Preparation Of Monoclonal Antibodies Research Articles

Monoclonal Antibodies, Bispecific Antibodies and Antibody-Drug Conjugates in Oncohematology.

The therapeutic outcomes and the prognosis of patients with various hematologic malignancies are not always ideal with the current standard of care. The aim of this study is to analyze the results of the use of monoclonal antibodies, bispecific antibodies and antibody-drug conjugates for the therapy of malignant hemopathies. A mini-review was achieved using the articles published in Web of Science and PubMed between January 2017 and January 2020 and the new patents were made in this field. Naked monoclonal antibodies have improved the therapeutic results obtained with standard of care, but they also have side effects and the use of some of them can lead to the loss of the target antigen through trogocytosis, which explains the resistance that occurs during therapy. The results obtained with naked monoclonal antibodies have been improved by a better monoclonal antibody preparation, the use of bispecific antibodies (against two antigens on the target cell surface or by binding both surface antigen on target cells and T-cell receptor complex, followed by cytotoxic T-lymphocytes activation and subsequent cytolysis of the target cell), the use of monoclonal or bispecific constructs in frontline regimens, combining immunotherapy with chemotherapy, including through the use of antibody-drug conjugates (which provides a targeted release of a chemotherapeutic agent). Immunotherapy and immuno-chemotherapy have improved the outcome of the patients with malignant hemopathies through a targeted, personalized therapy, with reduced systemic toxicity, which in some cases can even induce deep complete remissions, including minimal residual disease negativity.

A cost analysis study of the implementation of fixed-dosing of monoclonal antibodies in the Netherlands Cancer Institute.

Background There is a strong rationale for fixed-dosing of monoclonal antibodies in oncology. Although fixed-dosing of recently introduced monoclonal antibodies is well accepted, the rationale is also applicable for other monoclonal antibodies that already have been used for years, but are still body-size-based dosed in many hospitals. In the Netherlands Cancer Institute, Antoni van Leeuwenhoek (NKI-AVL), fixed-dosing has been implemented now for all monoclonal antibodies and, therefore, this site offers an ideal opportunity for a cost analysis study. Objective To investigate the financial impact of switching to fixed-dosing in the NKI-AVL. Setting The NKI-AVL. Method Information on the preparations of monoclonal antibodies was collected from August 2017 to February 2020. We compared the number of vials needed during preparation for fixed-dosing and body-size -based dosing strategies. The economic impact was calculated for 2 scenarios: scenario 1 assumed clustering of all preparations per day and scenario 2 assumed no clustering of preparations. Main outcome measure Number of saved vials and the correlating savings in health care costs. Results The implementation of fixed-dosing resulted in a substantial reduction in vials used for almost all monoclonal antibodies. The economic savings were calculated to be €0,8 and €3,1 million per year for scenario 1 and 2, respectively. Conclusion Fixed-dosing resulted in substantial savings in health care costs.

Targeting the cell wall: Preparation of monoclonal antibody for accurate identification of Alicyclobacillus acidoterrestris in apple juice

Alicyclobacillus acidoterrestris represents the predominant microorganism implicated in apple juice spoilage. Most of existing detection methods for this microorganism necessitate advanced equipment and skilled technicians, both of which are unsuitable for practical application. In this study, an effective approach for the identification of A. acidoterrestris was developed based on an indirect sandwich enzyme-linked immunosorbent assay (is-ELISA). This method utilizes the species-specific monoclonal antibody (mAb) targeting a cell wall protein of A. acidoterrestris. The is-ELISA was highly sensitive with a limit of detection (LOD) of 5.4 × 102 CFU/mL. Furthermore, the assay is capable of accurately differentiating between A. acidoterrestris and other species of genus Alicyclobacillus. The applicability of is-ELISA was also evaluated in artificially contaminated apple juice. The convenience, simplicity, sensitivity and specificity of this assay represents a robust method for the detection of A. acidoterrestris and the resultant prevention of the spoilage of fruit juices.

