Use of a monoclonal antibody specific for a protein epitope of Echinococcus granulosus antigen 5 in a competitive antibody radioimmunoassay for diagnosis of hydatid disease.

A monoclonal antibody (mAb) designated as EG 02 154/12, specific for the major antigen (antigen 5) of Echinococcus granulosus was produced, and used to study the binding sites recognized by anti-antigen 5 antibodies from patients with hydatid disease. The nature of the target epitope was partially characterized. The antibody reactivity was analyzed towards sheep hydatid fluid antigens (SHF Ag) using ELISA, immunoelectrophoresis (IEP), Western blotting (WB), and immunoprecipitation (IP). In IEP, EG 02 154/12 mAb gave a single precipitin of Ag 5. The mAb and human hydatid patient sera recognized a major antigen of 64 kDa, in SHF Ag analyzed in non-reducing conditions. Both types of antibodies revealed two components of 37 and 22 kDa in reducing conditions. Deglycosylation and delipidation of SHF Ag did not affect the mAb binding. These results, together with the observation of mAb binding to in vitro translation products from protoscoleces messenger RNA, suggest the protein nature of the epitope recognized on the antigen 5. Using competitive antibody radioimmunoassay (CRIA), a competition between this mAb and hydatid patient sera, for the same epitope or closely related sites on antigen 5, was observed. No such competition was detected with the sera from other helminthiasis. The sensitivity and specificity of CRIA was compared to that of ELISA and CRIA found to be an improved diagnostic test for hydatid disease.     

Amino acid sequence requirements for the epitope recognized by a monoclonal antibody reacting with the secreted antigen GP28.5 of Toxoplasma gondii.

We have characterized in detail, the epitope of the secreted antigen GP28.5 recognized by the mouse monoclonal antibody TG 17-179 using synthetic peptides and a truncated recombinant fusion protein. The screening of a T. gondii expression library with TG17-179 mAb led to the isolation of cDNAs clones, all encoding for the C-terminal region of GP28.5 and with one encoding for only the five last C-terminal residues. Competitive ELISA with longer peptides revealed that the immunoreactivity was retained for peptides of eight residues or longer, and lost when the peptide was reduced to the six last C-terminal residues or less. Experiments with the octapeptide lacking the carboxy-terminal glutamine residue showed it to be 64-fold less active. Moreover, neither addition of residues in the carboxy-end nor substitution of COOH function changed the immunoreactivity of the epitope. Competition experiments between TG17-179 mAb and sera from infected individuals demonstrates that the epitope defined by a mouse monoclonal probe is also a major epitope for human polyclonal antibodies. These results describe the sequence requirements within a probably linear epitope and give rise to some general question concerning experimental test for epitope mapping.     

Genetic and biochemical characterization of antigens encoded by the Ly-24 (Pgp-1) locus

Three allele-specific monoclonal antibodies to Pgp-1 (Ly-24) were used to biochemically characterize the cell surface structures with which they reacted and to map the gene(s) coding for these antigens. The targets of these three monoclonal antibodies (mAb) were shown to be encoded by a gene situated on chromosome 2 close to beta 2m [gene order (Pgp-1-beta 2m-a)] and no recombination between the loci detected by the three antibodies was revealed by genetic analysis. The genetic mapping of loci and tissue distribution of these antigens suggested that they might all correspond to a particular allelic form of the mouse phagocyte glycoprotein-1 (Pgp-1) antigen. Biochemical and serological analysis confirmed that this was indeed the case and revealed that all three mAbs were directed to one epitope. It is surprising that the tissue distribution defined by one mAb (Ly-24A) was different from that for the two other (Ly-24B) antibodies, despite the serological and biochemical identity of their respective targets. The possible reason for this unusual finding is discussed.     

Competitive ELISA for the measurement of tau protein in Alzheimer's disease.

