Human Rh D monoclonal antibodies (BRAD-3 and BRAD-5) cause accelerated clearance of Rh D+ red blood cells and suppression of Rh D immunization in Rh D- volunteers

The use of prophylactic anti-D to prevent Rh D immunization in Rh D- women and subsequent hemolytic disease in Rh D+ infants is widespread, but has led to shortages of the anti-D Ig. With the aim of substituting monoclonal anti-D for Rh D prophylaxis, we have compared the abilities of monoclonal and polyclonal anti-D to clear Rh D+ red blood cells (RBCs) infused into Rh D- male volunteers and to suppress Rh D immunization. Two human monoclonal antibodies (MoAbs), BRAD-3 (IgG3) and BRAD-5 (IgG1), produced from stable Epstein-Barr virus-transformed B-lymphoblastoid cell lines, were selected because of their proven in vitro activity in promoting RBC lysis in antibody-dependent cell- mediated cytotoxicity assays. RBC clearance was assessed by intravenous injection of 3 mL of 51chromium-labeled D+ RBCs into 27 volunteers 48 hours after intramuscular injection of monoclonal or polyclonal anti-D. Further 3-mL injections of unlabeled D+ cells were administered at 6 and 9 months to induce immunization. Blood samples were taken throughout the 12-month period of study for the serologic detection of anti-D. The mean half-life (t50%) of RBCs in 7 recipients of 300 micrograms BRAD-5 (5.9 hours) was similar to that in 8 recipients of 500 IU polyclonal anti-D (5.0 hours), whereas D+ cells were cleared more slowly in some of the 8 subjects injected with 300 micrograms BRAD- 3 (mean t50% 12.7 hours) and in 1 individual administered 100 micrograms BRAD-3 (t50% 41.0 hours). The rate of RBC clearance in both groups administered 300 micrograms monoclonal anti-D correlated with the amount of antibody bound per cell, determined by flow cytometry. There was no evidence of primary immunization having occurred in any subject after 6 months of follow-up. Five of 24 subjects produced anti- D after one or two further injections of RBCs, confirming that they were responders who had been protected by the monoclonal or polyclonal anti-D administered initially. Four of these responders were recipients of monoclonal anti-D (3 BRAD-3, 1 BRAD-5). One individual who received BRAD-5 produced accelerated clearance of D+ RBCs at the third unprotected RBC challenge but did not seroconvert. This study shows that the human MoAbs BRAD-3 and BRAD-5 can prevent Rh D immunization, and indicates that they may be suitable replacements for the polyclonal anti-D presently used in prophylaxis of Rh D hemolytic disease of the newborn.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantitation of Rh D Antigen Sites on Weak D and D Variant Red Cells by Flow Cytometry

Information on the number of D sites on weak D (D^u) and D variant cells is limited and incomplete. The aim of this study was to use a simple non-isotopic technique utilising a combination of flow cytometry and ELISA to quantitate the number of D sites on an extensive range of these cells. Five human monoclonal IgG anti-D (BRAD-7 (JAC10), BRAD-5, 2B6, BRAD-3, H27) and one affinity-purified polyclonal IgG anti-D were each used at a saturating concentration of 20 μg/ml. In general, BRAD-3, BRAD-5 and 2B6 gave the highest number of D sites per cell (SPC), H27 and the polyclonal anti-D were slightly lower, while BRAD-7 gave the lowest SPC with all D-positive cells tested except DFR. Interestingly, BRAD-7 gave the highest SPC with DFR cells. Rh D antigen density for R₂R₂ cells was approximately double that seen with either R₂r or R_o (presumed R_or) cells. R₁R₁ cells gave only moderately higher SPC than R₁r cells. Higher SPC were obtained with the R₁ haplotype if the C^w antigen was present. Weak D, Va and VI cells of the R₁ haplotype had higher SPC than those of the R₀ or R₂ haplotypes. The majority of D variant cells were found to have lower SPC than normal cells, and for polyclonal anti-D, which was the only anti-D to react with all D variants, SPC decreased in the order IIIc > IIIa > HMii > IVa > Va > DFR > DBT > IVb > VII > II > HMi > VI. The number of molecules of IgG anti-D bound to D variant cells varied by up to 10 times with reactive monoclonal antibodies. The highest SPC on weak D and D variant cells were obtained with BRAD-7 (DFR, 9,500), BRAD-3 (Va, 12,500), BRAD-5 (II, 5,500; VII, 5,300; weak D, 1,300), H27 (III, 24,000; VI, 2,900; HMi, 3,000; HMii, 16,800) and polyclonal anti-D (IVa, 9,300; IVb, 4,000; DBT, 4,300).     

