation of oestrogen action. The differential expression of ERalpha and ERbeta in some of these tissues suggests a more complex control mechanism in oestrogenic potential than originally envisioned.

First Report of Banana Streak Virus Infecting Sugarcane and Arrowroot in Colombia

We have recently reported on the presence of banana streak virus (BSV) affecting plantains (Musa spp.) in Colombia (2). BSV is serologically related to sugarcane bacilliform virus and has been found to be transmitted by the pink mealybug (Saccharicoccus sacchari) from sugarcane to banana (1). In the vicinity of affected plantain crops in the localities of Andes (Antioquia) and Montenegro (Quindio), we observed sugarcane (Saccharum officinarum L.) plants with chlorotic streaks on their leaves, as well as arrowroot (Canna edulis Ker-Gawl.) plants with mild mosaic symptoms. The foliar tissue of symptomatic plants of these two species was tested for BSV and cucumber mosaic virus (CMV) by double antibody sandwich-enzyme-linked immunosorbent assay with commercial polyclonal antisera (Agdia Inc., Elkhart, IN). BSV was detected in samples of both plant species, whereas CMV was not detected in either one. Immunosorbent electron microscopy analysis of BSV-infected, symptomatic, foliar tissue of sugarcane showed the presence of viral-like bacilliform particles measuring approximately 150 × 30 nm, typical of BSV. This is the first report of BSV infecting Saccharum officinarum in Colombia and the first report of Canna edulis as a host for this virus.

Primer Reporte del Virus del Rayado del Banano (BSV) Afectando Plantaciones de Plátano (Musa AAB Simmonds), Caña de azúcar (Sacharum officinarum) y Achira (Canna edulis) en Colombia

<p>En noviembre de 1995, en los Municipios de Andes, Venecia e Hispania (Antioquia), La Tebaida y Montenegro (Quindío), se observaron hojas de plantas del don Dominico-Hartón (Musa AAB Simmonds) con rayas cloróticas y necróticas, síntomas que caracterizan la enfermedad del rayado del banano. En ocasiones las plantas presentaban síntomas de mosaico en el pseudotallo y el engrosamiento y/o rompimiento de la base del mismo. Además, en las cercanías de las plantaciones de plátano de los Municipios de Andes y Montenegro, se observaron respectivamente plantas de caña de azúcar (Saccharum officinarum) con síntomas de clorosis y de achira (Canna edulis) con síntomas de mosaico leve en sus hojas. Muestras de tejido foliar de éstas tres especies de plantas fueron analizadas para detectar la presencia del badnavirus del rayado del banano (BSV) y del virus del mosaico del pepino (CMV), mediante la prueba serológica DAS-ELISA, empleando anticuerpos policlonales comerciales (AGDIA Inc., Elkhart, IN). En el caso del plátano, en la región de Antioquia únicamente se detectó el BSV en el so% de las plantas sintomáticas analizadas, mientras que en el Quindío, el 6oo/o de las plantas estuvieron infectadas simultáneamente por BSV y CMV. El BSV se detectó también en muestras de tejido foliar de caña de azúcar y achira, pero en ningún caso resultaron positivas para el CMV, según la prueba DAS-ELISA. El análisis mediante microscopía electrónica de immunoabsorbancia (ISEM) del tejido foliar de plátano y caña de azúcar infectado, indicó la presencia de partículas baciliformes típicas del BSV de aproximadamente 30 x 110 nm. Hasta donde conocemos, este es el primer reporte sobre la presencia del BSV afectando plátano, caña de azúcar y achira en Colombia y es la primera vez que se reporta a la achira como hospedero de éste virus en el mundo.</p><p><strong><br /></strong></p><p><strong>First Report of Banana Streak Virus (BSV) Infecting Plantain (Musa AAB Simmonds), Sugar cane (Sacharumofficinarum) and Achira (Canna edulis) in Colombia</strong></p><p>Viruslike symptoms of yellow striate mosaic to necrotic streaks were observed on plantain leaves of the cultivar Dominico-Hartón (Musa AAB Simmonds) in the municipalities of Andes, Venecia and Hispania (Antioquia) and Tebaida and Montenegro (Quindío), Colombia. Symptoms sometimes included mosaic and swelling at the base of the pseudostem. Furthermore, in the neighborhood of the plantain crops, at the localities of Andes and Montenegro, it was observed respectively plants of sugarcane (Saccharum officinarum L.) with chlorotic foliar tissue and of achira (Canna edulis L.) with symptoms of mild mosaic in their leaves. The foliar tissue of symptomatic plants of these three species, was tested for banana streak virus (BSV ) and cucumber mosaic virus (CMV ) by double antibody sandwich enzime-linked immunosorbent assay (DAS-ELISA) with commercial polyclonal antisera (Agdia Inc., Elkhart, IN). BSV was detected in samples of all plant species. In Plantain, so% of the examined plants from the Antioquia region were infected by BSV, but not CMV; whereas, at the Quindío region, 6o% of the plants were simultaneously infected by both viruses, as detected by DAS- ELISA. CMV was not detected in foliar tissue of either sugarcane or achira plants. Immunosorbent electron microscopy analysis (ISEM) of BSV infected foliar tissue of plantain and sugarcane, showed the presence of viral bacilliform particles measuring ca. 30 x 110 nm, typical of BSV. Up to our knowledge, this is the first report of BSV infecting Musa spp., Saccharum officinarum and Canna edulis in Colombia, and the first time that Canna edulis is reported as a host for this virus.</p>

