Hinge region of human IgG2 protein: conformational studies with monoclonal antibodies.

We previously produced three anti-human IgG2 mAbs with high specificity and found that they recognize distinct epitopes in the hinge region and neighboring residues in human IgG2: HG2-6A was reactive with the hinge region (Glu216-Pro230); HG2-56F with the Pro234 residue and HG2-30F with the Val235 residue. In this study, we evaluated the reactivities of those three mAbs with human IgG2 protein under various conditions. The results obtained using HG2-6A mAb indicated that the hinge region was concealed in the native form, but exposed after heat treatment at 63 degrees C, or chemical treatment with 3 M KSCN, 3 M guanidine, 30% CH3CN, 8 M urea or acid at pH 2.0 as well as by adsorption onto polystyrene beads. The IgG2 hinge region was also exposed after binding to specific antigens. The Pro234 residue recognized by HG2-56F mAb was exposed under all conditions studied. The neighboring Val235 residue recognized by HG2-30F, however, was completely concealed in the native and antigen-bound states. Only treatment with 3 M guanidine and acid at pH 2.0, or physical adsorption induced conformational changes to partially expose the Val235 residue.     

Monoclonal antibody-based immunoenzymetric assays for quantification of human IgG and its four subclasses

A panel of 5 immunoenzymetric assays (IEMAs) has been developed for quantification of the total and four subclasses of immunoglobulin G (IgG) in human serum using IUIS/WHO documented monoclonal antibodies (MoAb). Human IgG specific MoAb was adsorbed to microtiter plates and used to capture IgG from serum. Peroxidase conjugated forms of polyclonal mouse anti-human IgG Fc or a mixture of 4 MoAb (anti-kappa, anti-lambda, anti-IgG Fc PAN and anti-IgG Fd PAN) were used as detection antibodies. Use of monoclonal antibody in chromatographically purified form was required for acceptable assay sensitivity (S) and working ranges (WR). All 5 IEMAs displayed good precision (intra-assay %CV less than 5%, inter-assay %CV less than 12%) and parallelism (inter-dilutional CV less than 20%). Both HP6069 or HP6070 (anti-IgG1 Fc) worked well alone or together as capture antibodies in the IgG1 IEMA: WR = 20-1250 ng/ml, S = 15 ng/ml. HP6002 (anti-gG2 Fc) alone or in combination with HP6014 (anti-IgG2 Fd) produced an IgG2 IEMA with a WR of 5-200 ng/ml and S of 5 ng/ml. HP6047 (anti-IgG3 hinge) alone generated a sensitive IgG3 IEMA with a narrow working range: WR = 2-50 ng/ml, S = 1.6 ng/ml. Both HP6025 and HP6023 (anti-IgG4 Fc) worked equally well alone and together to produce a useful IgG4 IEMA: WR = 8-250 ng/ml, S = 7.8 ng/ml. HP6017 (anti-IgG PAN Fc) was combined in an equal molar ratio with HP6046 (anti-IgG PAN Fd) to produce a total IgG PAN IEMA with a WR of 5-530 ng/ml and a sensitivity of 5 ng/ml. All 5 IEMAs fulfilled requirements for robust clinical immunoassays that permit the quantitation of human IgG and its 4 subclasses.     

Evaluation of monoclonal antibodies having specificity for human IgG subclasses: results of the 2nd IUIS/WHO collaborative study.

Following the 1st IUIS/WHO Collaborative Study of monoclonal anti-IgG subclass antibodies, a panel of WHO Specificity Reference Reagents (SRR) was established [Jefferis, R., et al. (1985) Immunol. Lett., 10, 223]. At the time, the hope was expressed that further reagents particularly for IgG2, and other allotypic specificities would become available which could be applied in a wide range of assay protocols. The 2nd study reports the evaluation of nineteen anti-subclass and seven anti-allotype monoclonal antibodies. The anti-IgG1 antibody HP6187 was equivalent in performance to the SRR. Others, that were not of the mouse IgG1 isotype, may be useful for particular applications. The anti-IgG2 antibody HP6200 could be a valuable addition to the WHO SRR; it is specific for an epitope in the Fab region but does not have the light chain bias of HP6014. Antibodies of putative allotype specificity exhibited the claimed specificity when used within protocols similar to those employed by the originating laboratory. It appears to be inherent in the nature of the epitopes (allotopes) recognized that it will take several years before reagents applicable to a wide range of techniques will become available.     