In vitro Functional Activity of IgG1 and IgG3 Polyclonal and Monoclonal anti-D

Background and objectives: IgG anti-D is generally restricted to IgG1 and IgG3; it mediates red cell destruction through interactions with IgG Fc receptors (FcγR) on effector cells. The relative ability of these two IgG subclasses of anti-D to mediate haemolysis in vitro by monocytes and K cells was investigated. Materials and methods: Anti-D was affinity purified from 5 preparations of prophylactic anti-D immunoglobulin, and IgG subclasses quantified by ELISA; mean levels were 86.5% IgG1, 1.4% IgG2, 11.6% IgG3 and 0.4% IgG4. IgG1 and IgG3 polyclonal anti-D were further purified separately from some of the anti-D by removal of either IgG3 using magnetic beads coated with anti-IgG3, or of IgG1 using protein A. These preparations were compared with monoclonal anti-D (BRAD-3 and BRAD-5) for their ability to lyse red cells in antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Results: Monocyte-mediated lysis of red cells coated with IgG3 anti-D was approximately twice that of cells coated with IgG1 anti-D at similar sensitization levels, and anti-D preparations containing 10% or more IgG3 gave similar lysis. By contrast, in the K cell ADCC, IgG1 anti-D was 2–4 times more haemolytic than IgG3 anti-D. Polyclonal IgG1 and IgG3 anti-D promoted about 20% more lysis than BRAD-5 (IgG1) and BRAD-3 (IgG3), respectively, in the K cell ADCC, although no difference was observed between polyclonal and monoclonal anti-D in the monocyte ADCC. Conclusions: These experiments demonstrated a functional dichotomy between these two subclasses of anti-D; IgG3-coated red cells were lysed preferentially by monocytes mediated predominantly through FcγRI interactions, whereas haemolysis of IgG1-sensitized cells was mediated mainly by FcγRIII on K cells.     

Functional in vivo characterization of human monoclonal anti-D in NOD-scid mice

The prophylaxis of the hemolytic disease of the newborn requires significant amounts of plasma-derived polyclonal human anti-D. Because of procurement problems, there is a growing interest in replacing plasma-derived anti-D by in vitro-produced human monoclonal anti-D. Hundreds of monoclonal anti-D have been prepared, but the selection of the most potent for in vivo use is difficult because it cannot be predicted by in vitro characterization. This study evaluated the possibility of using nonobese diabetic/severe combined immunodeficient (NOD-scid) mice for the in vivo evaluation of human monoclonal anti-D. Human red blood cells (RBCs) were found to circulate normally in the blood of NOD-scid mice previously injected with a physiologic amount of human immunoglobulin G (10 mg). The addition of a small amount of anti-D (1 to 5 microg) resulted in the clearance of Rh D(+) RBCs within 4 hours. The comparative testing of 8 monoclonal anti-Ds showed a wide range of potency (15% to 87%) relative to plasma-derived polyclonal anti-D. There was no strong correlation between the in vivo potency index and the immunoglobulin G isotype, affinity, or fine specificity of the antibodies. These results show the usefulness of NOD-scid mice for the initial in vivo screening of human monoclonal anti-D before testing the most active antibodies in clinical trials done in human volunteers.     

An in-vitro assessment of the functional activity of monoclonal anti-D

The response of human monocytes to red cells sensitized with known levels of monoclonal antibody to the Rh antigen D (anti-D) was compared with that of polyclonal anti-D. Monocyte response was determined by measuring red cell adherence, erythrophagocytosis, monocyte-mediated red cell lysis and luminol-dependent chemiluminescence. By all criteria, monoclonal and polyclonal antibodies showed comparable activity, with IgG3 antibodies promoting a greater monocyte-red cell interaction than IgG1 antibodies. It is suggested that monoclonal anti-D may be effective in the prophylaxis of haemolytic disease of the newborn, providing such material is clinically acceptable.     