Monoclonal antibodies in the treatment of multiple sclerosis: from clinical research to practical application

Multiple sclerosis (MS) is a chronic autoimmune inflammatory demyelinating and neurodegenerative disease with a multifactorial etiology of development. MS in most cases has a wave-like course (periods of exacerbations and remissions), over time, the disease becomes progressive, which worsens the quality of life of patients. The drugs disease-modifying therapies (DMT) has been actively used in clinical practice for more than 30 years to prevent exacerbations and progression of MS. In patients with MS, in which the disease occurs with frequent exacerbations and signs of radiological activity of the demyelinating process, according to magnetic resonance imaging (MRI) of the brain and spinal cord, it is recommended to use monoclonal antibody preparations. The only drug registered for the treatment of primary progressive MS is ocrelizumab. In addition, ocrelizumab is indicated for patients with remitting and secondary progressive MS. Ocrelizumab is a humanized monoclonal antibody that selectively depletes a population of CD20+ B cells. The article presents data from clinical studies of OPERA I and OPERA II and describes a clinical case from the practice of a neurologist. Depletion of the B cell population is achieved by several mechanisms, including antibody-dependent cell-mediated phagocytosis, antibody-dependent T cell-mediated cytotoxicity, complement-dependent cytotoxicity, and apoptosis induction. The issues of efficacy and safety of ocrelizumab therapy in patients with MS are considered.

Preparation, identification, and functional analysis of monoclonal antibodies against atypical porcine pestivirus NS3 protein.

Atypical porcine pestivirus (APPV) had been detected in many countries. However, to date, a commercial detection kit is not available because of a lack of specific monoclonal antibodies (mAbs) to APPV. We generated 7 mAbs targeting the NS3 protein of APPV. Isotyping results indicated that all of these mAbs are IgG1 with a kappa light chain. We analyzed the epitopes recognized by mAbs 2B6, 6G11, 8D1, 8D3, and 8F12, which recognized the same linear epitope (GRIKSAYSDE); the 6H3 and 7E10 mAbs recognized 2 different conformational epitopes. Applications of these antibodies were verified by ELISA, western blot, indirect immunofluorescence assay, and flow cytometry. The antibodies were functionally workable for these immunoassays except for 8F12, which could not be used in flow cytometry.

Monoclonal Antibody Against N Terminal 439-451 Epitopes of Anti-Mullerian Hormone and Its Properties

To prepare the specific monoclonal antibody against the N-terminal specific epitope peptide of anti-mullerian hormone (AMH) and to identify its specificity. Using bioinformatics analysis software to predict the specific peptide fragment of AMH. Then synthesized four antigenic epitope peptide segments of mature N-terminal region of AMH as the screening target antigen. Synthesized AMH wholegene.Using the prokaryotic expression system to abtain recombinant AMH protein. Immunized BALB/c mice with the recombinant AMH, and prepared mouse spleen cells for fusing with SP/20 cells. Preparation of AMH monoclonal antibody by hybridoma technology. The monoclonal antibodies against AMH were screened by using four N-terminal epitope peptides (1: 439-451 RGRDPRGPGRAQ, 2: 273-285 PPRPSAELEESPP, 3: 42-54 DLDWPPGSPQEPL, 4: 494-506 WPQSDRNPRYGNH) as antigens, and indirect ELISA and Western blot were used to identify the antigen binding characteristics of the selected monoclonal antibodies. Two hybridoma cell lines with stable anti-AMH-1 and anti-AMH-2 antibody activities were screened. The two antibodies were named anti-AMH-1 and anti-AMH-2 respectively. The antibody titers were 1∶12 000 and 1∶1 600 after purification. Western blot confirmed that the two McAbs recognized different antigens. Anti-AMH-1 could not only recognize the N-terminal 439-451 epitope peptide of AMH, but also recognize the amino acid sequence of recombinant AMH, as well as the ovarian tissue. Anti-AMH-2 could recognize recombinant AMH and ovarian tissue. Two monoclonal antibodies against N-terminal specific epitopes of human AMH were successfully constructed.

