Development of donor specific antibodies after SARS-CoV-2 vaccination:What do we know so far?

Vaccination against Coronavirus disease-19(COVID-19)was pivotal to limit spread,morbidity and mortality.Our aim is to find out whether vaccines against COVID-19 lead to an immunological response stimulating the production of de novo donor specific antibodies(DSAs)or increase in mean fluorescence intensity(MFI)of pre-existing DSAs in kidney transplant recipients(KTRs).This study involved a detailed literature search through December 2nd,2023 using PubMed as the primary database.The search strategy incorporated a combination of relevant Medical Subject Headings terms and keywords:"COVID-19","SARS-CoV-2 Vaccination","Kidney,Renal Transplant",and"Donor specific antibodies".The results from related studies were collated and analyzed.A total of 6 studies were identified,encompassing 460 KTRs vaccinated against COVID-19.Immunological responses were detected in 8 KTRs of which 5 had increased MFIs,1 had de novo DSA,and 2 were categorized as either having de novo DSA or increased MFI.There were 48 KTRs with pre-existing DSAs prior to vaccination,but one study(Massa et al)did not report whether pre-existing DSAs were associated with post vaccination outcomes.Of the remaining 5 studies,35 KTRs with pre-existing DSAs were identified of which 7 KTRs(20%)developed de novo DSAs or increased MFIs.Overall,no immunological response was detected in 452(98.3%)KTRs.Our study affirms prior reports that COVID-19 vaccination is safe for KTRs,especially if there are no pre-existing DSAs.However,if KTRs have pre-existing DSAs,then an increased immunological risk may be present.These findings need to be taken cautiously as they are based on a limited number of patients so further studies are still needed for confirmation.

Construction of Multi-Specific Antibody by Genetic Engineering and Its Progress in Tumor Therapy

Targeted treatment of cancer with monoclonal antibodies increases the benefit for patients. In order to improve the anti-tumor activity of monoclonal antibodies, multi-specific antibodies have entered the research field. The emergence of various techniques to produce multi-specific recombinant antibody molecules has led to the selection of target combinations in various forms. To date, only a few multi-specific constructs have entered phase III clinical trials, in contrast to classical monoclonal antibodies. Some of the format options are outlined from a technical point of view. We focus on the achievements and prospects of the underlying technologies for generating biand multispecific antibodies.

Application of Current Hapten in the Production of Broad Specificity Antibodies Against Organophosphorus Pesticides

Diethylphosphono acetic acid （DPA） was used as a current hapten to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. Six New Zealand white rabbits were immunized with immunogens synthesized by the active ester method （AEM） or 1-ethyl-3-（3-dimethylaminopropyl）-carbodimide method （EDC）. The titers of antisera reached 25 600 by AEM and 6 400 by EDC, respectively. Polyclonal antibodies raised against DPA were screened and selected for the competitive indirect enzyme-linked immunosorbent assay （CI-ELISA）. A CI-ELISA for DPA was developed with a detection limit of 3.536 ng mL^-1and an I50 value of 0.182 μg mL^-1. The assay specificity was evaluated by obtaining competitive curves for several structurally related compounds as competitors. The antiserum showed high affinities to chlorpyrifos, diazinon, omethoate, parathion-ethyl and profenofos with I50 of 0.12, 0.15, 0.21, 0.88, 0.97 and 2.5 μg mL^-1, respectively. The results indicate that the assay could be a screening tool for quantitation and semiquantitation determination of the above former five organophosphorus pesticides.

