A lateral flow-based portable platform for determination of reproductive status of cattle.

Our objective was to develop and validate a tool integrating a disposable fluorescence-based lateral flow immunoassay (LFIA) coupled with a portable imaging device for estimating circulating plasma concentrations of progesterone (P4). First, we developed and optimized a competitive LFIA test strip to measure P4 in bovine plasma. The LFIA design included a sample pad, a conjugate pad that stores R-phycoerythrin-anti-P4 conjugates, a glass-fiber spacer pad, a nitrocellulose membrane with printed test and control lines, and a cellulose-fiber absorbent pad. To perform a test, 20 µL of plasma and 50 µL of running buffer were added on the sample pad. After 3 min, 45 µL of running buffer was added to initiate sample flow. After allowing 15 min to stabilize the colorimetric signal, strips were introduced in an LFIA portable reader wirelessly linked to a laptop to determine P4 concentration based on test-to-control-line signal (T/C ratio). In a series of experiments (n = 6), the ability of the LFIA to differentiate plasma samples with ≥1 or <1 ng/mL of P4 was evaluated. For each experiment, a calibration curve was constructed using plasma with known concentrations of P4 (0.1 to 3.7 ng/mL; n = 5). The resulting linear equation was then used to determine a T/C ratio cutoff to differentiate samples with ≥1 or <1 ng/mL of P4. In addition, to evaluate the ability of the platform to assign samples to P4 concentration groups without a calibration curve for individual batches, we performed a receiver operating characteristic analysis to identify a single cutoff value for T/C ratio that could potentially be used for all batches. Overall, calibration curves showed a linear relationship between T/C ratio and P4 levels (mean coefficient of determination = 0.74; range 0.42 to 0.99). Next, plasma samples from lactating dairy cows (n = 58) were tested in triplicate to determine the ability of the LFIA system to differentiate plasma samples with ≥1 or <1 ng/mL of P4 using a RIA for P4 as reference test. Overall, the LFIA assay correctly classified 90% of the samples, with 97% sensitivity, 83% specificity, 85% positive predictive value, and 96% negative predictive value. Agreement between the tests was substantial (kappa = 0.79; 95% confidence interval 0.64 to 0.95). When using a single cutoff value for T/C ratio selected by receiver operating characteristic analysis, sensitivity and specificity to determine CL presence were 97 (95% confidence interval 82 to 99) and 79% (95% confidence interval 60 to 92), respectively. These data suggest that the developed portable LFIA system can accurately differentiate plasma samples with ≥1 or <1 ng/mL of P4.

Mechanisms involved in the p62-73 idiopeptide-modulated delay of lupus nephritis in SNF(1) mice.

The F(1) progeny of the (SWR × NZB) cross develop a lupus-like disease with high serum titers of autoantibodies, and increased frequency and severity of immune complex-mediated glomerulonephritis in females. In previous work, we found that an idiotypic peptide corresponding to aa62-73 (p62-73) of the heavy chain variable region of autoantibody 540 (Id(LN)F(1)) induced the proliferation of p62-73 idiotype-reactive T cell clones. Further, monthly immunization of pre-nephritic SNF(1) female mice with p62-73 resulted in decreased nephritis and prolonged life spans. Here we show that this treatment modulated proliferative responses to Id(LN)F(1) antigen, including a reduction in the population of idiopeptide-presenting antigen-presenting cells (APCs), as early as two weeks after immunization (10 weeks of age). Th1-type cytokine production was increased at 12 weeks of age. The incidence and severity of nephritis was reduced by 14 weeks compared to controls. Clinical indicators of nephritis, specifically histological evidence of glomerulonephritis and urine protein levels, were reduced by 20 weeks. Together these data suggest that events involved in the mechanism(s) whereby p62-73 immunization delayed nephritis occurred early after immunization, and involved modulation of APCs, B and T cell populations.

17ß-Estradiol (E-2) administration to male (NZB × SWR)F1 mice results in increased Id(LN)F1-reactive memory T-lymphocytes and accelerated glomerulonephritis.

