人红细胞非凝集型单抗(自身全血凝集试验试剂)的研制及鉴定

应用杂交瘤技术 ,混合人O型红细胞免疫纯系BALB/C小鼠 ,包被的红细胞作为固相抗原 ,ELISA法筛选出识别红细胞表面共同抗原的抗体 ,然后利用Coombs原理 ,加入抗球蛋白试剂。用玻片法筛选出非凝集型抗体 ,得到 2株稳定分泌上述抗体的杂交瘤并进行初步鉴定 ,单抗上清血凝效价可达 1∶5 0～ 1∶10 0 ,腹水血凝效价为 1∶8× 10 4 ～ 1∶16× 10 4 。另外 ,本文特别讨论了凝集型单抗的Fab段作为非凝集型抗体应用的可能性。     

Enzyme-linked immunosorbent assay (ELISA) using a glycolipid antigen for the serodiagnosis of melioidosis

Abstract The serodiagnosis of melioidosis is commonly performed with tests using protein or polysaccharide as antigen. However, due to the low sensitivity, specificity and difficulty in the preparation of the antigens, more simple, precise and reproducible diagnostic tests were required. A purified glycolipid antigen (GL) which is a specific lipid component of Burkholderia pseudomallei has been used in an ELISA. With this antigen, specific immunoglobulin G (IgG) was detected in 49 out of 50 melioidosis sera. IgG was also detected in 2 out of 185 (Japanese) and 16 out of 181 (Vietnamese) control sera. Thus, the sensitivity was 98.0%, and specificity was 98.9% and 91.1% in the Japanese and Vietnamese sera, respectively. When the ELISA and indirect haemagglutination (IHA) tests were combined, a sensitivity of 100% and specificity of 97.8% were achieved. The advantages of the glycolipid antigen are ease of preparation, stability, high sensitivity and specificity.     

Characterization of the binding specificity ofAnguilla anguilla agglutinin (AAA) in comparison toUlex europaeus agglutinin I (UEA-I)

Using immunochemical and immunohistochemical methods, the binding site ofAnguilla anguilla agglutinin (AAA) was characterized and compared with the related fucose-specific lectin fromUlex europaeus (UEA-I). In solid-phase enzyme-linked immunoassays, the two lectins recognized Fuc1-2Gal-HSA. AAA additionally cross-reacted with neoglycolipids bearing lacto-N-fucopentaose (LNFP) I [H type 1] and II [Le^a] and lactodifucotetraose (LDFT) as glycan moieties. UEA-I, on the other hand, bound to a LDFT-derived neoglycolipid but not to the other neoglycolipids tested. Binding of AAA to gastric mucin was competitively neutralized by Le^a-specific monoclonal antibodies. UEA-I binding, on the other hand, was reduced after co-incubation with H type 2- and Le^y-specific monoclonal antibodies. According to our results, AAA reacts with fucosylated type 1 chain antigens, whereas UEA-I binds only to the 1-2-fucosylated LDFT-derived neoglycolipid. In immunohistochemical studies, the reactivity of AAA and UEA-I in normal pyloric mucosa from individuals with known Lewis and secretor status was analysed. AAA showed a broad reaction in the superficial pyloric mucosa from secretors and non-secretors, but AAA reactivity was more pronounced in Le^(a+b-) individuals. On the other hand, UEA-I stained the superficial pyloric mucosa only from secretor individuals. A staining of deep mucous glands by the lectins was found in all specimens. Both reacted with most human carcinomas of different origin. Slight differences in their binding pattern were observed and may be explained by the different fine-specificities of the lectins.     

Expression of murine early embryonic antigens, SSEA-1 and antigenic determinant of EMA-1, in embryos and ovarian follicles of a teleost medaka (Oryzias latipes)

Stage-specific embryonic antigen-1 (SSEA-1) and the antigenic determinant of monoclonal antibody EMA-1 are expressed in a stage-specific manner in mouse early embryos. To study whether these antigens generally exist in fish, expression of the antigens was examined in embryos, ovarian follicles, and adult tissues of a teleost medaka (Oryzias latipes), using immunohistochemical techniques. In 1-cell-stage embryos, these carbohydrate antigens were found in numerous cytoplasmic granules in the blastodisc and the cortical cytoplasm. These granules gradually decreased in number as the embryos developed. In 4-cell-stage embryos, the antigens appeared on the cleavage planes and were located on the cleavage planes within the blastoderm in the following cleavage stages. In blastula-stage embryos, the expression was ubiquitously found on the cell surface of blastomeres. At the mid-gastrula stage, the antigens were restricted to the enveloping layer, yolk syncytial layer, and cortical cytoplasm, but were rarely found in deep cells that contribute to formation of the embryonic body. In later-stage embryos and adult fish, the antigens were located in various tissues. In ovarian follicles, the antigens were found in granules of oocytes and granulosa cells. These observations were basically consistent with those in mice; however, expression in 1-cell-stage embryos and ovarian follicles has not been observed in mice. This unexpected finding suggests that the antigens are produced in granulosa cells and transferred to 1-cell-stage embryos via oocytes, and that the antigens involved in the early developmental process are maternally prepared in teleosts.     

