A one-step monoclonal antibody typing procedure that simplifies HLA class I and class II typing

Abstract: We have developed monoclonal antibodies to most HLA specificities, making it possible for us to devise a simple, rapid, one-step micro-cytotoxicity test. The test is performed by adding 1 μl of cells to 1 μl of antibody-complement mixture predotted on the microtest tray. The reactions are read following a 1-hour incubation period (30 minutes in some instances). The analysis of reactions seen on testing 105 class I antibodies and 50 class II antibodies is shown. A comparison of typing by the standard NIH method and the new one-step procedure showed a > 96% concordance in the 500 T cells and 200 B cells we examined. Class I and class II typing could be performed using B cells, thus obviating the need to isolate both T and B cells for HLA typing.     

An HLA class I specific monoclonal antibody that fails to bind to all HLA-A antigens

A monoclonal antibody, MHM.5, specific for HLA class I antigens, bound to lymphocytes of all donors tested and was thought to bind to a monomorphic determinant. When the antibody was used to precipitate 35S methionine labeled HLA class I molecules from lymphoid cells, which were then isoelectric focused, it was found that the HLA-A1,A2 and A3 antigens were not precipitated. Similarly, MHM.5, which is IgG1, failed to block complement mediated lysis by alloantisera specific for HLA-A1, 2 and 3, and most other HLA-A antigens. HLA-Aw24, A25, and A32, and all other HLA-B and C typing reactions tested were blocked. Thus the antibody binds to an epitope that is lacking on most A antigens, but present on Aw24, A25, A32 and all B and C locus antigens. Comparison of the published amino acid sequences of HLA-A2, A3, Aw24, A28, Cw3, B7, and B40 suggests some possible sites for this epitope.     

Luminex technology for HLA typing by PCR-SSO and identification of HLA antibody specificities

Luminex technology is a new flow cytometry technology enabling us to analyse numerous reactions in a unique tube or well. It is a multiplexed data acquisition and analysis platform of microsphere-based assays that performs simultaneous measurements of up to 100 different analytes. In the histo- compatibility field, individual sets of microspheres are modified with reactive components such as antigens in order to perform HLA antibodies identification, or with oligonucleotides in order to perform HLA typing after reverse PCR-SSO. Thus microspheres are the equivalent of a panel of HLA typed lymphocytes (for PRA determination and antibody identification) or equivalent to a large set of probes selected to assign HLA typing. This new tool can be very useful in HLA laboratories since it is very easy to use and the results are concordant with those obtained with reference technics.     

HLA monoclonal antibody registry: a proposal

A review of the literature and discussions at the preliminary meeting of the 9th International Histocompatibility Workshop (Munich, March 1982) have highlighted the growing importance of HLA monoclonal antibodies (McAb) for immunogenetic, biochemical and functional studies. Thus it would seem necessary to establish a registry to provide permanent information on the subject.     

A monoclonal antibody (GF 22.1) detecting different patterns of HLA class I cross-reactivities at different dilutions

A murine cytotoxic monoclonal antibody (GF 22.1) was produced by Balb/c immunization with GRA human lymphoblastoid cells. HLA-A, B and C blanketing and SDS-PAGE analysis of the immunoprecipitate demonstrated MHC class I structures as the targets. Cytotoxic assays were performed with peripheral blood lymphocytes from 84 unrelated donors and from members of 15 families at different antibody dilutions. Statistical analysis was performed by Fisher test on each dilution separately and by Mann-Whitney U test on the dilutions taken all together. The data suggest the detection of a cross-reacting group (B15, A32, B17, B40/w41 and B21) with high affinity and the inclusion of other antigens (B12, B35, A2, B13, A11 and A24) with a lower affinity.     

Description of a monoclonal antibody defining an HLA allotypic determinant that includes specificities within the B5 cross-reacting group

The selection and characterization of a cloned murine monoclonal antibody (4D12) that defines an HLA allotypic determinant is described. Antibody 4D12 immunoprecipitates HLA heavy and light chains. From studies on 4D12 reactivity with a large panel of well-characterized target cells, antibody 4D12 defines a public HLA determinant that includes specificities within but not identical to the B5 cross-reactive group. Antibody 4D12 should be useful in the study of HLA-B5-related supratypic antigens.     