Reaction of rheumatoid factors with IgG3 monoclonal anti-Rh(D) antibodies: more frequent reactivity to a monoclonal antibody of the Gm allotype G3m(5) in rheumatoid patients negative for G3m(5).

Human monoclonal anti-Rh(D) antibodies of known IgG isotype and Gm allotype were bound to erythrocytes and then used as the target IgG antigens for rheumatoid factors (RFs) in a direct haemagglutination test. When serum samples from patients with rheumatoid arthritis (RA) were tested for RF specificity towards these IgG monoclonal anti-D antibodies the incidence and titre of reactivity towards an IgG3 monoclonal anti-D antibody was considerably greater than for a polyclonal anti-D antibody of the same Gm allotype, G3m(5). This difference was not explained by the amount of each anti-D antibody which bound to erythrocytes. Furthermore, when patients with RA were divided into groups according to their Gm phenotype, sera from a greater proportion of patients negative for the phenotype G3m(5) reacted to the G3m(5) monoclonal anti-D antibodies than sera from those patients positive for this allotype. Analysis of RF reactivities towards two IgG3 and three IgG1 monoclonal anti-D antibodies, each with different Gm allotypic epitopes, indicated, however, that individual serum samples contained RFs with a spectrum of specificities; some sera appeared to react to a single set of Gm alleles, whereas others also reacted to isotypic or iso-allotypic epitopes, or both. Our data suggest that RFs with specificity for Gm allotypes do not arise in patients who carry that particular allotype owing to tolerance induced in fetal-neonatal life. Conversely, RFs with apparent specificity for a Gm allotype formed in patients negative for that allotype may be reacting to a closely related but different epitope. Final proof requires precise specificities for each RF formed, and IgG3 monoclonal anti-D antibodies would be useful reagents for this purpose.     

Selection of Monoclonal Antibodies for the Identification of D Variants: Ability to Detect Weak D and to Split epD2, epD5 and epD6/7

Red cells from known D variant donors were tested with 41 monoclonal anti-D reagents, 26 IgG and 15 IgM, with the view to selecting a panel to aid the identification of unusual D types. These antibodies gave reaction patterns which allowed the identification of most of the known D category cells, recognizing epD2, epD5, epD6/7, epD8 and epD9, but were unable to distinguish category III from normal D-positive cells. Reactivity with HMi, HMii, DFR, DBT and R_o^Har cells split epD2, epD5 and epD6/7 into two, three and eight groups, respectively. A panel comprising 15 monoclonal anti-D, 11 IgG and four IgM, was selected as representative of the antibodies tested. Reactivity of monoclonal anti-D was dependent on antibody concentration and antibody avidity. An antibody concentration of at least 12 pg/ml was required for optimum reactivity of the two monoclonal antibodies tested. A simple calculation of division of the titre by the antibody concentration provided a relatively simple means of establishing the reactivity performance of the antibody and correlated well with ability to detect weak D (D^u) cells. A characteristic variable reduction in reaction strength with all the IgG anti-D was observed with weak D cells. The IgM antibodies, except the high avidity RUM-1, T3D2T6, D9A4 and BS226, performed poorly in detecting weak D. The majority of the IgM antibodies tested reacted with R_o^Harr cells, while only one IgG antibody was positive.     

Efficacy of RhD monoclonal antibodies in clinical trials as replacement therapy for prophylactic anti-D immunoglobulin: more questions than answers

Prophylactic anti-D is a very safe and effective therapy for the suppression of D-immunization and prevention of haemolytic disease of the foetus and newborn. The primary mode of action of anti-D is rapid clearance of fetal D-positive red cells from the maternal circulation, mediated by interactions with immunoglobulin G Fc receptors on macrophages in the spleen. Many anti-D monoclonal antibodies (mAb) have been produced by a variety of methods. Twelve anti-D mAbs were tested in eight studies for their ability to mediate clearance of autologous red cells, and 13 antibodies studied in seven trials of the clearance of D-positive red cells injected into D-negative subjects. Antibodies produced by human B-cell lines, mouse-human heterohybridomas and Chinese hamster ovary cells varied in their activity with none being quite as effective as polyclonal anti-D. However, clearance mediated by recombinant anti-D produced by rat YB2/0 cells was extremely rapid, faster than polyclonal anti-D, but with haemolysis and some hepatic accumulation of red cells observed in one study. Two human anti-D mAbs prevented D-immunization. In contrast, anti-D mAbs from heterohybridomas increased the incidence and rapidity of anti-D responses. It is hypothesised that unnatural glycosylation of monoclonal anti-D produced by some cell lines may have caused these unexpected results. In some antibodies, unusual oligosaccharides on anti-D may have affected binding to Fc receptors resulting in reduced red cell clearance. For others, non-human glycoforms of anti-D might have bound to innate immune recognition molecules promoting pro-inflammatory reactions. These extensive data on the clinical activity of monoclonal anti-D produced by cell lines derived from four species will inform the future development of monoclonal anti-D for RhD prophylaxis.     