While it has been shown that estradiol treatment accelerates the onset of lupus nephritis with autoantibody production and kidney damage in both male and female lupus-prone mice, the specific mechanism(s) involved are unknown. Our previous work has shown that alterations in Id(LN)F(1)-reactive T cells and Id(LN)F(1)+ antibodies correlated closely with the onset of autoimmune nephritis in female F(1) progeny of SWR and NZB (SNF(1)) mice, supporting a critical role for the Id(LN)F(1) idiotype in the development of disease. Since male SNF(1) mice normally do not develop nephritis, we tested whether administration of 17ß-estradiol (E-2) to male SNF(1) mice would increase Id(LN)F(1) IgG levels and autoreactive T cells, and further, induce nephritis. We found that E-2-treated male SNF(1) mice developed nephritis with the same time course and mean survival as normal female SNF(1) mice. Moreover, it appeared that the mechanism involved increased serum Id(LN)F(1)(+)IgG and its deposition in kidney glomeruli, preceded by a striking twofold increase in T-lymphocytes expressing the memory phenotype (CD44(+)CD45RB(lo)) predominantly in the Id(LN)F(1)-reactive T-cell population. In addition, we noted that cells with this phenotype were increased in the nephritic kidneys of treated mice, suggesting a direct involvement of those cells in the renal pathology. E-2 treatment also induced increased numbers of pathogenic Id(LN)F(1)+ antibody-producing B cells and elevated presentation of pathogenic Id(LN)F(1)+ peptide. Taken together, these results suggest a mechanism of E-2-induced acceleration of autoimmune disease in lupus-prone mice may involve expansion of autoreactive idiotypic T and B-cell populations.

Antibody and skin-test responses of sheep vaccinated against Johne's Disease.

Current vaccines against Mycobacterium avium subsp. paratuberculosis (MAP, Johne's Disease) may cause animals to react positively when tested for Mycobacterium bovis (Bovis). Therefore, the effects of vaccination on MAP serum Ab and skin-test responses to MAP and Bovis PPD were compared in 25 ewes vaccinated against MAP with 24 control ewes in an infected flock 3 years post-vaccination. MAP-specific Ab levels were higher (P<0.001) in vaccinated ewes than in control ewes. All increases in skinfold-thickness from 0 to 48h were greater (P<0.0001) than zero while increases in skinfold-thickness from 48 to 72h were greater (P<0.05) than zero for Johnin but not for Bovis PPD. The Vaccine x PPD x Time interaction for skinfold-thickness was significant (P<0.001) with greater increases to Johnin than to Bovis, but with much greater increases in vaccinated ewes. These data suggest that administration of vaccines against MAP developed from whole organisms increase the likelihood that animals will be classified as "responders" to a Bovis screening test and negative by the follow-up comparative cervical tuberculin test, but they also show that vaccination initiates both humoral and cell-mediated MAP-specific responses.

Characteristics of auto anti-idiotypic antibodies reactive with antibodies expressing the pathogenic idiotype, IdLNF1, in the (NZB x SWR)F1 model for lupus nephritis and its parental strains.

The F1 cross between SWR and NZB mice, SNF1, develops severe immune complex glomerulonephritis, in a similar manner to humans with systemic lupus erythematosus (SLE). Our previous data indicate that the idiotypically-related family of antibodies, IdLNF1 may play a role in the pathogenesis of this nephritis. The sera of SNF1 mice, but not NZB or SWR, contained high titers of IdLNF1+ IgG antibodies, which peaked at 22-24 weeks, coinciding with an increase in the CD4 to CD8 ratio of IdLNF1-reactive T cells and IdLNF1 Ig (IgG + IgM) deposition in the kidney glomerulus. Here, auto anti-IdLNF1 antibody levels were quantitated as the mice aged and were found to be significantly different in the three strains, particularly after 20 weeks of age. Moreover, auto anti-IdLNF1 antibody levels were decreased only in SNF1 mice at 20-24 weeks of age. Auto anti-IdLNF1 antibodies were purified by affinity chromatography; anti-IdLNF1 antibodies derived from SNF1 appeared to be of the Ab2 beta or gamma type, while those from SWR mice were Ab2 alpha. Thus, differences in the specificity of auto anti-idiotypic antibodies may be critical in the regulation of the IdLNF1 idiotype in SWR and SNF1 mice, and the development of nephritis in SNF1 mice.

