Goat Immunoglobulin Research Articles

Preparation and use of hyperimmune serum for prophylaxis and therapy of Ebola virus infections.

To obtain hyperimmune serum appropriate for the treatment of filovirus infection, methods were developed to immunize nonsusceptible animals with live Ebola (EBO) virus preparations. Immune plasma with high ELISA and neutralization-specific antibody titers was obtained by multiple immunization of sheep and goats with preparations of live EBO virus. Goat immunoglobulin was prepared by Cohn's method and tested on guinea pigs, using an EBO virus strain that is highly pathogenic for guinea pigs. Prophylaxis with these immunoglobulins within 48 h after infection was effective in challenge experiments, with a log10 prophylaxis index as high as 1.92+/-0.52. Other studies have shown that equine anti-EBO virus immunoglobulins worked well in baboons. The goat immunoglobulins were also tested in preclinical trials on laboratory animals; after being positively evaluated, they were administered to volunteers in clinical trials for biologic safety and reactivity, and they were administered to researchers suspected of becoming infected with EBO during their experimental work. These immunoglobulins may be useful for the emergency treatment of persons accidentally infected with EBO.

Five homologous repeats of the protein G-related protein MIG cooperate in binding to goat immunoglobulin G.

Protein MIG, from Streptococcus dysgalactiae, binds alpha2-macroglobulin and immunoglobulin G (IgG). MIG-derived fusion proteins with one to five IgG-binding repeats differed up to 72,000- fold in avidity for goat IgG, indicating a considerable cooperativity of the repeats. Significant sequence variation in the IgG-binding repeats was recognized. Protein MIG interacted with goat IgG1 via both the Fc and Fab parts.

Cultured human airway smooth muscle cells stimulated by interleukin-1beta enhance eosinophil survival.

Airway smooth muscle may be an important cellular source of proinflammatory mediators and cytokines and may participate directly in airway inflammation. In this study we have examined whether airway smooth muscle cells could contribute to mechanisms of eosinophil accumulation by prolonging their survival. To investigate this possibility, conditioned medium from human airway smooth muscle cells stimulated with interleukin (IL)-1beta was examined on the in vitro survival of highly purified human peripheral blood eosinophils. After 7 d, when cultured in control medium, less than 1 +/- 0.2% of the initial eosinophil population remained viable. In contrast, culture in medium conditioned for 96 h by human airway smooth muscle cells stimulated with IL-1beta (1 pg-100 ng/ml) resulted in a concentration-dependent increase in eosinophil survival. (The concentration that produced 50% of this effect was 0.03 ng/ml IL-1beta.) Maximum eosinophil survival occurred at 1 to 3 ng/ml IL-1beta. This effect was also time-dependent and was readily detected in airway smooth muscle cell-conditioned medium after just 3 h of stimulation with IL-1beta (1 ng/ml). It continued to increase before reaching a plateau around 24 h, with no decrease in activity for up to 120 h of stimulation. Conditioned medium from unstimulated airway smooth muscle cells did not enhance eosinophil survival. The survival-enhancing activity was completely inhibited (the concentration that inhibited 50% [IC50] was 6.9 microg/ml) by a polyclonal goat antihuman antibody to granulocyte-macrophage colony stimulating factor (GM-CSF) (0.3-100 microg/ml), but antibodies (10-100 microg/ml) to IL-3 and IL-5, and a normal goat immunoglobulin G control had no effect on the eosinophil survival-enhancing activity. GM-CSF levels in culture medium from smooth muscle cells were markedly increased by IL-1beta and were maximum at 30 ng/ml (0.037 ng/ml/10(6) cells versus 3.561 ng/ml/10(6) cells, unstimulated versus 30 ng/ml IL-1beta). The IL-1 receptor antagonist inhibited both the production of GM-CSF (IC50 19. 1 ng/ml) and the eosinophil survival-enhancing (IC50 53.7 ng/ml) activity stimulated by IL-1beta. Release of GM-CSF elicited by IL-1beta was inhibited by dexamethasone but not by indomethacin. These data indicate that cultured human airway smooth muscle cells stimulated with IL-1beta support eosinophil survival through production of GM-CSF and thus may contribute to the local control of inflammatory cell accumulation in the airways.

