Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

Download Full-text

The Ability of Zika virus Intravenous Immunoglobulin to Protect From or Enhance Zika Virus Disease

Frontiers in Immunology ◽

10.3389/fimmu.2021.717425 ◽

2021 ◽

Vol 12 ◽

Author(s):

Amelia K. Pinto ◽

Mariah Hassert ◽

Xiaobing Han ◽

Douglas Barker ◽

Trevor Carnelley ◽

...

Keyword(s):

Virus Infection ◽

Polyclonal Antibody ◽

Zika Virus ◽

Virus Disease ◽

Polyclonal Antibodies ◽

Antibody Therapeutics ◽

The World ◽

Flavivirus Infection

The closely related flaviviruses, dengue and Zika, cause significant human disease throughout the world. While cross-reactive antibodies have been demonstrated to have the capacity to potentiate disease or mediate protection during flavivirus infection, the mechanisms responsible for this dichotomy are still poorly understood. To understand how the human polyclonal antibody response can protect against, and potentiate the disease in the context of dengue and Zika virus infection we used intravenous hyperimmunoglobulin (IVIG) preparations in a mouse model of the disease. Three IVIGs (ZIKV-IG, Control-Ig and Gamunex®) were evaluated for their ability to neutralize and/or enhance Zika, dengue 2 and 3 viruses in vitro. The balance between virus neutralization and enhancement provided by the in vitro neutralization data was used to predict the IVIG concentrations which could protect or enhance Zika, and dengue 2 disease in vivo. Using this approach, we were able to define the unique in vivo dynamics of complex polyclonal antibodies, allowing for both enhancement and protection from flavivirus infection. Our results provide a novel understanding of how polyclonal antibodies interact with viruses with implications for the use of polyclonal antibody therapeutics and the development and evaluation of the next generation flavivirus vaccines.

Download Full-text

Development of Murine Monoclonal Antibodies for the Immunohistochemical Diagnosis of Systemic Bovine Aspergillosis

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879600800111 ◽

1996 ◽

Vol 8 (1) ◽

pp. 68-75 ◽

Cited By ~ 31

Author(s):

H. E. Jensen ◽

B. Aalbaek ◽

P. Lind ◽

H. V. Krogh ◽

P. L. Frandsen

Keyword(s):

Monoclonal Antibodies ◽

Polyclonal Antibodies ◽

Fungal Species ◽

Cross Reactivity ◽

Water Soluble ◽

Enzyme Linked Immunosorbent Assay ◽

Immunohistochemical Techniques ◽

Immunohistochemical Diagnosis ◽

Murine Monoclonal Antibodies

Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and-4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and-2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions ( n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.

Download Full-text

Effects of chronic corticotrophin treatment on aldosterone metabolism in the rat

Journal of Endocrinology ◽

10.1677/joe.0.1370445 ◽

1993 ◽

Vol 137 (3) ◽

pp. 445-455 ◽

Cited By ~ 8

Author(s):

D. R. E. Abayasekara ◽

N. I. Onyezili ◽

B. J. Whitehouse ◽

S. M. Laird ◽

G. P. Vinson

Keyword(s):

