Identification of Two New D Variants, DHMi and DHMii Using Monoclonal Anti-D

Fifteen examples of red cells are described which showed discrepant reactivity on routine grouping with two monoclonal anti-D typing reagents, HAM-A being negative and MAD-2 strongly positive. The reaction profile of these cells with a number of monoclonal anti-D characterised the samples into two new variant groups, D^HMi and D^HMii. D^HMi was characterised by a negative reaction with BRAD-8 and D^HMii by a negative reaction with BRAD-5. D^HMi cells were found to have a papain-sensitive site, identified by Mab 21 G6, which was not detectable in D^HMii. The presence of allo-anti-D in one case of D^HMi suggests a partial D in these individuals. The positive reaction of HAM-A with sialidase- and endo-F-treated D^HMii cells suggests that sialic acid and/or N-glycans could possibly be involved in blocking the reaction with untreated cells. All samples typed as C-E+. One was e negative while the rest had variable depression of the e and f antigens. Marginal depression of E was seen with those D^HMi cells tested, but not with D^HMii cells. Data from family studies suggest that the variant D in both D^HMi and D^HMii is inherited as a cDE gene complex and is controlled by the RH locus.     

蟑螂主要过敏原Blag7单克隆抗体的制备与鉴定

目的 制备蟑螂主要过敏原Bla g7的单克隆抗体(mAb).方法 用含编码Bla g7基因的大肠杆菌菌株Origami/pGS21a,诱导表达并纯化出Bla g7蛋白,以该蛋白为抗原免疫BALB/c小鼠,运用杂交瘤技术制备抗Bla g7 mAb;通过ELISA、Western blot技术鉴定mAb的特异性.结果 获得...     

The Generation and Application of Monoclonal Antibodies to Detect SAPCD2 Expression in Precancerous and Malignant Gastric Lesions

Background: Suppressor APC domain containing 2 (SAPCD2) is involved in cell cycle regulation and its mRNA levels are higher in cancer tissues. But, the function of SAPCD2 in cancer development remains unclear.Objective: To generate mouse monoclonal antibodies (mAbs) specific to SAPCD2 and thus clarify the function of SAPCD2 in the development of gastric carcinoma (GC).Methods: Purified SAPCD2-GST immunized BALB/c mouse spleen cells were collected and fused with myeloma cells to obtain monoclonal antibody hybridoma. A group of monoclonal antibodies exhibiting high specificity and sensitivity against SAPCD2 has been generated and characterized by IHC, WB, IP, IF, and ELISA. By immunohistochemical analysis, the SAPCD2 expression was evaluated in 228 clinical samples of gastric mucosal lesions, including precancerous lesions and GC samples.Results: We identified a highly specific and sensitive clone of s12 in eukaryotic cells and performed an IHC analysis. We found that 81.3% of 107 GC tissues were SAPCD2-positive, compared with the 26.2% in the matched adjacent normal-appearing tissues (P<0.001). Furthermore, among the 121 gastritis tissues, SAPCD2 was overexpressed in precancerous gastric lesions such as dysplasia (Dys, 78.9%), intestinal metaplasia (IM, 44.7%), and chronic atrophic gastritis (CAG, 6.1%) compared with that in chronic non-atrophic gastritis (CNAG, 3.2%) (P<0.001). The SAPCD2-positivity rate was 81.3% in GC, suggesting that the expression of SAPCD2 increased with the severity of the lesion (P<0.001).Conclusion: In summary, we have described novel monoclonal antibodies against SAPCD2, which are highly expressed in GC tissues and may serve as the basis for an early clinical marker for GC development.     

Resistance of SARS-CoV-2 Omicron BA.1 and BA.2 Variants to Vaccine-Elicited Sera and Therapeutic Monoclonal Antibodies

The recent emergence of the Omicron BA.1 and BA.2 variants with heavily mutated spike proteins has posed a challenge to the effectiveness of current vaccines and to monoclonal antibody therapy for severe COVID-19. After two immunizations of individuals with no history of previous SARS-CoV-2 infection with BNT162b2 vaccine, neutralizing titer against BA.1 and BA.2 were 20-fold decreased compared to titers against the parental D614G virus. A third immunization boosted overall neutralizing titers by about 5-fold but titers against BA.1 and BA.2 remained about 10-fold below that of D614G. Both Omicron variants were highly resistant to several of the emergency use authorized therapeutic monoclonal antibodies. The variants were highly resistant to Regeneron REGN10933 and REGN10987 and Lilly LY-CoV555 and LY-CoV016 while Vir-7831 and the mixture of AstraZeneca monoclonal antibodies AZD8895 and AZD1061 were significantly decreased in neutralizing titer. Strikingly, a single monoclonal antibody LY-CoV1404 potently neutralized both Omicron variants.     

