Lambda monoclonal free light chain abnormalities detected by a serum immunofixation electrophoresis assay are underrepresented by quantitative serum free light chain results

Protein and immunofixation (IFIX) electrophoresis are used to diagnose and monitor monoclonal gammopathies. While IFIX detects clonal production of intact immunoglobulins and free light chains (FLC), the latter can also be quantified using a serum free light chain (SFLC) assay, in which polyclonal antisera detects epitopes specific for free kappa (KFLC) or lambda light chains (LFLC). An abnormal KFLC: LFLC ratio (KLR) serves as a surrogate for clonality. While the SFLC assay is highly sensitive, normal LFLC (<2.63mg/dL) and KLR results (>0.26 & <1.65) were found in samples with distinct lambda monoclonal free light chains visualized by IFIX (X-LMFLC). To investigate this discordance, contemporaneous SFLC or KLR values were evaluated for their ability to accurately classify monoclonal FLCs identified by IFIX. We performed a retrospective analysis of serum and urine IFIX (Sebia Hydrasys) and SFLC (Freelite®, Binding Site) results from our institution between July 2010 through December 2020, using R 4.0.2 and Tidyverse packages. From among 9,594 encounters in which a single monoclonal component was initially identified by IFIX, 157 X-LMFLC and 131 X-KMFLC samples were analyzed. Elevated LFLC with normal KFLC was identified in 105/157 X-LMFLC samples (67%), while both LFLC and KFLC were elevated in 42/157 samples (27%). Concordance between X-KMFLC and KFLC was markedly higher, where 122/131 samples (93%) displayed elevated kappa FLC (>1.94mg/dL) with normal LFLC, and only 7/131 X-KMFLC samples (5%) possessed both elevated KFLC and LFLC. The use of KLR to identify pathogenic monoclonal free light chains improved lambda concordance to 85%; however, 19/157 (12%) of X-LMFLC samples still exhibited normal KLR. High concordance of 98% was again observed for X-KMFLC with abnormal KLR. When samples were segregated according to normal or impaired renal function (eGFR > or ≤60mL/min/1.73m², respectively), this disparate identification of X-LMFLC and X-KMFLC by the SFLC assay persisted, suggesting that renal dysfunction (as measured by eGFR) does not underlie this phenomenon. Lastly, we corroborated the above findings in a larger sample population by examining patients with urine Bence Jones FLC identified by IFIX who had free or intact monoclonal components in serum (N=724), grouped by lambda or kappa light chain involvement. The cause(s) of the discrepant performance by the Freelite® SFLC assay, relative to the Sebia Hydrasys IFIX assay, for identifying lambda FLC components is currently unclear. Possible contributory factors include assay reference range cutoffs, other patient disease parameters, and differences in assay-specific polyclonal antisera. Future analyses of these factors will help to further characterize SFLC assay performance and elucidate how interpretation of composite serum FLC test results can be improved to better guide patient management.

Making Weak Antigens Strong: Preparing Immune Complexes for Injection

If antibodies against a particular antigen are available, that antigen can be purified and used for further immunizations, and antigens thus purified can show enhanced immunogenicity. Purified immune complexes can be injected directly, or while coupled to beads; the presence of antibodies and/or beads stimulates phagocytosis and usually will not influence the response. This method provides a useful means of antigen enrichment for a variety of applications, such as using antibodies raised against a denatured antigen to harvest a native protein for further immunizations, or when using a monoclonal antibody as an intermediate to the preparation of polyclonal antisera. Injecting antibody-coated antigens has also been used to mask a particularly immunodominant epitope on an antigen, and thereby develop a response against other epitopes. The amount of antigen needed to elicit a strong response using immune complexes will vary from one compound to another. Doses as low as 50 ng of antigen have been used successfully when delivered this way.

Evidence for Unknown Sarcocystis-Like Infection in Stranded Striped Dolphins (Stenella coeruleoalba) from the Ligurian Sea, Italy

Two striped dolphins (SD1, SD2), stranded along the Ligurian coast of Italy, were diagnosed with a nonsuppurative meningoencephalitis associated with previously undescribed protozoan tissue cysts. As tissue cysts were morphologically different from those of Toxoplasma gondii, additional histopathological, immunohistochemical, ultrastructural, and biomolecular investigations were performed, aiming to fully characterize the organism. Histopathology revealed the presence of large Sarcocystis-like tissue cysts, associated with limited inflammatory lesions in all CNS areas studied. IHC was inconclusive, as positive staining with polyclonal antisera did not preclude cross-reaction with other Sarcocystidae coccidia. Applied to each animal, 11 different PCR protocols precluded a neural infection by Sarcocystis neurona, Sarcocystis falcatula, Hammondia hammondi, and Neospora caninum. T. gondii coinfection was confirmed only in dolphin SD2. Sarcocystis sp. sequences, showing the highest homology to species infecting the Bovidae family, were amplified from SD1 myocardium and SD2 skeletal muscle. The present study represents the first report of Sarcocystis-like tissue cysts in the brain of stranded cetaceans along with the first description of Sarcocystis sp. infection in muscle tissue of dolphins from the Mediterranean basin.

