Comparison of immunomagnetic beads coated with protein A, protein G, or goat anti-mouse immunoglobulins. Applications in enzyme immunoassays and immunomagnetic separations.
Immunomagnetic beads were prepared using either protein A (PA) or protein G (PG) coupled to magnetic beads for binding antibodies at their Fc region. The performance of these beads was compared with commercially available beads coated with goat anti-mouse (G alpha M) immunoglobulins. Both the PA- and PG-beads possessed a higher binding capacity than the G alpha M-beads for the monoclonal antibodies tested, although, PA bound weakly with some IgG1 antibodies. PA-beads were compared with G alpha M-beads in a magnetic enzyme immunoassay for the detection of mouse immunoglobulins as an alternative to a conventional capture ELISA.
The magnetic enzyme immunoassay was characterized by a detection time of less than 60 min and a linear assay range from 5-10 to 500 ng/ml for G alpha M-beads and 5-10 to 1000 ng/ml for PA-beads. The capture ELISA was linear from 10 to 250 ng/ml. For immunomagnetic separation of Salmonella with immunomagnetic beads, PA-beads were superior to both PG- and G alpha M-beads. For specific isolation of bacteria from heterogeneous suspensions by immunomagnetic separation, PA- and PG-beads are preferable since G alpha M-beads crossreact with bacteria possessing proteins with Fc-binding activity.
Secondary immunoglobulin responses of BALB/c mice previously stimulated with goat anti–mouse IgD
Intravenous injection of goat antibodies to mouse IgD (GAMD) into BALB/c mice has been shown to induce vigorous T-cell dependent immunoglobulin responses, particularly of the IgG1 and IgE isotypes. We have confirmed these findings and show that IgA responses are also triggered in this model. Since the study of IgE regulation in allergic individuals is concerned with secondary and subsequent T- and B-cell responses, we boosted GAMD-primed mice with goat antibodies to IgE or IgA in an attempt to specifically retrigger IgE- and IgA-bearing memory B cells. However, we found that secondary IgG1, IgE and IgA production could be elicited equally well by either antibody preparation or by normal goat IgG (GIg). As with the primary response, GIg primed and boosted mice produced very low or undetectable IgG1, IgE and IgA responses.
These data suggest that GAMD is very efficient at priming T cells specific for GIg epitopes and that once primed they can be readily re-triggered by GIg. Spleen cells taken 7 days after boosting GAMD-primed mice were found to spontaneously produce much higher levels of interleukin-6 (IL-6) in culture than cells from unboosted or GIg primed and boosted mice.
In contrast to primary responses, where IgE levels return to background (less than 40 ng/ml) very quickly, circulating IgE levels in boosted mice initially declined before reaching a plateau level (approximately 1 microgram/ml) which was maintained for at least 148 days. IgG1 and IgA levels continued to fall over this same time period. Mice which had been primed (but not boosted) 10 months earlier were all found to have detectable IgE in their blood, despite the fact that following priming IgE becomes undetectable within 2-3 weeks. Since only a part of the IgE response was directed towards the antigen (GIg), these observations suggest the possibility that B cells initially primed to make IgE can be non-specifically retriggered in vivo.
Polyclonal activation of the murine immune system by an antibody to IgD. VI. Influence of doses of goat anti–mouse delta chain and normal goat IgG on B lymphocyte proliferation and differentiation
The injection of mice with 800 micrograms of an affinity-purified goat antibody to mouse IgD (GaM delta) induces early, T-independent polyclonal increases in the expression of B cell surface Ia, and B cell size and DNA synthesis, as well as later, T-dependent polyclonal increases in spleen cell number and Ig secretion. We have now studied the effects of varying the doses of injected GaM delta on all phases of B cell activation, as well as the effects of supplementing GaM delta with varying quantities of normal goat IgG (GIgG). We have found that while 12.5 micrograms of GaM delta modulates most of the IgD from the surface of splenic B lymphocytes, it fails to activate these cells. Increases in the expression of B cell surface Ia are first seen when 50 micrograms of GaM delta is injected, while increases in B cell DNA synthesis usually require the injection of 200 micrograms of GaM delta and peak with doses of approximately 800 micrograms.
Increases in splenic B cell number and DNA synthesis during the T-dependent phase of GaM delta-induced B cell activation are seen only in those mice that were injected with sufficient quantities of GaM delta to induce DNA synthesis during the T-independent phase. Supplementing the dose of GaM delta injected with additional GIgG has no significant effect on B cell DNA synthesis or B cell number but dramatically increases polyclonal IgG1 secretion.