Development of an ELISA for quantification of human protein S in cell culture fluids using commercial polyclonal antisera.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure protein S antigen released into cell culture fluids. We used readily available commercial polyclonal antisera to develop the assay. This assay was sensitive with a detection limit of about 0.086 ng/ml. Between-assay precision (coefficient of variation) at levels of 0.2, 1.1, and 13.9 ng/ml was 14%, 15%, and 11% respectively. Specificity and accuracy were demonstrated from the use of: 1) culture fluids from 3-primary endothelial cell cultures and 7-cell lines known to constitutively produce protein S; 2) 2-cell lines not synthesizing protein S; and 3) from selected samples of normal and protein S deficient plasma. The ELISA described here was about 12-fold more sensitive and 40-fold more cost effective when compared to a commercial ELISA kit. Thus the assay provided a sensitive, specific, precise and economical method useful for the measurement of the nanogram amounts of protein S commonly encountered in cell culture fluids.

Evidence that a 550,000-dalton cartilage matrix glycoprotein is a chondrocyte membrane-associated protein closely related to ceruloplasmin.

Cartilage matrix glycoprotein (CMGP) is a disulfide-bonded 550,000-dalton protein that is synthesized by chondrocytes and ciliary epithelial cells. We have purified the protein from bovine and porcine articular cartilage and have sequenced two peptides, which both have significant homology with human ceruloplasmin, a copper-binding oxidase. Immunolocation analysis indicates that a commercial polyclonal antiserum to human ceruloplasmin reacts with bovine cartilage CMGP. Chelating columns made with copper bind CMGP from bovine cartilage extracts. CMGP is present in bovine chondrocyte membrane preparations purified from sucrose density gradients. Oligonucleotide probes have been synthesized based on the published sequence of the 3'-untranslated region and a portion of the C terminus of human ceruloplasmin and have been used to amplify a cDNA fragment from bovine cartilage and human liver libraries. CMGP demonstrates oxidase activity towards p-phenylenediamine similar to that of ceruloplasmin. These studies suggest that CMGP is closely related to, if not identical with, ceruloplasmin. It is possible that CMGP may be involved in metal transport into and/or within the chondrocyte.

MON-150, a versatile monoclonal antibody against involucrin: characterization and applications.

A monoclonal antibody, designated MON-150, was found serendipitously to react strongly with the granular layer of normal human epidermis and with the upper spinous layers of psoriatic epidermis. From analysis by flow cytometry of cultured human keratinocytes, it appeared that the percentage of MON-150-positive cells strongly increased when the cells reached confluence and the growth fraction declined. To identify the antigen recognized by MON-150, a lysate of human keratinocytes was subjected to affinity chromatography using a MON-150 Sepharose column. This yielded a single protein of approximately 350 kDa as measured on Superose 6 FPLC gel permeation chromatography using non-denaturing conditions. In Western blot analysis under denaturing and reducing conditions, a 140-kDa protein was detected. The subcellular localization and the molecular weight of the antigen recognized by MON-150 suggested that the antigen involved might be involucrin. This was confirmed using a commercial polyclonal antiserum against involucrin. We conclude that MON-150 is a new, versatile antibody against human involucrin.

