Vaccination of African catfish with Vibrio anguillarum O2: II. Oral intubation with vaccine-containing pellets

The aim of this study was to investigate the success of oral vaccination application in African catfish using Vibrio anguillarum O2 bacterins. The antigen uptake was followed by competitive enzyme‐linked immunosorbent assay (ELISA). Serum antibody response was measured using an indirect ELISA. Several in vivo administration methods were investigated. Intraperitoneal injection gave the highest absorption rate, with high antibody levels in the systemic circulation. Oral intubation of bacterin‐layered pellets resulted in low antigen uptake and low antibody levels. The addition of absorption enhancers increased the serum antigen levels. An enteric coating applied on the pellets containing vaccine did not improve the immune response.     

Oral vaccination of African catfish with Vibrio anguillarum O2: effect on antigen uptake and immune response by absorption enhancers in lag time coated pellets

The impact on antigen uptake and antibody response by the addition of absorption enhancers to Vibrio anguillarum O2 antigen was studied in oral vaccination trials of African catfish (Clarias gariepinus). Oral vaccination was achieved by feeding lag time coated pellets. The lag time coat prevents premature release of the encapsulated vaccine in the tank, before ingestion of the pellets by the fish. To monitor the antigen uptake, a competitive ELISA was used. The antibody response was measured using an indirect ELISA. Feeding of bacterin-layered pellets without absorption enhancers resulted in a rather low antigen uptake and antibody levels. The addition of absorption enhancers such as sodium salicylate, sodium caprate and vitamin E TPGS increased the serum antigen levels and specific antibody levels in the systemic circulation. Skin mucus antibody levels were higher after oral vaccination compared to the IP and control group. The addition of absorption enhancers in the oral groups further increased the antibody levels obtained in the skin mucus.     

Mucosal response in African catfish after administration of Vibrio anguillarum O2 antigens via different routes

The aim of this study was to investigate the possibility of mucosal vaccination in African catfish (Clarias gariepinus) with Vibrio anguillarum O2 bacterins. The antigen was administered via different routes: anal intubation, oral administration, intraperitoneal injection and immersion. To monitor the antigen uptake, a competitive ELISA was used. The antibody response was measured using an indirect ELISA. Increased antibody levels were found in bile and mucus upon anal intubation, which was not the case after intraperitoneal injection. The data indicate that oral vaccination of fish may be possible when antigens can reach the second gut segment in sufficient quantities and in the right form as confirmed by the recorded substantial induction of systemic and mucosal immunity. The results obtained are a strong indication for mucosal immune response and the two compartmental models for immune response in fish.     

Genetic fusion protein 3×STa‐ovalbumin is an effective coating antigen in ELISA to titrate anti‐STa antibodies

Heat‐stable toxin type I (STa)‐ovalbumin chemical conjugates are currently used as the only coating antigen in ELISA to titrate anti‐STa antibodies for ETEC vaccine candidates. STa‐ovalbumin chemical conjugation requires STa toxin purification, a process that can be carried out by only a couple of laboratories and often with a low yield. Alternative ELISA coating antigens are needed for anti‐STa antibody titration for ETEC vaccine development. In the present study, we genetically fused STa toxin gene (three copies) to a modified chicken ovalbumin gene for genetic fusion 3×STa‐ovalbumin, and examined application of this fusion protein as an alternative coating antigen of anti‐STa antibody titration ELISA. Data showed fusion protein 3×STa‐ovalbumin was effectively expressed and extracted, and anti‐STa antibody titration ELISA using this recombinant protein (25 ng per well) or STa‐ovalbumin chemical conjugates (10 ng/well) showed the same levels of sensitivity and specificity. Furthermore, mice immunized with this fusion protein developed anti‐STa antibodies; induced antibodies showed in vitro neutralization activity against STa toxin. These results indicate that recombinant fusion protein 3×STa‐ovalbumin is an effective ELISA coating antigen for anti‐STa antibody titration, enabling a reliable reagent supply to make standardization of STa antibody titration assay feasible and to accelerate ETEC vaccine development.