Characteristics of Viral Shedding in Respiratory Samples and Specific Antibodies Production in 564 COVID-19 Patients

Positive nucleic acid(NA)results have been found in recovered and discharged COVID-19 patients,but the proportion is unclear.This study was designed to analyze the recurrent positive rate of NA results after consecutively negative results,and the relationship between the specific antibody production and positive NA rate.According to Strengthening the Reporting of Observational Studies in Epidemiology guidelines,data of inpatients in Sino-French New City Branch of Tongji Hospital between Jan.28 and Mar.6,2020 were collected.A total of 564 COVID-19 patients over 14 years old who received the examinations of NA and antibodies against SARS-CoV-2 were included.Days of viral shedding and specific antibodies were recorded and assessed.Among NA tests in respiratory samples(throat swabs,nasopharyngeal swabs,sputum and flushing fluid in alveoli),the patients with all-negative NA results accounted for 17.20%,those with single-positive results for 46.63%,and those with multiple-positive results for 36.17%respectively.Besides,the recurrent positive NA results after consecutively negative results appeared in 66 patients(11.70%).For multiple-positive patients,median viral shedding duration was 20 days(range:1 to 57 days).Of the 205 patients who received 2 or more antibody tests,141(68.78%)had decreased IgG and IgM concentrations.IgM decreased to normal range in 24 patients,with a median of 44 days from symptom onset.Viral shedding duration was not significantly correlated with gender,age,disease severity,changes in pulmonary imaging,and antibody concentration.It is concluded that antibody level and antibody change had no significant correlation with the positive rate of NA tests and the conversion rate after continuous negative NA tests.In order to reduce the recurrent positive proportion after discharge,3 or more consecutive negative NA test results with test interval more than 24 h every time are suggested for the discharge or release from quarantine.

Isolation and Characterization of Recombinant Variable Domain of Heavy Chain Anti-idiotypic Antibodies Specific to Aflatoxin B_1

Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain （VHH）. The recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. In particular, they might be very useful for molecular mimicry. The present study demonstrated an alpaca immunized with the F（ab＇）z fragment of anti-aflatoxin B1 mAb and developed an important anti-idiotypic （anti-ld） responses. Antigen-specific elution method was used for panning private anti-ld VHHs from the constructed alpaca VHH library. The selected VHHs were expressed, renatured, purified, and then identified by a competitive enzyme-linked immunosorbent assay （ELISA）. Our findings indicated that the VHH would be an alternative tool for haptens mimicry studies.

THE SPECIFIC PHOTODYNAMIC EFFECTS OF MONOCLONAL ANTIBODIES CONJUGATED WITH HEMATOPORPHYRIN DERIVATIVE ON GASTRIC CANCER IN VITRO AND IN VIVO

Murine monoclonal antibody (MoAb) BB4.3, raised against the human gastric cancer cell line BGC823, was puriffied with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. It was then conjugated with a hematoporphyrin derivative (HPD) by using carbodiimide. The qualitative analysis of this conjugate showed that the amount of free HPD was negligible and there were no IgG aggregates among the conjugates. The conjugate retained both the antibody and photochemical activity of HPD.In vitro, the phototoxic effect of this HPD-BB4.3 conjugate on target cells was about 15 times higher than that of free HPD. The quality of selective photocytotoxicity was proven by the greater cytotoxi-city this conjugate showed than that of corresponding normal mouse IgG (NIgG) conjugated with HPD. It showed less cytotoxicity to colon cancer cell line B-80 (negative reaction to MoAb BB4.3) than to BGC825. Moreover, its cytotoxicity to BGC823 cells could be blocked specifically by excess BB4.3 antibody, but not by another MoAb 3G9, which combines with BGC823 at different binding sites from MoAb BB4.3.Nude mice inoculated with 2 × 10- BGC823 cells were given HPD-BB4.3, HPD, HPD-NIgG, HPD plus BB4.3 and PBS, respectively then exposed to light. Four out of six animals treated with the HPD-BB4.3 conjugate remained tumor-free for a long period. Although two developed tumors, there was a significant difference between the HPD-BB4.3-treated group and all the control groups in tumor induction time, tumor growth rate, and survival time (p<0.001). The HPD-BB4.3 conjugate inhibited the growth of established tumors by more than 40% in comparison with control groups (p<0.05).

