Herpes Simplex Virus Infection Alters the Immunological Properties of Adipose-Tissue-Derived Mesenchymal-Stem Cells.

The proper functioning of mesenchymal stem cells (MSCs) is of paramount importance for the homeostasis of the body. Inflammation and infection can alter the function of MSCs, which can also affect the regenerative potential and immunological status of tissues. It is not known whether human herpes simplex viruses 1 and 2 (HSV1 and HSV2), well-known human pathogens that can cause lifelong infections, can induce changes in MSCs. In non-healing ulcers, HSV infection is known to affect deeper tissue layers. In addition, HSV infection can recur after initially successful cell therapies. Our aim was to study the response of adipose-derived MSCs (ADMSCs) to HSV infection in vitro. After confirming the phenotype and differentiation capacity of the isolated cells, we infected the cells in vitro with HSV1-KOS, HSV1-532 and HSV2 virus strains. Twenty-four hours after infection, we examined the gene expression of the cells via RNA-seq and RT-PCR; detected secreted cytokines via protein array; and determined autophagy via Western blot, transmission electron microscopy (TEM) and fluorescence microscopy. Infection with different HSV strains resulted in different gene-expression patterns. In addition to the activation of pathways characteristic of viral infections, distinct non-immunological pathways (autophagy, tissue regeneration and differentiation) were also activated according to analyses with QIAGEN Ingenuity Pathway Analysis, Kyoto Encyclopedia of Genes and Genome and Genome Ontology Enrichment. Viral infections increased autophagy, as confirmed via TEM image analysis, and also increased levels of the microtubule-associated protein light chain 3 (LC3B) II protein. We identified significantly altered accumulation for 16 cytokines involved in tissue regeneration and inflammation. Our studies demonstrated that HSV infection can alter the viability and immunological status of ADMSCs, which may have implications for ADMSC-based cell therapies. Alterations in autophagy can affect numerous processes in MSCs, including the inhibition of tissue regeneration as well as pathological differentiation.

Diabetes complications and extracellular vesicle therapy.

Diabetes is a chronic disorder characterized by dysregulated glycemic conditions. Diabetic complications include microvascular and macrovascular abnormalities and account for high morbidity and mortality rates in patients. Current clinical approaches for diabetic complications are limited to symptomatic treatments and tight control of blood sugar levels. Extracellular vesicles (EVs) released by somatic and stem cells have recently emerged as a new class of potent cell-free therapeutic delivery packets with a great potential to treat diabetic complications. EVs contain a mixture of bioactive molecules and can affect underlying pathological processes in favor of tissue healing. In addition, EVs have low immunogenicity and high storage capacity while maintaining nearly the same regenerative and immunomodulatory effects compared to current cell-based therapies. Therefore, EVs have received increasing attention for diabetes-related complications in recent years. In this review, we provide an outlook on diabetic complications and summarizes new knowledge and advances in EV applications. Moreover, we highlight recommendations for future EV-related research.

Production of monoclonal antibody against recombinant bovine sex-determining region Y (SRY) and their preferential binding to Y chromosome-bearing sperm.

Separation of X and Y chromosome-bearing sperm is an appropriate method for the selection of desired sex of offspring to increase the profit in livestock industries. The purpose of this study was the production of a monoclonal antibody against recombinant bovine sex-determining region Y protein for separation Y sperm. The hybridoma cells from splenocytes of immunized female's balb/C mice and Sp2/0 cells were made. The binding affinity of our monoclonal antibody (mAbSRY2) was compared with mouse monoclonal SRY-15. The Western blot method indicated that mAbSRY2 successfully detected the rbSRY protein. The specificity and sensitivity of mAbSRY2 is comparable to SRY-15 commercially ones. The SRY gene in 100% of bull semen contains the Y chromosome that had the strongest binding affinity to mAbSRY2 was synthesized. In other words, the binding affinity of semen contains the X sperms near the negative control. In general, this immunological method can help to separate X from Y sperms. However, the mAbSRY2 is bind to Y-bearing sexed sperm, but in the future; the sexed sperms need to apply in farms.

