A Comparative Study of "Grafting to" and "Grafting from" Conjugation Methods for the Preparation of Antibody-Temperature-Responsive Polymer Conjugates.

Early diagnosis of infectious diseases is still challenging particularly in a nonlaboratory environment or limited resources areas. Thus, sensitive, inexpensive, and easily handled diagnostic approaches are required. The lateral flow immunoassay (LFIA) is commonly used in the screening of infectious diseases despite its poor sensitivity, especially with low pathogenic loads (early stages of infection). This article introduces a novel polymeric material that might help in the enrichment and concentration of pathogens to overcome the LFIA misdiagnosis. To achieve this, we evaluated the efficiency of introducing poly(N-isopropylacrylamide) (PNIPAAm) into immunoglobulin G (IgG) as a model antibody using two different conjugation methods: grafting to (GT) and grafting from (GF). The IgG-PNIPAAm conjugates were characterized using SDS-PAGE, DLS, and temperature-responsive phase transition behavior. SDS-PAGE analysis revealed that the GF method was more efficient in introducing the polymer than the GT method, with calculated polymer introduction ratios of 61% and 34%, respectively. The GF method proved to be less susceptible to steric hindrance and more efficient in introducing high-molecular-weight polymers into proteins. These results are consistent with previous studies comparing the GT and GF methods in similar systems. This study represents an important step toward understanding how the choice of polymer incorporation method affects the properties of IgG-PNIPAAm conjugates. The synthesized polymer allowed binding and enrichment of mouse IgG that was used as a model antigen with a clear LFIA band. On the basis of our findings, this system might help in improving the sensitivity of simple diagnostics.

Prominin-1 deletion results in spermatogenic impairment, sperm morphological defects, and infertility in mice.

Purpose: Spermatogenesis is a complex process orchestrated by several essential genes. Prominin-1 (Prom1/PROM1) is a gene that is expressed in the testis but with a poorly understood role in spermatogenesis. Methods: We used Prom1 knockout (Prom1 KO) mice to assess the role of Prom1 in spermatogenesis. To this end, we performed immunohistochemistry, immunofluorescence, western blotting, ß-galactosidase staining, and apoptosis assay. Additionally, we analyzed the morphology of sperm and assessed litter sizes. Results: We observed that PROM1 is localized to the dividing spermatocytes in seminiferous epithelial cells, sperm, and columnar epithelium in the epididymis. In the Prom1 KO testis, an aberrant increase in apoptotic cells and a decrease in proliferating seminiferous epithelial cells were observed. Cellular FLICE-like inhibitory protein (c-FLIP) and extracellular signal-regulated kinase 1/2 (ERK1/2) expression were also significantly decreased in Prom1 KO testis. In addition, a significantly increased number of epididymal spermatozoa with abnormal morphology and less motility was found in Prom1 KO mice. Conclusions: PROM1 maintains spermatogenic cell proliferation and survival via c-FLIP expression in the testis. It is also involved in sperm motility and fertilization potential. The mechanism underlying the effect of Prom1 on sperm morphology and motility remains to be identified.

Preparation of Temperature-Responsive Antibody-Nanoparticles by RAFT-Mediated Grafting from Polymerization.

Herein, we report the preparation of temperature-responsive antibody-nanoparticles by the direct polymerization of N-isopropylacrylamide (NIPAAm) from immunoglobulin G (IgG). To this end, a chain transfer agent (CTA) was introduced into IgG, followed by the precipitation polymerization of NIPAAm in an aqueous medium via reversible addition-fragmentation chain transfer polymerization above the lower critical solution temperature (LCST). Consequently, antibody-polymer particles with diameters of approximately 100-200 nm were formed. Owing to the entanglement of the grafted polymers via partial chemical crosslinking, the antibody-nanoparticles maintained their stability even at temperatures below the LCST. Further, the dispersed nanoparticles could be collected by thermal precipitation above the LCST. Additionally, the antibody-nanoparticles formulation could maintain its binding constant and exhibited a good resistance against enzymatic treatment. Thus, the proposed antibody-nanoparticles can be useful for maximizing the therapeutic potential of antibody-drug conjugates or efficacies of immunoassays and antibody recovery and recycling.