The glycosylation of red cell autoantibodies affects their functional activity in vitro

Summary. Factors governing the functional activity of red cell autoantibodies are poorly defined. Here we report the presence of qualitative differences in the glycosylation of IgG autoantibodies which affect in vitro interactions with FCγRIII. The following antibodies were affinity-purified by adsorption and elution from normal red cells: IgG eluted from the red cells of 27 haemolysing or non-haemolysing patients, anti-D in sera from 11 pregnant women, and IgGl and IgG 3 human monoclonal anti-D. Monoclonal antibodies with differing levels of agalactosyl IgG were produced by culturing cell lines at high or low cell density. The % IgG with oligosaccharides lacking terminal galactose residues (agalactosyl IgG) of antibodies was designated as low, medium or high according to their reactivity with a monoclonal antibody to terminal N-acetylglucosamine. FcγRIII-mediated functional activity was assessed by measuring the K-cell-mediated lysis of red cells in eluates diluted to achieve comparable levels of red cell sensitization. All eluates containing allo-anti-D were lytic (range 74-100%). In contrast, lysis by autoantibodies varied from 0 to 100%; 11/13 eluates from red cells of haemolysing patients promoted <5% lysis compared to 2/7 eluates from red cells of non-haemolysing patients (P<0.02). The ability of autoantibodies to promote K-cell-mediated red cell lysis correlated inversely with their level of agalactosyl IgG (r=-0-56, P<0.01, n=23). Further, monoclonal anti-D antibodies with very low levels of agalactosyl IgG were comparatively more lytic than the same antibodies containing more agalactosyl IgG. Analysis of the ratio of κ:λ light chains suggested that autoantibodies from 6/19 patients were monoclonal or oligoclonal in nature. The data indicate that IgG red cell autoantibodies from different patients are functionally heterogenous, and that this may be due, at least in part, to qualitative differences in the Fc region glycosylation reflected by differences in the proportion of agalactosyl IgG. This heterogeneity is consistent with the clonally-restricted nature of the autoantibodies in some patients.     

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

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

Diversity of Human Anti-D Monoclonal Antibodies Revealed by Reactions with Chimpanzee Red Blood Cells

Fifty-three human anti-D monoclonal antibodies (mAbs) revealed a striking diversity of reactions in tests with panels of chimpanzee red blood cells (RBCs) of various R-C-E-F blood group phenotypes (counterparts of the human Rh-Hr groups). The reactivities of these antibodies, which depended on the agglutination technique used, could be classified into four main types. These patterns of reactivity of anti-D mAbs with chimpanzee RBCs showed only limited correlation with types of reactions observed with human D variant RBCs. Primate red cells may, therefore, constitute an independent test system for subclassification of human monoclonal antibodies. Comparison of reactivities of human anti-D mAbs with chimpanzee and human D variant RBCs confirms the homology between the chimpanzee R^c, and the human D antigens. The chimpanzee R^c shares with human D the epitopes epD5, epD6/7 and epD8, but lacks epitopes epD1, epD2, epD3 and epD4 of the Rh mosaic, thus resembling the human D variants IVb and Vc.     