New Murine Monoclonal Antibodies Directed against Glycophorins C and D, Have Anti-Ge2 Specificity

Background and Objectives : The Gerbich blood group system comprises three high-incidence antigens which have been localized on glycophorins C (GPC) and D (GPD) molecules. Murine monoclonal antibodies (mAbs) directed against Ge4 and Ge3 epitopes have already been obtained. We describe two murine mAbs (Z151.17 and J6) with Ge2 specificity. Materials and Methods : The mAbs were investigated by serological methods and immunoblotting, with common, Ge-negative and enzyme-modified RBCs. Results : Both mAbs behaved like anti-Ge2 in serological tests, but bound to GPC and GPD on immunoblots. Z151.17 did not recognize the GPC^Yus or GPC^Ge variants, whereas J6 revealed a 25-kD component from Ge:-2,-3,4 membranes, which is identical to the GPC^Ge molecule. Conclusion : These two mAbs are directed to slightly different epitopes. The Z151.17 and J6 epitopes are common to GPC and GPD, thus distinct from the Ge2 antigen defined by human alloantibodies.     

Functional Cooperation among Human IgG-Specific Murine Monoclonal Antibodies for the Detection of Weak Blood Group Antibodies in Routine Agglutination Tests

Seven monoclonal antibodies (MAbs) selected from a large panel of human IgG-specific murine MAbs were characterized serologically and studied for their ability to cooperate in routine antihuman globulin agglutination tests. In binding inhibition experiments, three of these MAbs were shown to bind simultaneously to immobilized human IgG molecules. Cooperation among these MAbs increased significantly the capacity of the individual MAbs to agglutinate red cells sensitized with weak blood group antibodies. These results demonstrate the usefulness of selected MAb blending for the preparation of potent antihuman IgG reagents from murine monoclonal antibodies.     

Functional Cooperation among Human IgG-Specific Murine Monoclonal Antibodies for the Detection of Weak Blood Group Antibodies in Routine Agglutination Tests

Seven monoclonal antibodies (MAbs) selected from a large panel of human IgG-specific murine MAbs were characterized serologically and studied for their ability to cooperate in routine antihuman globulin agglutination tests. In binding inhibition experiments, three of these MAbs were shown to bind simultaneously to immobilized human IgG molecules. Cooperation among these MAbs increased significantly the capacity of the individual MAbs to agglutinate red cells sensitized with weak blood group antibodies. These results demonstrate the usefulness of selected MAb blending for the preparation of potent antihuman IgG reagents from murine monoclonal antibodies.     

New Tests to Detect Antiphospholipid Antibodies: Antiprothrombin (aPT) and Anti-Phosphatidylserine/Prothrombin (aPS/PT) Antibodies

Antiprothrombin antibodies have been proposed as potential new biomarkers for thrombosis and/or pregnancy morbidity in the setting of the antiphospholipid syndrome (APS). Antiprothrombin antibodies are commonly detected by ELISA, using prothrombin coated onto irradiated plates (aPT), or prothrombin in complex with phosphatidylserine (aPS/PT), as antigen. Although these antibodies can co-exist in the same patient, aPT and aPS/PT seem to belong to different populations of autoantibodies. Early research explored the role of antibodies to prothrombin as potential antigenic targets for the lupus anticoagulant (LA). To date their clinical significance is being investigated and their potential role in identifying patients at higher risk of developing thrombotic events or pregnancy morbidity is being probed.     

