The use of antibody-based therapies for the treatment of high consequence viral pathogens has gained interest over the last fifteen years.
Here, we sought to evaluate the use of unique camelid-based IgG antibodies to prevent lethal hantavirus pulmonary syndrome (HPS) in Syrian hamsters.
Using purified, polyclonal IgG antibodies generated in DNA-immunized alpacas, we demonstrate that post-exposure treatments reduced viral burdens and organ-specific pathology associated with lethal HPS.
Antibody-treated animals did not exhibit signs of disease and were completely protected.
The unique structures and properties, particularly the reduced size, distinct paratope formation and increased solubility of camelid antibodies, in combination with this study support further pre-clinical evaluation of heavy–chain only antibodies for the treatment of severe respiratory diseases, including HPS.
Immune subtraction for improved resolution in serum protein immunofixation electrophoresis and antibody isotype determination in a patient with autoantibody
Heavy chain isotypes of low level monoclonal immunoglobulins are sometimes obscured in serum immunofixation electrophoresis (SIFE) by a heavy background of polyclonal immunoglobulins.
However, accurate determination of the heavy chain isotype is essential for a complete diagnosis, as isotype determination of autoantibodies may have relevance in determining therapeutic procedures.
Immune subtraction (IS) was employed in a patient with neuropathy and GD1a autoantibody.
IS allowed identification of the cognate heavy chain related to a lambda light chain restriction noted on initial SIFE as well as isotype determination of the autoantibody.
Antisera specific to individual heavy and light chains were used for depletion of specific immunoglobulin types.
Depletion of kappa light chain associated immunoglobulins allowed unequivocal determination of the isotype of lambda light chain-associated low level monoclonal band to be IgG Lambda.
Selective depletion of kappa, lambda, gamma and mu heavy chain immunoglobulins was employed to determine IgG Kappa isotype of the auto-antibody.
Immunofluorescence Staining for Immunoglobulin Heavy Chain/Light Chain on Kidney Biopsies is a Valuable Ancillary Technique For the Diagnosis of Monoclonal Gammopathy-Associated Kidney Diseases
Heavy chain/light chain (HLC) antibodies target conformational epitopes at the junctions of the heavy chain and light chain constant regions (CH1 and CL) of serum IgGκ, IgGλ, IgAκ, IgAλ, IgMκ, and IgMλ to provide quantitation of intact HLC pairs.
Here, we developed an HLC tissue immunofluorescence protocol to test if it can complement conventional immunofluorescence in the diagnosis of monoclonal gammopathy-associated kidney diseases.
HLC immunofluorescence was performed on archived frozen tissue of 104 kidney biopsies.
The sensitivity and specificity of HLC immunofluorescence was confirmed by testing cases of lupus nephritis, other polyclonal immunoglobulin nephropathies, and light chain nephropathies (light chain amyloidosis and deposition disease).
Testing of ten cases of the IgG variant of proliferative glomerulonephritis with monoclonal immunoglobulin deposits excluded monoclonal deposits in two by revealing positivity for IgGκ and IgGλ.
Testing of 12 cases of monotypic IgA nephropathy excluded monoclonal deposits in six by revealing staining for IgAκ and IgAλ.
Testing of six cases of monotypic fibrillary glomerulonephritis excluded monoclonal deposits in three by revealing positivity for IgGκ and IgGλ. None of 14 cases of glomerulonephritis in which HLC immunofluorescence unmasked polytypic deposits were associated with a serum or urine monoclonal immunoglobulins matching the conventional immunofluorescence results.
HLC immunofluorescence outperformed paraffin immunofluorescence and IgG subclass staining in 10/13 (77%) of cases.
Testing of 18 cases of cryoglobulinemic glomerulonephritis showed better correlation with serum cryoprecipitate immunofixation than conventional immunofluorescence with regards to the type of cryoglobulin in 47% of cases.
Thus, HLC immunofluorescence is a valuable ancillary technique in kidney pathology for the diagnosis of monoclonal gammopathy-associated nephropathies, and could be utilized to confirm or exclude the monoclonal nature of deposits.
A Novel Total Drug Assay for Quantification of Anti-C5 Therapeutic Monoclonal Antibody in the Presence of Abundant Target
SKY59 or RO7112689 is a humanized monoclonal antibody against complement protein C5 with pH-dependent C5-binding and neonatal Fc receptor-mediated recycling capabilities, which result in long-lasting neutralization of C5.
We developed and validated a novel total drug assay for quantification of target-binding competent SKY59 in the presence of endogenous C5 in cynomolgus monkey plasma. The target-binding competent SKY59 was determined after complex formation by the addition of recombinant monkey C5 using goat anti-human IgG–heavy chain monkey-adsorbed polyclonal antibody as a capture antibody and rabbit anti-C5 monoclonal antibody (mAb) non-competing with SKY59 for detection.