     

<Emphasis Type="Italic">Blastomyces Dermatitidis</Emphasis> Antigen Detection in Urine Specimens from Dogs with Blastomycosis Using a Competitive Binding Inhibition ELISA

A competitive binding inhibition enzyme linked immunosorbent assay (ELISA) was used to detect Blastomyces dermatitidis antigens in urine specimens from dogs with blastomycosis. Sera from rabbits immunized with B. dermatitidis killed whole yeast cells were used as the primary antibody in the competitive ELISA. This initial study was performed to determine if B. dermatitidis antigen detection was possible and to test the efficacy of the rabbit sera as a primary antibody. An indirect ELISA was also performed to compare antigen detection in urine to antibody detection in the sera of the infected dogs. The results indicate 100% (36/36 specimens) detection of both antigen and antibody. Cross reactivity with Histoplasma capsulatum, as well as non-specific binding with the normal urine specimens, was observed with the competitive binding inhibition ELISA.     

Growth response of African catfish,Clarias gariepinus (B.), larvae and fingerlings fed protease‐incorporated diets

African catfish, Clarias gariepinus (B.), is one of the promising freshwater fish species in African aquaculture but the expansion of its farming needs more production of its larvae. The use of live food organisms at first feeding for larvae is still obligatory. That increases the cost of larvae production. Hence, the incorporating of exogenous enzymes especially protease in artificial microdiets may provide affordable alternatives for enhancing the larvae performance. The present study was carried out to evaluate the growth and survival of larvae or fingerlings of African catfish fed artificial diets incorporated with different protease levels. Four artificial diets were formulated and enriched with protease enzyme at levels of 0.0, 750, 1,000, and 1,250 unit/kg diet; after that diets were made into crumbles (100–200 µm diameter). After absorption of the yolk sac, diets were offered to fish larvae (3.6 ± 0.2 mg) in triplicates as a starter feed up to apparent satiation every two hours for 30 days. In another treatment, fish larvae were fed on newly hatched Artemia nauplii (2,500 Artemia/L) as a starter food. In another experiment, African catfish fingerlings (10.1 ± 1.6 g) were fed on the same diets up to satiation twice a day for 2 months. It was noticed that the dietary protease improved larval growth and survival but not as Artemia nauplii did where fish larvae fed on Artemia nauplii showed highest growth and survival followed by those fed a diet enriched with 1,250 unit/kg diet of protease. The mortality of larvae fed protease‐enriched diets as well as the control diet was occurred mostly at the first week reaching its maximum at the third week. The poor growth was observed with fish larvae fed the control diet. Meanwhile, catfish fingerlings fed protease‐enriched diets showed higher growth over those fed the control diet. The larvae survival (11.0%–41.7%) was enhanced by increasing protease levels and it was lower than that of fingerlings (95.6%–100.0%). Furthermore, protein retention and digestibility were significantly improved with protease supplementation over the control diet especially at a level of 1,000 unit/kg diet. As compared with the previous studies, live food should be used in larvae rearing for the first week after that a starter diet enriched with protease at levels of 1,250 unit/kg diet should be used. In case of fish fingerlings, the dry diets should be enriched with 1,100 unit/kg diet to improve diet digestibility and subsequently enhance their growth.     

SCREENING OF PHOTOSYNTHETIC O2‐EVOLVING PROKARYOTES FOR AN INSULIN‐LIKE ANTIGEN1

Diabetes mellitus (DM), a metabolic disorder, is becoming a major health problem worldwide. Insulin is the single hope for management of type 1 diabetes, but it is not always available or suitable. For finding additional bioresources, the present study was performed. ELISA‐based preliminary screening of cyanobacterial biomass using antihuman insulin antibody have detected an insulin‐like antigen in Spirulina platensis S‐5, Spirulina NCCU‐482, and Spirulina NCCU‐483. Their similarity with insulin‐like antigen was further confirmed by electrophoretic mobility using bovine insulin as marker.     

Resistin concentrations in murine adipose tissue and serum measured by a new enzyme immunoassay

