Exploration of hapten-induced atopic dermatitis murine models for non-clinical pharmacodynamics study of drugs / 药学学报

Atopic dermatitis (AD) is a chronic, relapsing, inflammatory dermatosis with a variety of clinical manifestations and difficult to cure. Currently, many AD drug candidates have entered the research and development pipeline. In order to provide technical specifications for the clinical development of AD drugs, the Center for Drug Evaluation of National Medical Products Administration released the "Technical Guidelines for Clinical Trials of Drugs for AD Treatment" (Draft for Comments) in November 2022. Non-clinical pharmacodynamics evaluation is an important research before the drug enters clinical trials. Oxazolone (OXA)- and 2,4-dinitro-fluorobenzene (DNFB)-induced models are the most popular classical hapten-induced AD murine models, but variations of modeling are existing in the methods from different studies, including sensitization sites, haptens' dosages, the period of challenges, and the skin lesions severity evaluation as well. In this study, the investigation of OXA- and DNFB-induced AD murine models with various conditions of modeling was performed to compare the characteristics of hapten-induced AD murine models in the pathological process and severity according to the appearance of AD patients, and the guidance of pharmacodynamics evaluation of AD-therapeutic drugs in clinical trials as well, which may provide a proposal for AD treatment drug candidates in the non-clinical pharmacodynamics evaluation. All animal experiments were approved by the Animal Care & Welfare Committee of Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (approval No.: 00007782 and 00007784).

Allergy to carminic acid: in vitro evidence of involvement of protein-binding hapten

We previously described a rare case of anaphylaxis presumably induced by carminic acid in cochineal dye used as a food additive. In this study, highly pure carminic acid was added to an albumin-containing buffer at various concentrations, followed by serial dilution. Varying the mixing ratio of carminic acid and albumin affected the extent of histamine release from passively sensitized basophils. Similar basophil histamine release occurred with carminic acid-globulin solutions. These results provide experimental evidence indicating that basophil activation is dependent on hapten (carminic acid) and carrier (protein) interaction.

Synthesis of Novel Hapten and Development of Monoclonal Antibody-based Enzyme-Linked Immunosorbent Assay for Malachite Green in Fish Samples / 分析化学

To produce specific antibodies against malachite green ( MG) , one special hapten was synthesized and characterized, and conjugated to carrier protein as immunogen. The immunogen showed excellent reactogenicity and immunogenicity. One specific monoclonal antibody (mAb, named MG-DA4-C7) with high sensitivity and specificity for MG in indirect competitive enzyme-linked immunoassay ( icELISA ) was screened. The isotype was IgG1 and the light chain was κ type. After optimization of ELISA conditions, the proposed icELISA showed a 50% inhibition value ( IC50 ) of 0. 96 μg/L, a linear range ( IC20-IC80 ) of 0. 1-8. 1 μg/L and a limit of detection ( LOD, IC10 ) of 0. 05 μg/L for determination of MG. The assay showed cross-reactivity of 18. 1%, 26. 5% with crystal violet and brilliant green, respectively, and negligible cross-reactivity with other metabolites of MG (<0 . 1%) . The average recoveries of MG from spiked fish samples were from 87. 3% to 107. 3%. Good correlation (R2=0. 999) was obtained between the results of icELISA and those of liquid chromatography-tandem mass spectrometry analysis. The proposed icELISA is suitable for the determination of MG in fish samples in a simple and sensitive manner.