Tissue transglutaminase autoantibody enzyme-linked immunosorbent assay in detecting celiac disease

Background & Aims: Tissue transglutaminase has been reported to be the target for endomysial antibodies in celiac disease. We sought to establish whether immunoglobulin (Ig) A class tissue transglutaminase autoantibodies can be considered specific for celiac disease. Methods: Serum samples from 136 patients with untreated celiac disease (diagnosed according to the criteria of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition) and 207 disease controls were studied. Enzyme-linked immunosorbent assay (ELISA) and Western blots were performed using calcium-treated and untreated tissue transglutaminase as antigen. Reticulin, endomysial, and mouse monoclonal tissue transglutaminase antibodies were studied by an indirect immunofluorescence method and gliadin antibodies with ELISA. Results: The calcium-activated tissue transglutaminase autoantibody ELISA was highly sensitive (129 of 136) and specific (194 of 207) in detecting celiac disease. The new autoantibody ELISA test correlated well with the endomysial antibody test. Tissue transglutaminase autoantibody ELISA showed a clearly better predictive potential than the IgA class gliadin antibody ELISA. Immunoblots and ELISA blocking studies showed that calcium is needed for the specific antigen-antibody reaction to occur. Double immunofluorescence staining in human umbilical cord with sera from patients with celiac disease and with monoclonal tissue transglutaminase antibodies showed complete overlap. Conclusions: Calcium-activated tissue transglutaminase autoantibody ELISA is highly accurate in detecting untreated celiac disease. Tissue transglutaminase seems to be the target self-antigen for endomysial antibodies.GASTROENTEROLOGY 1998;115:1322-1328     

Production and Characterization of Murine Monoclonal Antibodies Recognizing Conformational and Linear Epitopes Localized on Human IgA2 Molecules

Background: There are two subclasses of human IgA (IgA1 and IgA2) that differ in antigenic properties and in chemical composition. The constant domains of α1 and α2 heavy chains have >95% sequence homology though major structural differences exist in the hinge region. Quantitation of IgA subclass levels depends on the availability of monoclonal antibodies (MAbs) specific for conserved conformational or linear epitopes restricted to each subclass. Objective: To produce, select and characterize monoclonal antibodies specific for human IgA2. Methods: Splenocytes from BALB/C mice immunized with a human IgA2 myeloma protein were fused with SP2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by limiting dilution assay. Antibody (Ab) secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of purified myeloma proteins and some animal sera by ELISA and immunoblotting. The affinity constant (Kaff) was also determined by ELISA. Results: Four murine hybridoma clones designated 2F20G5, 2F20B5, 3F20E3 and 6F20H11 were obtained that secreted MAbs specific for the human IgA2. 2F20G5 and 6F20H11 MAbs react with linear epitope(s) while 2F20B5 and 3F20E3 react with conformational epitope(s) located to human IgA2 subclass. 2F20G5 MAb displays a weak cross-reactivity with monkey and rabbit sera and a strong cross-reactivity with cat and dog sera while the other three MAbs showed no cross-reactivity with the animal sera tested. Conclusion: These MAbs, especially 6F20H11 with high affinity constant (6.03 ×109 M-1) are useful tools for quantitation of human IgA2 subclass levels in various diseases. Cross-reactivity of 2F20G5 MAb with some animalsera suggests phylogenic conservation ofthe epitope recognized by this MAb.     

脂蛋白脂肪酶单克隆抗体的制备及特性

为了研究脂蛋白脂肪酶的构效关系及其在动脉粥样硬化中的病理作用机制，用牛奶纯化的脂蛋白脂肪酸免疫Balb/c小鼠通过杂交瘤技术建立了30余株分泌抗牛脂蛋白脂肪酸单克隆抗体的杂交瘤细胞。对其中2E5、2G10、6D7、8D2、7F4进行特征分析发现：①其抗体类别均为IgG，亚类分别为IgG1、IgG2b和IgG2b、IgG1、IgG1；②抗体效价为5×10-2～2×10-3；③5种单克隆抗体可认为识别三个不同的抗原表位；④West－ern－blot及Dot－blot结果显示各单克隆抗体均能识别纯化的脂蛋白脂肪酸，与人奶均无交叉反应，与牛奶显示不同的结合特征。     

An Enzyme-Linked Immunosorbent Assay for the Quantitative Determination of Anti-D in Plasma Samples and Immunoglobulin Preparations

Here we present an enzyme-linked immunosorbent assay (ELISA) on microtiter plates for the quantitative determination of anti-D. This method is based on the solubilization of red blood cells sensitized with anti-D and the subsequent measurement of immunoglobulin G by ELISA. The measuring range of the assay is 40–5,000 IU/ml and the lowest quantifiable concentration in plasma is 0.5 IU/ml. The interassay relative standard deviation for concentrations above 130 IU/ml ranges from 3.2 to 8.1% and below 50 IU/ml from 10.5 to 19.7%. Comparison of ELISA with automated hemagglutination shows that the results of the two assays correlate well: r = 0.992, n = 26. The assay was validated for donor plasma samples and anti-D immunoglobulin preparations and it can also be used in assessing the severity of Rh (D) hemolytic disease during pregnancy.     