Objective: In an attempt to clarify the conflicting data on resistin mRNA expression and protein analysis by western blotting in adipose tissue and serum, we developed a sensitive enzyme‐linked immunosorbent assay (ELISA) for direct measurement of mouse resistin. Research Methods and Procedures: We developed polyclonal antibodies directed to the N (21 to 40) and C (79 to 91) termini of mouse resistin. Then, affinity‐purified anti‐C‐terminal resistin immunoglobin G (IgG) was biotinylated. ELISA was based on the sandwiching of antigen between antibody IgG coated on polystyrene plates and biotinylated antibody IgG. The bound biotinylated antibody was quantified with streptavidin‐linked horseradish peroxidase. Results: New ELISA can measure a concentration as low as 0.5 ng/mL of recombinant mouse resistin and is sensitive and specific enough to measure resistin protein in various adipose tissues and in sera. In normal mice, decreases in resistin concentrations in both white adipose tissue and serum were age dependent during 6 to 24 weeks of development. Resistin concentrations were significantly higher in omental adipose tissue in comparison with perirenal and abdominal adipose tissues and were 2‐ to 5‐fold higher in females than males during the growth period. ob/ob mice had significantly lower resistin concentrations than the control mice in both sera and the white adipose tissues, particularly in the omental fat. The treatment by testosterone, but not progesterone or β‐estradiol, in cultured adipocytes reduces resistin protein levels in a dose‐dependent manner. Discussion: New sensitive ELISA for mouse resistin clarified that the resistin concentrations in normal mice were markedly elevated in the omental adipose depots as compared with the perirenal and abdominal adipocyte depots and significantly elevated compared with adipose tissues in genetically obese mice.     

One-step immunoaffinity purification and partial characterization of hypophyseal growth hormone from the African catfish, Clarias gariepinus (Burchell)

Growth hormone (GH) was purified from African catfish (Clarias gariepinus) pituitary extracts in a single step by use of immunoaffinity chromatography. A monoclonal antibody to chicken GH, which labels the catfish hypophyseal somatotropes in immunocytochemistry, was coupled to CNBr-activated Sepharose, and crude alkaline pituitary extracts were run over the immunoadsorbent. Reversed-phase high-performance liquid chromatography analysis of the eluted material suggested heterogeneity, whereas silver staining upon SDS-polyacrylamide gel electrophoresis showed one single band with an estimated molecular weight between 22,000 and 23,000 Da. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the same preparation revealed the presence of several components with molecular weights ranging from 20,170 to 20,900 Da. The amino terminus of the protein was homogeneous, and the first 50 residues matched the proposed sequence of GH from two other siluran species (Ictalurus punctatus and Pangasius pangasius), except for one substitution at position 3. These data unequivocally confirm the identity of the purified molecule as suggested by immunochemical evidence. The bioactivity of the GH preparation was demonstrated by the short-term effect of GH on T₃ plasma levels in juvenile catfish.     

A comparative study on the efficacy of a pest-specific and prey-marking enzyme-linked immunosorbent assay for detection of predation

The efficacy of two different antigen–antibody combinations to detect predation on eggs of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) was compared. The first method was an indirect enzyme‐linked immunosorbent assay (ELISA) using monoclonal antibody‐based gut content analysis that detects H. armigera egg protein. The second method was a sandwich ELISA that detects an exotic protein [rabbit immunoglobulin G (IgG)] applied as an external marker to H. armigera eggs. The target predators were the predatory beetles Dicranolaius bellulus (Guerin‐Meneville) (Coleoptera: Melyridae) and Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae). Beetles were fed with H. armigera eggs that had been marked with rabbit IgG and then held at various intervals after prey consumption. Each individual beetle was then assayed by both ELISA techniques to identify the prey remains in their guts. The two ELISA methods were further tested on field‐collected predators. Specifically, protein‐marked egg masses were strategically placed in a cotton field. Then, predators from surrounding cotton plants were collected at various time intervals after the marked eggs were exposed and assayed by both ELISAs to detect the frequency of predation on the marked eggs. The rabbit IgG‐specific sandwich ELISA had a higher detection rate than the H. armigera‐specific indirect ELISA under controlled and field conditions for both predator species. Moreover, a greater proportion of field‐collected D. bellulus tested positive for predation than H. variegata. The advantages and disadvantages of using prey‐marking ELISAs instead of pest‐specific ELISA assays are discussed.     