Expression, purification and immunochemical characterization of recombinant bovine beta-lactoglobulin, a major cow milk allergen.

The immunological characteristics of a recombinant beta-lactoglobulin were studied using monoclonal antibodies, polyclonal antiserum and sera from allergic patients. Recombinant beta-lactoglobulin (rBLG) was expressed in Escherichia coli strain DH5alpha and purified as described previously [Cho et al. (1994) J. Biol. Chem. 269, 11 102-11 107]. The method has been modified by adding an immunoaffinity purification step. A quantity of 5-10mg of purified rBLG per liter of medium culture can be produced. rBLG shared the same molecular weight as the natural BLG (nBLG) and also possessed at least one intrachain disulfide bridge. In HPLC, rBLG appeared as a single peak, and the purity was estimated to be greater than 95%. All the monoclonal antibodies (mAbs) used in this study recognized different epitopes of the BLG and presented compatible binding. No differences could be detected between rBLG and nBLG when tested in a Western blot with rabbit polyclonal antiserum or with three mAbs that bound preferentially the reduced and S-carboxymethylated form of BLG. In a competitive enzyme immunoassay (EIA) using either a rabbit polyclonal antiserum or four mAbs that recognized conformational epitopes, we could not distinguish between rBLG or nBLG. In direct ELISA using nBLG or rBLG as the immobilized allergen, we measured a similar concentration of specific anti-BLG IgE in five sera from allergic patients. The results of this study indicate that we have obtained a rBLG with biochemical and immunological properties very similar to nBLG.

Regional localization of the putative cell surface receptor for HTLV-I to human chromosome 17q23.2-17q25.3.

The gene for the cell surface receptor for HTLV-I, the etiologic agent of adult T-cell leukemia and HTLV-I-associated myelopathy, has been localized to distal human chromosome 17q. A panel of somatic cell hybrids containing fragments of human 17q as the only human genetic component was mapped with a set of 10 chromosome 17 probes and utilized to regionally localize the gene. When compared to the murine fibroblast fusion partner, L-M(TK-), and a hybrid cell line containing human chromosome 20, human 17q-containing hybrid cells bound high levels of both HTLV-I virions and the monoclonal antibody, Mab 34-23, which may be directed against the putative HTLV-I receptor. Additional experiments revealed that the human 17q-containing hybrids could also be more efficiently infected by cell-free HTLV-I virions than could the control cell lines. Western blot analyses of cell lysates showed that recombinant HTLV-I envelope gp46 protein and Mab 34-23 both bound to proteins of approximate MW 30 and 31 kDa which were found only in the hybrid cell lines which contained human chromosome 17q. The data suggest that the gene for the HTLV-I receptor is located on the distal region of human chromosome 17q demarcated by the tk-1 locus (17q23.2-17q25.3).

IdLNF1-specific T cell clones accelerate the production of IdLNF1 + IgG and nephritis in SNF1 mice.