Antigenic change in a human IgG4-specific CH3 epitope upon binding of a monoclonal antibody against a neighboring IgG4-specific epitope.

Two sets of monoclonal antibodies (mAbs) probably reacting with two different epitopes in the CH3 domain of the human IgG4 molecule were studied. We observed that the commercially available mAb HP 6011 inhibited the antigen binding of the three mutually inhibitable mAbs, 40-A2, 41-E8 and 43-F11 (40-series), made by us. However, the 40-series mAbs, including those with similar affinity such as mAb HP6011, were not able to inhibit mAb HP 6011. When the 40-series mAbs were preincubated with IgG4, the mAb HP 6011 could partially displace these antibodies. This one-way inhibition indicates that upon binding mAb HP 6011 changes the antigenic structure of the IgG4 molecule by disrupting the epitope for the 40-series mAbs. A steric hindrance of this epitope by mAb HP 6011 is more unlikely, since the small Fab fragment of mAb HP 6011 also inhibited the reaction of the 40-series mAbs.     

Clinical significance of IgG subclasses of anti-Sm and U1 ribonucleoprotein antibodies in patients with systemic lupus erythematosus and mixed connective tissue disease

IgG subclasses of anti-Sm and anti-U1 ribonucleoprotein (U1 RNP) antibodies were determined using a new clone of the anti-IgG2 antibody (HG2-56F). Although the predominance of IgG1 coincided with previous reports, IgG2 anti-Sm and U1 RNP antibodies were detected in numerous patients. IgG3 anti-Sm antibody significantly correlates with joint involvement and a high titer of anti-DNA antibody. On the other hand, IgG4 anti-U1 RNP antibody significantly correlated with esophageal dilation and muscular involvement. These results may suggest that some IgG subclasses are related to a specific clinical feature or manifestation.     

Complement activation by individual and combinations of monoclonal antibodies to Actinomyces viscosus T14V fimbriae: a probe for epitope distribution on these polymeric proteins.

The spatial requirements for IgG activation of the classical complement pathway has provided a basis for utilizing complement consumption by individual and pairs of monoclonal antibodies (mAbs) to compare the repeating epitope patterns of the type 1 and type 2 fimbriae of Actinomyces viscosus T14V and to examine the co-operative effects of mAbs against these polymeric proteins. Three of five mAbs specific for the type 1 fimbriae consumed complement when assayed individually. Four patterns of complement consumption were detected with pairs of these mAbs: inhibition, addition, enhancement or synergy. Inhibition occurred when both members of a pair reacted with the same epitope but only one consumed complement. A strictly additive effect was observed if both mAbs consumed complement and, in addition, recognized the same epitope. Complement consumption by mAbs against certain epitopes was enhanced by non-complement consuming mAbs that reacted with different epitopes. Synergy was observed with extremely low concentrations of two mAbs each of which reacted with a different epitope and consumed complement. In contrast to the anti-type 1 mAbs, only one of seven mAbs against the type 2 fimbriae consumed more than 20% of the available complement. Pairs of anti-type 2 mAbs exhibited only inhibition or synergy. The latter effect was particularly striking as pairs containing mAbs that reacted with different epitopes and failed to consume complement or were minimally active when assayed individually were extremely efficient. These data indicated that the spatial arrangements of individual mAbs bound to repeating epitopes in the type 1, but not the type 2, fimbriae were appropriate for activation of complement. Thus, the repeating epitope patterns of the two types of fimbriae apparently differ.     

the heterogeneity of bovine IgG2—IV. Structural differences between IgG2a molecules of the A1 and A2 allotypes

The major product of digestion of bovine IgG2a(A1) with immobilized pepsin is F(ab')₂ while similar treatment of IgG2a(A2) yields Fab, Fc and other products. It is postulated that structural differences in the hinge region, including the absence of the most N-terminal disulfide bridge in IgG2a(A2) and a displacement of the primary pepsin cleavage site toward the Fd, can explain this effect. The immunodominant A1 allotope(s) appears to be located in the CH3 domain of IgG2a(A1) while the A2 allotopes are located elsewhere and are apparently affected by digestion. The allotypic bias of rabbit anti-IgG2a is also present in anti-IgG2a raised in goats. However, the A2 allotypic determinants of bovine IgG2a are recognized by goat precipitins although precipitins of this specificity are not detectable in rabbits immunized with IgG2a(A2). Rabbit anti-A2 antibodies are detectable using the ELISA in rabbits immunized with IgG2a(A2).     