Anti-inflammatory effect of chymotrypsin to autoimmune response against CNS is dose-dependent.

Multiple sclerosis is an inflammatory autoimmune disease of central nervous system (CNS) in which inflammatory cells release pro-inflammatory cytokines, proteases, and other toxic mediators. Proteases are involved in many aspects of inflammatory process. There are many reports regarding the effect of proteases on inflammation. Chymotrypsin is a serine protease with anti-inflammatory effect. We investigated chymotrypsin effect on experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. Intra-CSF injection with 0.1 mg/ml, 0.2 mg/ml chymotrypsin, or saline was done on day 7 after EAE induction. Our study demonstrated that 0.1 mg/ml chymotrypsin treatment did not decrease clinical signs, but 0.2 mg/ml chymotrypsin ameliorated clinical signs and manipulated immune response in both brain and spinal cord. Administration of 0.1 mg/ml or 0.2 mg/ml chymotrypsin led to decreased IL-17 along with increased IL-4 and FoxP3 in 0.2 mg/ml chymotrypsin-treated animals. Presumably, chymotrypsin acts in a dose-dependent manner and concentrations of chymotrypsin more than 0.2 mg/ml may have more beneficial effect.

Investigating the influence of taurochenodeoxycholic acid (TCDCA) on pancreatic cancer cell behavior: An RNA sequencing approach.

Pancreatic cancer (PC) poses a substantial global health challenge, ranking as the fourth leading cause of cancer-related deaths due to its high mortality rate. Late-stage diagnoses are common due to the absence of specific symptoms. Pancreatic ductal adenocarcinoma (PDAC) accounts for the majority of PC cases. Recent research has suggested a potential link between elevated serum levels of bile acids (BAs) and tumorigenesis of PDAC. This study aims to understand how taurochenodeoxycholic acid (TCDCA), a secondary BA, influences PDAC using RNA sequencing techniques on the Capan-1 cell line. We identified 2,950 differentially expressed genes (DEGs) following TCDCA treatment, with 1,597 upregulated and 1,353 downregulated genes. These DEGs were associated with critical PDAC pathways, including coagulation, angiogenesis, cell migration, and signaling regulation. Furthermore, we reviewed relevant literature highlighting genes like DKK-1, KRT80, UPLA, and SerpinB2, known for their roles in PDAC tumorigenesis and metastasis. Our study sheds light on the complex relationship between BAs and PDAC, offering insights into potential diagnostic markers and therapeutic targets. Further research is needed to unravel these findings' precise mechanisms and clinical implications, potentially improving PDAC diagnosis and treatment.

Three-dimensional bioprinting of functional ß-islet-like constructs.

256Diabetes is an autoimmune disease that ensues when the pancreas does not deliver adequate insulin or when the body cannot react to the existing insulin. Type 1 diabetes is an autoimmune disease defined by continuous high blood sugar levels and insulin deficiency due to ß-cell destruction in the islets of Langerhans (pancreatic islets). Long-term complications, such as vascular degeneration, blindness, and renal failure, result from periodic glucose-level fluctuations following exogenous insulin therapy. Nevertheless, the shortage of organ donors and the lifelong dependency on immunosuppressive drugs limit the transplantation of the entire pancreas or pancreas islet, which is the therapy for this disease. Although encapsulating pancreatic islets using multiple hydrogels creates a semi-privileged environment to prevent immune rejection, hypoxia that occurs in the core of the capsules is the main hindrance that should be solved. Bioprinting technology is an innovative process in advanced tissue engineering that allows the arranging of a wide array of cell types, biomaterials, and bioactive factors as a bioink to simulate the native tissue environment for fabricating clinically applicable bioartificial pancreatic islet tissue. Multipotent stem cells have the potential to be a possible solution for donor scarcity and can be a reliable source for generating autograft and allograft functional ß-cells or even pancreatic islet-like tissue. The use of supporting cells, such as endothelial cells, regulatory T cells, and mesenchymal stem cells, in the bioprinting of pancreatic islet-like construct could enhance vasculogenesis and regulate immune activity. Moreover, scaffolds bioprinted using biomaterials that can release oxygen postprinting or enhance angiogenesis could increase the function of ß-cells and the survival of pancreatic islets, which could represent a promising avenue.