Detection of filarial antigen by inhibition enzyme linked immunosorbent assay using fractionatedBrugia malayi microfilarial excretory secretory antigen

Detection of filarial antigen in blood or urine samples would provide an accurate indication of active infection. The absence of a simple, well established animal model and limitations in getting the required amount of parasite material from human sources have been the main obstacles for the diagnosis ofWuchereria bancrofti infection. An inhibition ELISA has been developed for detection of filarial antigen using a partially purifiedBrugia malayi mf ES antigen (BmE DE1) and its affinity purified antibodies. Filarial antigen was detected in the sera of 88% of microfilaraemic, 60% of chronic filarial, 17% of endemic normal and none of the non- endemic normal subjects. The sensitivity and specificity of the assay were 88% and 89% respectively. Moreover, undiluted urine samples from 82% of microfilaraemic and 17% of endemic normal, but none of the non- endemic normal samples showed the presence of filarial antigen. With the limitations on the availability of sufficient homologous parasite materials, the inhibition ELISA using BmE DE1 and anti BmE DE1 antibodies shows promise for the detection of active infection in bancroftian filariasis in man. Moreover, its detection in urine makes it more possible to test patients in field areas.     

Characterization of an enterobacterial common antigen (ECA) epitope recognized by anti-ECA-tetanus toxoid conjugate serum

The antigenic reactivity of both native and chemically modified enterobacterial common antigen (ECA) with anti-ECA-tetanus toxoid (TT) conjugate serum was investigated. The results obtained suggest that reduction of the carboxyl group of the mannosaminuronic acid component of ECA diminishes, but does not destroy its antigenic reactivity. Each of the sugar components were found to contribute to its reactivity with anti-ECA-TT conjugate serum, indicating that the trisaccharide repeating unit represents the ECA epitope. A nonasaccharide (trimer of the ECA repeating unit) inhibited antibody binding better than the hexasaccharide dimer, a finding which suggests that oligosaccharide conformation also makes a contribution to its inhibitory activity.     

A rat oocyte differentiation antigen (OA-1) detected by a monoclonal antibody

Cell surface antigenic changes associated with differentiation of the rat oocyte and early embryo have been demonstrated with a monoclonal antibody (anti-OA-1). Antigen is first detectable coincident with initiation of oocyte growth, is a constant feature of all growing oocytes and displays a redistribution during meiotic maturation. Following fertilization, antigen is detectable on the surface of the embryo through the four-cell stage. This first monospecific marker for the rat oocyte and embryo should prove useful in probing structure/function relationships in oocyte growth, meiotic maturation fertilization, and/or early embryonic development.     

Identification of BCP-20 (FBXO39) as a cancer/testis antigen from colon cancer patients by SEREX

Cancer/Testis (CT) antigens are considered promising target molecules for immunotherapy. To identify potential CT antigens, we performed immunoscreening of a testis cDNA library with sera from colon cancer patients by SEREX. We isolated 114 positive cDNA clones comprising 90 different antigens, designated BCP-1 through BCP-90. Quantitative real-time and conventional RT-PCR analysis showed that BCP-20, -33, and -41 antigens were expressed strongly only in a normal testis and detected in 22 cases (39%), 12 cases (21%), and 17 cases (30%), respectively, from 57 colon tumors. BCP-20 was also detected in various cancer cell lines including breast, colon, hepatoma, renal, thyroid anaplastic, ovary, sarcoma, and lung. By ELISA analysis, anti-BCP-20 antibody was detected in 3 of 50 colon cancer and 1 of 24 gastric cancer patients while healthy donors were three positive (3/50). But the BCP-20 antibody levels of patients with colon cancer showed significantly higher titers than those of healthy donors. These data suggest that the BCP-20 gene is a new CT antigen and may be useful for diagnosis and immunotherapy.     