Antigenic characterization of an abnormal isoform of prion protein using a new diverse panel of monoclonal antibodies

We established a panel of monoclonal antibodies (mAbs) against prion protein (PrP) by immunizing PrP gene-ablated mice with the pathogenic isoform of prion protein (PrPSc) or recombinant prion protein (rPrP). The mAbs could be divided into at least 10 groups by fine epitope analyses using mutant rPrPs and pepspot analysis. Seven linear epitopes, lying within residues 56-90, 119-127, 137-143, 143-149, 147-151, 163-169, and 219-229, were defined by seven groups of mAbs, although the remaining three groups of mAbs recognized discontinuous epitopes. We attempted to examine whether any of these epitopes are located on the accessible surface of PrPSc. However, no mAbs reacted with protease-treated PrPSc purified from scrapie-affected mice, even when PrPSc was dispersed into a detergent-lipid protein complex, to reduce the size of PrPSc aggregates. In contrast, denaturation of PrPSc by guanidine hydrochloride efficiently exposed all of the epitopes. This suggests that any epitope recognized by this panel of mAbs is buried within the PrPSc aggregates. Alternatively, if the corresponding region(s) are on the surface of PrPSc, the region(s) may be folded into conformations to which the mAbs cannot bind. The reactivity of a panel of mAb also showed that the state of PrPSc aggregation influenced the denaturation process, and the sensitivity to denaturation appeared to vary between epitopes. Our results demonstrate that this new panel of well-characterized mAbs will be valuable for studying the biochemistry and biophysics of PrP molecules as well as for the immuno-diagnosis of prion diseases.     

HLAMatchmaker-based analysis of human monoclonal antibody reactivity demonstrates the importance of an additional contact site for specific recognition of triplet-defined epitopes

Five HLA-A3 reactive human monoclonal antibodies (mAbs) originating from a parous woman were screened against HLA-typed panels by means of complement-dependent lymphocytotoxicity, high-definition ELISA, and flow cytometry with single antigen beads. Antibody reactivity profiles were compared with triplet amino acid sequence polymorphisms identified by HLAMatchmaker, and a three-dimensional structural modeling program (Cn3D of the National Center for Biotechnology Information) was used to determine the topography of epitopes recognized by each mAb. These mAbs originated from a woman who during pregnancy developed antibodies to the paternal HLA-A3 antigen of her child. Each mAb was specific for one mismatched triplet on HLA-A3, and the reactivity patterns of these IgM-type mAbs were practically the same in lymphocytotoxicity and antigen-binding assays. One mAb was specific for 163dT, a unique triplet present only on A3. The other mAbs reacted with 62Qe, 142mI, or 144tKr; these triplets are present on different groups of HLA-A alleles, some of which, however, did not react. Topographic modeling of triplet-defined epitopes identified clusters of polymorphic surface residues that were shared between reactive alleles. These clusters may serve as primary contact sites for the specificity-determining complementarity-determining region (CDR) loops of antibody. The reactivity with these mAbs required also the presence of self-sequence elsewhere on the HLA molecular surface as a critical secondary contact site for antibody, likely through another CDR loop. For instance, the reactivity of the 62Qe-specific mAb required the presence of a glycine residue in position 56 and the reactivity of the 142mI-specific mAb required the presence of the GTLRG sequence in positions 79-83. Conversely, there were many other amino acid differences between the mAb-reactive alleles and HLA-A3 that did not prevent antibody binding. For instance, the 62Qe-specific mAb-reactive alleles had 35 and the 142mI-reactive alleles had 50 of such "permissive" residue differences. An HLAMatchmaker-based analysis of the reactivity of human mAbs will increase our understanding of the structural definition of HLA epitopes and their reactivity with alloantibodies.     

Monoclonal anti-human IgG antibodies for quantitation of allergen-specific IgG in human sera

Eight monoclonal anti-human IgG antibodies were fully characterized and evaluated as possible reagents in solid phase radioimmunoassay for quantitating allergen-specific IgG antibody. Four monoclonal antibodies (HG24D, HG2-14, HG2-18, and HG2-25) recognize CH2 domain of human IgG and bind to human IgG fixed to microtiter plate with high affinities. These monoclonal antibodies were more suitable than polyclonal rabbit anti-human IgG antibody in Phadebas RAST for honey bee venom-specific IgG antibody. Nonspecific binding was much lower, and the slopes of standard curves were much steeper. In contrast to polyclonal antibody, the standard curve was hardly influenced by human serum IgG in sample diluent. These advantages of monoclonal antibodies that recognize CH2 domain of human IgG made it possible to quantitate egg white- and Dermatophagoides pteronyssinus-specific IgG antibodies with use of allergen disks prepared for IgE RAST. This property allows a single system to be used for measurement of IgG and IgE antibodies against clinically relevant allergens.     