Unraveling Transcriptome Profile, Epigenetic Dynamics, and Morphological Changes in Psoriasis-like Keratinocytes: "Insights into Similarity with Psoriatic Lesional Epidermis".

Keratinocytes are one of the primary cells affected by psoriasis inflammation. Our study aimed to delve deeper into their morphology, transcriptome, and epigenome changes in response to psoriasis-like inflammation. We created a novel cytokine mixture to mimic mild and severe psoriasis-like inflammatory conditions in cultured keratinocytes. Upon induction of inflammation, we observed that the keratinocytes exhibited a mesenchymal-like phenotype, further confirmed by increased VIM mRNA expression and results obtained from confocal microscopy. We performed RNA sequencing to achieve a more global view, revealing 858 and 6987 DEGs in mildly and severely inflamed keratinocytes, respectively. Surprisingly, we found that the transcriptome of mildly inflamed keratinocytes more closely mimicked that of the psoriatic epidermis transcriptome than the severely inflamed keratinocytes. Genes involved in the IL-17 pathway were a major contributor to the similarities of the transcriptomes between mildly inflamed KCs and psoriatic epidermis. Mild and severe inflammation led to the gene regulation of epigenetic modifiers such as HATs, HDACs, DNMTs, and TETs. Immunofluorescence staining revealed distinct 5-hmC patterns in inflamed versus control keratinocytes, and consistently low 5-mC intensity in both groups. However, the global DNA methylation assay detected a tendency of decreased 5-mC levels in inflamed keratinocytes versus controls. This study emphasizes how inflammation severity affects the transcriptomic similarity of keratinocytes to psoriatic epidermis and proves dynamic epigenetic regulation and adaptive morphological changes in inflamed keratinocytes.

Efficient conjugation of anti-HBsAg antibody to modified core-shell magnetic nanoparticles (Fe3O4@SiO2/NH2).

Introduction: Further development of magnetic-based detection techniques could be of significant use in increasing the sensitivity of detection and quantification of hepatitis B virus (HBV) infection. The present work addresses the fabrication and characterization of a new bio-nano composite based on the immobilization of goat anti-HBsAg antibody on modified core-shell magnetic nanoparticles (NPs) by (3-aminopropyl) triethoxysilane (APTES), named Fe3O4@SiO2/NH2, and magnetic NPs modified by chitosan (Fe3O4@CS). Methods: At the first step, Fe3O4 was modified with the silica and APTES (Fe3O4@SiO2/NH2) and chitosan (Fe3O4@CS) separately. The goat anti-HBsAg antibody was activated by two different protocols: Sodium periodate and EDC-NHS. Then the resulted composites were conjugated with activated goat anti-HBsAg IgG. An external magnet collected Bio-super magnetic NPs (BSMNPs) and the remained solution was analyzed by the Bradford method to check the amount of attached antibody to the surface of BSMNPs. Results: The findings indicated that activation of antibodies by sodium periodate method 15-17 µg antibody immobilized on 1 mg of super magnetic nanoparticles (SMNPs). However, in the EDC-NHS method, 8-10 µg of antibody was conjugated with 1 mg of SMNPs. The resulting bio-magnetic NPs were applied for interaction with the HBsAg target using enzyme-linked immunosorbent assay (ELISA). About 1 µg antigen attached to 1 mg SMNPs, which demonstrated that the fabricated materials are applicable in the detection scope of HBsAg. Conclusion: In the present study, we developed new antibody-conjugated magnetic NPs for the detection of HBsAg using an efficient conjugation strategy. The results demonstrated that the binding capacity of Fe3O4@SiO2/NH2 was comparable with commercially available products. Our designed method for conjugating anti-HBsAg antibody to a magnetic nanoparticle opens the way to produce a high capacity of magnetic NPs.