Tau protein is a major component of paired helical filaments (PHFs) which constitute the characteristic neurofibrillary tangle lesions observed in Alzheimer's disease. Two tau mAbs have been produced which show distinct patterns of immunoreactivity with intact human tau and with tau incorporated in PHFs. The mAb 423 recognises PHFs but not human tau on immunoblots whereas mAb 7/51 reacts with human tau but its epitope is buried within the PHF and is only exposed after formic acid treatment. A competitive ELISA has been developed for both of these mAbs and these have been used to quantify the two distinct tau epitopes in PHFs. Samples containing antigen are incubated with horseradish peroxidase-conjugated mAb at 4 degrees C for 16 h and non-adsorbed antibody then measured by binding, at 37 degrees C for 1 h, to a fragment of tau coated on microtitre plates. Bound enzyme-labelled antibody is measured kinetically using a spectrophotometer capable of automatically mixing the samples throughout a 2-min incubation with substrate and chromogen. The interfacing of the plate reader with a computer permits competitive curves to be plotted automatically using Softmax. Curves are fitted using a 4-parameter logistic algorithm which allows one to determine the relative immunoreactivity for different samples. The application of these assays to monitoring biochemical fractions and quantifying distinct immunochemical presentations of tau protein with these two mAbs is described.     

A Schwann cell matrix component of neuromuscular junctions and peripheral nerves

Molecules localized to the synapse are potential contributors to processes unique to this specialized region, such as synapse formation and maintenance and synaptic transmission. We used an immunohistochemical strategy to uncover such molecules by generating antibodies that selectively stain synaptic regions and then using the antibodies to analyse their antigens. In this study, we utilized a monoclonal antibody, mAb 6D7, to identify and characterize an antigen concentrated at frog neuromuscular junctions and in peripheral nerves. In adult muscle, immunoelectron microscopy indicates that the antigen is located in the extracellular matrix around perisynaptic Schwann cells at the neuromuscular junction and in association with myelinated and nonmyelinated axons in peripheral nerves. The maintenance of the mAb 6D7 epitope is innervation-dependent but is muscle-independent; it disappears from the synaptic region within 2 weeks after denervation, but persists after muscle damage when the nerve is left intact. mAb 6D7 immunolabelling is also detected at the neuromuscular junction in developing tadpoles. Biochemical analyses of nerve extracts indicate that mAb 6D7 recognizes a glycoprotein of 127 kDa with both N- and O-linked carbohydrate moieties. Taken together, the results suggest that the antigen recognized by mAb 6D7 may be a novel component of the synaptic extracellular matrix overlying the terminal Schwann cell. The innervation-sensitivity of the epitope at the neuromuscular junction suggests a function in the interactions between nerves and Schwann cells.     

The monoclonal antibody DCGM4 recognizes Langerin, a protein specific of Langerhans cells, and is rapidly internalized from the cell surface.

We generated monoclonal antibody (mAb) DCGM4 by immunization with human dendritic cells (DC) from CD34+ progenitors cultured with granulocyte-macrophage colony-stimulating factor and TNF-alpha. mAb DCGM4 was selected for its reactivity with a cell surface epitope present only on a subset of DC. Reactivity was strongly enhanced by the Langerhans cell (LC) differentiation factor TGF-beta and down-regulated by CD40 ligation. mAb DCGM4 selectively stained LC, hence we propose that the antigen be termed Langerin. mAb DCGM4 also stained intracytoplasmically, but neither colocalized with MHC class II nor with lysosomal LAMP-1 markers. Notably, mAb DCGM4 was rapidly internalized at 37 degrees C, but did not gain access to MHC class II compartments. Finally, Langerin was immunoprecipitated as a 40-kDa protein with a pI of 5.2 - 5.5. mAb DCGM4 will be useful to further characterize Langerin, an LC-restricted molecule involved in routing of cell surface material in immature DC.     

Functional evidence that the HECA-452 antigen is involved in the adhesion of human neutrophils and lymphocytes to tumour necrosis factor-alpha-stimulated endothelial cells.

The migration of leucocytes into tissues is a process mediated by leucocyte endothelial interactions, in which adhesion receptors play a crucial role. Recently, it was found that 80-90% of T cells in inflammatory skin diseases were reactive to the monoclonal antibody (mAb) HECA-452+ in contrast to inflamed non-cutaneous tissues. It was suggested that the HECA-452 antigen is a homing receptor for lymphocyte migration into skin. This receptor was designated cutaneous lymphocyte-associated antigen or CLA and subsequently identified as a group of related sugar moieties. E-selectin, formerly known as ELAM-1 expressed by the endothelium has been implicated to be a counter-receptor for CLA. In this study, we investigated the adhesion of HECA-452+ leucocytes, i.e. freshly isolated neutrophils and B-cell line BV173 to tumour necrosis factor-alpha (TNF-alpha)-stimulated (E-selectin+) endothelial cells. We found that the adhesion of these cells could be inhibited significantly by mAb HECA-452, in a similar fashion to CSLEX1, a mAb specific for E-selectin ligand sialyl Lewisx. This inhibiting effect of both mAb on the adhesion of polymorphonuclear leucocytes (PMN) and BV173 could only be demonstrated when the assay was performed at 4 degrees, but not at 37 degrees. Furthermore, using immunohistochemical analysis we found that the mAb HECA-452-reactive epitope is different from that recognized by CSLEX1. The present results give direct evidence that the antigen recognized by HECA-452 is involved in the adhesion of leucocytes to endothelial cells, although this antigenic epitope is different from that reactive to CSLEX1.     