Immunoturbidimetric assay for estimating free light chains of immunoglobulins in urine and serum.

An immunoturbidimetric assay for the assessment of free kappa and lambda light chains of immunoglobulins was developed using a commercial polyclonal antiserum with reactivity towards epitopes on the light chains, which are not expressed when they are bound to heavy chains. The assay, on a centrifugal analyser, is simple and rapid. The limit of detection is 5 mg/l of free light chain, with an assay range of 5-120 mg/l, intrabatch precisions from 1.5-6.4%, and interbatch precisions from 6.5-8.9%. The assay was only slightly less sensitive than colloidal gold staining of cellulose acetate electrophoreses for the detection of Bence-Jones protein in urine. For the serial monitoring of response to chemotherapy in patients with myeloma, the assay correlated well with serum paraprotein estimates obtained by densitometric scanning of Ponceau stained cellulose acetate electrophoreses, but not with serum beta-2 microglobulin measurements, even after correction for the effects of creatinine. These assays may prove to be of use for the monitoring of tumour response in the treatment of Bence-Jones myeloma.

NON-SPECIFIC MESANGIAL STAINING WITH ANTIBODIES AGAINST CYTOMEGALOVIRUS IN IMMUNOGLOBULIN-A NEPHROPATHY

The glomerular staining of a commercial polyclonal antiserum against cytomegalovirus (CMV) and of a mixture of 13 monoclonal anti-CMV antibodies in twelve patients with immunoglobulin-A (IgA) nephropathy was examined. The polyclonal antiserum stained the mesangium in all patients. The CMV staining coincided only partially with that of IgA in five patients and the staining distributions were discordant in six. The monoclonal antibodies stained mesangium in only one patient. Serum levels of IgA and IgG anti-CMV antibodies did not differ between patients and controls. In western blot analysis, the polyclonal antiserum reacted with three proteins in uninfected human fibroblasts. Absorption of the antiserum with uninfected fibroblasts reduced the reactivity on western blot analysis and eliminated glomerular staining in five of six renal biopsy samples. It is concluded that the previously reported binding of anti-CMV antibodies to glomeruli from patients with IgA nephropathy may be the result of contaminating antibodies to human antigens.

Immunohistologic Analysis of a Case of Metastatic Prostate Cancer: Method Considerations and Interpretive Pitfalls

AbstractMorphologic analysis of a needle biopsy of the prostate was suggestive but not diagnostic of neoplasia. Subsequent cytologic examination of a pelvic soft tissue mass was suggestive of a poorly differentiated adenocarcinoma. Immunocytochemical studies with the two most commonly used markers for prostate carcinoma, prostate specific acid phosphatase (PSAP) and prostate-specific epithelial antigen (PSA), confirmed the prostatic origin of the pelvic mass. We concluded that it is practical to use commercial polyclonal antisera for PSAP and PSA detection, but we do not recommend the use of similar grade reagents for detection of carcinoembryonic antigen (CEA). Pitfalls related to use of certain immunological reagents and techniques as well as misinterpretation of results due to nonspecific sequestration of serum proteins are discussed. (The J Histotechnol 11: 55,1988.)

A screening assay for monoclonal antibodies based on the availability of a polyclonal antiserum

A double-sandwich enzyme-linked immunospecific assay (ELISA) has been developed for screening hybridoma clones for monoclonal antibodies against human α-fetoprotein (AFP). The assay uses commercial polyclonal antiserum to bind α-fetoprotein from biological fluids, and the complex then binds the specific monoclonal antibodies in hybridoma culture supematants. The sandwich is completed with commercial peroxidase-linked anti-mouse immunoglobulin. In exploring the variables of this system, it was found that the quality of the polyclonal anti-α-fetoprotein antiserum was the most critical factor. The assay functioned with as little as 10 ng α-fetoprotein from cord serum or amniotic fluid. A variety of commercial peroxidase-linked second antibodies was satisfactory, and both IgG and IgA class monoclonals were detected. Comparison of the ELISA with a standard radioimmunoassay showed the former to perform very adequately as a general hybridoma screening assay.