Impact of donor-specific antibodies on the outcomes of kidney graft:Pathophysiology, clinical, therapy

Allo-antibodies, particularly when donor specific, are one of the most important factors that cause both early and late graft dysfunction. The authors review the current state of the art concerning this important issue in renal transplantation. Many antibodies have been recognized as mediators of renal injury. In particular donorspecific-Human Leukocyte Antigens antibodies appear to play a major role. New techniques, such as solid phase techniques and Luminex, have revealed these antibodies from patient sera. Other new techniques have uncovered alloantibodies and signs of complement activation in renal biopsy specimens. It has been acknowledged that the old concept of chronic renal injury caused by calcineurine inhibitors toxicity should be replaced in many cases by alloantibodies acting against the graft. In addition, the number of patients on waiting lists with preformed anti-human leukocyte antigens(HLA) antibodies is increasing, primarily from patients with a history of renal transplant failure already been sensitized. We should distinguish early and late acute antibody-mediated rejection from chronic antibody-mediated rejection. The latter often manifets late during the course of the posttransplant period and may be difficult to recognize if specific techniques are not applied. Different therapeutic strategies are used to control antibody-induced damage.These strategies may be applied prior to transplantation or, in the case of acute antibody-mediated rejection, after transplantation. Many new drugs are appearing at the horizon; however, these drugs are far from the clinic because they are in phase Ⅰ-Ⅱ of clinical trials. Thus the pipeline for the near future appears almost empty.

Development and Characterization of Monoclonal Antibody Specifically Against TSP50

Testis-specific protease 50（TSP50） has been identified as a testis-specific protein that is expressed abnormally in most human breast cancer samples,which makes it an attractive molecular marker and a potential target for diagnosis and therapy.In the present study,we prepared a panel of monoclonal antibodies（mAbs） with high specificity and sensitivity against TSP50 by hybridoma method and characterized them by ELISA,Western blot,immunofluroescence and immunohistochemical analyses.The results show that all of the 9 different clones can specifically bind to TSP50.The mAbs against TSP50 we generated could be good tools for both basic and clinical studies.

SPECIFIC UPTAKE OF MONOCLONAL ANTIBODY-CONJUGATED METHOTREXATE BY HUMAN LYMPHOCYTIC LEUKEMIC B CELLS

Objective: To analysis the uptake of free MTX and MTX conjugated to tumor specific monoclonal antibody by target and nontarget cells. Methods: The folate antagonist methotrexate (MTX) was conjugated to two monoclonal antibodies (Mab) directed against human chronic lymphocytic leukemia (CLL), Dal B01 and Dal B02, by an active ester method. Both conjugates were more cytotoxic toward the target tumor cell line D101 than to the nontarget cell line MOLT3, and Dal B02MTX conjugate was more inhibitory to D101 cells than free MTX in a 6 h pulse exposure assay. Results: Drug uptake studies revealed that D101 cells took up much more Dal B01 and Dal B02conjugated MTX than free MTX. The amounts of drug taken up by D101 cells incubated with Dal B01 and Dal B02conjugated MTX were always 3 to 5fold higher than that taken up by MOLT3 cells, although the latter took up more drug when incubated with free MTX. Furthermore, tumor cells incubated with Dal B01 or Dal B02conjugated MTX retained much larger amounts of drug for a prolonged period of time than those incubated with free MTX. Conclusion: The enhanced specific cytotoxicity of Dal B01 and Dal B02MTX conjugates toward target tumor cells is therefore likely due to (I) delivery of larger amounts of MTX to target cells when the drug is conjugated to Mab; (ii) longer retention of Mabconjugated MTX by target cells; and (iii) slow, prolonged release of MTX from the surfacebound or endocytosed conjugates, rendering them into a sustained release dosage form.