Use of the DAF Assay to Assess the Functional Properties of Polyclonal and Monoclonal Rh D Antibodies

The mechanism whereby passive Rh (D) immunoglobulins suppress the feto-maternal alloimmunization is still unclear. New in vitro tests are needed to better characterize the functional properties of polyclonal anti-Ds. The DAF assay was developped to monitor the antibody-dependent cell-mediated cytotoxicity (ADCC) and the phagocytosis of anti-Rh (D)-sensitized RBCs by effector cells. The principle of this test is based on the oxydization of the 2,7-diaminofluorene (DAF) by the pseudoperoxidase activity of free hemoglobin. The reaction is proportional to the hemoglobin concentration. This test was performed to determine and emphasize the efficacy of different polyclonal anti-D immunoglobulin preparations to mediate lysis and phagocytosis of sensitized RBCs by human peripheral mononuclear cells. The functional properties of different human RhD monoclonal antibodies were also analyzed and compared. The test was found to be convenient to perform and allowed the avoidance of radioactive labelling of RBCs for ADCC studies. It is mainly useful for the direct quantitation of phagocytosis.     

抗重组日本血吸虫P38抗原单克隆抗体的制备与鉴定

目的?摇在大肠埃希菌中可溶性表达日本血吸虫P38（SjP38）抗原分子，并以其纯化产物为抗原，制备抗SjP38单克隆抗体(McAb)。 方法 将pET32(a)-P38重组质粒转化大肠埃希菌BL21(DE3)，在1 mmol/L异丙基-β-D-硫代半乳糖苷诱导下表达，超声破菌后获得可溶性表达产物，通过镍柱一步法纯化，以纯化的重组SjP38为抗原，免疫BALB/ｃ小鼠，采用杂交瘤技术制备McAb，用ELISA和有限稀释法筛选出高分泌滴度McAb杂交瘤细胞株，测定其免疫球蛋白亚类及其效价，蛋白质印迹法（Western blotting）分析其特异性。 结果 筛选出能稳定分泌抗重组SjP38单克隆抗体的8株杂交瘤细胞株，均为IgG1； Western blotting 分析显示8株单抗能与日本血吸虫虫卵抗原中的天然P38发生特异性结合。 结论 制备的抗P38杂交瘤细胞株能分泌高滴度、高特异性的McAb。     

Molecular basis of the D variant phenotypes DNU and DII allows localization of critical amino acids required for expression of Rh D epitopes epD3, 4 and 9 to the sixth external domain of the Rh D protein

The discovery of Rh partial D variant red cells by discrepant reactions with different monoclonal anti-D has demonstrated the range of Rh D epitopes that have arisen due to alterations in Rh D protein structure. There are two current classification systems, one which uses a nine epitope model (epD1–epD9) whereas a more recent model proposes 30 different epitopes. We describe here the molecular basis of two D variants which lack epD4 and epD9 namely the DNU and D^II phenotypes. These would have both been originally classified as D^II phenotype individuals, but we have revealed subtle differences in the serological profile of these erythrocytes. Such a differential reactivity and determination of the molecular bases of these phenotypes allows us to predict critical amino acids for epD3, epD4 and epD9 expression. The DNU phenotype arises from a single point mutation in the RHD gene resulting in a single amino acid change (Gly353Arg). Sequence analysis of exon 7 of the RHD gene derived from the D^II propositus indicates that there is a single point mutation in this exon resulting in a single amino acid change (Ala354Asp). It is likely that this point mutation gives rise to the D^II phenotype. Both mutations result in the change to Rh D-specific residues. Our results indicate that the following amino acids are crucial for epD3a (Asp³⁵⁰), epD3b (Asp³⁵⁰+Gly³⁵³), epD4a (Gly³⁵³ + Ala³⁵⁴), epD4b (Ala³⁵⁴), epD9a (Asp³⁵⁰ + Gly³⁵³ + Ala³⁵⁴) and epD9b (Asp³⁵⁰ + Ala³⁵⁴) expression. All of these amino acids reside on the predicted sixth external domain of the Rh D protein, so it is possible that epD3, 4 and 9 are continuous epitopes.     