Polyclonal Antibodies ◽

Cross Reactivity ◽

Plasma Aldosterone ◽

Adrenal Vein ◽

Metabolic Clearance ◽

Sprague Dawley ◽

Peripheral Plasma ◽

Acth Treatment

ABSTRACT Chronic treatment with high doses of ACTH leads to marked reduction in aldosterone biosynthesis and secretion both in vivo and in vitro. In contrast, it has been reported that peripheral plasma aldosterone levels may be elevated following prolonged ACTH treatment. The present study attempts to determine the reason(s) for this apparently paradoxical finding. ACTH treatment (40 μg/100 g body weight) of male Sprague–Dawley rats for 7 days caused a decrease of more than 90% in aldosterone secretion into the adrenal vein in vivo and aldosterone production by intact adrenal capsules incubated in vitro. In contrast, peripheral plasma aldosterone levels appeared to be increased when measured by radioimmunoassay using two different polyclonal antibodies (antibody 1 (AB1) raised against aldosterone-3-carboxymethyloxime–bovine serum albumin (BSA) and antibody 2 (AB2) raised against aldosterone-21-hemisuccinate–BSA). However, when a highly specific monoclonal antibody (raised against aldosterone-3-carboxymethyloxime–BSA and showing low cross-reactivity to aldosterone metabolites) was used, peripheral plasma aldosterone levels appeared to be reduced in ACTH-treated rats. Following chromatographic fractionation of peripheral plasma, significantly more material with aldosterone-like immunoreactivity, but which was less polar than authentic aldosterone in chromatographic mobility, was detected in the fractions using antibodies AB1 and AB2. The absence of this material from fractions of adrenal vein plasma leads us to infer that this material is generated in the peripheral circulation, probably as a result of hepatic metabolism. In addition, the overall metabolic clearance rate (MCR) of [3H] aldosterone was found to be significantly decreased following prolonged ACTH treatment. We conclude that the seemingly discrepant findings with regard to the effects of chronic ACTH treatment on peripheral plasma aldosterone levels and the secretion of aldosterone in vivo can be reconciled by (1) the changes in the overall MCR of aldosterone and (2) the generation of increased quantities of aldosterone metabolites such as 5α-dihydroaldosterone and 3α,5β-tetrahydroaldosterone which show significant cross-reactivity with some aldosterone antibodies. Journal of Endocrinology (1993) 137, 445–455

Download Full-text

Detection of Parechovirus A1 with Monoclonal Antibody against Capsid Protein VP0

Microorganisms ◽

10.3390/microorganisms8111794 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1794

Author(s):

Ming-Hsiang Kung ◽

Ming-Wei Jan ◽

Jih-Jung Chen ◽

Yi-Chien Shieh ◽

Tsung-Hsien Chang

Keyword(s):

Monoclonal Antibody ◽

Polyclonal Antibody ◽

Cross Reactivity ◽

Immunofluorescence Assay ◽

Human Parechovirus ◽

Mouse Immunization ◽

The Common ◽

Common Infection ◽

Conserved Epitope ◽

Mab Production

Parechovirus A (PeV-A; human parechovirus) causes mild infections and severe diseases such as neonatal sepsis, encephalitis, and cardiomyopathy in young children. Among the 19 types of PeV-A, PeV-A1 is the most common type of infection. We have previously established an immunofluorescence assay for detecting multiple PeV-A types with a polyclonal antibody against the conserved epitope of VP0. Although the polyclonal antibody is useful for PeV-A diagnosis, it could not distinguish the PeV-A genotypes. Thus, the development of a specific monoclonal antibody for identifying the common infection of PeV-A1 would be beneficial in clinical diagnosis practice. In this study, the recombinant full-length PeV-A1 VP0 protein was used in mouse immunization; a total 10 hybridomas were established. After evaluation by immunoblotting and fluorescence assays, six hybridoma clones with monoclonal antibody (mAb) production were confirmed. These mAbs, which specifically recognize viral protein PeV-A1 VP0 without cross-reactivity to PeV-A3, will prove useful in research and PeV-A1 diagnosis.

Download Full-text

Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat αs1-Casein

Molecules ◽

10.3390/molecules25112622 ◽

2020 ◽

Vol 25 (11) ◽

pp. 2622

Author(s):

Aliah Zannierah Mohsin ◽

Rashidah Sukor ◽

Jinap Selamat ◽

Anis Shobirin Meor Hussin ◽

Intan Hakimah Ismail ◽

...

Keyword(s):