Although mice which have been injected with 50 micrograms of GaM delta or with 800 micrograms of GIgG alone have few polyclonal IgG1-secreting cells, substantial increases in the number of IgG1-secreting cells are seen in mice injected with 50 micrograms of GaM delta plus 750 micrograms of GIgG. GIgG and larger doses of GaM delta similarly act synergistically to increase polyclonal IgG1 secretion.
In contrast to the induction of polyclonal IgG1 secretion, the stimulation of polyclonal IgM secretion requires the injection of mitogenic doses of GaM delta and is not enhanced by the injection of additional GIgG. These observations suggest that, in this model system, stimulatory signals that activate B cells through their surface Ig are limiting for the induction of polyclonal proliferation and IgM secretion, while the generation of T helper lymphokines that do not directly interact with B cells through their surface Ig may be more limiting for the stimulation of polyclonal IgG1 secretion.
Clinical utility of abdominal multidetector CT in 85 goats with suspected abdominal disease
Diagnosing the cause of abdominal disease in goats can be challenging. Clinical history, physical investigation, and laboratory findings do not always allow definitive identification of intra-abdominal disease or the underlying cause. Multidetector CT (MDCT) has become more readily available and now often replaces or augments other abdominal imaging techniques. The objective of this retrospective, observational, descriptive study was to investigate the clinical utility of MDCT for evaluation of the abdomen in goats with suspected abdominal disease involving the urinary, gastrointestinal, reproductive tracts and abdominal wall. Medical records (1/2009-12/2017) were reviewed for all goats undergoing an abdominal MDCT. Signalment, clinical history, examination and MDCT findings and outcome were recorded and categorized by abdominal organ system and wall lesion.
IC IgG1/IgG1/ CD45 | ||||
ICIGG1FPE45PP1-50T | ImmunoStep | 50 test | 574.8 EUR | |
IgG1, Fc | ||||
71456 | BPS Bioscience | 200 µg | 265 EUR | |
Mouse IgG1 | ||||
C040307-10mg | Unibiotest | 10mg | 2148 EUR | |
Mouse IgG1 | ||||
C040307-1mg | Unibiotest | 1mg | 728.4 EUR | |
Human IgG1 | ||||
C040330-10mg | Unibiotest | 10mg | 2148 EUR | |
Human IgG1 | ||||
C040330-1mg | Unibiotest | 1mg | 728.4 EUR | |
Mouse IgG1 | ||||
31C-CH1004 | Fitzgerald | 1 mg | 452.4 EUR | |
Human IgG1 | ||||
31R-1087 | Fitzgerald | 1 mg | 333.6 EUR | |
IgG1 antibody | ||||
70R-49901 | Fitzgerald | 100 ul | 292.8 EUR | |
IgG1 Antibody | ||||
DF8170 | Affbiotech | 200ul | 420 EUR | |
Calretinin (IgG1) | ||||
MO22165 | Neuromics | 100 ul | 522 EUR | |
IC IgG1 / IgG1 / CD45 Antibody (FITC, PE, PerCP) | ||||
abx200630-50tests | Abbexa | 50 tests | 678 EUR | |
Mouse IgG1 (FITC) / Rat IgG1 (RPE) Isotype Control | ||||
abx405006-50tests | Abbexa | 50 tests | 744 EUR | |
Mouse IgG1 (FITC) / Mouse IgG1 (RPE) Isotype Control | ||||
abx405001-50tests | Abbexa | 50 tests | 760.8 EUR | |
Mouse IgG1 (FITC) / Mouse IgG1 (RPE) Isotype Control | ||||
abx405005-100tests | Abbexa | 100 tests | 744 EUR | |
Mouse IgG1 (HRP) | ||||
65C-CH1006 | Fitzgerald | 100 µg | 309.6 EUR | |
Mouse IgG1 (FITC) | ||||
65C-CH1005 | Fitzgerald | 100 µg | 271.2 EUR | |
IgG1 Antibody (PE) | ||||
abx159555-100ul | Abbexa | 100 ul | 427.2 EUR |
×
Clinical problems and MDCT findings were compared in the various abdominal categories. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic odds ratio (OR) were calculated for MDCT, using clinical examination findings as the reference standard. A total of 85 goats underwent an abdominal MDCT examination. The sensitivity of MDCT for detecting urinary, gastrointestinal, reproductive tract, and abdominal wall abnormalities in goats with clinical problems related to these body systems was high at 94.7 %, 78.3%, 94.1%, and 100%, and the specificity was high at 95.6%, 96.7%, 93.9%, and 100%, respectively. The PPV was 94.7%, 90.0 %, 80.0%, and 100.0%, the NPV was 95.6%, 92.1%, 98.4%, and 100%, and the OR were 387.0, 104.4, 248.0, and infinite. In conclusion, findings supported the use of MDCT as an adjunct diagnostic test for assessing goats with abdominal disease.