Enhancement of molecular fluorescence near the surface of colloidal metal films.

Fluorescence enhancement was studied on silver colloidal metal films (CMFs) using two systems: (1) Langmuir--Blodgett monolayers of fluorescein-labeled phospholipids separated from the surface of the films by spacer layers of octadecanoic acid and (2) biotin--fluorescein conjugates captured by avidin molecules adsorbed on top of a multilayer structure formed by alternating layers of bovine serum albumin--biotin conjugate (BSA--biotin) and avidin. The dependence of fluorescence intensity on the number of lipid or protein spacer layers deposited on the surface of the CMF was investigated. The results demonstrate the requirement for adsorbate location within the region between Ag particles for maximal enhancement. The density of avidin molecules on the surface of the BSA--biotin/avidin multilayers adsorbed on the CMF was also determined. A procedure for forming a rigid, uniform silica layer around the Ag particles on the CMF is described. The layer protects the particles from undesirable chemical reactions such as etching by halide ions, for example, and provides the requisite stability for bioanalytical applications. Colloidal films composed of Ag particles covered by approximately 10-nm-thick silica layers were tested for fluorescence enhancement using goat immunoglobulin and a conjugate of rabbit anti-goat immunoglobulin with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino)hexanoate. An enhancement factor of approximately 20 was obtained.

Species-specific β- N-acetylgalactosaminylation of serum IgG proteins

Lectin blot analysis of bovine, goat, human, rabbit and mouse serum immunoglobulin G (IgG) samples revealed that Wisteria floribunda agglutinin (WFA) binds to the heavy chains of bovine, goat and human serum IgG proteins but not those of the rabbit and mouse proteins. WFA-positive light chain bands were also detected in bovine, goat and human serum IgG samples only after the filters were treated with Arthrobacter ureafaciens sialidase. The WFA-binding to these IgG proteins was abolished by treatment of the filter with sialidase and then β- N-acetylhexosaminidase or N-glycanase prior to incubation with the lectin. WFA-agarose column chromatography of the oligosaccharides released by hydrazinolysis from the IgG samples followed by reduction with NaB 3H 4 revealed that 0.15, 0.09 and 0.07% of the total oligosaccharides from bovine, goat and human serum IgG samples bind to the column, respectively. Partial characterization of WFA-positive bovine IgG oligosaccharides by Bio-Gel P-4 column chromatography suggested that the major oligosaccharide is of non-fucosylated biantennary complex-type. These results indicate that β- N-acetylgalactosaminylation occurs to N-linked sugar chains of heavy and light chains of IgG proteins in a species-specific manner.

Indirect Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for Plasma Apolipoprotein E

We have developed an indirect sandwich ELISA for measuring plasma apolipoprotein E (apo-E), using commercially available antibodies. A monoclonal anti-apo-E was used as the capture antibody and the captured apo-E detected with polyclonal anti-apo-E antiserum (goat). The detecting antibody was quantitated using horseradish peroxidase-conjugated rabbit immunoglobulin to goat immunoglobulins. There was no detectable cross-reactivity between the three antisera. Interference with the assay by apolipoprotein A-1, bilirubin and haemoglobin was not significant up to 1.7 g/L, 1250 mumol/L and 13.0 g/dL, respectively. The ELISA method showed high correlation with an established immunonephelometric method (ELISA apo.E = 1.04 Immunonephelometric apo-E + 16; r2 = 0.954, P < 0.0001, n = 39). The assay has a measuring range between 5 and 560 mg/L. The coefficient of duplicates was 2.0%, within-run coefficients of variation (CV) ranged from 3.7 to 6.0% and between-run CV from 6.1 to 15.1%. The reference range determined for 168 normotriglyceridaemic subjects was 20 to 130 mg/L. In an analysis of the lipoprotein subfractions isolated by ultracentrifugation as the fraction of density less than 1.25 g/mL and separated by gel permeation chromatography, apo-E was found to be associated with very low-density lipoprotein and large high-density lipoprotein.