Practical recommendations for choosing an immunobiological preparation for the treatment of severe bronchial asthma of T2-endotype

Biological therapy of bronchial asthma (BA) is a modern method of treating severe forms of the disease, that are uncontrolled by traditional pharmacotherapeutic approaches. Currently, 5 monoclonal antibody (AT) preparations are registered in the world for the treatment of severe bronchial asthma (SBA) of the T2 endotype (T2-SBA) – antibodies, binding to immunoglobulin (Ig) E (anti-IgE – omalizumab), interleukin antagonists (IL)-5 (anti-IL-5 – mepolizumab, resizumab) and its receptor (anti-IL-5Rα – benralizumab), as well as antibodies, that selectively bind to the IL-4 and -13 receptor (anti-IL-4 /13Rα – dupilumab). The article presents data on the effectiveness of these drugs in relation to the key characteristics of SBA, formulates clinical and laboratory criteria, the study of which in real practice can potentially predict the likelihood of a clinical response to a particular type of biological therapy. An algorithm is proposed for choosing a targeted therapy strategy for patients with SBA, clinically associated with allergies, for patients with severe non-allergic eosinophilic BA and for patients with eosinophilic BA of a combined phenotype.

Preparation of sensitive monoclonal antibodies against triptolide and establishment of a rapid and cost-effective icELISA method for authentication of Tripterygium wilfordii and related products

Triptolide is a characteristic chemical component and quality marker of Tripterygium wilfordii Hook. F., a widely used traditional Chinese medicine with various pharmacological activities. Triptolide-14-succinate is a derivative of triptolide that can be combined with bovine serum albumin and ovalbumin to form an immunogen and coating antigen. Monoclonal antibody specific to triptolide-14-succinate conjugated with ovalbumin was produced and named mAb 2D10 1H8. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was established by using mAb 2D10 1H8. The 50% inhibition concentration and working range of triptolide for the icELISA were 0.28 and 0.09–0.87 μg mL−1, (based on 20–80% binding inhibition of mAb 2D10 1H8 to triptolide, R2 = 0.9997, y = -0.571Logx + 0.246), respectively. The icELISA displayed crossreactivity values of 48.6, 20.5, 3.27, and < 1.00% for triptonide, triptophenolide, tripterifordin, and other analogs of triptolide, respectively. The average recovery of triptolide from the Tripterygium wilfordii samples as determined by icELISA ranged from 90.05 to 106.6%. Triptolide was detected in 38 batches of Tripterygium wilfordii samples by icELISA, which was validated by ultra-fast liquid chromatography combined with electrospray ionization tandem-mass spectrometry. In conclusion, the icELISA method established in the present study is applicable for rapid authentication of Tripterygium wilfordii and related products.

Immunotherapy for cancer of the oral cavity and oropharynx. The use of monoclonal antibodies

The purpose is to review the literature and analyze the use of monoclonal antibodies in the treatment for cancer of the mouth and oropharynx.Conclusions. 1. Today, cetuximab is the only monoclonal antibody that has received FDA approval for the treatment for oral and oropharyngeal cancer. Clinically used for more than 10 years, and shows good results. 2. Clinical trials have confirmed the efficacy of moAbs targeting immune checkpoint receptors, including anti-CTLA-4 and anti-PD-1. Immune checkpoint blockade may improve the existing clinical effects of therapy on the background of low toxicity and may contribute to long-term effects through immunological memory. 3. The use of immune checkpoint inhibitors is currently only approved for advanced stages of cancer. 4. There is insufficient clinical data to demonstrate the feasibility of combining several different monoclonal antibody preparations and combining them with radiation therapy or chemotherapy.