单克隆多克隆抗体酶联免疫吸附法测定血清脂蛋白(a)的比较

应用淋巴细胞杂交瘤技术，建立了抗人血清脂蛋白（ａ）杂交瘤细胞株，对制备的单克隆抗体进行了特异性鉴定和抗原位点测定。建立了多克隆双抗体夹心酶联免疫吸附法、载蛋白（ａ）、载脂蛋白Ｂ双位点酶联免疫吸附法、单克隆单株、混合株双抗体夹心酶联免疫吸附法对脂蛋白（ａ）进行了检测，并对结果进行了分析。认为样本、参考品间载脂蛋白（ａ）的多态型不同可引起结果间的差异。     

Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins

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Monoclonal antibodies to conserved regions of the major core protein (gag24) of HIV-1 and HIV-2.

Five mouse monoclonal antibodies were raised against a recombinant protein comprising the complete sequence of gag24 protein from HTLV-IIIB. All monoclonal antibodies recognized the native protein in enzyme-linked immunosorbant assay (ELISA) and Western blots. All monoclonal antibodies also cross-reacted with an human immunodeficiency virus type 2 (HIV-2) strain in western blots, whereas only one antibody detected HIV-2 p25 in ELISA. By using synthetic peptides, cross-reacting epitopes were mapped and three regions were defined. The conserved immunogenic sites were located in the carboxyterminal region of the protein. Inhibition experiments with human sera showed that this region is also immunogenic in humans.     

Monoclonal anti-D for immunoprophylaxis

Routine antenatal prophylaxis with anti-D has become accepted as desirable, but concerns have been expressed about the adequacies of supply and safety of polyclonal anti-D. Human monoclonal anti-D has been produced using Epstein-Barr virus (EBV)-transformed peripheral B cells, sometimes coupled with fusions to myeloma cell lines. More recently, molecular biology techniques have been used to produce human monoclonal anti-D in a variety of different ways. Many monoclonal antibodies (mAbs) have been characterized for fine specificity and in vitro functional activity in International Workshops. Two mAbs have been shown to cause red cell clearance and immunosuppression in male volunteers. Considerations for the future development of monoclonal anti-D for prophylactic use are reviewed.     

Enzyme-linked immunosorbent serum assay specific for the 7S domain of Collagen Type IV (P4NP 7S): A marker related to the extracellular matrix remodeling during liver fibrogenesis

Aim: The present study describes the ability of a newly developed N‐terminal pro‐peptides of type IV collagen 7S domain (P4NP 7S) competitive enzyme‐linked immunosorbent assay (ELISA) for describing liver fibrosis. The assay applies a monoclonal antibody specific for a PIVNP 7S epitope 100% homologous in the human, rat, and mouse species. Methods: Monoclonal antibodies were raised against selected P4NP 7S specific sequences. Antibodies were screened and a competitive ELISA assay was developed using a selected antibody. The assay was evaluated in relation to technical performance, and in two preclinical liver fibrosis models; the bile duct ligation model (BDL) and the carbon tetrachloride model (CCL4) both performed in rats. Results: A technically robust P4NP 7S ELISA assay using a monoclonal antibody was produced. In the BDL and CCL4 liver fibrosis models it was observed that the P4NP 7S levels were significantly elevated in rat with liver fibrosis as seen by histology (CCL4: 283% elevated in the highest quartile of total hepatic collagen compared with controls, P = 0.001; BDL: 183% elevated at week 4 compared with sham, P < 0.001) and correlated to the amount of hepatic type IV collagen expression in BDL rats (r = 0.49, P < 0.05) in contrast to sham (r = ?0.12). P4NP 7S also correlated to total collagen in CCL4 treated livers (P < 0.001, r = 0.67), however, not in controls (r = 0.04). Conclusions: This newly developed serum assay specific for P4NP 7S was highly related to liver fibrosis and correlated to extent of hepatic fibrosis. This assay may improve fibrosis quantification.