We have shown that antibodies bearing a nephritogenic idiotype (IdLNF1) are important in the pathogenesis of autoimmune glomerulonephritis in the (NZB x SWR)F1 hybrid, SNF1. A significant shift in the ratio of CD4+ to CD8+ IdLNF1-specific T lymphocytes, in favour of CD4+ IdLNF1-specific T cells, occurred at 22-24 weeks of age in the SNF1 and correlated with an increase in serum IdLNF1 + IgG and its deposition in the kidney. Recently, we reported the characteristics of six IdLNF1-specific T cell clones (CD3+CD4+CD8-V beta 17a+) derived from 22-week-old SNF1 mice which proliferated in response to IdLNF1 + Ig and augmented IdLNF1 + IgG production when mixed with SNF1 B cells in vitro. No increase in the production of anti-DNA antibodies was seen. Here we report results of the adoptive transfer of three of these clones, A1, B6 and D2, into 6-8-week-old SNF1 mice. Serum immunoglobulin (Ig) (IgG and IgM) levels did not differ from those of age-matched unmanipulated SNF1 up to and including 35 days post-injection. Similarly, total IdLNF1 + Ig levels did not vary significantly among the groups until 28 days post-injection. However, elevated levels of IdLNF1 + IgG were observed as early as 7 days post-injection in mice receiving clone B6 and 21 days post-injection in mice receiving clone D2. This was in sharp contrast with the results obtained in mice treated with clone A1 or unmanipulated SNF1 mice, which did not express IdLNF1 + IgG during this period. Digital image analysis of the kidney glomeruli of mice receiving T cell clones B6 or D2 demonstrated a significantly (P < 0.05) higher level of IdLNF1 + Ig deposition and glomerulonephritis than age-matched unmanipulated SNF1 mice or mice which had received clone A1. Interestingly, there was no statistically significant difference in the production of anti-ssDNA or dsDNA antibodies with the treatments. Mean survival for mice treated with T cell clones B6 and D2 was significantly (P < or = 1 x 10(-6)) shorter compared to unmanipulated SNF1 mice, while that of mice which had received T cell clone A1 was significantly (P < or = 3 x 10(-6)) longer, as determined by chi-squared analysis. The overall survival of mice treated with clones B6 and D2 did not differ from that of unmanipulated mice; however, mice treated with clone A1 survived significantly (P < or = 0.05) longer.(ABSTRACT TRUNCATED AT 400 WORDS)

Treatment with antibody reactive with the nephritogenic idiotype, IdLNF1, suppresses its production and leads to prolonged survival of (NZB x SWR)F1 mice.

The F1 progeny of the cross between SWR and NZB mice (SNF1) develop severe immune complex glomerulonephritis, similar to that seen in human SLE. An idiotypically-related family of nephritic antibodies (IdLNF1) has been shown to be important in the pathogenesis of autoimmune glomerulonephritis in these mice. Interestingly, the majority of IdLNF1+ antibodies do not bind DNA. Here, we sought to examine whether regulation of the expression of this idiotype was important in the development of lupus nephritis and to identify the mechanisms regulating its expression. In the present study, biweekly injections of SNF1 mice with 100 micrograms of rabbit anti-IdLNF1 antibodies, beginning at 8 to 10 weeks of age, resulted in significant P < or = 0.05) suppression of IdLNF1+ Ig(G+M) and IgG production. The decrease appeared to be mediated via significant (P < or = 0.05) decreases in the percentage of IdLNF1-expressing B cells and CD4+ IdLNF1-specific T cells in the treated SNF1 mice compared to the controls. This was accompanied by a significant (P < or = 0.005) increase in survival with delayed onset of glomerulonephritis. Surprisingly, there was no difference in the incidence of anti-DNA antibody production between the treated and control SNF1 mice. These results support the hypothesis that dysregulation of pathogenic idiotypes, not confined to anti-DNA antibody idiotypes as had been shown in previous studies, may contribute to the development of SLE.

Characterization of IdLNF1-specific T cell clones from the (NZB x SWR)F1 murine model for systemic lupus erythematosus.