Comparison of the Reactions of the Rh-Related Murine Monoclonal Antibodies BS58 and R6A

The murine monoclonal antibodies BS58 and R6A are known to recognize epitopes related to the human Rh system: neither antibody reacts with Rhnull cells and the BS58 antigen is not expressed by -D- or .D. cells. It is shown here that the numbers of BS58 and R6A antigen sites vary with Rh phenotype. Both epitopes are well represented on cells of the CDe/CDe, CDe/cDE and CDe/cde phenotypes; BS58 sites are markedly reduced on cde/cde and cDE/cde and are only just detectable on cDE/cDE cells when compared with R6A sites. The number of R6A sites per red cell ranged between 20,000 and 150,000. The evidence indicates that the BS58 epitope is not on the polypeptides carrying D or R6A, nor is it uniquely on one of the polypeptides carrying either C, c, E or e. It is suggested that the BS58 epitope is either common to all the CcED polypeptides or that it is present on a polypeptide which has not yet been identified biochemically.     

抗环子孢子蛋白保守II+区单克隆抗体的制备及鉴定

目的　获得针对恶性疟原虫环子孢子蛋白 (CSP)保守II⁺ 区 (RegionII⁺ )的单克隆抗体。　方法　采用恶性疟原虫保守II⁺ 区十二肽 (EWSPCSVTCGNG)免疫BALB/c小鼠 ,经融合 ,ELISA 3次筛选 ,获得 3株分泌针对保守II⁺ 区单克隆抗体的杂交瘤细胞株。　结果　ELISA检测结果显示单抗能与重组表达的恶性疟原虫CSP片段及天然CSP特异性反应。间接荧光抗体检测显示 ,单抗不仅能识别恶性疟原虫子孢子 ,也能识别约氏疟原虫子孢子。　结论　成功地获得了针对恶性疟原虫CSP保守II⁺ 区的单克隆抗体。     

Evolution of SARS-CoV-2: BA.4/BA.5 Variants Continues to Pose New Challenges

The acquisition of a high number of mutations, notably, the gain of two mutations L452R and F486V in RBD, and the ability to evade vaccine/natural infection-induced immunity suggests that Omicron is continuing to use “immune-escape potential” as an evolutionary space to maintain a selection advantage within the population. Despite the low hospitalizations and lower death rate, the surges by these variants may offset public health measures and disrupt health care facilities as seen recently in Portugal and the USA. Interestingly these BA.4/BA.5 variants have been found to be more severe than the earlier-emerged Omicron variants. We believe that aggressive COVID-19 surveillance using affordable testing strategies might actually help understand the evolution and transmission pattern of new variants. The sudden dip in reporting of new cases in some of the low- and middle-income countries is an alarming situation and needs to be addressed as this could lead to undetected transmission of future variants of interest/concern of SARS-CoV-2 in large population settings, including advent of a ‘super’ virus. It would be interesting to examine the possible role/influence, if any, of the two different kinds of vaccines, the spike protein-based versus the inactivated whole virus, in the evolution of BA.4/BA.5.     

The Cloning and Expression of Human Monoclonal Antibodies: Implications for Allergen Immunotherapy

Allergic responses are dependent on the highly specific effector functions of IgE antibodies. Conversely, antibodies that block the activity of IgE can mediate tolerance to allergen. Technologies that harness the unparalleled specificity of antibody responses have revolutionized the way that we diagnose and treat human disease. This area of research continues to advance at a rapid pace and has had a significant impact on our understanding of allergic disease. This review will present an overview of humoral responses and provide an up-to-date summary of technologies used in the generation of human monoclonal antibodies. The impact that monoclonal antibodies have on allergic disease will be discussed, with a particular focus on allergen immunotherapy, which remains the only form of treatment that can modulate the underlying immune mechanisms and induce long-term clinical tolerance.     

抗环子孢子蛋白保守II~ 区单克隆抗体的制备及鉴定

目的　获得针对恶性疟原虫环子孢子蛋白 (CSP)保守II 区 (RegionII )的单克隆抗体。　方法　采用恶性疟原虫保守II 区十二肽 (EWSPCSVTCGNG)免疫BALB/c小鼠 ,经融合 ,ELISA 3次筛选 ,获得 3株分泌针对保守II 区单克隆抗体的杂交瘤细胞株。　结果　ELISA检测结果显示单抗能与重组表达的恶性疟原虫CSP片段及天然CSP特异性反应。间接荧光抗体检测显示 ,单抗不仅能识别恶性疟原虫子孢子 ,也能识别约氏疟原虫子孢子。　结论　成功地获得了针对恶性疟原虫CSP保守II 区的单克隆抗体     