Temperature Responsive Polymer Conjugate Prepared by "Grafting from" Proteins toward the Adsorption and Removal of Uremic Toxin.

In this study, temperature-responsive polymer-protein conjugate was synthesized using a "grafting from" concept by introducing a chain transfer agent (CTA) into bovine serum albumin (BSA). The BSA-CTA was used as a starting point for poly(N-isopropylacrylamide) (PNIPAAm) through reversible addition-fragmentation chain transfer polymerization. The research investigations suggest that the thermally responsive behavior of PNIPAAm was controlled by the monomer ratio to CTA, as well as the amount of CTA introduced to BSA. The study further synthesized the human serum albumin (HSA)-PNIPAAm conjugate, taking the advantage that HSA can specifically adsorb indoxyl sulfate (IS) as a uremic toxin. The HSA-PNIPAAm conjugate could capture IS and decreased the concentration by about 40% by thermal precipitation. It was also revealed that the protein activity was not impaired by the conjugation with PNIPAAm. The proposed strategy is promising in not only removal of uremic toxins but also enrichment of biomarkers for early diagnostic applications.

Temperature responsive smart polymer for enabling affinity enrichment of current coronavirus (SARS-CoV-2) to improve its diagnostic sensitivity.

The current commercially available SARS-CoV-2 diagnostic approaches including nucleic acid molecular assaying using polymerase chain reaction (PCR) have many limitations and drawbacks. SARS-CoV-2 diagnostic strategies were reported to have a high false-negative rate and low sensitivity due to low viral antibodies or antigenic load in the specimens, that is why even PCR test is recommended to be repeated to overcome this problem. Thus, in anticipation of COVID-19 current wave and the upcoming waves, we should have an accurate and rapid diagnostic tool to control this pandemic. In this study, we developed a novel preanalytical strategy to be used for SARS-CoV-2 specimen enrichment to avoid misdiagnosis. This method depends on the immuno-affinity trapping of the viral target followed by in situ thermal precipitation and enrichment. We designed, synthesized, and characterized a thermal-responsive polymer poly (N-isopropylacrylamide-co-2-hydroxyisopropylacrylamide-co-strained alkyne isopropylacrylamide) followed by decoration with SARS-CoV-2 antibody. Different investigations approved the successful synthesis of the polymeric antibody conjugate. This conjugate was shown to enrich recombinant SARS-CoV-2 nucleocapsid protein samples to about 6 folds. This developed system succeeded in avoiding the misdiagnosis of low viral load specimens using the lateral flow immunoassay test. The strength of this work is that, to the best of our knowledge, this report may be the first to functionalize SARS-CoV-2 antibody to a thermo-responsive polymer for increasing its screening sensitivity during the current pandemic.

Conjugation of antibody with temperature-responsive polymer via in situ click reaction to enable biomarker enrichment for increased diagnostic sensitivity.

Early diagnosis of infectious diseases is one of the current prevalent challenges, especially in low and limited resource settings where simple, fast, portable, cheap, and sensitive diagnostic approaches are needed. Lateral flow immunoassay (LFIA) is a common, rapid screening assay. However, the low assay sensitivity limits the utility of LFIA for specimens with low pathogenic loads (early infection stages). Antibodies conjugated with stimulus-responsive polymers have been previously utilized to improve assay sensitivity for detection of biomarkers at low concentrations. However, the loss of antibody affinity after polymer conjugation remains a significant challenge. In this study, we developed poly(N-isopropylacrylamide-co-N-(2-hydroxyisopropyl)acrylamide-co-strained alkyne-isopropylacrylamide), a novel polymer for biomarker enrichment, by polymer conjugation after antibody-antigen recognition. We employed and promoted the click chemistry in situ, to facilitate highly specific conjugation between novel temperature-responsive polymers and antibody-antigen complexes. This method could suppress the decrease in the binding constant associated with polymer conjugation (>20-fold). The conjugation was successfully demonstrated in body fluids such as urine and saliva. We achieved >5-fold antigen enrichment via thermal precipitation by conjugating polymers to the antibodies after antigen recognition. Concentrated biomarkers resulted in improved LFIA detection. This approach can potentially be utilized to improve diagnostic tests for infectious diseases in low and limited resource settings.