Update on Advances in Research on Idiosyncratic Drug-Induced Liver Injury

Drug-induced liver injury (DILI) is a major concern for public health, as well as for drug development in the pharmaceutical industry, since it can cause liver failure and lead to drug withdrawal from the market and black box warnings. Thus, it is important to identify biomarkers for early prediction to increase our understanding of mechanisms underlying DILI that will ultimately aid in the exploration of novel therapeutic strategies to prevent or manage DILI. DILI can be subdivided into 'intrinsic' and 'idiosyncratic' categories, although the validity of this classification remains controversial. Idiosyncratic DILI occurs in a minority of susceptible individuals with a prolonged latency, while intrinsic DILI results from drug-induced direct hepatotoxicity over the course of a few days. The rare occurrence of idiosyncratic DILI requires multicenter collaborative investigations and phenotype standardization. Recent progress in research on idiosyncratic DILI is based on key developments in 3 areas: (1) newly developed high-throughput genotyping across the whole genome allowing for the identification of genetic susceptibility markers, (2) new mechanistic concepts on the pathogenesis of DILI revealing a key role of drug-responsive T lymphocytes in the immunological response, and (3) broad multidisciplinary approaches using different platform "-omics" technologies that have identified novel biomarkers for the prediction of DILI. An association of a specific human leukocyte antigen (HLA) allele with DILI has been reported for several drugs. HLA-restricted T-cell immune responses have also been investigated using lymphocytes and T-cell clones isolated from patients. A microRNA, miR-122, has been discovered as a promising biomarker for the early prediction of DILI. In this review, we summarize recent advances in research on idiosyncratic DILI with an understanding of the key role of adaptive immune systems.

Preparation and immunogenicity and specificity characterization of Emodin-BSA-PVDF membrane artificial antigen / 中国免疫学杂志

Objective:To study feasibility of preparing artificial antigen by membrane coated with hapten-carrier.To compare the Emodin-BSA membrane antigen immunogenicity and specificity against the liquid antigen.Methods:Emodin-BSA-PVDF membrane was prepared by the method that BSA was coated on PVDF membrane and the BSA was coupled with Emodin-couplint agent derivative.Rats were immunized by subcutaneous implantation.The immunogenicity and antibody specificity were characterized using Emodin-CA or Chrysophanol-CA or Physcion-CA membrane immunoassay. Results: The immunogenicity of Emodin-BSA coated membrane antigen was higher than Emodin-BSA liquid antigen;the specificity for three anthraquinones was almost the same(P>0.05). Conclusion:Emodin antiserum generated using Emodin-BSA coated membrane antigen has a high immunogenicity and specificity to Emodin.The results show it is feasible that membrane coated with hapten-carrier is used as artificial antigen.

Synthesis and Identification of Artificial Antigen for Alkaloids from Traditional Chinese Medicine / 世界科学技术-中医药现代化

Alkaloids are a kind of natural nitrogen compounds which are widespread in the nature, with special and significant activities in pharmacology. In recent years, immunoassay techniques have been applied for the qualitative and quantitative analysis of alkaloids from traditional Chinese medicine (TCM). However, preparation and identifica-tion of artificial antigen with good immunogenicity are necessary and important for establishment of immunoassay for alkaloids from TCM. This paper summarized the designing ideas and procedures for preparation of artificial antigen and identification for the conjugates of alkaloids and carrier protein, in order to provide reference point for establish-ment and application of immunoassay techniques.

Preparation and identification of norepinephrine complete antigen and study on its immunogenicity in mice / 中华微生物学和免疫学杂志

Objective To construct and identify norepinephrine ( NE) complete antigen for the preparation of high sensitive and specific anti-NE monoclonal antibody .Methods Glutaraldehyde ( GA) and 1-Ethyl-3-(3-Dimethylaminopropyl ) carbodiimide ( EDC) were used to cross-link NE with carrier pro-teins (BSA, OVA) for NE complete antigen preparation under conditions of pH 4.5 or pH9.0.Three assays including UV scanning , SDS-PAGE and FeCl3 color reaction were performed for identification of NE com-plete antigen.Serum antibody titers were evaluated in mice model induced by intraperitoneal immunization with NE complete antigen .Results NE complete antigens were successfully prepared as indicated by the three identification assays .The coupling ratio was significantly increased in a time-depended manner under the condition of pH9.0 in comparison to that in the condition of pH 4.5.Indirect ELISA results showed that , when coating antigens and serum antibodies were prepared with the same cross -linking method , the serum antibody titers were significantly higher than those with different methods .Conclusion Anti-NE antibodies were successfully prepared by immunizing mice with NE complete antigens .

Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification

We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 x anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine-HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein.