Evaluation of a newly developed HIV antigen test

A new monoclonal antibody-based enzyme immunoassay (Innogenetics) for the detection and quantification of p24 core antigens of HIV-1 (group M and group O) and of HIV-2 was evaluated on 2745 serum samples and 18 culture supernatants and compared with a reference (Coulter) HIV-1 p24 antigen assay. Positive results were confirmed by neutralization with the reagents of the respective tests. As demonstrated by dilution series of HIV cocultures, the new test recognizes p24 antigen of the most common HIV genetic subtypes, including group O and HIV-2. Titres ranged from 729 to 531441. Therefore p24 antigen assay is but very weakly reactive with HIV-2 (titres from 9 to 81). The new test is considerably more sensitive than the reference. In a population of 365 follow-up samples from 86 different patients, representing all stages of infection, the new test detected p24 antigen at least once in 52% (45/86) of these patients, whereas the reference was positive in 31% (27/86). The newly designed test detected antigen in 40% (145/365) of the samples, while the reference was positive in 21% (75/365). In a group of PCR and/or culture positive neonates, 33% (9/27) of the samples were positive with the new test versus 18% (5/27) with the reference. The specificity of the new test, as determined on 2,000 blood donor samples, was 99.65% (initially), 99.80% (after repetition), and 100% (with neutralization). The reference scored 99.95%, 100%, and 100%, respectively. In 300 seronegative samples from persons at risk, the initial specificity of the new test was 98.67% (the reference, 99.00%). With neutralization, both assays were 100% specific. J. Med. Virol. 53:31–35, 1997. © 1997 Wiley-Liss, Inc.     

Evolution of HLA class I epitopes defined by murine monoclonal antibodies: Distribution in macaques

Murine anti-HLA monoclonal antibodies (MoAbs) to monomorphic and polymorphic epitopes were compared for their reactivity in humans vs. pigtailed macaques (Macaca nemestrina). Five MoAbs to monomorphic class I epitopes in humans displayed distinct patterns in macaques: two were unreactive, one reacted with 93% of animals tested, another with 17%, and one with only 8% of animals tested. Thus, epitopes that are monomorphic in one species can be highly polymorphic in another. Most of the 23 MoAbs (91%) against polymorphic epitopes in humans also detected polymorphisms in macaques. The epitopes detected by MoAbs could be divided roughly into two groups: (1) epitopes that were expressed at the same frequency in both species, i.e., monomorphic, public, or private epitopes in both species, or (2) epitopes that had quite different expression in the two species, e.g., a “public” epitope in one species expressed as a “private” epitope in the other. The genes encoding some of these polymorphisms were shown to segregate in families and thus some anti-HLA MoAbs are useful typing reagents for macaques. Two MoAbs thought to detect the same specificity in humans were found to react in macaques with different animals. Thus, reactivity patterns of anti-HLA class I MoAbs in primate populations enabled MoAbs to closely associated epitopes to be distinguished.     

抗MHC I类单抗对NK细胞杀伤能力的影响

探讨ＮＫ细胞杀务与靶细胞表面ＭＨＣＩ类分子的关系。方法用ＨＬＡ－ＡＢＣ单抗培养上清或腹水封闭靶细胞Ｋ５６２表面的ＨＬＡ－ＡＢＣ分子后,分别观察了外周血新鲜ＮＫ细胞、纯化ＮＫ细胞、ＬＡＫ细胞和ＣＤ３ＡＫ细胞对Ｋ５６２细胞杀伤能力的变化。     

Structural and genetic analyses of HLA class I molecules using monoclonal xenoantibodies

The monoclonal antibodies B1.23.2 and B9.12 were developed to characterize human class I major histocompatibility gene products. They detect two different epitopes and define on a given lymphoblastoid B cell line two subsets of beta 2 microglobulin-complexed HLA class I molecules. One subset reacts with B9.12 and B1.23.2 and the other one expresses only the B9.12 epitope. Binding assays were performed on C3H mouse L cells which had been transformed with various single HLA-class I genes and the two detected molecular subsets were shown to be encoded by different genetic loci. Unlike B1.23.2, B9.12 detects all the beta 2m-complexed molecules expressed in human B cell lines and recognizes an epitope different from the one defined by W6/32. In contrast to the vast majority of the other reported anti-class I monoclonal antibodies (including B9.12), B1.23.2 recognizes an epitope expressed on both beta 2m-associated and -free HLA class I heavy chains.     