5株鼠抗人CD28单克隆抗体的研制及生物学特性的研究

目的：制备鼠抗人CD28分子功能性单克隆抗体，研究其对T细胞活化、增殖及信号转导等方面的生物学效应。方法：以天然高表达CD28分子的人多发性骨髓瘤细胞株U266和小鼠淋巴瘤细胞转人CD28基因细胞株CD28-T分别作为免疫原和检测细胞株，采用B淋巴细胞杂交瘤技术进行单抗的研制；以快速定性试纸法鉴定单抗亚类；腹水诱生法和免疫亲和层析法进行单抗的制备和纯化；经间接免疫荧光法分析单抗对不同细胞膜表面CD28分子的识别；采用竞争抑制法分析单抗识别的抗原位点；利用^3H-TdR掺入法分析单抗对PBTC的刺激效应和免疫荧光法分析PBTC活化前后的表型变化。结果：成功获得5株鼠抗人CD28功能性单克隆抗体，分别命名为2D5、2F5、3136、3F8和8G8，其中2D5为IgG2a亚类，其余均为IgG1亚类；流式细胞仪分析结果显示，5株单抗均能良好识别CD28-T、U266、XGI和Jurkat细胞表面的CD28分子；竞争抑制试验表明，2D5和8G8能完全阻断标准抗人CD28单抗与U266膜CD28分子的结合，其余3株为部分阻断；^3H-TdR掺入法实验结果表明，单抗8G8联合激发型CD3单抗能明显促进PBTC的增殖，刺激指数为7．4，活化细胞CD4、CD25、ICOS、4IBB及OX40分子的表达上调。结论：5株单抗均为抗人CD28单克隆抗体，具有重要的基础研究及潜在的临床应用价值。     

C1q binding to chimeric monoclonal IgG3 antibodies consisting of mouse variable regions and human constant regions with shortened hinge containing 15 to 47 amino acids

We have constructed four different deletion mutants of a chimeric mouse-human IgG3 anti-(4-hydroxy-3-nitrophenyl)acetyl/(5-iodo-4 hydroxy-3 nitrophenyl) acetyl (NP/NIP) antibody lacking one or more of the four exons coding for the hinge region. The mutant variants all retained intact hinge region epitopes since they all reacted with IgG3 hinge-specific antibodies. Surprisingly, all the deletion mutants bound C1q equally well or even better than the wild type. Thus the high C1q binding activity of IgG3 compared to IgG1 is apparently not due to the total length of the IgG3 hinge, which is 62 amino acids, nor is it due to the length of the upper hinge which is the stretch from the end of CH1 to the first inter-heavy chain disulfide bond.     

The antigen binding domain of non-idiotypic human anti-F(ab')2 autoantibodies: study of their interaction with IgG hinge region epitopes.

In previous studies we described a natural human IgG-anti-F(ab')2 autoantibody family with immunoregulatory properties. Genes coding for the variable regions of the heavy and light chains of the Abs were isolated from a natural Ig gene library and scFv Abs were expressed in E. coli. The scFv Abs bound to F(ab')2 but not to Fab fragments. This points to an epitope located in the hinge region since Fab fragments are lacking most of the hinge. In order to verify our hypothesis, double chain peptides comprising the lower-, middle-, and part of the upper hinge subregion of IgG1-IgG4 were synthesized on cellulose membranes and tested for binding to the Abs. The results show binding of Abs to IgG1 and IgG4 hinge region peptides. In order to identify the key residues of the discontinuous epitopes we carried out complete substitutional analyses in which each amino acid of the wt peptides was substituted by all other amino acids except cysteine. The exchange of proline in the IgG1 or IgG4 middle hinge region abrogated the binding, revealing the importance of this subregion for epitope expression. No binding to the IgG2 or IgG3 hinge was detected. These results indicate that scFv anti-F(ab')2 Abs recognize the hinge region of IgG1 and IgG4 and that the expression of the epitope depends on an intact middle hinge subregion.     