A family of nephritogenic antibodies bearing an idiotype, IdLNF1, has been shown to be important in the pathogenesis of autoimmune glomerulonephritis in the (NZB x SWR)F1 hybrid, SNF1. Previously, we have reported that a significant shift in the ratio of CD4+ to CD8+ IdLNF1-specific T lymphocytes, in favor of CD4+ IdLNF1-specific T cells, occurred at 20 to 24 weeks of age in the SNF1 and correlated with an increase in serum IdLNF1+ IgG and deposition of IdLNF1 Ig in the kidney glomeruli. Six IdLNF1-specific CD3+CD4+CD8- T cells clones have been derived from 22-week-old SNF1 mice. All six proliferated in response to Con A and anti-CD3. Three of the clones reacted with monoclonal antibody for V beta 8.1,8.2: B3, B5, and TA5 and proliferated specifically in response to IdLNF1 Ig in an Ia-restricted manner. The other three clones, A1, B6, and D2, reacted with anti-V beta 17a+ monoclonal antibody and appeared to be not Ia-restricted. T cell clones B6, D2, and TA5 promoted the production of very high levels of IdLNF1+ IgG by SNF1 B cells in vitro, while B3 and B5 induced the production of only low levels. Interestingly, clone A1 did not induce any IdLNF1+ Ig production. Furthermore, these T cell clones did not induce the production of anti-DNA antibody by SNF1 B cells.

Treating activated CD4+ T cells with either of two distinct DNA methyltransferase inhibitors, 5-azacytidine or procainamide, is sufficient to cause a lupus-like disease in syngeneic mice.

Human antigen-specific CD4+ T cells become autoreactive after treatment with various DNA methylation inhibitors, including 5-azacytidine, procainamide, and hydralazine. This suggests a mechanism that could contribute to the development of some forms of autoimmunity. In this report we have asked whether T cells treated with DNA methylation inhibitors can induce autoimmunity. Murine CD4+ T cells were treated with 5-azacytidine or procainamide and were shown to respond to syngeneic antigen-presenting cells, similar to CD4+ human T cell clones treated with these drugs. Functional characterization demonstrated that cells treated with either drug spontaneously lysed syngeneic macrophages and secreted IL-4, IL-6, and IFN-gamma. Adoptive transfer of 5-azacytidine- or procainamide-treated cells into unirradiated syngeneic recipients induced an immune complex glomerulonephritis and IgG anti-DNA and antihistone antibodies. These experiments demonstrate that T cells treated with either of two distinct DNA methyltransferase inhibitors are sufficient to induce a lupus-like disease. It is possible that the lysis of macrophages, together with the release of cytokines promoting B cell differentiation, contributes to the autoantibody production and immune complex deposition. These results suggest that environmental agents that inhibit DNA methylation could interact with T cells in vivo to produce a lupus-like illness, a mechanism that could have relevance to drug-induced and idiopathic lupus.

Identification of a putative cellular receptor for HTLV-I by a monoclonal antibody, Mab 34-23.

The ability of HTLV-I to infect cells is presumed to be dependent, in some part, on the attachment of the virus to a target cell via a specific cell surface receptor which is, as yet, unknown. Here we present evidence that a monoclonal antibody, Mab 34-23, inhibits the binding of HTLV-I to IL-2 and phytohemagglutinin-activated peripheral blood mononuclear cells and also inhibits virus entry into these cells. Analysis of a variety of target cells, including a human:mouse somatic hybrid which contains only human chromosome 17q, indicates that the binding of Mab 34-23 correlates with HTLV-I adsorption and entry. SDS-PAGE and Western blot analysis show that Mab 34-23 binds to four major proteins of MW 31, 45, 55, and 70 kDa and this binding can be inhibited by HTLV-I and not HIV proteins. HTLV-I virions bind to proteins of similar molecular weight and virus-binding to these proteins can be inhibited by preincubation with Mab 34-23. These data suggest that Mab 34-23 may identify a specific cell surface receptor(s) for HTLV-I.

The onset of nephritis in the (NZB x SWR)F1 murine model for systemic lupus erythematosus correlates with an increase in the ratio of CD4 to CD8 T lymphocytes specific for the nephritogenic idiotype (IdLNF1).