Impact of preformed donor-specific antibodies against HLA class Ⅰ on kidney graft outcomes:Comparative analysis of exclusively anti-Cw vs anti-A and/or-B antibodies

AIM To analyze the clinical impact of preformed antiH LA-Cw vs antiH LA-A and/or-B donor-specific antibodies(DSA) in kidney transplantation.METHODS Retrospective study, comparing 12 patients transplanted with DSA exclusively antiH LA-Cw with 23 patients with preformed DSA antiH LA-A and/or B.RESULTS One year after transplantation there were no differencesin terms of acute rejection between the two groups(3 and 6 cases, respectively in the DSA-Cw and the DSA-A-B groups; P = 1). At one year, eG FR was not significantly different between groups(median 59 mL /min in DSA-Cw group, compared to median 51 mL /min in DSA-A-B group, P = 0.192). Moreover, kidney graft survival was similar between groups at 5-years(100% in DSA-Cw group vs 91% in DSA-A-B group, P = 0.528). The sole independent predictor of antibody mediated rejection(AMR) incidence was DSA strength(HR = 1.07 per 1000 increase in MFI, P = 0.034). AMR was associated with shortened graft survival at 5-years, with 75% and 100% grafts surviving in patients with or without AMR, respectively(Log-rank P = 0.005).CONCLUSION Our data indicate that DSA-Cw are associated with an identical risk of AMR and impact on graft function in comparison with "classical" class I DSA.

Detection of cardiac myosin binding protein-C (cMyBP-C) by a phospho-specific PKD antibody in contracting rat cardiomyocytes

Protein phosphorylation plays an important role in physiological processes, such as muscle contraction. Phospho-specific antibodies have become powerful tools to study these processes. Cardiac myosin binding protein-C (cMyBP-C) is one of the proteins that make up the contractile apparatus of cardiomyocytes. Phosphorylation of cMyBP-C is essential for normal cardiac function, since dephosphorylation of this protein leads to its degradation and has been associated with cardiomyopathy. One of the upstream kinases, which phosphorylate cMyBP-C, is protein kinase D (PKD). While studying the role of PKD in cMyBP-C phosphorylation, we tried to analyze phosphorylation of PKD with a phospho-specific PKD-Ser744/748 antibody. Contrary to the expected 115 kDa, a signal was found for a 150-kDa protein. By MALDI-TOF mass spectrometry, we identified this protein to be cMyBP-C. These data were confirmed by immunostaining using the p-PKD-Ser744/748 antibody, which displayed a striated pattern similar to the one observed for a regular cMyBP-C antibody. To our knowledge there are no antibodies commercially available for phosphorylated cMyBP-C. Thus, the p-PKD-Ser744/748 antibody can accelerate research into the role of cMyBP-C phosphorylation in cardiomyocytes.

Development of rabbit monoclonal and polyclonal antibodies for detection of site-specific histone modifications and their application in analyzing overall modification levels

除了 DNA 顺序信息，地点特定嘘一修正是在一个真核细胞的有机体的基因表示的另一个重要决定因素。我们选择了四个修正地点在普通嘘被知道显著地影响染色质功能并且产生的单音同种细胞或认出每那些地点特定的修正的 polyclonal 抗体。我们使用了这些抗体证明地点特定嘘一个修正层次在一样的有机体的不同机关仍然保持相对不变。我们也比较了层次选择嘘在几个代表性的有机体之中的一修正并且发现地点特定的修正在不同有机体之中是高度可变的，提供新卓见进进化分叉特定嘘一修正。

Inhibitory Effect of Anti-HER-2 Anti-CD3 Bi-specific Antibody on the Growth of Gastric Carcinoma

To evaluate the effect of anti-HER-2 × anti-CD3 bi-specific antibody（BsAb） on the growth of HER-2/neu-expressing human gastric carcinoma in vitro and in vivo, an MTT assay was carried out to test the inhibitive rates of herceptin, anti-CD3 and BsAb antibodies on SGC-7901 gastric carcinoma cells. Immunocytochemistry methods were used to test the HER-2 level of SGC-7901. Nude mice models were employed to test the effect of HER-2 CD3 BsAh combined with effector ceils（ peripheral blood lymphatic cells of healthy human beings） on the growth of tumors in animals. Compared with that of the untreated control group, the tumor cell growth rates in vitro and in vivo will both be significantly inhibited when treated with effector cells combined with anti-CD3 McAb, herceptin or HER2 CD3 BsAb （p 〈0. 05）, and the growth inhibition is the most remarkable in the group treated with HER2 CD3 BsAb combined with effector cells. The growth of tumor xenografts will also be significantly inhibited in the group treated with HER2 CD3 BsAb combined with effector cells when compared with that in the group treated with anti-CD3 McAb or the group treated with herceptin combined with effector cells（p 〈0. 05）. We can conclude that HER-2/neu is possibly a useful target for immunotherapy of gastric carcinoma, and anti-HER2 × anti-CD3 BsAb has evident anti-tumor efficacy both, in vitro and in vivo.