A Novel IgM‐capture enzyme‐linked immunosorbent assay using recombinant Vag8 fusion protein for the accurate and early diagnosis of Bordetella pertussis infection

An ELISA that measures anti‐PT IgG antibody has been used widely for the serodiagnosis of pertussis; however, the IgG‐based ELISA is inadequate for patients during the acute phase of the disease because of the slow response of anti‐PT IgG antibodies. To solve this problem, we developed a novel IgM‐capture ELISA that measures serum anti‐Bordetella pertussis Vag8 IgM levels for the accurate and early diagnosis of pertussis. First, we confirmed that Vag8 was highly expressed in all B. pertussis isolates tested (n = 30), but little or none in other Bordetella species, and that DTaP vaccines did not induce anti‐Vag8 IgG antibodies in mice (i.e. the antibody level could be unaffected by the vaccination). To determine the immune response to Vag8 in B. pertussis infection, anti‐Vag8 IgM levels were compared between 38 patients (acute phase of pertussis) and 29 healthy individuals using the anti‐Vag8 IgM‐capture ELISA. The results revealed that the anti‐Vag8 IgM levels were significantly higher in the patients compared with the healthy individuals (P < 0.001). ROC analysis also showed that the anti‐Vag8 IgM‐capture ELISA has higher diagnostic accuracy (AUC, 0.92) than a commercial anti‐PT IgG ELISA kit. Moreover, it was shown that anti‐Vag8 IgM antibodies were induced earlier than anti‐PT IgG antibodies on sequential patients’ sera. These data indicate that our novel anti‐Vag8 IgM‐capture ELISA is a potentially useful tool for making the accurate and early diagnosis of B. pertussis infection.     

Cloning,expression, and identification of anti‐carbofuran single chain Fv gene

Phage display method was used to clone anti‐carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF‐specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly₄Ser)₃. The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti‐CBF antibody libraries were then constructed. After one round of panning with CBF‐ovalbumin (CBF‐OVA) as a conjugate, antigen‐binding positive recombinant phage clones were successfully selected by enzyme‐linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS‐PAGE and showed HRP‐anti‐E‐tag antibody‐recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC₅₀ value of 1.07 ng/mL. The cross reactivity studies showed that the anti‐CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Development of an enzyme‐linked immunosorbent assay (ELISA) for Luteinizing Hormone (LH) in the elephant (Loxodonta africana and Elephas maximus)

A simple, rapid enzyme‐linked immunosorbent assay (ELISA) for the measurement of LH in plasma and serum of elephants (Loxodonta africana and Elephas maximus) has been developed, validated, and used for comparative studies. Purified elephant LH (eleLH) diluted in elephant plasma was used as standards (0.78–50 ng/ml). A monoclonal antibody against the β‐subunit of bovine LH (518B₇) was used as the capture antibody. The second antibody (a polyclonal rabbit anti‐human LH antibody), conjugated to horseradish peroxidase, cleaved a substrate (tetramethyl benzidine), resulting in a color change. The total assay time was approximately 2½ hr, with incubations at room temperature. Sensitivity was found to be 1.56 ng/ml. Cross‐reactivities to elephant FSH and TSH were low: 0.9% and 0.15%, respectively. The accuracy of the assay was demonstrated by comparing the ELISA with a validated eleLH radioimmunoassay (RIA), progesterone data, and ultrasound observations. Blood samples from 18 Asian and African elephant cows were analyzed with the ELISA and RIA, and an additional 11 cows were used to describe endocrine parameters for LH and progesterone using only RIA. No difference was found in LH peak concentrations between the ELISA and RIA. The time from the progesterone decline to the first LH peak, and the time between the two peaks were similar between species. Asian cows had higher LH peaks than African cows. Ultrasound confirmed the time of ovulation occurring with the second LH peak. Three cows were inseminated and confirmed to be pregnant using this ELISA as a timing device. Instrumentation is not always required, as LH peaks approximating 3 ng/ml can be visually observed. In conclusion, this ELISA can be used as a field test to determine time of ovulation for artificial insemination (AI) or natural breeding of both species of elephants, and thus is an important tool for the preservation of captive populations worldwide. Zoo Biol 23:65–78, 2004. © 2004 Wiley‐Liss, Inc.     

Comparison of stool antigen immunoassay and serology for screening for Helicobacter pylori infection in intellectually disabled children