Preparation and characterisation of monoclonal antibodies against deoxynivalenol

To prepare broad-spectrum and specific monoclonal antibodies (mAb) against deoxynivalenol (DON), DON-BSA was synthesised by the carbonyl diimidazole method as an artificial antigen to immunise Balb/C mice, and the coated DON-OVA was synthesised by the carbodiimide method to detect anti-DON antibodies. Monoclonal antibodies were screened by indirect ELISA and indirect competitive ELISA. A hybridoma cell line (4F3) capable of stably secreting DON antibody was obtained. The titre of antibody in the culture supernatant was 1:1.28 × 103, and the titre of the ascites antibody was 1:3.2 × 105. The monoclonal antibody homology was IgG1/λ. The half inhibitory concentration (IC50) of DON was 9.84 ng/mL. The cross-reaction rates with 3-Ac-DON and 15-Ac-DON were 60.44% and 52.04%, respectively, but no cross-reaction with other mycotoxins was observed. The results showed that the anti-DON monoclonal antibodies prepared in this experiment could recognise not only DON but also 3-Ac-DON and 15-Ac-DON, which could provide materials for the next step to establish a method to detect DON and its similar compounds. Highlights This study demonstrated that an artificial antigen was synthesised by carbonyl diimidazole method, and a novel broad-spectrum, high affinity monoclonal antibody was developed. The anti-DON mAb developed in this study has better sensitivity, moreover the antibody was sensitive to DON and its derivatives. Therefore, it could be used for simultaneous monitoring of three mycotoxins. Results of this study will further lay the foundation for the establishment of immunological assays for the total amount of similar compounds.

Characterization of the monoclonal antibody specific to the ORF72 protein of koi herpesvirus and cellular distribution analysis of the viral protein.

Koi herpesvirus (KHV) is an emerging pathogen of koi and common carp that causes a severe disease and mass mortality of infected fish. The KHV ORF72 protein is an important capsid protein that has been suggested to be a candidate for the development of diagnostic reagents and KHV vaccines. The purpose of this study was to clone and express the KHV ORF72 gene for further preparation of a specific monoclonal antibody (mAb) and to analyse cellular distribution of the viral protein. The mAb 3E1 could specifically recognize the expressed ORF72 protein of transfected cells by indirect immunofluorescence, and the antigenic site recognized by the mAb 3E1 was mapped to the region of N-terminal 124 residues of KHV ORF72. This mAb was further demonstrated to specifically detect the KHV-infected fish tissue by immunohistochemistry, thereby suggesting its high diagnostic potential. In addition, the cellular distribution analysis of the KHV ORF72 protein revealed that the region of amino acid residues 125-247 was related to mitochondrial localization and proliferation. Furthermore, a putative nuclear export signal (NES) of ORF72 at the residues 201-212 was confirmed on the basis of its function associated with NES activity.

Preparation of Monoclonal Antibody Against EMA-1 and Development of Rapid Serological Detection Method for Theileria equi Infection, Xinjiang, China.

The erythrocytic-stage surface protein equi merozoite antigen 1 (EMA-1) of Theileria equi is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In this study, BALB/c mice were immunized with purified recombinant EMA-1 to prepare monoclonal antibody (mAb) against T. equi EMA-1, and 1 mAb 5H2 was obtained that showed good reaction with infected red blood cells (RBC) in the indirect immunofluorescence assay (IFA). To develop a rapid serological detection method for T. equi infection in Xinjiang Uygur Autonomous Region, China, recombinant EMA-1 originating from the local T. equi strain and the mAb to EMA-1 were employed to develop an immunochromatographic test (ICT) to detect antibodies to T. equi in horse sera. The ICT showed high sensitivity and specificity and no cross-reaction with Babesia caballi. Ninety-two horse serum samples collected from Ili, Xinjiang, were tested by ICT and compared with the detection results of a commercial ELISA kit. The results showed that 56 of 92 (61%) serum samples were seropositive according to the ICT assay, and 50 (54%) samples were seropositive according to the ELISA kit. The ICT had a high coincidence (91.3%) but was more sensitive than the reference ELISA kit. To confirm whether the horses were infected by T. equi, 30 blood DNA samples from 92 horses were examined by PCR. The results showed that 14 of 30 (47%) horses were confirmed to be infected with T. equi by PCR, while 16 of 30 (53%) horses were seropositive by ICT. All PCR-positive horses were ICT-positive. The findings indicate that T. equi is endemic in Ili, Xinjiang, and that the ICT is reliable as a serological diagnosis method. The ICT developed in this study could be an efficient diagnostic tool to detect T. equi infection in horses in the Xinjiang area.