Demonstration of Seven Epitopes on the Rh Antigen D Using Human Monoclonal Anti-D Antibodies and Red Cells from D Categories

The agglutination patterns have been established for the reaction between 29 monoclonal antibodies with specificity for the Rh antigen D and red cells of D categories IIIa, IIIc, IVa, IVb, Va, Vc, VI and VII, which are known to lack certain epitopes on the D polypeptide. Six different agglutination patterns were recognized and interpreted to indicate the recognition of seven different epitopes. These epitopes are termed epD1 through to epD7. The separate existence of epD6 and epD7 is deduced from previous observations in inhibition studies using purified 125I-labelled antibodies; they cannot yet be distinguished in agglutination tests. The number of epitopes lacking from cells of each category varied between two and five. As all the antibodies agglutinated cells of categories IIIa, IIIc and VII and cells of categories II, IIIb and Vb were not available, it is probable that there are epitopes other than the seven presently recognized. Eighteen out of the 29 antibodies which were examined recognized epitopes epD6/7 and it is suggested either that antibodies recognizing these epitopes predominate in polyclonal anti-D sera, or that the lymphocytes producing these antibodies are preferentially selected during establishment of cell lines.     

A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology

Marek’s disease virus (MDV) is an important oncogenic α-herpesvirus that induces Marek’s disease (MD), characterized by severe immunosuppression and rapid-onset T-cell lymphomas in its natural chicken hosts. Historically, MD is regarded as an ideal biomedical model for studying virally induced cancers. Monoclonal antibodies (mAbs) against viral or host antigenic epitopes are crucial for virology research, especially in the exploration of gene functions, clinical therapy, and the development of diagnostic reagents. Utilizing the CRISPR/Cas9-based gene-editing technology, we produced a pp38-deleted MDV-1 mutant—GX0101Δpp38—and used it for the rapid screening and identification of pp38-specific mAbs from a pool of MDV-specific antibodies from 34 hybridomas. The cross-staining of parental and mutated MDV plaques with hybridoma supernatants was first performed by immunofluorescence assay (IFA). Four monoclonal hybridomas—namely, 4F9, 31G7, 34F2, and 35G9—were demonstrated to secrete specific antibodies against MDV-1’s pp38 protein, which was further confirmed by IFA staining and confocal analysis. Further experiments using Western blotting, immunoprecipitation (IP), liquid chromatography–tandem mass spectrometry (LC–MS/MS), and immunohistochemistry (IHC) analysis demonstrated that the pp38-specific mAb 31G7 has high specificity and wide application potential for further research in MD biology. To the best of our knowledge, this is the first demonstration of the use of CRISPR/Cas9-based gene-editing technology for efficient screening and identification of mAbs against a specific viral protein, and provides a meaningful reference for the future production of antibodies against other viruses—especially for large DNA viruses such as herpesviruses.     

Monoclonal Antibodies to S and N SARS-CoV-2 Proteins as Probes to Assess Structural and Antigenic Properties of Coronaviruses

Antibodies targeting the spike (S) and nucleocapsid (N) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential tools. In addition to important roles in the treatment and diagnosis of infection, the availability of high-quality specific antibodies for the S and N proteins is essential to facilitate basic research of virus replication and in the characterization of mutations responsible for variants of concern. We have developed panels of mouse and rabbit monoclonal antibodies (mAbs) to the SARS-CoV-2 spike receptor-binding domain (S-RBD) and N protein for functional and antigenic analyses. The mAbs to the S-RBD were tested for neutralization of native SARS-CoV-2, with several exhibiting neutralizing activity. The panels of mAbs to the N protein were assessed for cross-reactivity with the SARS-CoV and Middle East respiratory syndrome (MERS)-CoV N proteins and could be subdivided into sets that showed unique specificity for SARS-CoV-2 N protein, cross-reactivity between SARS-CoV-2 and SARS-CoV N proteins only, or cross-reactivity to all three coronavirus N proteins tested. Partial mapping of N-reactive mAbs were conducted using truncated fragments of the SARS-CoV-2 N protein and revealed near complete coverage of the N protein. Collectively, these sets of mouse and rabbit monoclonal antibodies can be used to examine structure/function studies for N proteins and to define the surface location of virus neutralizing epitopes on the RBD of the S protein.