Species-specific long range interactions between receptor/ligand pairs

Total internal reflection microscopy (TIRM) monitors Brownian fluctuations in elevation as small as 1 nm by measuring the scattering of a single sphere illuminated by an evanescent wave when the sphere is levitated by colloidal forces such as electrostatic double-layer repulsion. From the Boltzmann distribution of elevations sampled by the sphere over time, the potential energy profile can be determined with a resolution of approximately 0.1 of the thermal energy kT. Thus, the interaction between a receptor-coated (goat, horse, or rabbit immunoglobulin G (IgG)) latex sphere and a protein A (SpA)-coated glass microscope slide was studied. A typical TIRM potential energy profile measured between a bare sphere and a bare glass plate, where the sphere fluctuates around the secondary potential energy minimum formed between double-layer repulsion and gravitational attraction, agrees well with DLVO theory. The interactions measured between IgG-coated spheres and SpA-coated slides, on the other hand, displayed a weaker repulsion compared with that observed between bare surfaces under the same conditions. Analysis of the results obtained between the coated surfaces suggests an additional attractive force. The decay length of this attraction correlates with the known dissociation constants for the binding of IgG with SpA in free solution.

Rapid one-step purification of goat immunoglobulins by immobilized metal ion affinity chromatography

A rapid, single step purification of immunoglobulins from goat serum was achieved using immobilized metal ion affinity chromatography (IMAC) on a new high capacity gel, Novarose, coupled to tris(2-aminoethyl)amine (TREN) chelated with copper. When goat serum was adsorbed to this gel in buffer pH 7 at 11 cm/h (8.6 ml/h), the immunoglobulin fraction was recovered in a decreasing linear pH gradient at about pH 5.5. When the adsorption buffer was adjusted to pH 6.0 and the linear velocity increased to 110 cm/h (221 ml/h), an immunoglobulin fraction of greater than 95% homogeneity was obtained. Protein purity was assessed by silver-stained native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The capacity of the gel for immunoglobulins was 17 mg immunoglobulin/ml at the low flow rate with adsorption at pH 7 and 15 mg immunoglobulin/ml at the high flow rate with adsorption at pH 6. No problems of back pressure or gel compression were observed at the higher linear velocity. The mild elution pH, high flow rate, and synthetic nature of the ligand support make this new metal-chelating gel a powerful alternative to the use of other currently available commercial gels commonly used for immunoglobulin purification.

Plasminogen activators in tissue extract of aural cholesteatoma

Using a biochemical technique, the authors characterized and identified the plasminogen activator (PA) derived from tissue extracts of six aural cholesteatomas. The results of fibrin zymography indicated that the tissue extracts of two cholesteatomas demonstrated two lytic zones on fibrin-agarose plates. One of the lytic zones was at about 72 kd, while the other zone was at about 64 kd. Using various goat immunoglobulin G (IgG)-containing antibodies (anti-human uterine tissue type PA (t-PA), anti-human low-molecular-weight (LMW) urokinase, and nonspecific goat IgG) and plasminogen-free fibrin-agarose plates, we confirmed that the cholesteatoma tissue extracts contained 72 kd t-PA and 64 kd urokinase type PA (u-PA). Furthermore, we measured the t-PA and u-PA activities in the tissue extracts selectively by parabolic rate assay. In order to estimate the PA activity, we developed optimal conditions for this assay. The specific t-PA activity ranged from 0.03 to 0.43 mIU/micrograms-protein and the specific u-PA activity ranged from 0 to 0.35 mIU/microgram-protein. The highest percentage of u-PA with respect to the total PA activity was 44.9%. However, in four of the six cases, we failed to detect u-PA activity. In the present study, we thus clarified the presence of PAs in tissue extracts of aural cholesteatomas. Furthermore, we confirmed that measureable u-PA occurred in some tissue extracts. We anticipate that the u-PA in inflammatory tissues plays an important role in the degradation of the extracellular matrix via the formation of plasmin and collagenases.