Binding Affinity ◽

Analytical Methods ◽

Polyclonal Antibodies ◽

Sequence Similarity ◽

High Specificity ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

High Sequence Similarity ◽

High Affinity

The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of αs1-casein. Detection and quantification of αs1-casein requires high-specificity methods to overcome high-sequence similarity between this protein and others in the casein family. Unavailability of antibodies with high affinity and specificity towards goat αs1-casein hinders the development of immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors. Here, we report the generation of polyclonal antibodies (or immunoglobulins, IgGs) raised towards goat αs1-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat αs1-casein were immunized in rabbits for the generation of antisera, which were purified using protein G affinity chromatography. The binding affinity of the antisera and purified IgGs were tested and compared using indirect ELISA, where peptide-BSA conjugates and goat αs1-casein were used as the coating antigens. The Nter antiserum displayed higher titer than Cter antiserum, at 1/64,000 and 1/32,000 dilutions, respectively. The purification step further yielded 0.5 mg/mL of purified IgGs from 3 mL of antisera. The purified Nter IgG showed a significantly (p < 0.05) higher binding affinity towards peptide-BSA and goat αs1-casein, with lower Kd value at 5.063 × 10−3 μM compared to 9.046 × 10−3 μM for the Cter IgG. A cross-reactivity test showed that there was no binding in neither Nter nor Cter IgGs towards protein extracts from the milk of cow, buffalo, horse and camel. High-quality antibodies generated will allow further development of immuno-based analytical methods and future in vitro studies to be conducted on goat αs1-casein.

Download Full-text

Reverse Vaccinology Approach for a Potential Rhinovirus Vaccine

Avicenna Journal of Clinical Microbiology and Infection ◽

10.34172/ajcmi.2021.12 ◽

2021 ◽

Vol 8 (2) ◽

pp. 66-73

Author(s):

Shuaibu Abdullahi Hudu ◽

Saadatu Haruna Shinkafi ◽

Shuaibu Umar ◽

Babazhitsu Makun

Keyword(s):

Recombinant Proteins ◽

Cross Reactivity ◽

Immunoglobulin M ◽

Capsid Proteins ◽

Reverse Vaccinology ◽

Chronic Obstructive ◽

Obstructive Pulmonary Disease ◽

Acute Exacerbations ◽

The Common

Background: Rhinoviruses (RVs) represent the most important aetiological agents of the common cold and are responsible for about two-thirds of acute exacerbations of chronic bronchitis, asthma, and chronic obstructive pulmonary disease (COPD) in both children and adults. This study aimed to design a pan-serotypic vaccine capable of inducing cross-reactive antibodies against most of the RV by using a reverse Vaccinology approach. Methods: Bioinformatics analysis was carried out to characterise the capsid proteins (VP1, VP2, VP3, and VP4) of all known RV serotypes and to predict potential immune motifs. Conserved motifs consisting at least nine-mers common across all RV-A or B serotypes were selected and synthesized chemically. Four tagged full-length genes coding the capsid proteins of an ideal strain (HRV-74), VP1, VP2, VP3, and VP4 were constructed and cloned in vitro. Upon expression , the purified recombinant proteins were also administered subcutaneously to other groups of rabbits. The responses and cross-reactivity of the specific immunoglobulin M (IgM) and G (IgG) to the peptides, proteins, and whole viruses were measured. Results: The obtained anti-peptide antibodies exhibited a cross-neutralizing activity for different RV strains in vitro. Antibodies raised to the synthetic peptides exhibited cross-reactivity against the corresponding recombinant proteins and antigenically distinct RV strains coated on plates via ELISA assay. Conclusions: The study findings indicated that the peptides corresponding to the conserved region of the RV capsid proteins were potent immunogenic; moreover, the findings showed that their combination was crucial for extending the cross-protection against variant RVs.

Download Full-text

XAV-19, a novel swine glyco-humanized polyclonal antibody against SARS-CoV-2 spike, efficiently neutralizes B.1.1.7 British and B.1.351 South-African variants.

10.1101/2021.04.02.437747 ◽

2021 ◽

Author(s):

Bernard Vanhove ◽

Stephane Sylvain Marot ◽

Benjamin Gaborit ◽

Gwenaelle Evanno ◽

Isabelle Malet ◽

...

Keyword(s):