An idiotypically related family of nephritogenic antibodies (IdLNF1) has been shown to be important in the pathogenesis of autoimmune glomerulonephritis in the (NZB x SWR)F1 hybrid, SNF1. Idiotype-specific T lymphocytes which modulate expression of antibody bearing that idiotype may be important in the pathogenesis of systemic lupus erythematosus (SLE). Here, IdLNF1-reactive T lymphocytes were not only found to be present in the NZB, SWR, and SNF1, but a significantly (P < or = 0.05) greater number of IdLNF1-reactive Thy 1.2+ splenic lymphocytes were observed as early as 12 weeks of age in the SNF1. Further, a significant shift in the ratio of CD4+ to CD8+ IdLNF1-reactive T lymphocytes in favor of CD4+ IdLNF1-reactive T cells was observed at 20 to 24 weeks of age only in the SNF1. This shift correlated with an increase in IdLNF1+IgG, and deposition of IdLNF1 bearing immunoglobulin in the kidney glomeruli. These observations suggest a role for idiotype-specific T lymphocytes in the induction of glomerulonephritis in this murine model of SLE.

Infection of peripheral blood mononuclear cells and cell lines by cell-free human T-cell lymphoma/leukemia virus type I.

Previous studies of in vitro infection by human T-cell lymphoma/leukemia virus type I (HTLV-I) have required cocultivation of target cells with HTLV-I cell lines or vesicular stomatitis virus pseudotypes containing HTLV-I envelope proteins. We report here the development of a cell-free infection assay for HTLV-I. Target cells were incubated with purified, DNase-treated HTLV-I virions for 4 h at 37 degrees C. Target cell DNA was then analyzed for the presence of newly synthesized HTLV-I proviral DNA by the highly sensitive polymerase chain reaction. Using this assay system, we have been able to consistently detect in vitro infection of a variety of cellular targets by different HTLV-I isolates. Optimal infection required the presence of 10 micrograms of DEAE-dextran per ml. The assay was dose dependent with respect to virus input. In general, the amount of proviral DNA detected correlated with the level of HTLV-I receptors present on the surface of the target cells, as measured by fluorochrome-labelled HTLV-I binding. Finally, the specificity of the assay was confirmed by demonstrating that the cell line, L1q, a somatic cell hybrid containing human chromosome 17q, to which the gene for the HTLV-I receptor has been mapped, was susceptible to infection by HTLV-I, while the parental mouse cell line from which it was derived, LMTK-, which lacks human chromosome 17q, was not.

Inactivation of human immunodeficiency virus type 1 by ozone in vitro.

A device was designed to deliver a constant source of given concentrations of ozone to fluids containing human immunodeficiency virus type 1 (HIV-1). Ozone was found to inactivate HIV-1 virions in a dose-dependent manner. Greater than 11 log inactivation was achieved within 2 hours at a concentration of 1,200 ppm ozone. Similar concentrations of ozone had minimal effect on factor VIII activity in both plasma and immunoaffinity-purified preparations of factor VIII treated for the same time period. The data indicate that the antiviral effects of ozone include viral particle disruption, reverse transcriptase inactivation, and/or a perturbation of the ability of the virus to bind to its receptor on target cells. Ozone treatment offers promise as a means to inactivate human retroviruses in human body fluids and blood product preparations.

Non-H-2 genes alter the H-2 determined susceptibilities in immune complex nephritis.

These experiments examined the effects of genes outside of the H-2 region on disease susceptibility and pathogenesis. Four strains of mice with the susceptible H-2 type, H-2d, but different non-H-2 genes were studied. B10, D2, Balb/c, NZB, and DBA/2J mice were injected with 4 mg of apoferritin i.p. q.d. for 28 days. B10, D2 and Balb/c mice developed proliferative and crescentic glomerulonephritis. NZB mice developed proliferative and crescentic glomerulonephritis with wire loop lesions suggestive of lupus. DBA/2J mice developed only minimal mesangial proliferation without crescents or necrosis. Electron microscopy showed subepithelial and mesangial deposits in B10, D2, moderate subepithelial and mesangial deposits in Balb/c, and marked mesangial, subendothelial and subepithelial deposits in NZB. Immunofluorescence demonstrated the presence of IgG, IgM, C3 and apoferritin in these deposits. The DBA/2J mice had only minimal mesangial deposits by immunofluorescence and electron microscopy. These experiments demonstrate that non-H-2 genes alter the H-2d determined disease susceptibility seen in H-2 congenic mice. NZB genes can alter the disease so that lupus-like lesions develop and DBA/2J genes can substantially ameliorate the disease.

