Combined use of ELISA and Western blot with recombinant N protein is a powerful tool for the immunodiagnosis of avian infectious bronchitis

Background

The avian infectious bronchitis virus (IBV) remains a significant source of loss in the poultry industry and early diagnosis is required to prevent the disease from spreading. This study examined the combined use of an ELISA and Western blot (WB) to detect antibodies against the nucleocapsid protein (N) of IBV. The coding sequence for N was amplified by RT-PCR and expressed in Escherichia coli. A soluble recombinant N protein (rN) of approximately 50?kDa was obtained. A total of 389 sera were tested against the rN in ELISA and the results were compared with those of the commercial IDEXX IBV Ab test. ELISA-rN achieved a 90.34% sensitivity and 90.16% specificity. WB confirmed all false negative sera in ELISA-rN or IDEXX test as truly positive. The current study indicate that the combined use of rN in ELISA and WB is a powerful tool for the immunodiagnosis of avian infectious bronchitis.

Methods

Constructed recombinant pAE/n expression vectors were used to transform E. coli BL21(DE3) Star competent cells (Invitrogen). The rN of infectious bronchitis virus was purified by affinity chromatography using HisTrap HP 1?mL columns pre-packed with pre-charged Ni Sepharose in the ÄKTAprime Automated Liquid Chromatography system (GE Healthcare). A total of 389 serum samples from chickens were used to develop and evaluate the ELISA-rN test. To standardize the indirect ELISA development, serum dilutions (1:100, 1:200 and 1:400) and different concentrations of purified rN antigen (50, 100 and 200?ng/well) were tested. Positive and negative sera for IBV were used as controls. The results were compared with those obtained from a commercial kit. Serum samples scored as negative with the commercial kit but as positive with the ELISA-rN were further analysed by Western blot analyses using the rN protein as an antigen. The results of the ELISA-rN were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software GraphPad Prism version 6.0 for Windows (GraphPad Software, USA).

Results

The expected cDNA fragment of approximately 1240?bp was successfully amplified by PCR using primers designed to select for the coding region of the N protein. The rN was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of 10?mg/L of rN was obtained. The SDS-PAGE results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 and 50 KDa. Out of 244 sera that scored positive in the commercial ELISA IDEXX IBV Ab Test, 220 were also positive in the ELISA-rN, yielding an ELISA-rN test sensitivity of 90.16%. Out of 145 sera that scored negative in the IDEXX IBV Ab Test, 131 also scored negative in the ELISA-rN, indicating a specificity of 90.34%. Sera that tested negative in the ELISA-rN and positive in the commercial test also reacted with the rN protein in Western blot.

Conclusions

The association between the ELISA and Western blot techniques developed in this study with a subunit of IBV (rN) were able to detect antibodies that the commercial ELISA did not detect suggesting that the ELISA-rN has greater sensitivity.

     

The myosin filament. XIII. The sensitivity of LMM assembly to Mg.ATP

An LMM fragment (Mr 62,000) of myosin has been prepared which has aggregation properties that are sensitive to the presence of Mg.ATP. Aggregation of the LMM by reducing the ionic strength in the presence of 1 mM Mg.ATP produces non-periodic aggregates which gradually rearrange to paracrystals with a 43 nm axial repeat pattern. This fragment includes the C-terminal end of the myosin rod starting at residue 1376. Therefore, at least one of the Mg.ATP binding sites responsible for this effect is located somewhere along this region of the myosin rod. Although assembly of the rod fragment of myosin into paracrystals does not show sensitivity to Mg.ATP, assembly of intact myosin molecules to form filaments does show sensitivity to Mg.ATP. For myosin filaments, assembly initially gives a broad distribution around a mean length of 1.5 microns, which sharpens around the mean length with time. The rearrangement of the LMM rods and intact myosin molecules both induced by the presence of Mg.ATP are probably related. These findings highlight the complexity of the cooperative interactions between different portions of the myosin molecule that are involved in determining the assembly properties of the intact molecule.     