Synthesis and Identification of Hapten and Complete Antigen of Norketa mine / 分析化学

Under low temperature conditions, the hapten carboxyl-norketamine was synthesized by reacting norketamine and succinaldehyde acid.Identification result using electrospray ionization mass showed the hap ten was successfully synthesized.The artificial antigen confirmed by infrared spectroscopy was developed by conjugating hapten to carrier proteins with carbodiimide(EDC) method.Matrix-assisted laser desorption ioni zation time of flight mass spectrometry(MALDI-TOF-MS) showed that the ratio of hapten to BSA was 11:1.The antibody with high titer(5.12 × 10~4) was produced after immuning to rabbits.

Synthesis and Identification of the Antigens for Ciprofloxacin / 中国兽医学报

Hapten-carrier protein conjugates were made using ciprofloxacin (CPFX) and two carrier proteins by 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC) method. Ultraviolet spectrophotometry were used to demonstrated that the molecule conjugate ratio of CPFX to ovalbumin (OVA) and bovine serum albumin (BSA) are 6:1 and 13:1 respectively. Nondenaturing gel electrophoresis results revealed that the conjugate band migrates differently from that of the carrier protein alone and of the EDC-treated protein when as few as 6 molecules of CPFX are attached to the carrier protein. The results indicate that nondenaturing gel electrophoresis and ultraviolet spectrophotometry can be employed to analyze the molecule coupling ratio of CPFX to carrier proteins qualitatively and quantitatively.

Synthesis and identification of antigenic conjugates of podophyllotoxin / 药学学报

Aim To synthesize and identify artificial antigen of podophyllotoxin for the production of podophyllotoxin polyclonal antibody. Methods The hapten was synthesized by two different chemical approaches and characterized by TLC, IR, NMR, and MS. Mixed anhydride reaction (MAR) and active ester method (AEM) were used to couple the podophyllotoxin to carrier proteins (BSA and OVA). Characterization of artificial antigens was done by using spectroscopy and electrophoresis. The anti-podophyllotoxin polyclonal antibodies were obtained through immunizing rabbits. Results The results from IR, NMR and MS showed that 4-O-succinoyl podophyllotoxin (hapten) was successfully synthesized. The coupling molar ratios of the hapten and carrier proteins were 88.6 for Hapten-BSA1, 40.3 for Hapten-BSA2, 17.8 for Hapten-OVA1, and 54.2 for Hapten-OVA2. Hapten conjugates coupled with BSA yielded two sets of the specific and affinitive polyclonal antibodies. One set of antibodies showed an IC50 value of 2.21 μg·mL -1 with a detection limit of 0.12 μg·mL -1. Conclusion Antigenic conjugates were artificially synthesized, and based on these artificial antigens, polyclonal antibodies against podophyllotoxin were raised from rabbits immunized with two different immunogens and characterized with an indirect ELISA format.

Synthesis of huperzine A-bovine serum albumin conjugate and its immunogenicity in mice / 第三军医大学学报

Objective To prepare huperzine A-carrier protein conjugate and determine its immunogenicity for later production of monoclonal antibody against HupA and establishing an enzyme-linked immunosorbent assay ( ELISA) -based detection method for HupA. Methods HupA was conjugated with bovine serum albumin ( BSA) using glutaraldehyde ( GA) method to obtain the HupA-GA-BSA conjugate,and the hapten number in the conjugate was determined by matrix-assisted laser absorption ionization time-of-flight mass spectrometry ( MALDI-TOF-MS) . BALB /c mice were immunized with HupA-GA-BSA to prepare the antiserum against HupA. The serum titer and specificity of the HupA antibodies were detected by indirect ELISA and competitive ELISA,respectively. Results The conjugation ratio of HupA and the carrier protein BSA was 8∶ 1. The antiserum against HupA with a titer of 1∶ 62 500 was obtained after immunizing the mice with the conjugate,and the antiserum reacted specifically to HupA. Conclusion The synthesized HupA-GA-BSA conjugate possesses good immunogenicity in mice,suggesting the feasibility of preparing the monoclonal antibody against HupA using this conjugate.

A sandwich method of enzyme immunoassay for serum FT_4 / 中华内分泌代谢杂志

T_4 was coupled to human serum albumin(HSA)to make the conjugate T_4-HSA,a polyvalent T_4 analogue rather than monovalent T_4 per se alone.A new strategy for sandwich enzyme immunoassay model system of small molecular hapten hormones FT_4 was developed,such as intra-assay CV,inter-assay CV,average recovery, normal and abnormal values were fit for clinical application.The sensitivity was as much as 10 times higher than conventional competitive enzyme immunoassay.