具有抗—HBs结合活性的单克隆抗—抗独特型抗体的制备和鉴定

B_3为我室建立的带有HBsAg表位内影像的单克隆抗独特型抗体(mAb_2)。本文采用脾脏内和尾静脉内注射途径,用B_3免疫BALB/c鼠,获得了具有抗-HBs结合活性的同系单克隆抗-抗独特型抗体(mAb_3)3B_8株。这一结论基于以下两点观察:(1)3B_8能与纯化的HBsAg结合。纯化的HBsAg或抗-HBs均能以剂量依赖形式抑制这一结合;(2)3B_8能以剂量依赖形式抑制抗-HBs与B_3的结合。小鼠在从未接触HBsAg的情况下,单独用B_3免疫,获得了具有抗-HBs结合活性的单克隆抗-抗独特型抗体3B_8,进一步证明我室过去建立的mAb_2B_3株,确实带有HBsAg表位内影像。     

GENETIC AND BIOCHEMICAL CHARACTERIZATION OF ANTIGENS ENCODED BY THE LY-24 (Pgp-1) LOCUS1

Three allele-specific monoclonal antibodies to Pgp-1 (Ly-24) were used to biochemically characterize the cell surface structures with which they reacted and to map the gene(s) coding for these antigens. The targets of these three monoclonal antibodies (mAb) were shown to be encoded by a gene situated on chromosome 2 close to β₂m [gene order (Pgp-1-β₂m-a)] and no recombination between the loci detected by the three antibodies was revealed by genetic analysis. The genetic mapping of loci and tissue distribution of these antigens suggested that they might all correspond to a particular allelic form of the mouse phagocyte glycoprotein-1 (Pgp-1) antigen. Biochemical and serological analysis confirmed that this was indeed the case and revealed that all three mAbs were directed to one epitope. It is surprising that the tissue distribution defined by one mAb (Ly-24A) was different from that for the two other (Ly-24B) antibodies, despite the serological and biochemical identity of their respective targets. The possible reason for this unusual finding is discussed.     

Monoclonal antibodies specific for human monocytes, granulocytes and endothelium

Four monoclonal antibodies against antigens of human myeloid cells have been produced and thoroughly characterized in terms of their reactions with peripheral blood cells, cell lines, nine lymphoid and non-lymphoid tissues and the polypeptides with which they react. UCHM1 and SmO identify antigens present on the majority of blood monocytes and a variable, but lower, proportion of tissue macrophages. From their morphology and location in tissues, these cells appear to be recirculating monocytes. SMO antigen is also present on platelets. In addition, both antibodies stained endothelial cells, SMO in all tissues examined and UCHM1 variably. Biochemical investigation indicated that the UCHM1 antigen is a protein of 52,000 MW while the SMO antigen could not be indentified. The antibodies TG1 and 28 identify antigens mainly present on granulocytes. While mAb 28 reacted with neutrophils, TG1 also stained eosinophils and stained strongly a proportion of monocytes. TG1 also reacted variably with some non-haemopoietic cell lines. Both antibodies reacted predominantly with granulocytes in tissue sections. MAb TG1 precipitated a single polypeptide of 156,000 MW from monocytes and granulocytes, while mAb 28 precipitated non-convalently associated polypeptides of 83,000 and 155,000 MW from granulocytes but only a single molecule from monocytes, corresponding to the lower MW chain of 83,000. The epitope with which mAb 28 reacts appears not to be exposed on the surface of intact monocytes. This suggests that a similar or identical 83,000 MW molecule is made by both neutrophils and monocytes, but that its expression differs according to cell type.     

Variant specific epitopes of Giardia lamblia.