Diagnosis of active Helicobacter pylori infection in intellectually disabled (ID) children is problematic because they are unable to cooperate with performance of invasive tests. In this study, the non‐invasive methods of measuring serum IgG antibody concentrations and performing stool antigen tests were used to screen for H. pylori infection in ID children. Eighty‐seven children with intellectual disabilities were studied. The amount of serum IgG antibody against H. pylori was measured by the ELISA method. Stool samples were examined using an amplified IDEIA HpStAR kit. To assess categorical variables, X², Fisher's exact and Kappa tests were used. The stool antigen tests showed that 93.1% of the children had H. pylori antigen and the serology test that 85.1% of children were positive for H. pylori IgG antibodies. Agreement between results of H. pylori stool antigen (HpSA) testing and IgG antibody serology was 82.8%; however, according to the kappa measure of agreement this agreement is not statistically significant (value, 0.128; P = 0.19). Discordant results were observed for 15 children (17.2%): 11 (12.6%) who were positive on HpSA test but negative by serology and 4 (4.6%) who were IgG seropositive but had negative HpSA tests. This study showed a notably higher rate of H. pylori infection in ID children than has been reported by others for non‐ID children from the same geographical area. The HpSA test is a valid method for primary screening for H. pylori infection in ID children; it detects the specific antigens shed during active infections and has less cross‐reactivity than serological tests that detect antibodies. HpSA is a sensitive non‐invasive method for detecting infection in ID children and may serve as an accurate alternative to serology.     

Reduction in Listeria monocytogenes and spoilage bacteria on smoked catfish using X-ray treatments

Aims: To determine the efficacy of X‐ray processes in inactivating L. monocytogenes levels in smoked catfish during storage at 5°C and to determine the effects of X‐ray doses on controlling the growth of spoilage bacteria on smoked catfish during storage at 5°C for up to 5 weeks. Methods and Results: Smoked catfish fillets inoculated with L. monocytogenes were treated with 0·0–2·0 kGy X‐ray and stored at 5°C for 5 weeks. The negative controls (uninoculated/untreated) and uninoculated samples treated with the lowest (0·1 kGy) and highest (2·0 kGy) doses were stored at 5°C and tested for psychrotrophs count during the 5 weeks of storage. The initial L. monocytogenes population on smoked catfish was significantly (P < 0·05) reduced to undetectable level by a treatment of 1·0 kGy or higher. The initial psychrotrophs count on smoked catfish was significantly reduced from 4·7 CFU g^?1 to below the detectable level by a treatment with 2·0 kGy. Conclusions: Smoked catfish treated with 2·0 kGy X‐ray had no detectable L. monocytogenes throughout 35 days of storage at 5°C. A treatment with 2·0 kGy X‐ray also kept the levels of psychrotrophs in the smoked catfish within the acceptable level until 35 days. Significance and Impact of the Study: The results of this investigation indicate that X‐ray at 2·0 kGy can eliminate L. monocytogenes and extend the shelf life of smoked catfish stored at refrigeration temperature.     

Application of quantitative structure‐activity relationship analysis on an antibody and alternariol‐like compounds interaction study

The antigen‐antibody interaction determines the sensitivity and specificity of competitive immunoassay for hapten detection. In this paper, the specificity of a monoclonal antibody against alternariol‐like compounds was evaluated through indirect competitive ELISA. The results showed that the antibody had cross‐reactivity with 33 compounds with the binding affinity (expressed by IC₅₀) ranging from 9.4 ng/mL to 12.0 μg/mL. All the 33 compounds contained a common moiety and similar substituents. To understand how this common moiety and substituents affected the recognition ability of the antibody, a three‐dimensional quantitative structure‐activity relationship (3D‐QSAR) between the antibody and the 33 alternariol‐like compounds was constructed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods. The q² values of the CoMFA and CoMSIA models were 0.785 and 0.782, respectively, and the r² values were 0.911 and 0.988, respectively, indicating that the models had good predictive ability. The results of 3D‐QSAR showed that the most important factor affecting antibody recognition was the hydrogen bond mainly formed by the hydroxyl group of alternariol, followed by the hydrophobic force mainly formed by the methyl group. This study provides a reference for the design of new hapten and the mechanisms for antibody recognition.     

Lacustrine radiations in African Synodontis catfish

Synodontis catfish are a species‐rich, tropical pan‐African genus that predominately occur in fluviatile environments, but which also form a small radiation within Lake Tanganyika (LT). Here we estimate Synodontis relationships, based on mitochondrial and nuclear DNA, greatly expanding previous sampling. Data were analysed using different methods of phylogenetic inference: Bayesian (also testing compositional heterogeneity), likelihood and parsimony, in order to investigate biogeographic history and the extent of intralacustrine speciation within this group. Bayesian‐relaxed clock analyses were used to estimate timings of radiations. Our analyses reveal a single origin of the LT flock with the inclusion of the nonendemic S. victoriae, and that these taxa evolved relatively recently (5.5 Ma), considerably later than the formation of LT (9–12 Ma). Two internal endemic clades diversified at a similar time (2–2.5 Ma), corresponding to a period of climate change, when lake levels dropped. We find evidence for a further species flock, composed of riverine southern African taxa, the diversification of which is very rapid, 0.8 Ma (95% HPD: 0.4–1.5) and infer a similar scenario for the diversification of this flock to southern African serrachromine cichlids in that they radiated in the now extinct lake Makgadikgadi. We also reveal that the biogeographic history of Synodontis catfish is more complex than previously thought, with nonmonophyletic geographic species groupings.     