抗rhNDPK-A单克隆抗体的制备及鉴定

目的 :研制抗rhNDPK A (recombinanthumannucleosidediphosphatekinase A)单克隆抗体 (mAb) ,并鉴定其特性。 方法 :以纯化的rhNDPK A免疫BALB/c小鼠 ,采用杂交瘤技术制备抗rhNDPK AmAb ;用免疫双扩散鉴定Ig亚类 ;West ernblot鉴定mAb的特异性 ;间接ELISA检测mAb的腹水效价、亲和常数 ,并进行表位分析。结果 :获得 6株可分泌特异性mAb的抗rhNDPK A的杂交瘤细胞系 2D9、8C7、13E2、15D9、15E3和 2 0D9,Ig亚类均为IgG1;其效价为 1× 10 -4～5× 10 -6;亲和常数为 4 .5× 10 -9～ 2 .8× 10 -10 mol/L ;共有3个抗原表位。结论 :获得抗rhNDPK A的mAb ,为进一步用于临床诊断和实验研究创造了条件     

Determination of homozygosity or heterozygosity for the G2m(n) allotype by a monoclonal, precipitating antibody

Monoclonal antibody SH 21 was found to be a good anti-IgG2m(n) reagent both for the conventional typing (HA inhibition) and for precipitation in polyethylene glycol containing gel. Antibody SH 21, together with monoclonal antibody HP 6014 (anti-IgG2), was used for the discrimination between G2m(+n) homozygotes and heterozygotes in a double diffusion assay, where the two antibodies and a serum sample were placed in corners of a triangle. In the case of a homozygote SH 21 was able to stop the diffusion of IgG2 to a precipitation line, but some IgG2 of a heterozygote was able to diffuse beyond the precipitate, forming a crossed precipitation pattern. The validity of the typing was checked with serum samples from 54 families. All the 33 'obligatory heterozygotes' (G2m(+n) parent(s) of a G2m(-n) child, or the other way round) exhibited the crossed precipitate. The frequency of the G2mn allele in a Finnish population of 297 unrelated adults was 0.409.     

Rapid immunoassays for the measurement of immunoglobulin G subclass concentration in immunoglobulin preparations and human serum.

Enzyme immunoassays (EIA) capable of determining total IgG1, IgG2, IgG3 and IgG4 subclass concentrations in human serum preparations have been developed. Subclass-specific monoclonal antibodies (mAbs) are bound to polyacrylamide bead-conjugated anti-mouse immunoglobulin antibodies. Bound immunoglobulins are detected with a peroxidase-conjugated anti-IgG antibody or a biotin-conjugated anti-IgG antibody followed by peroxidase streptavidin. The standard curves were found to be linear in the regions 16.0-2.0 micrograms/ml for IgG1, 4.0-0.5 micrograms/ml for IgG2, 0.4-0.06 micrograms/ml for IgG3 and 0.25-0.05 micrograms/ml for IgG4. Coefficient of variation (CV) values range from 0.32-7.32% for IgG1, 0.66-4.85% for IgG2, 1.62-6.85% for IgG3 and 0.05-6.47% for IgG4 standard curves. The inter-assay variability for the control human serum samples was 9.6% for IgG1, 6.7% for IgG2, 9.5% for IgG3 and 6.8% for IgG4.     

Arizona cypress (Cupressus arizoniea) pollen allergens. Identification of crossreactive periodate-resistant and -sensitive epitopes with monoclonal antibodies

Species of the Cupressaceae family are a worldwide cause of respiratory allergies. We used monoclonal antibodies (mAbs) to investigate the presence and the nature of cross-reacting epitopes shared by various components within Cupressus arizoniea pollen extract (CaE) or by CaE and pollen extract from C. sempervirens (CsE). mAbs were produced in mice immunized with whole CaE (4A6 and 5E6) or with the major allergen components (2D5). Their reactivity was investigated by ELISA and immunoblotting before and after CaE periodate treatment. Cross-reactivity was evaluated by ELISA inhibition and immunoblotting. mAbs 2D5 and 4A6 recognized periodate-resistant epitopes, whereas the mAb 5E6 reacted with a periodate-sensitive determinant. The former mAbs recognized epitopes present on CaE major allergen and also shared by other components. mAb 5E6 showed a spread reactivity on CaE, with exclusion of the major allergen. When the three mAbs were tested with CsE, a restricted pattern of reactivity to mAbs 2D5 and 4A6 was obtained, whereas mAb 5E6 maintained a spread reactivity. The CaE major allergen is represented by two components recognized by human IgE and sharing common epitopes, as proven by mAbs reactivity. The use of these mAbs demonstrates that cross-reactivity within CaE components and between CaE and CsE is due to the presence of periodate-sensitive as well as -resistant epitopes.     