South African ◽

Receptor Binding ◽

Polyclonal Antibody ◽

Polyclonal Antibodies ◽

S Protein ◽

Angiotensin Converting Enzyme 2 ◽

African Form ◽

The Uk ◽

The Impact

Amino acid substitutions and deletions in spike (S) protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can reduce the effectiveness of monoclonal antibodies (mAbs). In contrast, heterologous polyclonal antibodies raised against S protein, through the recognition of multiple target epitopes, have the potential to maintain neutralization capacities. We report on XAV-19, a swine glyco-humanized polyclonal antibody (GH-pAb) raised against the receptor binding domain (RBD) of the Wuhan-Hu-1 spike protein of SARS-CoV-2. XAV-19 target epitopes are distributed all over the RBD and particularly cover the receptor binding motives (RBM), on direct contact sites with the Angiotensin Converting Enzyme-2 (ACE-2). Using spike/ACE2 interaction assays, we analyzed in vitro the impact of punctual and grouped mutations in the S protein corresponding to the B.1.1.7 (British form; UK) and B.1.351 (South-African form, SA) variants and recorded that neutralization by XAV-19 exhibited little if any sensitivity to these mutations. These results were confirmed by two independent tissue culture infective doses assays (TCID) showing 100% neutralization of the variants at close concentrations. XAV-19, which is currently evaluated in patients hospitalized for coronavirus disease 2019 (Covid-19) in the phase 2a-2b of the POLYCOR study (ClinicalTrial.gov, NCT04453384), may provide a novel effective therapeutic tool to combat coronavirus disease 2019 (Covid-19), caused by the original Wuhan form and by the UK/SA variants of concern.

Download Full-text

In Vitro Immunological Cross-Reactivity of Thai Polyvalent and Monovalent Antivenoms with Asian Viper Venoms

Toxins ◽

10.3390/toxins12120766 ◽

2020 ◽

Vol 12 (12) ◽

pp. 766

Author(s):

Janeyuth Chaisakul ◽

Muhamad Rusdi Ahmad Rusmili ◽

Jaffer Alsolaiss ◽

Laura-Oana Albulescu ◽

Robert A. Harrison ◽

...

Keyword(s):

Polyclonal Antibodies ◽

Southeast Asian ◽

Cross Reactivity ◽

Red Cross ◽

Bovine Plasma ◽

Specific Variation ◽

Calloselasma Rhodostoma ◽

Protein Components ◽

Viper Venoms

The intravenous administration of polyclonal antibodies known as antivenom is the only effective treatment for snakebite envenomed victims, but because of inter-specific variation in the toxic components of snake venoms, these therapies have variable efficacies against different snake species and/or different populations of the same species. In this study, we sought to characterize the in vitro venom binding capability and in vitro cross-neutralizing activity of antivenom, specifically the Hemato Polyvalent antivenom (HPAV; The Queen Saovabha Memorial Institute (QSMI) of the Thai Red Cross Society, Thailand) and three monovalent antivenoms (QSMI) specific to Daboia siamensis, Calloselasma rhodostoma, and Trimeresurus albolabris venoms, against a variety of South Asian and Southeast Asian viper venoms (Calloselasma rhodostoma, Daboia russelii, Hypnale hypnale, Trimeresurus albolabris, Trimeresurus purpureomaculatus, Trimeresurus hageni, and Trimeresurus fucatus). Using ELISA and immunoblotting approaches, we find that the majority of protein components in the viper venoms were recognized and bound by the HPAV polyvalent antivenom, while the monospecific antivenom made against T.albolabris extensively recognized toxins present in the venom of related species, T. purpureomaculatus, T. hageni, and T. fucatus. In vitro coagulation assays using bovine plasma revealed similar findings, with HPAV antivenom significantly inhibiting the coagulopathic activities of all tested viper venoms and T. albolabris antivenom inhibiting the venoms from Malaysian arboreal pit vipers. We also show that the monovalent C. rhodostoma antivenom exhibits highly comparable levels of immunological binding and in vitro venom neutralization to venom from both Thailand and Malaysia, despite previous reports of considerable intraspecific venom variation. Our findings suggest that Thai antivenoms from QSMI may by useful therapeutics for managing snake envenomings caused by a number of Southeast Asian viper species and populations for which no specific antivenom currently exists and thus should be explored further to assess their clinical utility in treating snakebite victims.

Download Full-text

Comparative Study of Two Insulinlike Proteases in Cryptosporidium parvum

Microorganisms ◽

10.3390/microorganisms9040861 ◽

2021 ◽

Vol 9 (4) ◽

pp. 861

Author(s):

Wei He ◽

Cong Lai ◽

Fuxian Yang ◽

Yu Li ◽

Na Li ◽

...