Evaluation of allergen-specific IgE antibodies by a newly developed mast allergy system

MAST, which stand for multiple antigen simultaneous test, uses enzyme-linked anti-human IgE and chemiluminogenic substate to determine IgE. This system is characterized by simultaneous analysis of multiple allergen items, up to 35, together with total IgE determination. We evaluated usefulness of this MAST system using 191 serum samples obtained from patients with bronchial asthma, allergic rhinitis and/or atopic dermatitis. It was found that there were statistically significant correlations between IgE antibody quantification by MAST and RAST in 24 out of 35 allergen items, with correlation coefficients more than r = 0.60. These included Dermatophagoides farinae and pteronyssinus, Japanese cedar pollen, orchard grass, Alternaria, Candida as inhalant allergens; egg white, milk, soybeans, wheat and rice as food allergens. It was also evaluated how many allergen-specific IgE antibodies could be detected in one serum sample. More than six allergen-specific IgE antibodies were simultaneously detected in 33% of 191 cases, indicating the importance of multiple-allergen analysis. These results indicate the clinical usefulness of the MAST allergy system in detecting IgE antibodies in allergic subjects.     

Characterization of three human monoclonal antibodies specific for polymorphic class I and class II HLA antigens

Three human monoclonal antibodies were derived from a single polytransfused patient awaiting renal transplantation. In microcitotoxicity assays, the patient's serum displayed strong positive reactions against> 90% of a panel of cells representing the known HLA specificities. The donor's peripheral blood lymphocytes were infected with Epstein-Barr virus, cloned, and supernatants of the virus transformed cultures were screened for the presence of IgG antilymphocyte reactivity utilizing an enzyme-linked immunosorbent assay method. Positive cultures were recloned and fused with the human-mouse heteromyeloma SHM. Supernatants from three clones were selected for alloreactivity and characterized by indirect immunofluorescent staining and fluoractivated cell sorter analysis on homozygous typing cells, including those from the Tenth International Histocompatibility Workshop core panel and on cell lines derived from selected families. Data obtained demonstrate that two human monoclonal antibodies have DQw1 specificity, one of them being reactive against several DQw7-positive cell lines, while one monoclonal antibody is specific for the A2 + A28 class I MHC antigens. Anti-DQw1 antibodies were of different light-chain subtypes.     

Expression of BAFF-R (BR3) in normal and neoplastic lymphoid tissues characterized with a newly developed monoclonal antibody

BAFF-receptor (BAFF-R) is required for the successful maturation and survival of B-cells. We developed an anti-human BAFF-R monoclonal antibody (mAb), 8A7. The reactivity of 8A7 in normal and neoplastic tissue was examined by performing immunohistochemistry on paraffin-embedded sections. 8A7 reacted with lymphocytes in the mantle and marginal zones, but not with lymphocytes in the interfollicular area. Lymphocytes in the germinal centers were found to be negative or occasionally weakly positive for 8A7. BAFF-R expression was found only in B-cell lymphoma (44/80, positive cases/examined cases): B-lymphoblastic lymphoma 0/3, B-chronic lymphocytic leukemia/small lymphocytic lymphoma 4/4, mantle cell lymphoma 9/11, follicular lymphoma 10/14, diffuse large B-cell lymphoma (DLBCL) 11/25, marginal zone B-cell lymphoma 8/10, lymphoplasmacytic lymphoma 2/2, plasma cell myeloma 0/2, and Burkitt lymphoma 0/9, but not in T/NK cell lymphomas (0/19) or Hodgkin lymphoma (0/10). BAFF-R was expressed in most low-grade B-cell neoplasms and a small number of DLBCL, suggesting that BAFF-R may play an important role in the proliferation of neoplastic lymphoid cells. Thus, the mAb is very useful for further understanding of both healthy B-cell biology and its pathogenic neoplasms.     