Giardia lamblia undergoes surface antigenic variation. The capability of different isolates to express certain epitopes on the surfaces of trophozoites from different isolates and clones was determined using 4 surface-reacting monoclonal antibodies (mAbs) to variants derived from WB or WB-like Giardia (mAbs 6E7, 5C1, and 3F6) and GS/M (mAb G10/4). Of 28 isolates, 11 possessed trophozoites reactive with mAbs 6E7, 5C1 and 3F6, 6 with mAb 3F6, 2 with Mab G10/4, 1 with mAb 6E7, and 8 showed no reactivity as determined by direct or indirect immunofluorescence. Newly established clones from different isolates generated small numbers of reactive trophozoites similar to their parents. Only one epitope was found on any single trophozoite. Southern blots hybridized to a probe encoding for the epitope recognized by mAb 6E7 revealed that the inability to express the antigen in most isolates was due to lack of the gene. Analysis of the surface antigens of mAb 6E7 reactive clones from 3 isolates revealed that mAb 6E7 reacted with surface antigens of different molecular masses.     

Major histocompatibility complex class II invariant chain expression in non-antigen-presenting cells.

In contrast to the generally accepted belief, the major histocompatibility complex (MHC) class II invariant chain (Ii) is commonly expressed intracellularly in cells that do not present exogenous antigens. Such cells include resting peripheral blood T cells and natural killer (NK) cells. In T cells, the Ii is associated with a 77 000 molecular-weight molecule (p77) that has yet to be identified. This molecule is co-precipitated with the anti-Ii monoclonal antibody (mAb) VCD-1, but not with mAb BU-45. This suggests that in the p77-Ii complex, the extracellular epitope of Ii recognized by BU-45 is hidden, whereas the Ii epitope for VCD-1 remains exposed. In antigen-presenting cells (APCs), p77 association with the Ii was minimal, if detectable. The p77-Ii association in non-professional APCs suggests that the Ii may have another, more general, function other than the one accepted in antigen presentation.     

筛选出一株可识别多种变异HBsAg的单克隆抗体及其应用

目的 制备筛选可识别变异表面抗原(hepatitis B surface antigen,HBsAg)的单克隆抗体(monoclonal antibody, mAb).用筛选出的单克隆抗体建立检测变异HBsAg的ELISA实验方法.方法 用血源HBsAg免疫Balb/c小鼠,通过杂交瘤细胞融合技术制备抗-HBs单克隆抗体.不同单克隆抗体包被酶标反应孔,检测真核细胞表达的野生及变异HBsAg,了解各种单克隆抗体的反应模式 .筛选出可以较好识别变异HBsAg的单克隆抗体Hb1,优化该抗体ELISA检测HBsAg的方法,与 8种HBs Ag检测试剂比较检测变异HBsAg的能力.结果 经过筛选,得到一种可以较好识别包括G145R在内大多数变异HBsAg的单克隆抗体.检测变异HBsAg的能力优于市售HBsAg 诊断试剂.结论 用本实验制备的单克隆抗体可以用于ELISA检测变异HBsAg,减少HBsAg变异株的漏检率.     

Differing processing requirements of four recombinant antigens containing a single defined T-cell epitope for presentation by major histocompatibility complex class II.

A set of predictive rules governing the likelihood of generating a particular peptide-major histocompatibility complex (MHC) class II complex from an intact antigen has not been fully elucidated. We investigated the influence of positional and structural constraints in the region of the epitope by designing a set of recombinant antigens that each contained the well-characterized T-cell epitope moth cytochrome c (MCC) (88-103), which is specifically recognized by the monoclonal antibody (mAb) D4 when complexed with H-2Ek. Our model antigens contained MCC(88-103) either peripherally, at or towards the C-terminus, or internally. Their abilities to bind directly to soluble H-2Ek, and the extent of D4 epitope formation from them by antigen processing-competent and -incompetent cell lines, were determined. Here we report that three of these four antigens yielded MCC(88-103)/H-2Ek complexes independently of the conventional MHC class II antigen-processing and presentation pathway, and in each case the epitope was carried peripherally; two bound directly as intact proteins, probably as a result of spatial separation of the epitope from the major globular domain, and one was processed to peptide by a cell-surface protease. One protein, which carried the epitope inserted into an internal loop, acted as a conventional processing-dependent MCC(88-103) delivery vehicle. Thus, this epitope has different presentation requirements depending on its context. These antigens constitute a panel whose framework could be modified to further define predictive rules for antigen processing for presentation through the different MHC class II complex-generating pathways.     