M BAND PROTEIN : Two Components Isolated from Chicken Breast Muscle

M band protein can be specifically extracted from fresh chicken breast muscle myofibrils suspended in 5 mM Tris-HCl pH 8.0. During discontinuous polyacrylamide gel electrophoresis the isolated protein separates into three bands which can be identified as two separate components (A, B) and a complex of the two. When partially purified fractions of the separated components are combined, an increase in the intensity of the band containing the complex can be shown. The polypeptide chain weights of the two components are 100,000 (A) and 40,000 (B) daltons as estimated by sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis. Antibody prepared against total M band protein stains only the M band of the myofibril and is completely absorbed by M band protein. M band protein also absorbs the M band staining specifically from antibody which stains both I and M bands. Immunodiffusion data indicate that anti-M band is a mixture of two specific antibodies, one against each component.     

Guanylate cyclase in rod outer segments of the toad retina. Effect of light and Ca2+

Guanylate cyclase activity was measured in disrupted rod outer segments of the toad retina. The experiments showed that cGMP is synthesized from GTP at a rate of 3 +/- 1 nmol/min per mg protein. In darkness this value is largely independent of the Ca2+ concentration, while it is enhanced by flashes of light of increasing intensity upon lowering Ca from 10-5 to 10-8 M. In view of recent observations that shortly after a flash of light calcium activity inside the photoreceptor cell decreases, it seems likely that calcium plays a regulatory role in cGMP metabolism in visual excitation.     

The utilization of placental substrates for cortisol synthesis by the baboon fetus near term

We have determined the proportion of the invivo cortisol (F) secretion rate (SRF) derived from circulating pregnenolone (P5) and progesterone (P4) in 9 baboon (Papiopapio) neonates, 5 (4 ♂, 1 ♀) delivered prior to the onset of labor by cesarean section (CS, 164–179 days) and 4 (2 ♂ , 2 ♀) delivered spontaneously at term (SD, 164–179 days; term = 184 days). The metabolic clearance rate (MCR; 1/d/kg) and production rates (PR; mg/d/kg) of P4 and P5 and transfer constants (p) for the reactions P5 → P4₁₄ P5 → F, and P4 → F, were determined by continuous infusion of [1,2-³H] P5 and [4-¹⁴C] P4.In CS animals the MCR-P5 (41.4 ± 5.6; X ± SE) was lower than the MCR-P4 (74.0 ± 8.5; P < 0.001). However, the pP5 → F (1.0% ± 0.3%) and pP4 → F (0.9% ± 0.3%) were similar although lower (P < 0.001) than pP5 → P4 (9.0% ±1.4%). The PR-P4 (7.0 ± 0.8) was 4–5 times greater than the PR-P5 (P < 0.01). In SD newborns, only the pP5 → F (3.4% ± 0.9%) and the PR-P4 (0.5 ± 0.2) were different (P < 0.01) from values in CS neonates. These results indicate that although the baboon fetus has the capacity to convert P5 to P4, the abundant quantities of P4 measured in fetal serum (95 ± 12 ng/ml) near term are primarily placental in origin. Moreover, the apparently low (< 5%) transfer constants for the conversion of P5 or P4 to F suggests minimal utilization of these circulating precursors for F synthesis. Indeed, the calculated proportion of the SR-F derived from circulating P4 was 5.3% ± 2.0% in CS neonates and only 0.3% ± 0.1% in SD newborns. In these animals, values for the utilization of circulating P5 were 1.4% ± 0.4% and 1.6% ± 0.4%, respectively.We conclude that endogenous adrenal substrates and not placental substrates are the primary precursors utilized for fetal adrenal F production in late baboon gestation.     