Purification of goat immunoglobulin G1 (IgG1) and IgG2 antibodies by use of Streptococcus dysgalactiae cells with Fc receptors

The application of stabilised streptococcal cells for the purification of both immunoglobulin 1 (IgG1) and IgG2 subclasses from goat sera was evaluated. Guanidinium chloride extracted, lyophilised cells of the Lancefield Group C Streptococcus dysgalactiae Sc 1 strain showed strong binding to goat IgG, reaching a capacity of approximately 1.4 mg IgG per 100 mg cells (dry weight). The IgG preparation obtained was of high quality. In immunoelectrophoretic analysis the preparation appeared to consist of pure IgG, whereas the high pressure liquid chromatography (HPLC) gel filtration and immunoblot analyses showed a very slight contamination (less than 1.3% of the total probe) with α 2-macroglobulin. The presence of both IgG subclasses in the preparation was verified by HPLC ion exchange chromatography. The adsorption procedure proved to be efficient and easy to perform without advanced technical equipment and the cells were reusable. As these streptococcal cells bind both IgG subclasses, this method presents an economical way for the small scale purification of goat IgG. Additionally the streptococcal cells may conveniently substitute Protein A bearing Staphylococcus aureus cells in various immunological assays.

Standardization of Flow Cytometric Crossmatch (FCXM) for Investigation of Unexplained Habitual Abortion

To define a positive flow cytometric crossmatch (FCXM), in terms of channel shift, for maternal IgG and IgM (n = 28) against paternal T and B lymphocytes. A reference range study. Mononuclear cells were obtained from 28 healthy volunteers using density gradient separation of heparinized blood, followed by pre-incubation with goat immunoglobulin. A total of twelve tubes were prepared for each volunteer. Primary incubation was with negative control serum, positive control sera (either IgG or IgM) and individual AB sera. Secondary incubation was with four combinations of fluorochromes: CD3 PE/IgG-Fc F(ab')2FITC, CD3 PE/IgM F(ab')2FITC, CD20 PE/IgG-Fc F(ab')2FITC and CD20 PE/IgM F(ab')2FITC. The cells were then analyzed with an EPICS Profile flow cytometer, using 256-channels and a four decade log scale. The linear mean channel fluorescence of the negative control serum was subtracted from the individual AB sera (channel shift) for each of the four combinations of fluorochromes. By determining the 95% one-sided upper reference limits of the negative control serum for each of the four trimmed data sets, we clinically defined a positive FCXM for bound IgG or IgM to T lymphocytes as a shift of 10 or more channels, and for bound IgG or IgM to B lymphocytes as a shift of 25 or more channels, above the linear mean channel shift of the negative control serum. Positive FCXMs were defined for maternal IgG and IgM against T and B lymphocytes, in terms of channel shift above the linear mean channel fluorescence of the negative control serum. By standardizing the dual-color FCXM methodology, the clinical significance of alloantibodies in the maintenance of pregnancy could be addressed in a collaborative manner.

A novel method for controlling the pepsin digestion of antibodies

We have discovered that 0.5–0.8 M ammonium sulfate has a beneficial effect on the peptic digestion of several antibodies. Initially, the project was begun to address the troublesome precipitation observed when pepsin was used to digest monoclonal antibody CYT-368 (C46), an IgG2a that binds carcinoembryonic antigen. High concentrations of various salts effectively blocked this precipitation, and also accelerated the rate of digestion. Of these, ammonium sulfate had the greatest effect. Therefore, it was tested on the peptic digestion of ten other murine IgG antibodies as well as rabbit, sheep and goat immunoglobulins. Besides preventing precipitation, ammonium sulfate usefully modulated the digestion rate, accelerating the digestion of some antibodies and reducing it for others. Finally, ammonium sulfate altered the specificity of pepsin during the digestion of antibody CYT-099 (B72.3) preventing the enzyme from cleaving in the F(ab′) 2 region, and thereby preserving the activity of the fragment. With ammonium sulfate we were able for the first time to use pepsin to obtain active F(ab′) 2 in good yield from this widely used antibody.

Growth hormone-releasing hormone antibodies suppress sleep and prevent enhancement of sleep after sleep deprivation.