Use of monoclonal antibodies in patients with multiple sclerosis in the practice of a neurologist

Multiple sclerosis (MS) ranks first for prevalence among diseases affecting the CNS white matter with 2.5 million cases estimated globally. InRussia, the number of cases is about 200 thousand. MS in most cases has a wavy course (periods of exacerbations and remissions), over time the progression of disease worses the quality of life of patients. The “gold standard” at the beginning of MS is first-line drugs disease-modifying therapies (DMT). If they are ineffective, it is necessary to strengthen the effect on the immune processes and the patient is prescribed second-line drugs (escalation of therapy). There is a method of induction therapy, when high activity of MS is recommended to start with drugs that have a strong immunosuppressive effect with a possible subsequent transition to soft supportive treatment. In patients with frequent exacerbations and signs of radiological activity of the disease, according to magnetic resonance imaging (MRI) of the brain and spinal cord, monoclonal antibody preparations are effectively used. Except of escalation and induction, it is also used the method of immune system reconstruction, which leads to a decrease in autoagression in MS. This article discusses a clinical case of using a drug of monoclonal antibodies that selectively bind to CD 52 on the surface of lymphocytes. The issues of efficacy and safety of alemtuzumab therapy in patients with MS are considered.

Preparation of monoclonal antibody against penicillic acid (PA) and its application in the immunological detection

The high content of Penicillic acid (PA) in the feed pose threat to human health and cause serious losses to economic wealth through the enrichment effect of the food chain. The reliable and rapidly detection of PA is of significant importance to ensure food safety. In this study, indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and immunochromatographic test strips (ICTS) were established for PA determination based on anti-PA mAb secreted by 4H9 cell line. The linear range of ic-ELISA detection was 0.12–1.95 μg/mL, and the limit of detection (LOD) was 0.03 μg/mL. Then, conventional gold nanospheres (AuNS) with the average diameter of 20 nm were synthetized and AuNS-based strip was developed for rapidly detection of PA. The visual LOD (vLOD) of the AuNS-based strip was 3.9 μg/mL and the assay time of visual evaluation was less than 10 min without any instrument. To enhance the signal sensitivity of the ICTS, the larger size (about 85 nm) of gold nanoflowers (AuNFs) was prepared in our work, and was used as higher signal reporter to establish the AuNF-based strip for PA determination. Fortunately, the vLOD of AuNF-based strip was 0.97 μg/mL, which was approximately 4-fold lower than that of traditional AuNS-based strip. In summary, the rapid and sensitive immunoassays established in this study could be applied to detect and analyze the contamination of PA toxin in real food samples.

Preparation of an Antidanofloxacin Monoclonal Antibody and Development of Immunoassays for Detecting Danofloxacin in Meat.