STUDY ON DETERMINATION AND APPLICATION OF THE SPECIFIC ANTIBODIES TO CYTOMEGALOVIRUS IN BONE MARROW TRANSPLANTATION

using enzymellnked lmmunosorheut assay (ELlsA), we had determined the speclrlc antibodies of IgM and lgA to CMV In 14 patients with BAT, 36 marrow donors and 682 blood donorsfrom 1991 to 1996. The antibodies detected were negative in 14 patients, 16. 16% POsitive in marrowdonors and 34. 31 % in blood donors respectively. These resultS suggested that there was a higher active or recent CMV Inrectlou in blood donors in XI'an area. In order to prevent transfusion-acquiredCMV infectlony it is nessessary ror us to screen out negative CMV antibodies donors in BAT, whichhas great value for clinical application.

Preliminary Identification of Human Nonserum Oviduct Specific Proteins by Using Electrophoresis and Immunoblotting Analysis

The present paper reported the preliminary results of identification of humannonserum oviduct specific proteins. The 1D-SDS-polyacrylamide gel electrophoresis (PAGE) and 2D-SDS-PAFE in conjunction with the immunoblotting assay were used in the present study. The results showed that the nonserum oviduct specific proteins with MW130, 100 and 80 kD existed in human oviduct fluid or oviductal extract. In addition, the antibody against pig oviduct antigens could more strongly cross-react with human oviduct antigens, mainly recognizing 130,116 and 100 kD proteins from human oviduct. It is suggested that in human oviduct there are some specific antigens possessing some similar epitopes to those in pig oviducts. This result seems to be consistent with predominant cross reactivity existing in antigens of porcine and human zona pellucida.

Blockade of γc Signals in Combination with Donor-specific Transfusion Induces Cardiac Allograft Acceptance in Murine Models

The γc cytokines play an important role in proliferation and survival of T cells. Blocking the γc signals can cause the activated donor-reactive T cells losing the ability to proliferate, and getting into apoptosis pathway, which contributes to induction of the peripheral tolerance. In this study, we induced the transplant tolerance through blocking the γc in combination with donor-specific transfusion (DST) in the cardiac transplantation. Following DST, on the day 2, 4 and 6, C57BL/6 recipients received anti-γc monoclonal antibodies (mAbs) injection, and those in control group were not given anti-γc mAbs. On the day 7, Balb/c cardiac allografts were transplanted. All recipients in experimental group accepted cardiac allografts over 30 days, and two of them accepted allografts without rejection until sacrifice on the 120 day. Animals only receiving DST rejected grafts within 5 days, and the mice receiving cardiac transplantation alone rejected grafts within 9 days. Our study showed that blockade of γc signaling combined with DST significantly prolonged allograft survival, which was probably associated with inhibition of antigen-specific T-cell proliferation and induction of apoptosis.