Enhanced chemiluminescence ELISA for Listeria specific antigen in cerebrospinal fluid using an FITC-anti-FITC system

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of a soluble Listeria monocytogenes serogroup 4 antigen in cerebrospinal fluid samples (CSFs). In the ELISA an anti-Listeria monoclonal antibody, immobilized onto assay wells, was used to capture antigen from CSFs. the captured antigen was then reacted with a fluorescein isothiocyanate (FITC) conjugate of the same anti-Listeria antibody, which was detected with a horseradish peroxidase conjugate of a monoclonal antibody to FITC. The presence of antigen was detected by an enhanced chemiluminescence assay using a camera luminometer. Antigen was detected in the CSFs taken from five out of seven patients with culture proven L. monocytogenes serogroup 4 central nervous system infections, and in none of the CSFs taken from 25 other patients.     

Insulin Sensitivity in African‐American and White Women: Association With Inflammation

Whether the contribution of inflammation to risk for chronic metabolic disease differs with ethnicity is not known. The objective of this study was to determine: (i) whether ethnic differences exist in markers of inflammation and (ii) whether lower insulin sensitivity among African Americans vs. whites is due to greater inflammatory status. Subjects were African‐American (n = 108) and white (n = 105) women, BMI 27–30 kg/m². Insulin sensitivity was assessed with intravenous glucose tolerance test and minimal modeling; fat distribution with computed tomography; body composition with dual‐energy X‐ray absorptiometry; markers of inflammation (tumor necrosis factor (TNF)‐α, soluble tumor necrosis factor receptor (sTNFR)‐1, sTNFR‐2, C‐reactive protein (CRP), and interleukin (IL)‐6) with enzyme‐linked immunosorbent assay (ELISA). Whites had greater intra‐abdominal adipose tissue (IAAT), insulin sensitivity, and concentrations of TNF‐α, sTNFR‐1, and sTNFR‐2 than African Americans. Greater TNF‐α in whites vs. African Americans was attributed to greater IAAT in whites. Among whites, but not African Americans, CRP was independently and inversely associated with insulin sensitivity, after adjusting for IAAT (r = ?0.29 P < 0.05, and r = ?0.13 P = 0.53, respectively). Insulin sensitivity remained lower in African Americans after adjusting for CRP (P < 0.001). In conclusion, greater IAAT among whites may be associated with greater inflammation. Insulin sensitivity was lower among African Americans, independent of obesity, fat distribution, and inflammation.     

An Improved Antiserum for Sensitive Serologic Detection of Chickpea chlorotic dwarf virus

A Syrian chickpea isolate of Chickpea chlorotic dwarf virus (CpCDV; genus Mastrevirus, family Geminiviridae) was purified and yielded 0.6–0.8 mg of purified virus per kg of infected chickpea tissue. The purified preparations were injected into a rabbit and an antiserum of good quality was obtained and used to evaluate different serological tests for the detection of CpCDV in infected chickpea leaf tissue and extracts. CpCDV was detected in sap dilutions of 1/640 by double‐antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) and dot‐blot ELISA, and in sap dilutions of 1/1280 by direct antigen‐coating (DAC)‐ELISA using CpCDV immunoglobulin G (IgG) at 0.5 μg/ml. The antiserum was also able to detect the capsid protein of CpCDV by Western blot using raw antiserum at a dilution of 1/2000. The CpCDV raw antiserum (third bleeding) produced had a titre of 1/320 000 when determined by tissue‐blot immunoassay (TBIA); whereas, coating ELISA plates with CpCDV IgG at a concentration of 0.004 μg/ml was enough to detect the virus by DAS‐ELISA in a sap dilution of 1/20 using an enzyme conjugate at a dilution of 1/2000.