Keyword(s):

Life Cycle ◽

Polyclonal Antibodies ◽

Parasite Load ◽

Cross Reactivity ◽

Binding Motif ◽

Apical Region ◽

Biological Functions ◽

Native Proteins ◽

Genes Encoding

Cryptosporidiumparvum is a common protozoan pathogen responsible for moderate-to-severe diarrhea in humans and animals. The small genome of C. parvum has 22 genes encoding insulinlike proteases (INS) with diverse sequences, suggesting that members of the protein family may have different biological functions in the life cycle. In this study, two members of the INS family, CpINS-4 and CpINS-6 with the Zn2+-binding motif “HXXEH” but different numbers of function domains, were expressed in Escherichia coli and used in the generation of polyclonal antibodies. In both recombinant and native proteins, CpINS-4 and CpINS-6 were spliced into multiple fragments. The antibodies generated recognized their respective recombinant and native proteins and the spliced products, but had minimum cross-reactivity with each other. Anti-CpINS-4 antibodies reacted with the middle region of sporozoites and merozoites, while CpINS-6 had the highest reactivity to the apical region. Polyclonal anti-CpINS-4 antibodies produced 36% reduction in parasite load in HCT-8 cultures at 24 h, while those against CpINS-6, which has one of the function domains missing, failed in doing so. The genes encoding both CpINS-4 and CpINS-6 had the highest expression in the invasion phase of in vitro C. parvum culture. These data suggest that CpINS-4 and CpINS-6 might be expressed in different organelles and play different biological functions in the life cycle of C. parvum.

Download Full-text

Immunohistochemical Diagnosis of Systemic Bovine Zygomycosis by Murine Monoclonal Antibodies

Veterinary Pathology ◽

10.1177/030098589603300207 ◽

1996 ◽

Vol 33 (2) ◽

pp. 176-183 ◽

Cited By ~ 24

Author(s):

H. E. Jensen ◽

B. Aalbæk ◽

P. Lind ◽

H. V. Krogh

Keyword(s):

Monoclonal Antibodies ◽

Lymph Nodes ◽

Rhizopus Oryzae ◽

Polyclonal Antibodies ◽

Cross Reactivity ◽

Water Soluble ◽

Enzyme Linked Immunosorbent Assay ◽

The Family ◽

Murine Monoclonal Antibodies

Murine monoclonal antibodies (Mabs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Rhizopus arrhizus (Rhizopus oryzae) were produced in vitro by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. Supernatants reacting only with homologous antigens in an enzyme-linked immunosorbent assay were subsequently screened for reactivity with homologous fungi in immunohistochemical techniques. All four Mabs raised against the WF of A. arrhizus failed to react on tissues. However, four of the Mabs raised against the WSSA of R. arrhizus (Mab-WSSA-RA-1 through Mab-WSSA-RA-4) revealed a high homologous reactivity on tissues and the cross-reactivity of these were subsequently evaluated on tissues containing other members of the family Mucoraceae and other unrelated fungi. On tissues and on immunoblots all four Mabs reacted identically and specifically with members of the family Mucoraceae, i.e., Absidia corymbifera, R. arrhizus, and Rhizomucor pusillus. The Mabs were all isotyped as IgM antibodies, were nonprecipitating, and reacted with homologous antigens with molecular masses from 14 to 110 kDa. With WSSA from A. corymbifera and R. pusillus the four Mabs were bound to antigens from 14 to 52 kDa and from 20 to 28 kDa, respectively. The diagnosis of 145 bovine lesions obtained by one of the specific Mabs (Mab-WSSA-RA-1) were compared to results obtained by heterologously absorbed polyclonal antibodies. In most lesions ( n = 140 [∼ 97%]) the Mab and the polyclonal antibodies reacted in a similar pattern, i.e., positively for zygomycosis in 89 lesions, negatively in 41 aspergillosis lesions, and negatively in 10 undiagnosed lesions. Hyphae within two of four lesions in lymph nodes, which were not stained by the polyclonal antibodies, reacted with the specific Mab. However, in another three lesions of lymph nodes stained by the polyclonal antibodies no reactivity was seen with the Mab-WSSA-RA-1. The immunoreactivity of the Mabs (Mab-WSSA-RA-1 through Mab-WSSA-RA-4) raised against WSSA of R. arrhizus justify their application for the in situ diagnosis of systemic bovine zygomycosis.

Download Full-text