Characterization of serum and mucosal antibody responses in white sturgeon (Acipenser transmontanus Richardson) following immunization with WSIV and a protein hapten antigen

Serum and cutaneous mucus antibodies were monitored in white sturgeon for 15 weeks following intraperitoneal immunization. Ten fish were immunized (50 microg) with white sturgeon iridovirus (WSIV) or white sturgeon gonad (WSGO) tissue culture cells emulsified with or without FCA. An additional group was immunized with FITC:KLH+FCA. Fish were booster immunized at 6 weeks. Fish immunized with FITC:KLH+FCA produced significant serum antibodies to FITC by 6 weeks and this response peaked at 12 weeks (average titer 31,000). Mucosal antibodies to FITC were first detected at 12 weeks and significantly elevated by 15 weeks (average titer 18). Anti-WSIV antibody titers were detected in the serum by 9 weeks in fish immunized with WSIV and WSIV+FCA, but only a small number responded to immunization. At 15 weeks, four fish immunized with WSIV produced serum antibodies (average titer 838) and one fish immunized with WSIV+FCA had a serum titer of 1600. Mucosal anti-WSIV antibody titers of 8 and 16 were observed in two fish from the WSIV group at 12 weeks while four different fish from this group responded at 15 weeks (average titer 4). Western Blot using a monoclonal antibody confirmed immunoglobulin in mucus, and specificity to WSIV was further demonstrated by immunocytochemistry using serum from fish immunized with WSIV. Specific antibody was not detected in mucus of fish immunized with WSIV+FCA, WSGO, or WSGO+FCA. Collectively, these experiments demonstrate that white sturgeon can generate a specific antibody response following immunization, and is the first report showing mucosal immunoglobulin is present in this species.     

Detection of a new non-MHC alloantigenic system (SLD) in pigs

A new, non-MHC cell membrane leucocyte alloantigen was detected in pigs by the complement dependent lymphocytotoxic technique. The new leucocyte system was designated SLD. Its product antigen SLD-1 was demonstrated to segregate independently of the SLA, SLB, SLC, A and E antigens. Family studies supplied evidence of a dominant inheritance of SLD-l. Since an allelic antigen could not be demonstrated, only two alleles for this locus are reported, namely SLD¹ and SLD^- . No evidence of linkage was detected between the above mentioned leucocyte alloantigenic systems and SLD. The antigen was detected on enriched suspensions of T and B cells from peripheral blood, but it was not detected on erythrocytes, granulocytes and thrombocytes.     

Slow-binding inhibition of peptide deformylase by cyclic peptidomimetics as revealed by a new spectrophotometric assay

A new spectrophotometric/fluorimetric assay for peptide deformylase (PDF) has been developed by coupling the PDF reaction with that of dipeptidyl peptidase I (DPPI) and using N-formyl-Met-Lys-AMC as substrate. Removal of the N-terminal formyl group by PDF renders the dipeptide an efficient substrate of DPPI, which subsequently removes the dipeptidyl units to release 7-amino-4-methylcoumarin as the chromophore/fluorophore. The PDF reaction is conveniently monitored on a UV-Vis spectrophotometer or a fluorimeter in a continuous fashion. The utility of the assay was demonstrated by determining the catalytic activity of PDF and the inhibition constants of PDF inhibitors. These studies revealed the slow-binding behavior of a previously reported macrocyclic PDF inhibitor. This method offers several advantages over the existing PDF assays and should be particularly useful for screening PDF inhibitors in the continuous fashion.     

pH6 antigen (PsaA protein) of Yersinia pestis, a novel bacterial Fc-receptor

Abstract It was found that recombinant pH6 antigen (rPsaA protein) forming virulence-associated fimbriae on the surface of Yersinia pestis at pH 6.7 in host macrophage phagolysosomes or extracellularly in abscesses such as buboes, is a novel bacterial Fc-receptor. rPsaA protein displays reactivity with human IgG1, IgG2 and IgG3 subclasses but does not react with rabbit, mouse and sheep IgG.     

Identification of a major hepatitis B core antigen (HBcAg) determinant by using synthetic peptides and monoclonal antibodies

To map the location of hepatitis B core and e Ag (HBcAg and HBeAg) on the hepatitis B virus core particle, we produced and analyzed four synthetic peptides which correspond to the most hydrophilic regions of the core P22 protein. Each peptide was tested in an ELISA for the ability to inhibit the binding between rHBcAg or rHBeAg and either polyclonal or monoclonal anti-HBc or anti-HBe antibodies. The former comprised 20 antisera positive for anti-HBc (anti-HBs and anti-HBe negative) and five antisera positive for anti-HBe and anti-HBc; the latter included three anti-HBc mAb developed in independent laboratories: G6F5, C51B10, and F8, as well as two anti-HBe mAb, E2 and E6. These experiments revealed the presence of a major HBcAg epitope expressed on C3, a peptide which covers amino acids 107-118 and reacted with all polyclonal and monoclonal antibodies tested. Another peptide, C2, sequence 73-85, reacted with 26% of human antisera but none of the anti-HBc mAb. None of the peptides seemed to express HBeAg activity because they do not cause any significant inhibition of the HBeAg/anti-HBe reaction. These data indicate the expression of an immunodominant HBcAg determinant on a linear dodecapeptide and argue against a strict conformation dependency of this Ag.     