抗人红细胞膜抗原非凝集型单克隆抗体的研制及特性鉴定

目的:制备抗人红细胞膜抗原的非凝集型单克隆抗体(mAb)并鉴定其特性。方法:应用杂交瘤技术,以人O型红细胞膜抗原免疫BALB/c小鼠。取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞融合,用聚凝胺试管法筛选识别红细胞表面共同抗原的抗体,玻片凝集实验剔除凝集型抗体(完全抗体),再将分泌非凝集型mAb(不完全抗体)的杂交瘤细胞株用有限稀释法克隆化3次。对杂交瘤细胞的稳定性和mAb的特性进行鉴定。结果:获得1株可稳定分泌mAb的杂交瘤细胞2E8。mAb2E8为IgG1类,可特异性地识别红细胞膜上的H抗原,没有种属交叉血凝反应。杂交瘤细胞的培养上清与人的A、B、AB和O型红细胞均能产生强凝集,凝集效价为1∶1024,腹水mAb的凝集效价达到1∶64×106。mAb的亲和力用凝集试验检测,出现血凝的时间为7s,3min以内凝块>1mm。结论:成功地制备了针对红细胞膜H抗原的非凝集性mAb,此mAb的凝集效价、相对亲和力及特异性均较良好,为构建双特异性抗体奠定了基础。     

免疫亲和层析纯化大肠癌相关抗原

目的 :从培养的大肠癌细胞Hce 86 93中分离纯化并鉴定大肠癌相关抗原。方法 :利用流式细胞术选择高表达大肠癌相关抗原的大肠癌细胞系 ,分别用单去污及三去污裂解液裂解细胞。然后用 5株抗大肠癌单克隆抗体 (mAb)CYL 1～CYL 5进行Westernblot,分析与大肠癌细胞结合反应最强的mAb。以此mAb作为配基进行亲和层析纯化大肠癌相关抗原 ,纯化结果利用Westernblot法进行鉴定。结果 :大肠癌细胞系Hce 86 93上相关抗原的表达量最高 ;抗大肠癌mAbCYL 2与Hce 86 93的结合力最强。纯化所获与CYL 2特异结合的大肠癌相关抗原 ,Mr 约为 13× 10 3 。该抗原由Mr 为 6 0× 10 3 和70× 10 3 两种亚基组成。结论 :利用mAbCYL 2进行亲和层析 ,从大肠癌细胞系Hce 86 93中获得纯化的肿瘤相关抗原。     

Characterization of Trichinella spiralis antigens sharing an immunodominant, carbohydrate-associated determinant distinct from phosphorylcholine.

The biochemical and immunochemical characteristics of T. spiralis molecules (group II antigens) sharing an immunodominant epitope were examined. Six major proteins, ranging from 43-68 kDa, and from pI 5.0-6.3, express the determinant. Together, they account for at least 3% by weight of the total protein in L1 larval homogenate. The antigens are glycosylated. Following periodate oxidation, they reacted with biotin aminocaproyl hydrazide, and treatment with trifluoromethanesulfonic acid decreased their Mr. Deglycosylated group II antigens lost immunoreactivity with a monoclonal antibody specific for the determinant, and oligosaccharides released by treatment with mild base blocked binding of the monoclonal antibody to native antigens. The determinant on one of the group II antigens (43 kDa) was removed by N-glycanase. Neither phosphorylcholine nor antibody to phosphorylcholine interfered with monoclonal antibody binding to native group II antigens. Together, these results suggest that the immunodominant group II antigen epitope is associated with N- and O-linked oligosaccharides, and that it is not phosphorylcholine.     

A carbohydrate neoepitope that is up-regulated on human mononuclear leucocytes by neuraminidase treatment or by cellular activation