In vitro and ex vivo antiangiogenic activity of Salvia officinalis.

Angiogenesis is a key process in the promotion of cancer and its metastasis. Herein, the antiangiogenic activity of Salvia officinalis extract and its fractions was investigated. S. officinalis aerial parts were extracted with ethanol and its successive hexane, ethyl acetate, n-butanol and aqueous fractions were evaluated for their antiangiogenic activities using human umbilical vein endothelial cells (HUVEC) capillary tube formation and rat aorta models in a three-dimensional collagen matrix. Furthermore, antimigrative effects of the fractions were assessed using a wound healing model. The ethanol extract of S. officinalis (ESO) potently inhibited capillary tube formation in HUVEC and rat aorta models of angiogenesis, and its hexane fraction (HSO) exerted the highest inhibitory effect. In addition, the ethanol extract of S. officinalis and its hexane fraction showed a dose-dependent inhibitory activity on the migration of the endothelial cells in the wound healing model. Furthermore, ESO inhibited endothelial cell proliferation at 50-200 µg/mL in a dose-dependent manner. These findings indicated some new pharmacological activities of S. officinalis such as antiangiogenic in vitro and ex vivo, and antimigrative activity in vitro. Therefore, S. officinalis could be a candidate as a useful herb with therapeutic or preventive activity against angiogenesis related disorders.

Goat Polyclonal Antibody Against the Sex Determining Region Y to Separate X- and Y-Chromosome Bearing Spermatozoa.

BACKGROUND: Sex selection of sperm by separating X- and Y-chromosome bearing spermatozoa is critical for efficiently obtaining the desired sex of animal offspring in the livestock industry. The purpose of this study was to produce a goat polyclonal antibody (pAb) against the bovine Sex Determining Region Y chromosome (bSRY) to separate female- and male-bearing spermatozoa. METHODS: To produce a goat polyclonal antibody against bSRY, a female goat was subcutaneously immunized with 27 kDa of recombinant bSRY (rbSRY) protein as the antigen. The anti-bSRY pAb was purified by ion-exchange chromatography. The purity of the pAb was determined using the SDS-PAGE method. The biological activity of the anti-bSRY pAb was examined using PCR to assess the binding affinity of pAb for the bSRY antigen and commercially sexed bull sperm. RESULTS: The total amount of purified anti-bSRY pAb was approximately 650 mg/goat serum (13 mg/mL). Interestingly, our data showed that the binding affinity of our pAb to the Y bearing was high, while the binding affinity of that to the X-chromosome bearing sperm was similar to the negative control. CONCLUSION: In conclusion, our findings show that the goat anti-SRY pAb specifically binds to Y-chromosome bearing sperm that suggesting its potential use for sex selection.

Kinetics of T cell response in the testes and CNS during experimental autoimmune encephalomyelitis: Simultaneous blood-brain and -testis barrier permeability?