THE FINE STRUCTURE OF THE VENTRAL INTERSEGMENTAL ABDOMINAL MUSCLES OF THE INSECT RHODNIUS PROLIXUS DURING THE MOLTING CYCLE : I. Muscle Structure at Molting

Rhodnius prolixus, a South American insect, molts five times in its development to an adult after emerging from the egg. Each molting cycle is triggered with a blood-meal. The ventral intersegmental abdominal muscles of Rhodnius develop during each molting cycle and are functional at molting. The fine structure of these fully developed muscles from fourth stage larval insects is studied. They have the characteristic structure of slow muscles. They have multiple motor nerve endings, and the myofibrils are poorly defined in cross-section. Longitudinal sections show long sarcomeres (8–10 µ), irregular Z-lines, and no apparent H zones. No M line is seen. Transverse sections through the A-band region show that each hexagonally arranged thick filament is surrounded by 12 thin filaments. Two thin filaments are shared by two neighboring thick filaments. The ratio of thin to thick filaments is 6:1. This structure is related to that found in vertebrate skeletal muscle and insect flight muscle.     

The myosin filament XIV backbone structure.

The substructure of the thick filaments of chemically skinned chicken pectoralis muscle was investigated by electron microscopy. Images of transverse sections of the myosin filaments were determined to have threefold symmetry by cross-correlation analysis, which gives an unbiased determination of the rotational symmetry of the images. Resolution, using the phase residual test (Frank et al. 1981. Science [Wash. DC]. 214:1353-1355), was found to be between 3.2 and 3.6 nm. Three arrangements of nine subfilaments in the backbone were found in all regions of the filament at ionic strengths of 20 and 200 mM. In the average images of two of these, there were three dense central subfilaments and three pairs of subfilaments on the surface of the thick filament. In the average image of the third arrangement, all of the protein mass of the nine subfilaments was on the surface of the filament with three of them showing less variation in position than the others. A fourth arrangement appearing to be transitional between two of these was seen often at 200 mM ionic strength and only rarely at 20 mM. On average, the myosin subfilaments were parallel to the long axis of the filament. The different arrangements of subfilaments appear to be randomly distributed among the filaments in a transverse section of the A-band. Relative rotational orientations with respect to the hexagonal filament lattice, using the three densest subfilaments as reference showed a major clustering (32%) of filaments within one 10 degrees spread, a lesser clustering (15%) at 90 degrees to the first, and the remainder scattered thinly over the rest of the 120 degrees range. There was no obvious pattern of distribution of the two predominant orientations that could define a superlattice in the filament lattice.     

Utilization of circulating androstenedione and testosterone for estradiol production during gestation in the rat

The placenta provides androgen precursors for ovarian estradiol (E2) production during the second half of gestation in the rat. However, no studies have measured E2 synthesis in vivo from circulating testosterone (T) or androstenedione (A) before or after Day 12 of gestation. In addition, it is not known whether the placenta near term continues to serve as the major source of androgens. Therefore, we measured the ovarian conversion of circulating T and A to E2 in vivo on Days 11, 16, and 21 of gestation (term = Day 23). Rats (N = 6-8/group) were anesthetized with pentobarbital and a constant infusion of [3H]T or [3H]A initiated via a jugular vein. After isotopic equilibrium was achieved at 60 min, blood samples were obtained from the contralateral jugular (J) vein and a uterine-ovarian (UO) vein, and the ovaries were removed. In a second group of rats on Day 16 of gestation, either the gravid uterus or both ovaries were removed after initiation of isotope infusion, and blood samples obtained 60 min later. Radiolabeled T, A, and E2 were isolated and purified by sequential paper chromatography. The concentration of [3H]E2 following infusion of either androgen was greater in the UO vein than in the J vein on Days 16 and 21 (p less than 0.02), but not on Day 11, of gestation. In animals infused with [3H]T, [3H]E2 (cpm/ml) in UO vein increased (p less than 0.001) from 84 +/- 33 (mean +/- SE) on Day 11 to 357 +/- 30 and 312 +/- 46 on Days 16 and 21, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)