The expression of cell-surface antigens can delineate specific leucocyte developmental or functional stages. For example, certain membrane glycoproteins are expressed selectively on leucocyte subsets only after activation. Leucocyte activation can also induce changes in carbohydrate epitopes expressed on surface antigens. In the present studies, we report on a novel monoclonal immunoglobulin M antibody (mAb 13.22) that recognizes a unique carbohydrate epitope expressed on human leucocyte membrane proteins. Characterization of mAb 13.22 specificity by immunoblotting showed that it recognized proteins of MW approximately 95 000 and 150 000, including both CD18 and CD11b. The mAb 13.22 epitope was removed by N-glycosidase F but not by endoglycosidase H or fucosidase, demonstrating that it is an N-linked carbohydrate antigen. Interestingly, immunoblot staining was enhanced after neuraminidase treatment, suggesting that the antibody epitope might also be partially masked by sialic acid. In resting leucocytes, the mAb 13.22 antigen was expressed strongly on neutrophils, while dull staining was present on monocytes, and no lymphocyte staining was observed. In marked contrast, treatment of leucocytes with neuraminidase resulted in exposure of a mAb 13.22 neoepitope on a subset of lymphocytes (primarily T lymphocytes and natural killer cells) as well as up-regulated staining more than 18-fold on monocytes. Activation of lymphocytes in culture with phytohaemagglutinin or concanavalin A also unmasked the mAb 13.22 neoepitope on approximately 37% of the CD45RO+ lymphocytes. Furthermore, analysis of leucocytes collected from the synovial fluid of patients with rheumatoid arthritis showed that approximately 18% of the lymphocytes present expressed the mAb 13.22 neoepitope. Taken together, our results suggest that the mAb 13.22 carbohydrate neoepitope could represent a physiologically relevant marker that is up-regulated on leucocyte subsets during the inflammatory response.     

The monoclonal antibody, UCHL1, recognizes a 180,000 MW component of the human leucocyte-common antigen, CD45.

The leucocyte-common antigen (L-CA or CD45) is a family of high molecular weight glycoproteins, ranging from 180,000 to 220,000 MW that are expressed only on cells of lymphoid and myeloid origin. CD45 monoclonal antibodies (mAbs) recognize epitopes present on all polypeptides of the family, while other mAbs, termed CD45R, recognize determinants found only on the 220,000 MW and 200,000 MW polypeptides. In contrast the mAb UCHL1 recognizes a 180,000 MW antigen. UCHL1-coupled Sepharose beads were used to absorb antigen from lysates of cell lines. CD45 mAbs bound to this immobilized antigen. Antigen immobilized with CD45 mAb-coupled Sepharose beads bound UCHL1. Antigen purified by absorption and elution from the MOLT-4 cell line with CD45 mAb-coupled beads yielded molecules of 180,000 and 190,000 MW. Reprecipitation of the eluted antigen with UCHL1 resulted in a 180,000 MW band only. In a reciprocal experiment, CD45 mAb reprecipitated a 180,000 MW molecule from purified UCHL1 antigen. UCHL1 and the CD45R mAb 2H4 showed a mutually exclusive pattern of reactivity with human T- and B-cell lines, but co-expression of the antigens was seen on two myeloid and one erythroleukaemic cell line. In contrast, epitopes recognized by other putative CD45R mAbs were co-expressed with UCHL1 both on myeloid, erythroid and many T- and B-cell lines. We conclude that UCHL1 recognizes an epitope present only on the 180,000 MW polypeptide of CD45. Expression of this antigen is essentially reciprocal to the epitope detected by the CD45R mAb 2H4.     

Induction of an immune response to the porin of Haemophilus influenzae type b by monoclonal anti-idiotypic antibodies.

Monoclonal anti-idiotypes were generated against monoclonal antibody (mAb) Hb-2 which recognized a highly conserved epitope on the outer membrane porin protein from Haemophilus influenzae type b (Hib). Four hybridomas reacting with F(ab') 2 fragments of Hb-2 were selected and characterized. Inhibition studies using syngeneic anti-anti-idiotypic antisera suggested that at least three different antigenic determinants on Hb-2 were recognized by these monoclonal anti-idiotypes. The binding of each anti-idiotype to Hb-2 was inhibited by Hb-2 whereas the reaction was not affected by any other anti-Hib mAb. Complete inhibition of the binding of anti-idiotype to the idiotype could be achieved with 10 micrograms of total outer membrane protein (OMP) from Hib suggesting that the anti-idiotypes might be directed against paratope-associated idiotypes. Outer membrane antigens not recognized by mAb Hb-2 did not inhibit the reaction. Furthermore, the pre-incubation of Hb-2 with each anti-idiotype specifically prevented the reaction of Hb-2 with its antigen. Antibodies with specificity for the porin were generated in guinea pigs immunized with anti-idiotypes AHb-22 and AHb-23. This study indicates that these particular monoclonal anti-idiotypes may be used as an antigen substitute for the porin of Hib in a xenogeneic species.