Potent neutralization of primary human immunodeficiency virus clade C isolates with a synergistic combination of human monoclonal antibodies raised against clade B.

OBJECTIVES: We investigated the ability of several human neutralizing monoclonal antibodies (mAbs), originally raised against human immunodeficiency virus (HIV) clade B isolates, to neutralize primary clade C isolates as single agents and in combination. STUDY DESIGN/METHODS: HIV clade C isolates from five different countries were tested for susceptibility to neutralization by anti-clade B mAbs in human peripheral blood mononuclear cells. Monoclonal antibody combinations were evaluated for possible synergy. RESULTS: All 20 primary HIV clade C isolates could be neutralized 97.5% to 100% by a quadruple combination of mAbs IgG1b12, 2G12, 2F5, and 4E10. These mAbs recognized conserved epitopes and were highly synergistic, resulting in strong cross-clade neutralization. CONCLUSIONS: In our previous experiment, a synergistic combination of human neutralizing mAbs protected all macaque neonates against oral challenge with a simian-human immunodeficiency virus encoding HIV env. Together, our data suggest that passive immunization with currently available anti-clade B mAbs could play a role in preventing HIV clade C transmission through breastfeeding.     

Presence of IgG4 on the membrane of human basophils. Histamine release is induced by monoclonal antibodies directed against the Fab but not the Fc region of the IgG4 molecule

We have studied the possible role of human IgG4 as an anaphylactic antibody. For that purpose, we have determined the induction of histamine release (HR) from human basophils by anti-IgE and anti-IgG4 monoclonal antibodies (MoAbs) recognizing different epitopes located at the Fc and Fab regions of the IgG4 molecule. The results show that anti-IgG4 (Fab) MoAb was able to induce HR in 93% of donors tested, with no differences between atopics and non-atopics. That HR is calcium dependent and is accompanied by the synthesis and release of leukotriene C4. In contrast, no HR could be induced by anti-IgG4(Fc) MoAbs in any individual, even in the presence of D2O or after a second challenge with a polyclonal goat anti-mouse IgG antibody. The results obtained suggest the presence of IgG4 on the basophil membrane and that the epitope recognized by the anti-IgG4 (Fc) MoAbs is probably hidden in cell-bound IgG4. This was demonstrated by immunofluorescence techniques: IgG4 bound to the basophil membrane could be detected with anti-IgG4(Fab) but not with anti-IgG4(Fc) MoAbs. In addition, we found that nine donors were unresponsive to an anti-IgE stimulus, while they released histamine efficiently after challenge with anti-IgG4(Fab), suggesting the existence of different receptors for both immunoglobulins.     

Production and characterization of monoclonal antibodies against binary ethylenimine inactivated Nipah virus

Nipah virus, a zoonotic paramyxovirus which emerged recently was chemically inactivated using binary ethylenimine (BEI). The inactivated virus was concentrated and purified by sucrose gradient centrifugation. The gradient fractions were examined by electron microscopy and Western immunoblot, and gradient fraction containing mainly Nipah matrix (M) and nucleocapsid (N) proteins was used for immunizing BALB/c mice to generate hybridomas. Screening of the resultant hybridoma clones identified five strongly positive clones producing IgG monoclonal antibodies (mAbs) reactive to the Nipah virus antigen. The protein specificity of these mAbs was determined by Western immunoblot using Nipah virus and recombinant Nipah virus proteins expressed in mammalian cells. Four mAbs reacted with Nipah N protein and one reacted with Nipah M protein. None of the mAbs neutralized Nipah virus infectivity in vitro. However, all mAbs recognized Nipah virus in ELISA and immunofluorescence assay. F45G2 mAb was most suitable for immunohistochemistry on long term formalin-fixed Nipah virus infected swine tissues. Three of the anti-nucleocapsid mAbs (F45G2, F45G3 and F45G6) showed cross-reactivity with closely related Hendra virus N protein in both immunofluorescence and Western Immunoblot assays. Two of the mAbs were specific for the Nipah virus only, F45G4 (anti-N) and F45G5 (anti-M), and could be used in the primary identification of Nipah virus. The use of these immunoreagents to develop new diagnostic assays is discussed.