Previous reports suggest that the hypothalamic growth hormone-releasing hormone (GHRH) promotes sleep, especially non-rapid-eye-movement sleep (NREMS). To evaluate the role of endogenous GHRH in sleep regulation, the effects of antibodies to rat GHRH (GHRH-ab) were studied on normal sleep, brain temperature (Tbr), and GH secretion in experiment I and on enhanced sleep after sleep deprivation in experiment II. In experiment I, affinity-purified GHRH-ab (50 and 200 micrograms) raised in goats and a control goat immunoglobulin G (IgG) preparation were injected intracerebroventricularly (icv) in rats 1 h before the onset of the light cycle, and sleep-wake activity and Tbr were recorded for the next 12 or 23 h. Both doses of GHRH-ab suppressed NREMS and REMS throughout the light cycle. Sleep durations at night were normal. Electroencephalographic (EEG) slow-wave activity, characterized by EEG slow-wave amplitudes, was reduced after GHRH-ab during both the light and the dark cycles. Plasma GH concentrations measured 6-12 h after injection of GHRH-ab (200 micrograms) were diminished. Both the control IgG and GHRH-ab elicited fever. In experiment II, the sleep-wake activity and Tbr of rats were recorded for 24 h in three experimental conditions: base-line with icv injection of IgG, 3-h sleep deprivation with icv IgG injection, and 3-h sleep deprivation with icv GHRH-ab (200 micrograms). After sleep deprivation (+IgG), a prompt increase in EEG slow-wave activity (power density analysis) and late increases in NREMS and REMS durations were found.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of urokinase-type plasminogen activator (u-PA) in tissue extracts of antrochoanal polyp.

Using a biochemical technique, the authors characterized and identified a plasminogen activator (PA) derived from tissue extracts of antrochoanal polyp (AP) and paranasal mucous membrane (PMM) with chronic sinusitis. The results of fibrin zymography indicated that the tissue extracts of AP revealed two lytic zones and that those of PMM revealed a single lytic zone on fibrin-agarose plates. One of the AP zones exhibited the same relative mobility as the PMM zone (molecular weight: 65 kd), while the other AP zone had a smaller molecular weight (about 54 kd). Goat immunoglobulin G (IgG) fraction of antihuman uterine tissue-type plasminogen activator (t-PA) inhibited the 65-kd lytic zones of AP and PMM. Antihuman low-molecular-weight urokinase inhibited only the 54-kd lytic zone of AP, and nonspecific goat IgG failed to inhibit any of the lytic zones. On the other hand, 10(-2) mol trans 4-(aminomethyl)cyclohexane-carboxylic acid (t-AMCHA) inhibited all of the lytic zones. No lytic zones could be observed on plasminogen-free fibrin-agarose plates. These findings confirmed that the tissue extracts of PMM contained t-PA, and that those of AP contained both t-PA and urokinase-type plasminogen activator (u-PA). In addition, it appeared that u-PA in inflammatory tissue was related to proliferative changes of the mucous membrane.

Identification of a mycoplasmal protein which binds immunoglobulins nonimmunologically

Immunoblotted protein samples from several strains of Mycoplasma hominis and from one strain of Mycoplasma arginini each contain a polypeptide of a molecular mass of 95,000 to 105,000 Da which binds immunoglobulin nonimmunologically. Immunoblots from these organisms were probed with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin, conjugated goat immunoglobulin G (IgG) Fab fragments, and conjugated goat IgG Fc fragments. The polypeptide bound the goat anti-rabbit molecules and the Fab fragments but not the Fc fragments. These reactions could be blocked with nonimmune unconjugated goat IgG and unconjugated human IgM. Controls probed with alkaline phosphatase alone did not stain. Binding of the conjugated preparations to whole mycoplasmal cells was dependent on concentrations of both conjugate and cells for the goat anti-rabbit preparation and for Fab. The mycoplasmal polypeptide may be a light-chain-specific reactant.