Danofloxacin (DAF), a third-generation fluroquinolone (FQ), is widely used as a broad-spectrum antibacterial drug to prevent diseases in livestock and poultry. In this study, a highly specific and sensitive monoclonal antibody (mAb) against DAF was prepared. Also, the mAb was used for the indirect competitive enzyme-linked immunosorbent assay (icELISA) and immunochromatographic strip for the detection of DAF residues in meat. The IC50 of the icELISA based on this mAb was 1.39 ng/mL, and the limit of detection was 0.2 ng/mL. According to the cross-reactivity (CR) experiment, the ELISA that we developed was highly specific and had low CR with other FQ analogues. Moreover, the cut-off of the immunochromatographic strip developed for detecting DAF in meat was 5 ng/mL. Overall, the developed ELISA and immunochromatographic strip based on the prepared mAb were proved reliable for the rapid detection of DAF in meat and can be considered as effective screening methods for food safety and quality management.

The emerging role of EGFL6 in angiogenesis and tumor progression.

Epidermal growth factor-like domain-containing protein 6 (EGFL6) belongs to the epidermal growth factor (EGF) superfamily. EGFL6 is expressed at higher levels in embryos and various malignant tumors than in normal tissues. Recent studies suggest that EGFL6 participates in the development of a variety of tumors. In this review, we summarize findings that support the role for EGFL6 in tumor proliferation, invasion and migration. Furthermore, our review results indicate the mechanism of EGFL6 activity angiogenesis. We also describe work toward the preparation of monoclonal antibodies against EGFL6. Altogether, the work of this review promotes understanding of the role of EGFL6 in tumor development, the mechanism of that action, and the potential of EGFL6 as a therapeutic target for tumor prevention and treatment.

Development of a Direct Competitive ELISA Kit for Detecting Deoxynivalenol Contamination in Wheat.

This study was conducted to develop a self-assembled direct competitive enzyme-linked immunosorbent assay (dcELISA) kit for the detection of deoxynivalenol (DON) in food and feed grains. Based on the preparation of anti-DON monoclonal antibodies, we established a standard curve with dcELISA and optimized the detection conditions. The performance of the kit was evaluated by comparison with high-performance liquid chromatography (HPLC). The minimum detection limit of DON with the kit was 0.62 ng/mL, the linear range was from 1.0 to 113.24 ng/mL and the half-maximal inhibition concentration (IC50) was 6.61 ng/mL in the working buffer; there was a limit of detection (LOD) of 62 ng/g, and the detection range was from 100 to 11324 ng/g in authentic agricultural samples. We examined four samples of wheat bran, wheat flour, corn flour and corn for DON recovery. The average recovery was in the range of 77.1% to 107.0%, and the relative standard deviation (RSD) ranged from 4.2% to 11.9%. In addition, the kit has the advantages of high specificity, good stability, a long effective life and negligible sample matrix interference. Finally, wheat samples from farms in the six provinces of Henan, Anhui, Hebei, Shandong, Jiangsu and Gansu in China were analyzed by the kit. A total of 30 samples were randomly checked (five samples in each province), and the results were in good agreement with the standardized HPLC method. These tests showed that the dcELISA kit had good performance and met relevant technical requirements, and it had the characteristics of accuracy, reliability, convenience and high-throughput screening for DON detection. Therefore, the developed kit is suitable for rapid screening of DON in marketed products.

Successful management of severe bronchial asthma: the right choice of biologic therapy in properly selected patients

Severe asthma along with the impact on the quality of life of those suffering from this disease leads to significant medical and social damage. Studies of the last decade indicate the leading role of eosinophilic inflammation of the bronchi as the basis of the pathogenesis of the T2 phenotype of bronchial asthma, which led to the development of targeted therapy. The most effective in this direction were preparations of humanized monoclonal antibodies directed against the main pro-inflammatory cytokines involved in respiratory tract inflammation in bronchial asthma, one of the most significant among which is interleukin 5. Refinement of the definition of severe asthma, selection of these patients among patients with difficult to treat bronchial asthma allows to clearly determine the contingent with a predicted positive effect these highly effective drugs precision therapy. On clinical examples, the difference between difficult to treat and severe bronchial asthma is discussed. The stages of clinical trials of the preparation of monoclonal antibodies against interleukin 5 Mepolizumab are analyzed in detail, the search for effective prognostic biological markers available in normal practice, allowing to select patients suitable for the treatment of patients with severe eosinophilic bronchial asthma. The effectiveness of the approach based on the allocation of two threshold values of the number of eosinophils in the peripheral blood is convincingly confirmed by the results indicating a significant reduction in the number of exacerbations, improved of lung function and an increase of the quality of life in patients, including with steroiddependent bronchial asthma, obtained not only in randomized clinical studies, but also in studies in real clinical practice.