Inhibition of cytotoxicity for screening a monoclonal antibody to HLA antigen

Cytotoxicity inhibition assay was established for the screening of a monoclonal antibody to HLA antigen. The assay involved the inhibition of typing cells with hybridoma culture supernatant and with F(ab')2 fragment of sheep anti-mouse IgG. Using the assay and the conventional microcytotoxicity test, an anti-HLA-A2 monoclonal antibody was screened.     

Identification of most frequent HLA class I antigen specificities in Botswana: relevance for HIV vaccine design

Since the mid-1990s, southern African countries have been experiencing an expansion of human immunodeficiency virus type 1 (HIV-1) infection caused by HIV-1 subtype C. To facilitate the design of an HLA-based HIV vaccine, we studied the distribution of the HLA class I antigen specificities in Botswana, a southern African country with a high prevalence of HIV infection. Botswana's highly efficient health care system and its central geographical location within southern Africa suggests that it might be an appropriate candidate site for future trials of an HLA-based HIV vaccine. Specificities of HLA class I genes have been investigated in DNA samples obtained from 161 persons of Botswana origin by polymerase chain reaction (PCR) with sequence-specific primers. We identified 4 HLA-A, 7 HLA-B, and 5 HLA-C specificities that were observed at high frequencies in the Botswana population: A30, A02, A23, A68, B58, B72, B42, B8, B18, B44, B45, Cw7, Cw2, Cw17, Cw6, and Cw4. HLA-A30, A02, A23, A68, B58, Cw2, Cw4, Cw6, Cw7, and Cw17 were observed at frequencies of more than 10%. The frequency of HLA-A30 was 27.3%. HLA-B58 (17.9%) was the most frequent generic HLA-B type. Other frequent antigen specificities detected for the HLA-B were B72 (9.6%), B42 (9.3%), B8 (7.4%), B18 (7.4%), B44 (7.4%), and B45 (6.4%). Analysis of haplotype frequencies revealed that haplotypes HLA-A30/HLA-B58 (6.7%), A30/B42 (6.1%), A30/B8 (4.1%), A30/B45 (3.2%), and A23/B58 (2.5%) were the most frequent among two-locus haplotypes. The comparison of HIV-positive patients and noninfected controls for HLA class I specificities confirmed the previously described association of A2/A6802 supertype with resistance to HIV. Our study suggested an increased resistance to HIV infection associated with A68 rather than A2. We also found that the generic HLA-B58 type was associated with increased susceptibility to HIV infection. Our findings suggest that the design of an HLA-based HIV vaccine that includes multiple CTL epitopes restricted by identified common HLA class I specificities might target up to 97.5% of the population in Botswana. The results of this study extend the HLA map to a southern African country that has high rates of HIV and also provide a database for the design of an HLA-based HIV vaccine.     

Evaluation of the iBeads assay as a tool for identifying class I HLA antibodies

In addition to antibodies targeting native class I human leukocyte antigens (HLA), the single antigen flow beads assay (SAFB) detects antibodies recognizing denatured forms (anti-dHLA). Acid treated SAFB and the modified SAFB reagent named iBeads are expected to distinguish anti-native (anti-nHLA) from anti-dHLA. Sera from 280 class I HLA-sensitized SAFB-positive kidney transplant candidates were retested with acid-treated SAFB and iBeads. Concordance between SAFB and iBeads, taking into account acid-treatment results, was described at global and locus levels. T-lymphocyte flow cytometry crossmatches (FCXM) were performed to identify an accurate iBeads MFI threshold allowing predicting FCXM results. Concordance between acid-treatment and iBeads assays was observed for 86.9% of alleles. The iBeads MFI were lower than for classical SAFB, especially for HLA-B and C alleles. Anti-dHLA identified with acid-treated SAFB were more frequently negative with iBeads for HLA-B and -C alleles. An iBeads MFI threshold of 1000 allowed predicting positive FCXM with 95.6% sensitivity, 91.6% negative predictive value and 0.08 negative likelihood ratio. The iBeads assay still has limitations, but might represent an invaluable alternative to SAFB for virtual crossmatch strategies in organ transplant allocation programs.