Cell Mediated Immunity in Tubulointerstitial Nephritis of Rats

To investigate the tubular major histocompatibility complex(MHC) expression and inflammatory phenotypes in tubulointerstitial nephritis, Lewis rats were inununized with azobenzen-earsonate-tyrosine in complete Freund adjuvant and challenged either foot pad or kidney, either by subcapsular injection or by ex vivo perfusion. The rats were sacrificed 2, 3, 5, 10 and 15 days after antigenic challenge. Foot pad swelling was significant at the antigenic challenge site (151.8 vs 6.8 x 10(-2) mm) at 24 hours. Tubulointerstitial nephritis was induced by both methods and the inflammatory infiltrate which first appeared on day 2, became prominent at day 5, then gradually subsided in ex vivo perfused rats, while inflannnation started on day 3 in subcapsular injected rats. The major site of inflammation was in the cortex and outer stripe of the outer medulla, with predominance of mononuclear cells throughout the course. The inflammatory cells showed mainly OX8 and ED1 positivity with OX19, W3/25 and CD5 positivity in minority. RT1B expression was diffuse in the cytoplasm of proximal tubules at day 2 and 5. These results suggest the involvement of cell mediated immunity in this experimental model, and the possibility that tubular epidielial cells process antigen and then become targets in immune injury.

Regulation of IgE antibody production in mice Ⅲ.IgE antibody response to hapten-carrier conjugate / 中国免疫学杂志

The anti-DNP and anti-OA IgE antibody responses of six strains of mice immunizedwith DNP-OA conjugate in presence of A1(OH)_3 adjuvant was investigated.Signifi-cant differences of the magnitude of serum IgE were found in anti-hapten and anti-carrier responses among strains.The primary and secondary anti-DNP and anti-OAIgE antibodies were elicited in NIH,BALB/c and DBA/2 mice after immunizationwith 1?g of DNP-OA.Among them NIH mice was the highest responder,whereasLACA,C3H and B6D2F1 mice were poor or nonresponders.In comparison the IgEresponse in BALB/c mice immunized with 1?g and 10?g of DNP-OA,the former de-monstrated a latency of primary response,but two groups had a very similar PCAtiters after 4 weeks of immunization.On the other hand,LACA mice gave a dose de-pendent IgE response.Profound primary and secondary IgE antibody responses can beonly seen in mice injected with 10?g of DNP-OA and PCA titer of anti-DNP washigher than that of anti-OA IgE antibody.However,no difference of IgE antibodylevels in NIH mice can be found between groups immunized with different doses.DNP_(4.7)-OA primed mice gave higher IgE response than that of mice immunized withDNP_3-OA.A prevailing anti-hapten IgE response was observed when higher dose ofDNP-OA conjugates was injected.

A simple radioimmunoassay used for detection of anti-hapten antibodies in screening of lymphocyte hybridomas / 中国免疫学杂志

A radioimmunoassay was designed to detect anti-hapten (Aflatoxin M1, AFM1)antibodies for screening of lymphocyte hybridoma clones. The advantages of the assay were follows.1.Through carriers and/or hapten competition binding tests antihapten antibodies in rat sera could be specifically and directly with ~(125)I labeled hapten-carrier (AFM1-BSA) which had been used as an antigen to immunize the rats. In this assay only one carrier was needed. It could circumvent the labor intension to use different carriers for immunization and detection respectively, especially when a complicated coupling method was required.2.Only a scanty amount of the hapten-carrier (as less as 5 ?g) labeled with ~(125)I was needed in the RIA, which was far less than that needed in ELISA. The advange was significant for a rare source of a hapten and a large of number of samples to be assayed,such as lymphocyte hybridoma supernatant in serial clonings.3.The anti-hapten antibodies in supernatant of lymphocyte hybridoma cultures could be specifically detected by the RIA without adding the competition binding carrier, because 10-15% BS in the medium was sufficient to neutralize all anti-carrier (BSA) antibodies. So the assay could be satisfactorily used to screen lymphocyte hybridoma clones secreting antihapten antibodies.The methodology of the assay was discussed in the paper.