Preparation of a new monoclonal antibody against subgroup A of avian leukosis virus and identifying its antigenic epitope

This study focuses on preparing the monoclonal antibody (MAb) against subgroup A of avian leukosis virus (ALV-A) and identifying its antigenic epitope. The ALV-A gp85 gene with a size of 1005bp was amplified and expressed into a recombinant protein with a size of 46KD in E.coli. The products expressed after purification were inoculated into BALB/c mice for preparing antibody-secreting splenic lymphocytes and further obtaining hybridoma cells. Finally, one new hybridoma cell (A18GH) secreting MAb against ALV-A was screened, and the MAb was able to detect ALV-A/K strains in an indirect immunofluorescence assay (IFA), but not ALV-B/J strains. A total of 14 overlapping truncated ALV-A gp85 protein segments were expressed and eight peptides containing different antigenic amino acids were artificially synthesized for analyzing the antigenic epitope of the MAb using a western blot or an ELISA, and the results indicate that the antigenic epitope consists of seven amino acids within the 146-ATRFLLR -152 region of the ALV-A gp85 protein. A biological information analysis shows that the antigenic epitope has a high antigenic index and develops a curved linear spatial structure. Further, its 7 amino acids are completely within the 17 representative ALV-A strains, 4 are within the 11 ALV-K strains, and fewer are within the ALV-B/J/E strains. This study will significantly assist in a further understanding of the protein structure and function of ALV-A, and in the establishment of specific ALV-A detection methods.

Preparation of monoclonal antibodies against human interleukin-35 and their application in a quantitative ELISA for interleukin-35 detection

To establish a quantitative ELISA for human interleukin-35 (IL-35) detection, we cloned cDNAs encoding the 2 subunits IL-27EBI3 and IL-12p35 of IL-35 by RT-PCR and transformed the cDNAs into Escherichia coli BL21 star (DE3) by recombinant DNA technology. IL-27EBI3 and IL-12p35 were expressed as recombinant proteins and used as immunogen to immunize Balb/c mice. Spleen cells from the positive serum mice were isolated and fused with SP-2/0 myeloma cells. We obtained the hybridoma cell lines stably secreting target antibodies by indirect ELISA screening of the cell supernatants with recombinant IL-27EBI3 and IL-12p35 as antigen and consecutive subcloning of the cells in the well with positive supernatant. Following further measurement of supernatant titers of the antibodies and identification of their antigen specificity, we obtained a hybridoma cell line 3B11 that stably secrets antibody against IL-27EBI3 and a hybridoma cell line 3A10 that secrets antibody against IL-12p35. Both monoclonal antibodies (mAbs) were identified as the subtype of IgG1. Finally, using the anti-IL-27EBI3 mAb from 3B11 as the capture antibody and the anti-IL-12p35 mAb from 3A10 as the secondary antibody, we established a quantitative double-antibodies sandwich ELISA for IL-35 detection with streptavidin-biotin amplification system. Results demonstrated that the quantitative assay had a detection range of 3.12-200 pg/mL, a detectability of 1.26 pg/mL, and a crossing-reactive rate of 0.1%. The intra-batch RSD and the inter-batch RSD of the quantitative assay were 5.1%-5.6% and 5.6%-7.2%, respectively, and the fortified recovery was 89%-103%. Therefore, the sandwich ELISA assay for IL-35 meets the qualification of quantitative analysis and laid a stable foundation for the development of quantitative ELISA kit for IL-35 detection.