High-Resolution Mapping of Human Norovirus Antigens via Genomic Phage Display Library Selections and Deep Sequencing

ABSTRACT Norovirus (NoV) infections are a leading cause of gastroenteritis. The humoral immune response plays an important role in the control of NoV, and recent studies have identified neutralizing antibodies that bind the capsid protein VP1 to block viral infection. Here, we utilize a NoV GI.1 Jun-Fos-assisted phage display library constructed from randomly fragmented genomic DNA coupled with affinity selection for antibody binding and subsequent deep sequencing to map epitopes. The epitopes were identified by quantitating the phage clones before and after affinity selection and aligning the sequences of the most enriched peptides. The HJT-R3-A9 single-chain variable fragment (scFv) antibody epitope was mapped to a 12-amino-acid region of VP1 that is also the binding site for several previously identified monoclonal antibodies. We synthesized the 12-mer peptide and found that it binds the scFv antibody with a KD (equilibrium dissociation constant) of 46 nM. Further, alignment of enriched peptides after affinity selection on rabbit anti-NoV polyclonal antisera revealed five families of overlapping sequences that define distinct epitopes in VP1. One of these is identical to the HJT-R3-A9 scFv epitope, further suggesting that it is immunodominant. Similarly, other epitopes identified using the polyclonal antisera overlap binding sites for previously reported monoclonal antibodies, suggesting that they are also dominant epitopes. The results demonstrate that affinity selection and deep sequencing of the phage library provide sufficient resolution to map multiple epitopes simultaneously from complex samples such as polyclonal antisera. This approach can be extended to examine the antigenic landscape in patient sera to facilitate investigation of the immune response to NoV. IMPORTANCE NoV infections are a leading cause of gastroenteritis in the United States. Human NoVs exhibit extensive genetic and antigenic diversity, which makes it challenging to design a vaccine that provides broad protection against infection. Antibodies developed during the immune response play an important role in the control of NoV infections. Neutralizing antibodies that act by sterically blocking the site on the virus used to bind human cells have been identified. Identification of other antibody binding sites associated with virus neutralization is therefore of interest. Here, we use a high-resolution method to map multiple antibody binding sites simultaneously from complex serum samples. The results show that a relatively small number of sites on the virus bind a large number of independently generated antibodies, suggesting that immunodominance plays a role in the humoral immune response to NoV infections.

Reagents for detection of Rift Valley fever virus infection in sheep

Rift Valley fever virus (RVFV) causes Rift Valley fever (RVF), resulting in morbidity and mortality in humans and ruminants. Evidence of transboundary outbreaks means that RVFV remains a threat to human health and livestock industries in countries that are free from the disease. To enhance surveillance capability, methods for detection of RVFV are required. The generation of reagents suitable for the detection of RVFV antigen in formalin-fixed, paraffin-embedded tissues from infected animals have been developed and are described herein. Recombinant nucleoprotein (rNP) was expressed in Escherichia coli and purified using immobilized metal ion affinity chromatography. Purified rNP was used as an immunogen to produce anti-NP polyclonal antisera in rabbits for use in detection of RVFV NP in experimentally infected animals by immunohistochemistry. Antisera raised in rabbits against rNP were able to recognize viral NP antigen in fixed infected Vero cell pellets and sheep liver. Therefore, the methods and reagents described herein are useful in assays for detection of RVFV infections in animals, for research and surveillance purposes.

Development of polyclonal antisera against movement proteins of the three poleroviruses infecting cucurbits and their serological relationships

Background: Cucurbit aphid-borne yellows virus (CABYV), Melon aphid-borne yellows virus (MABYV) and Suakwa aphid-borne yellows virus (SABYV) are three critical viruses infecting cucurbit crops. The preparation of specific antiserum against the virus is crucial for both the detection of virus and understanding the functions of the related genes. However, there is no report about detecting the three viruses using antisera against movement proteins (MP). Methods: In this study, we constructed prokaryotic expression vectors of the three viral movement proteins and transferred them into Escherichia coli strain Rosetta to purify the fusion proteins. Then the polyclonal antisera were obtained by immunizing New Zealand white rabbits. Western blotting was used to demonstrate the applicability of the three antisera. Results: We discovered that the titer of antiserum against MP CABYV reached to 1: 512000, and the titers of antisera against MP MABYV and MP SABYV reached to 1:256000. The optimized working concentration range for the three antisera was from 1:10000 to 1:64000. Both antisera against MP CABYV and MP MABYV could only react with the corresponding MP. The antiserum against MP SABYV not only had the strongest reaction with its MP but also could react with MP CABYV and MP MABYV at relative weaker levels and all the three antisera had no serological reactions with other poleroviruses tested. Furthermore, our results showed that the three antisera could specifically detect movement proteins both in Nicotiana benthamiana and cucumber leaves. Conclusions: We have established a sensitive system for detecting three poleroviruses infecting cucurbits by antisera against movement proteins, providing a material foundation for the future research on both the serological detection of viruses and the interaction mechanisms between the virus and host plants.