OBJECTIVES: Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are regarded as autoimmune diseases of the central nervous system (CNS). The CNS, testes, and eyes are immune privileged sites. It was initially presumed that ocular involvement in EAE and infertility in MS are neural-mediated. However, inflammatory molecules have been detected in the eyes of animals affected by EAE. It prompted us to investigate if the testes may also be targeted by immune response during EAE. MATERIALS AND METHODS: kinetics of T cell response was investigated in the CNS and testes in EAE at different clinical scores. IFN-γ, IL-4, IL-17, and FoxP3 mRNA expressions were considered as representatives of Th1, Th2, Th17, and Treg, respectively. RESULTS: In CNS, IL-17 and IFN-γ were initially up-regulated and attenuated at the late phase of the disease. IL-4 and FoxP3 were markedly down-regulated, but IL-4 was then up-regulated at the late phase of the disease. In the testes, IFN-γ and IL-17 were diminished but increased at the late phase of the disease. FoxP3 was gradually increased from the initial step to the peak of the disease. IL-17/ IFN-γ showed a similar pattern between the CNS and testes. However, FoxP3 and IL-4 expression appeared to have different timing patterns in the CNS and testes. CONCLUSION: Given the permeability in blood-retina/brain/CSF barrier by complete Freund's adjuvant, the pattern of T cells may be changed in the testes during EAE as a consequence of the blood-testis barrier permeability. More research is required to explore the connection between immune privileged organs.

Nontoxic silver nanocluster-induced folding, fibrillation, and aggregation of blood plasma proteins.

In recent years, concerns have been raised considering the potential risks of nanocluster (NC) for the environment and human health. Since the blood circulation system is probably the first entry route of NC into the human body, adsorption of blood proteins on NC may change cellular responses, including cellular uptake efficiency, bio distribution patterns, and nanotoxicity profiles, besides other biological effects. Therefore, the interaction of NCs with proteins and the cellular implications can be therapeutically of great importance. Adsorption of human blood proteins on NCs has been methodically investigated. In the present study, the first analysis of fibrillation was conducted between MBI-AgNCs and human serum (a complex biofluid). AgNCs were prepared by coating with 2-mercaptobenzimidazole. Then, interactions with human blood proteins, such as immunoglobulin, albumin, and insulin were investigated using various experimental approaches. Upon protein association, the fluorescence of proteins significantly decreased, accompanied by a blue shift in the AgNCs-human serum albumin (HSA) system and a red shift in the AgNCs-insulin/γ-globulin. Concomitantly, circular dichroism spectroscopy and atomic force microscopy were employed to investigate the effects of protein binding to NCs. We found that AgNCs induced γ-globulin aggregation. HSA at the AgNC surface was partially unfolded and could promote protein self-assembly into amyloid fibrils, while the surface morphology remained unchanged after insulin incubation. The atomic force microscopy (AFM) data and the ThT and CR analysis of the proteins, as well as circular dichroism (CD) and fluorescence findings, support the use of AgNCs as an indicator for monitoring the progress of HSA fibrillogenesis. Additionally, cytotoxicity assays were used to ensure the biocompatibility of nanoparticles within the applicable limits.

Construction and characterization of monoclonal antibodies against the extracellular domain of B-lymphocyte antigen CD20 using DNA immunization method.

To date, several new anti-CD20 monoclonal antibodies (mAbs) have been developed for potential efficacies compared with familiar mAb rituximab. Despite the recent advances in development of anti-CD20 mAbs for the treatment of B cell malignancies, the efforts should be continued to develop novel antibodies with improved properties. However, the development of mAbs against CD20 as a multi-transmembrane protein is challenging due to the difficulty of providing a lipid environment that can maintain native epitopes. To overcome this limitation, we describe a simple and efficient DNA immunization strategy for the construction of a novel anti-CD20 mAb with improved anti-tumour properties. Using a DNA immunization strategy that includes intradermal (i.d.) immunization with naked plasmid DNA encoding the CD20 gene, we generated the hybridoma cell line D4, which secretes functional mAbs against an extracellular epitope of CD20. Immunocytochemistry analysis and a cell-based enzyme-linked immunosorbent assay using a Burkitt's lymphoma cell line showed that D4 mAbs are capable of binding to native extracellular epitopes of CD20. Moreover, the binding specificity of D4 mAbs was determined by western blot analysis. Cell proliferation was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by the annexin V/propidium iodide staining and dye exclusion assay. The results showed that D4 anti-CD20 mAbs produced by DNA immunization exhibit potent growth inhibitory activity and have superior direct B-cell cytotoxicity compared to rituximab. We propose that antibody-induced apoptosis is one of the mechanisms of cell growth inhibition. Taken together, the data reported here open the path to DNA-based immunization for generating pharmacologically active monoclonal antibodies against CD20. In addition, the data support future in vivo animal testing and subsequent procedures to produce a potential therapeutic mAb.