Specific adsorption of HTLV-I to various target human and animal cells.

In this report, we describe a flow cytometric analysis of HTLV-I specific binding to fresh and cultured cells on a single cell basis. This assay uses rhodamine hydrocarbon tagged, purified HTLV-I virions according to the procedure originally described for avian retroviruses. Successful HTLV-I transmission was detected by analysis of integrated HTLV-I DNA, virion-associated reverse transcriptase, and/or intracellular HTLV-I core antigen p19 expression. Only a specific virus-cell interaction was detected because nonrhodamine-tagged homologous virus or related HTLV-II interfered with tagged HTLV-I binding. In contrast, an unrelated, nonlabeled animal retrovirus was unable to block tagged HTLV binding. Of the cell lines tested, 2 nonlymphoid mammalian and 3 human lymphoid bound significantly high to moderate levels of HTLV-I-tagged virions. The other three human lymphocyte cell lines were insensitive to HTLV-I adsorption. A direct correlation was observed between HTLV-I binding sites and infectivity of human lymphoid cells alone and not other nonlymphoid animal cells. Fresh normal human mononuclear cells bound low levels of HTLV-I virions. As expected, T lymphocytes demonstrated more binding than did the non-T cell population. Enhancement of HTLV-I cell binding in a subpopulation of mononuclear target cells was achieved with phytohemagglutinin (PHA) activation and interleukin 2 (IL2) stimulation, which correlates well with previously published infectivity studies.

The NZB X SWR model of lupus nephritis. I. Cross-reactive idiotypes of monoclonal anti-DNA antibodies in relation to antigenic specificity, charge, and allotype. Identification of interconnected idiotype families inherited from the normal SWR and the autoimmune NZB parents.

The incidence of lupus nephritis is low in autoimmune NZB mice, but when they are crossed with normal SWR mice, almost 100% of the female F1 hybrids (SNF1) develop lethal glomerulonephritis. In a previous study we showed that anti-DNA auto-antibodies produced by the SNF1 mice were qualitatively different from those made by the NZB parents with respect to their isotype, charge, and antigenic specificity patterns. Here we studied idiotypic cross-reactions among the 65 monoclonal anti-DNA antibodies that were derived from four NZB and seven SNF1 mice. A library of 15 anti-idiotypic antibodies were prepared by immunizing rabbits with 15 monoclonal anti-DNA antibodies selected from the panel of 65. We identified 10 cross-reactive idiotype (CRI) families among this large collection of autoantibodies. Five of these CRI families were restricted to cationic anti-DNA antibodies that were exclusively of SNF1 origin, and the strongly cross-reacting members were predominantly IgG2b auto-antibodies with the allotype of the normal SWR parent. The cationic anti-DNA CRI families could be grouped into an interrelated cluster called the Id564 cluster. The other five anti-DNA CRI families were not restricted to any particular parental allotype or charge, although two of these CRI were shared exclusively by SNF1-derived autoantibodies and four of these CRI families could also be grouped into an idiotypically interrelated cluster called the Id512 cluster. In the case of seven out of the 10 CRI families, the idiotypic determinants detected were close to the antigen-binding site of the anti-DNA antibodies. The results indicate that the idiotypic repertoire of anti-DNA autoantibodies produced by the SNF1 mice is different from the NZB parents, and potentially pathogenic (cationic) antibodies produced by the SNF1 mice that are encoded by genes from the normal SWR parent can be identified as distinct CRI families. In the accompanying paper we demonstrate the role of these anti-DNA CRI families in the development of lupus nephritis.