The total SKY59 assay was shown to be accurate and precise over the range of 0.05-3.2 μg/mL as well as be tolerant to more than 400 μg/mL of C5 (~ 3000-fold molar excess of target).
We also developed and validated a total C5 assay, confirmed selectivity and parallelism, and verified the utility of recombinant monkey C5 for the total C5 assay as well as the total SKY59 assay.
Furthermore, we used these validated methods to measure SKY59 and C5 concentrations in cynomolgus monkey plasma samples in a toxicology study.
This total drug assay can be applied not only to other antibody therapeutics against shed/soluble targets when a non-competing reagent mAb is available but also for clinical studies when a reagent mAb specific for engineered Fc region on a therapeutic mAb is available.
Inhibitory Potential of Polyclonal Camel Antibodies against New Delhi Metallo-β-lactamase-1 (NDM-1)
New Delhi Metallo-β-lactamase-1 (NDM-1) is the most prevalent type of metallo-β-lactamase, able to hydrolyze almost all antibiotics of the β-lactam group, leading to multidrug-resistant bacteria. To date, there are no clinically relevant inhibitors to fight NDM-1.
The use of dromedary polyclonal antibody inhibitors against NDM-1 represents a promising new class of molecules with inhibitory activity.
In the current study, immunoreactivities of dromedary Immunoglobulin G (IgG) isotypes containing heavy–chain and conventional antibodies were tested after successful immunization of dromedary using increasing amounts of the recombinant NDM-1 enzyme.
igg Heavy Chain Polyclonal Antibody |
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| A57584 | EpiGentek |
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igg Heavy Chain Polyclonal Antibody |
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| A57789 | EpiGentek |
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igg heavy chain Polyclonal Antibody |
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| 42160 | SAB | 100μg | 390 EUR |
igg heavy chain Polyclonal Antibody |
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| 42160-100ul | SAB | 100ul | 399.6 EUR |
igg heavy chain Polyclonal Antibody |
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| MBS9415818-01mg | MyBiosource | 0.1mg | 350 EUR |
igg heavy chain Polyclonal Antibody |
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| MBS9415818-5x01mg | MyBiosource | 5x0.1mg | 1420 EUR |
igg heavy chain Polyclonal Conjugated Antibody |
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| C42160 | SAB | 100ul | 476.4 EUR |
IgG Heavy Chain Polyclonal Conjugated Antibody |
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| MBS9450591-01mLAF350 | MyBiosource | 0.1mL(AF350) | 480 EUR |
IgG Heavy Chain Polyclonal Conjugated Antibody |
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| MBS9450591-01mLAF405 | MyBiosource | 0.1mL(AF405) | 480 EUR |
IgG Heavy Chain Polyclonal Conjugated Antibody |
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| MBS9450591-01mLAF488 | MyBiosource | 0.1mL(AF488) | 480 EUR |
IgG Heavy Chain Polyclonal Conjugated Antibody |
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| MBS9450591-01mLAF555 | MyBiosource | 0.1mL(AF555) | 480 EUR |
IgG Heavy Chain Polyclonal Conjugated Antibody |
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| MBS9450591-01mLBiotin | MyBiosource | 0.1mL(Biotin) | 480 EUR |
igg Heavy Chain Polyclonal Antibody, HRP Conjugated |
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| A57585 | EpiGentek |
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igg Heavy Chain Polyclonal Antibody, HRP Conjugated |
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| A57790 | EpiGentek |
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igg Heavy Chain Polyclonal Antibody, FITC Conjugated |
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| A57586 | EpiGentek |
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igg Heavy Chain Polyclonal Antibody, FITC Conjugated |
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| A57791 | EpiGentek |
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igg Heavy Chain Polyclonal Antibody, Biotin Conjugated |
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| A57587 | EpiGentek |
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Inhibition kinetic assays, performed using a spectrophotometric method with nitrocefin as a reporter substrate, demonstrated that IgGIgGIgG50) values ranging from 100 to 0.04 μM. Investigations on the ability of IgG subclasses to reduce the growth of recombinant Escherichia coli BL21(DE3)/codon plus cells containing the recombinant plasmid expressing NDM-1, L1, or VIM-1 showed that the addition of IgGs (4 and 8 mg/L) to the cell culture was unable to restore the susceptibility of carbapenems. Interestingly, IgGs were able to interact with NDM-1, L1, and VIM-1 when tested on the periplasm extract of each cultured strain.
The inhibitory concentration was in the micromolar range for all β-lactams tested.
A visualization of the 3D structural basis using the three enzyme Protein Data Bank (PDB) files supports preliminarily the recorded inhibition of the three MBLs.