Use of Polyclonal Antibody for the Diagnosis of Human African Trypanosomiasis

Human African trypanosomiasis (HAT), commonly known as sleeping sickness is one of the neglected tropical diseases (NTDs), which is fatal if left untreated. Its diagnosis is a challenge since the signs and symptoms of the primary phase are not specific, the existing diagnostic methods have low sensitivity and specificity, and the available drugs have some toxicity. New, robust, and cost-effective techniques are needed for the early identification of parasites. This study aimed to assess the sensitivity and specificity of two different types of polyclonal antibodies against T. b. gambiense using antigen detection ELISA. Polyclonal antibodies against the expressed proteins Tbg I2 and Tbg I17 were produced using New Zealand white rabbits. The antibody titer measured was greater than 32 g/L after the 3rd immunization for the expressed protein Tbg I2. For the expressed protein Tbg I17, the antibody titer measured was greater than 32 g/L after the 4th immunization. The sensitivity and specificity of the Tbg I2 polyclonal antibody confirmed with Polymerase Chain Reaction (PCR) as gold standard were respectively 89.5% and 80.6%, while for the Tbg I17 polyclonal antibody, the sensitivity and specificity were respectively 92.1% and 88.9%. The area under the curve for the Tbg I2 polyclonal antibody was 0.90 ± 0.032, while for the Tbg I17 polyclonal antibody, the area under the curve was 0.92 ± 0.0. The Tbg I17 polyclonal antibody produced in New Zealand white rabbits has good sensitivity and good specificity;it can be successfully used in the diagnosis of HAT.

原发与继发免疫性血小板减少症患者血小板膜糖蛋白特异性抗体及其与出血评分关系的研究

目的探讨原发与继发免疫性血小板减少症(ITP)患者抗GPⅡb/Ⅲa及GPΙb/Ⅸ抗体的表达、抗体阳性表达与出血评分的关系,以及不同抗体类型出血评分的差异,为原发与继发ITP患者的诊治和出血严重程度提供临床依据。方法选取2013年10月—2020年8月在新疆医科大学第一附属医院诊断为原发ITP患者(42例)及继发ITP患者(42例)为研究对象,所有患者入院时采用ITP出血评分量表行出血评分。应用改良MAIPA法检测患者抗GPⅡb/Ⅲa和抗GPΙb/Ⅸ抗体。结果原发与继发组患者抗GPⅡb/Ⅲa抗体、抗GPΙb/Ⅸ抗体阳性率比较,差异无统计学意义(P>0.05),原发组患者抗体表达以抗GPⅡb/Ⅲa抗体为主,继发组抗体表达以抗GPΙb/Ⅸ抗体为主,两者抗体阳性构成比较,差异有统计学意义(P<0.05)。继发组患者整体出血程度较原发组重(P<0.05)。抗体阳性表达与出血程度的关系:(1)抗GPⅡb/Ⅲa抗体阳性患者出血程度较抗体阴性患者重(P<0.05)。(2)抗GPΙb/Ⅸ抗体阳性患者出血程度较抗体阴性患者重(P<0.05),双抗体阳性患者出血程度较抗体阴性患者重(P<0.05)。结论抗GPⅡb/Ⅲa抗体、抗GPΙb/Ⅸ抗体阳性表达对原发与继发ITP无鉴别意义,但原发ITP患者抗体表达以抗GPⅡb/Ⅲa抗体为主,继发ITP患者抗体表达以抗GPΙb/Ⅸ抗体为主。继发ITP患者临床出血程度较原发ITP患者重。抗GPⅡb/Ⅲa抗体、抗GPΙb/Ⅸ抗体及抗体双阳性患者出血程度较抗体阴性患者重。

牛支原体NM001株单克隆抗体的制备与鉴定

为获得抗牛支原体NM001株单克隆抗体,并评价其特性,以牛支原体分离株NM001作为抗原免疫6周龄BALB/c小鼠,利用杂交瘤技术和间接ELISA方法筛选到2株能稳定分泌抗牛支原体的单克隆抗体细胞,命名为2C5和7G3。其细胞上清的间接ELISA抗体效价分别为6.4×10^(3)和1.2×10^(4)。经亚型测定,单抗2C5和7G3均属于IgG1类,轻链均是λ型。制备腹水并对单抗进行纯化和特性鉴定,两株单抗的间接ELISA抗体效价分别为1.02×10^(5)和4.09×10^(5),且两株单抗与无乳支原体、山羊支原体山羊肺炎亚种、丝状支原体山羊亚种、绵羊肺炎支原体、牛巴氏杆菌均无交叉反应。Western Blot结果显示,2株单抗均能特异性识别牛支原体全菌蛋白中的相应蛋白。试验表明,单抗2C5和7G3能够与牛支原体发生特异性反应,从而为牛支原体血清学检测提供一定的物质基础。