A direct surface plasmon—polariton immunosensor: Preliminary investigation of the non-specific adsorption of serum components to the sensor interface

Non-specific adsorption of serum protein components to a direct immunosensor constructed from a silvered diffraction grating coated with goat immunoglobulin G has been determined. Non-specific adsorption to a 'simplistic' sensor interface was found to be significant and would be expected to limit the sensitivity of reported immunosensors when used for the estimation of specific analytes in serum. This work highlights the need for complex sensor interfaces comprised of suitable chemical modifications and controlled, oriented, immobilisation of immunological components.

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The main purpose of this study is to examine the process of denture plaque formation and relationships between denture plaque and fibronectin.Plasma fibronectin has been detected in dental pellicle and dental plaque, and the capacity of the cells to bind gram-positive or gram-negative bacteria was shown. Three types of experimental denture plate were set in oral cavities of subject with normal dentition, for 1, 3 and 6 days. Obtained samples were staining with FITC labeled anti-aerobic bacteria rabbit serum, FITC labeled anti-anaerobic rabbit serum and FITC labeled anti-Candida rabbit serum and each of them were stained with RITC labeled anti-fibronectin goat immunoglobulins. They were observed by fluorescence microscopy. Fibronectin was found on all kinds of experimental denture plate, and existed positions are mainly equal to adherence of aerobic bacteria, anaerobic bacteria and Candida albicans.This study shows that fibronectin is significantly related to the formation of denture pellicle and denture plaque. The results play an important role that fibronectin might be correlated to the initial formation of the denture plaque.

Mouse Monoclonal Antibodies Detect an Allotypic Determinant Common to Several Ruminant Species

A monoclonal antibody against goat immunoglobulins recognizes an allotypic determinant (A1) which is common to goat, sheep, cattle and water buffalo. The frequency of the corresponding gene (A') is about the same in all four species, indicating the existence of a polymorphism that remained stable over a period of about 18-20 million years.

Distribution-analyzing latex immunoassay (DALIA): methods for determination of antigen and for elimination of non-specific reaction induced by rheumatoid factor.

A highly sensitive distribution-analyzing latex immunoassay method (DALIA), which is based on analysis of the volume distribution of latex particles including both the agglutinates and the residual non-agglutinating particles, has been established. Numbers of latex particles, which are sensitized with specific antibodies, are counted by using an electric particle counter with a personal computer and simultaneously the extent of agglutination was quantified by analyzing the volume distribution of the reacted latex particles. It was found that ultramicrospheres coated with normal goat immunoglobulin G can completely eliminate non-specific reactions induced by human serum containing rheumatoid factor (RF) in this DALIA method. The degree of absorption of RF activity by these ultramicrospheres was associated with the diameters of the ultramicrospheres. Moreover, by use of the combination of ultramicrospheres and latex particles coated with monoclonal antibodies against alpha-fetoprotein (AFP) or with polyclonal antibody specific to C-reactive protein, specific DALIA systems were able to be developed. Both DALIA systems exhibited the high sensitivity (1 to 7 ng/ml). The correlation coefficient (gamma) of the results of DALIA with those of enzyme-linked immunoadsorbent assay in measuring AFP was 0.994.

Effect of Microinjected Catalytic Fragment of Protein Kinase C on Morphological Change in Swiss 3T3 Cells

Although phorbol esters can enhance formation of an active, catalytic domain of protein kinase C (PKC) in intact cells, little is known about the actual importance of the proteolytic pathway in mediating cellular responses to the phorbol esters or other PKC activators. To explore this issue, we examined the effect of microinjected catalytic fragment of PKC on Swiss 3T3 cell morphology. In contrast to the dramatic, rapid response upon phorbol ester treatment, catalytic fragment microinjected in the presence of bovine serum albumin or normal goat immunoglobulin G as carrier protein had no effect. A morphological response similar but not identical to the effect of phorbol ester treatment was obtained, however, if catalytic fragment was microinjected in the presence of normal rabbit immunoglobulin G rather than the usual carrier proteins. The normal rabbit immunoglobulin by itself was inactive. Although the mechanism remains undefined, normal rabbit immunoglobulin but not other carrier proteins modulated PKC activity in vitro. We conclude that the generation of free catalytic fragment of PKC cannot account for the morphological response of Swiss 3T3 cells to the phorbol esters; secondary factors may, however, potentiate its action.