Neuroimmunomodulation by allogeneic seminal vesicle fluid in CNS is sex-independent.

OBJECTIVES: Thymus-arisen FoxP3 regulatory T cells (Tregs) are one of the most important immunoregulatory mechanisms in the central nervous system (CNS) and pregnancy. Multiple sclerosis (MS) is an inflammatory disease of CNS associated with a reduced frequency and/or function of Tregs. Previous works have shown that seminal vesicle fluid affects female immune system and causes expansion of Tregs pool in the female reproductive tissue upon mating. Accordingly, it has been demonstrated that intra-CSF administration of seminal vesicle fluid from Wistar rats can ameliorate clinical sign of a female Lewis rat model of experimental autoimmune encephalomyelitis (EAE), the animal model of MS. The results indicated an up-regulation of FoxP3 expression in the brain and spinal cord. However, there are sex-based differences in the CNS structure & composition, sex hormones influence immune system, and gender-based differences affect treatment response. Therefore, we decided to find out if anti-inflammatory effect of seminal vesicle fluid is sex-dependent or -independent. METHODS: EAE was induced in male Lewis rats using guinea pig spinal cord and complete Freund's adjuvant. Intra-CSF injection was done on day 7 after EAE induction and the animals were followed-up until on day 14 after EAE induction when sacrificed. Then, brain and spinal cord of the animals were isolated, total RNA was extracted, and expression of mRNA for IFN-γ, IL-4, IL-17, and FoxP3 was determined using real-time PCR where ß-actin was used as reference gene. RESULT: demonstrated that intra-CSF administration of seminal vesicle fluid from male Wistar rats ameliorated EAE in male Lewis rats and increased FoxP3 in the brain and spinal cord. CONCLUSION: This study suggests that seminal vesicle fluid from Wistar rats has anti-inflammatory effect on Lewis rats in a sex-independent manner. In addition, seminal vesicle fluid from Lewis rats had not beneficial effect on EAE in male Lewis rats. This is consistent with Tregs increase in allogeneic mating. More research is required to find out the immunologic aspect of allogeneic versus syngeneic administration of seminal vesicle fluid.

Immunomodulatory Effect of Chymotrypsin in CNS Is Sex-independent: Evidence of Anti-inflammatory Role for IL-17 in EAE.

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory autoimmune diseases of the central nervous system. Chymotrypsin is a serine protease with immunomodulatory effect in the peripheral organs. We previously demonstrated the immunomodulatory effect of chymotrypsin in ameliorating the EAE in female Lewis rats. However, there are sex-based differences in the immune system, drug activity, and CNS structure and composition. In addition, female gender is a better prognostic indicator of MS and males are more severely affected by EAE than females. Consequently, gender may have an important impact on therapeutic effect. Therefore, in this study we investigated the anti-inflammatory effect of chymotrypsin in male Lewis rat model of EAE. The disease was induced in male Lewis rats and the animals were evaluated for weight loss and clinical signs for 14 days. Intra-CSF injection of chymotrypsin was done on day 7 and expression of mRNA for IFN-γ, IL-4, IL-17, and FoxP3 in brain, spinal cord and deep cervical lymph node were determined using a two-step real-time PCR. Administration of 0.2mg/ml chymotrypsin ameliorated the disease by decreasing IFN-γ and increasing expression of IL-4 and IL-17 at the inflammatory foci. This is consistent with anti-inflammatory effect of IL-4 and IL-17 at high concentrations. We conclude that Immunomodulatory affect of chymotrypsin in CNS is sex-independent. Our result also provides more evidence on the anti-inflammatory role of IL-17. However more research is needed to elucidate the underlying immunomodulatory role of chymotrypsin and how to increase its beneficial effect by modification of dosage and/or regimen of administration.

Liver Damage and Mortality in a Male Lewis Rat of Experimental Autoimmune Encephalomyelitis.

BACKGROUND AND OBJECTIVES: Multiple sclerosis is an inflammatory disease of the central nervous system. This is due to migration of peripherally activated lymphocytes to central nervous system leading to inflammatory lesions. However, liver has an anti-inflammatory microenvironment. Myelin expression in the liver of transgenic mice suppresses inflammatory lesions within central nervous system. Considering the notion that the inflammatory events originate from periphery, we investigated if the liver was affected in an animal model for multiple sclerosis. METHODS: Experimental autoimmune encephalomyelitis was induced in male Lewis rats using guinea pig spinal cord and complete Freund's adjuvant. Weight, clinical score, and survival rate were evaluated for 14 days post immunization. Liver sections were taken and stained with Hematoxylin and Eosin and examined with an Olympus microscope. RESULTS: Mortality was accompanied by liver damage. Sinusoidal congestion, pycnotic nuclei within hepatocytes, hepatocyte necrosis, and severe widespread congestion along with fat accumulation within hepatocytes (fatty degeneration) were observed in liver tissue sections. CONCLUSION: Liver damage occurs in experimental autoimmune encephalomyelitis. The perpetuation of self antigen leading to continuous migration of extrahepatically activated T cells makes an inflammatory milieu in the liver. It follows migration and development of more inflammatory cells and may paralyses tolerance inducing mechanisms. Apart from central nervous system lesion, liver injury may act as synergistic factor for debilitation and mortality.

Efficient Conjugation of Aflatoxin M1 With Bovine Serum Albumin through Aflatoxin M1-(O-carboxymethyl) Oxime and Production of Anti-aflatoxin M1 Antibodies.

BACKGROUND: Aflatoxins are the most extensively studied group of mycotoxins produced by molds, especially the Aspergillus group, which are highly toxic to animals and humans. OBJECTIVES: Since immunoassay is a simple and rapid method for the analysis of many toxic substances in comparison to the chromatographic methods, it is necessary to produce specific and sensitive antibodies for detection of Aflatoxin M1 (AFM1). The current study was conducted to produce bioconjugate of Aflatoxin M1 (AFM1) with Bovine Serum Albumin (BSA) as well as to generate specific antibodies against AFM1 for immunoassay of the mycotoxin. MATERIALS AND METHODS: First, AFM1 was converted to AFM1-(O-carboxymethyl) oxime derivative. Then, AFM1-oxime was coupled with BSA and the product was assessed by UV-VIS spectrophotometry. In order to generate polyclonal antibodies against AFM1, rabbits were immunized with BSA-AFM1 conjugate. Produced antibodies were purified using ion exchange chromatography and BSA-Sepharose 4B affinity chromatography. The titers and specificity of the produced antibodies were determined by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: The results indicated that coupling of AFM1 with O-(Carboxymethyl) hydroxylamine hemihydrochloride was suitable and 12 moles of AFM1-oxime were successfully coupled to each mole of BSA. In addition, the titers and specificity of the prepared antibody were considerable compared to standard anti-AFM1 antibodies. The relative cross-reactivity of each toxin (relative to AFM1) with purified anti-AFM1 antibodies, as determined by the amount of aflatoxin necessary to cause 50% inhibition of enzyme activity, was 70, 105, 240, and 2500 ng/mL for AFB1, AFB2, AFG1, and AFG2, respectively. CONCLUSIONS: The prepared antibody can be used for the development of an ELISA kit to assay AFM1 in milk and other biological fluids.