Evaluation of a new apolipoprotein(a) isoform-independent assay for serum Lipoprotein(a)

The risk factor, Lipoprotein(a), [(Lp(a)], has been measured in numerous clinical studies by a variety of immunochemical assay methods. It is becoming apparent that for many of these assays antibody specificity towards the apolipoprotein(a) [apo(a)] repetitive component [the kringle 4 - type 2 repeats] and apo(a) size heterogeneity can significantly affect the accuracy of serum Lp(a) measurements. To address this issue, we investigated whether our current in house Lp(a) [Mercodia] assay showed such bias compared to a recently available assay [Apo-Tek], claiming to possess superior capability for isoform-independent measurement of Lp(a). Levels of Lipoprotein(a) by both Apo-Tek and Mercodia assays correlated inversely with apo(a) isoform sizes. No significant differences were observed between assays in ranges of Lp(a) concentration within each isoform group. The Mercodia assay exhibited similar isoform-independent behaviour to that of Apo-Tek for e quantitation of serum Lipoprotein(a). Essentially identical results were obtained by the two methods, suggesting that Mercodia assay's capture monoclonal antibody also (as is the case for Apo-Tek) does not recognize the kringle 4-type 2 repetitive domain of apo(a). Correlation of Lp(a) concentrations in patient specimens between Apo-Tek and Mercodia assays showed good agreement, although an overall higher degree of imprecision and non-linearity was noted for the Apo-Tek procedure. A change-over to the Apo-Tek assay would therefore not improve on our current assessment of risk contribution from Lp(a) for atherosclerotic vascular disease in individuals with measurable levels of circulating Lipoprotein(a).     

Characterization of a monoclonal antibody reactive with a glycolipid antigen expressed by tumorigenic and certain immortalized,non-tumorigenic rat esophageal epithelial cell lines

A monoclonal antibody (mAb 5G) was produced against a tumorigenic rat esophageal epithelial cell line, designated B2T. Using an enzyme-linked immunosorbent assay, immunofluorescence assay (IFA), thin-layer chromatography (TLC) and immunoperoxidase staining, it was found that mAb 5G reacted specifically with a glycolipid antigen expressed by three tumorigenic rat esophageal epithelial cell lines, and two out of the three nontumorigenic, immortalized rat esophageal epithelial cell lines tested; but did not react with primary cultures of normal rat esophageal epithelial cells or fibroblasts. mAb 5G did not bind to rat respiratory tract carcinoma cell lines, to immortalized rat tracheal epithelial cell lines, or to primary cultures of normal rat tracheal epithelial cells. In addition, mAb 5G did not react with any of the human or mouse cell lines tested. In IFA experiments, mAb 5G stained imprints prepared from in vivo propagated B2T tumor tissues, but did not react with normal rat esophageal, tracheal, lung, liver, and kidney tissues. The antigen was identified by TLC as a neutral glycolipid, consisting of two bands, withR _F = 0.45 and 0.41, which migrated in proximity to the ceramide trihexoside standard on TLC plates. Densitometric scanning of the antigen bands indicated that the tumorigenic rat esophageal cell lines possessed 50%–90% more mAb-5G-reactive antigen than the nontumorigenic esophageal cell lines. The results show that mAb 5G reacts specifically with a glycolipid antigen expressed by tumorigenic and certain non-tumorigenic, immortalized rat esophageal epithelial cell lines that might be at the late stages of transformation and early malignancy.     

Characterization of a human granulocyte differentiation antigen (CDw15) commonly recognized by monoclonal antibodies

Many different anti-human granulocyte monoclonal antibodies recognize the same carbohydrate antigen which contains the trisaccharide 3-fucosyl-N-acetyllactosamine. The antigen is expressed mainly on two cell surface glycoproteins of molecular weights around 105 K and 160 K which are apparently not members of the LFA-1 family of proteins. Although specific for granulocytes in blood, the antigen is expressed on a wide range of non-haemopoietic cell types.     

Solid-phase synthesis of a pentavalent GalNAc-containing glycopeptide (Tn antigen) representing the nephropathy-associated IgA hinge region

Incomplete or aberrant glycosylation leading to Tn antigen (GalNAcα1-Ser/Thr) expression on human glycoproteins is strongly associated with human pathological conditions, including tumors, certain autoimmune diseases, such as the idiopathic IgA nephropathy, and may modulate immune homeostasis. In addition, the Tn antigen is highly expressed by certain pathogens and plays a role in host-pathogen interactions. To enable experimental approaches to study interactions of the Tn antigen with the immune system and analyze anti-Tn antibody responses in infection or disorders, we generated a Tn-expressing resource that can be used for high-throughput screening. In consideration of IgA nephropathy in which the hinge region is incompletely glycosylated, we used this hinge sequence that encodes five potential glycosylation sites as the ideal template for the synthesis of a Tn antigen-expressing glycopeptide. Inclusion of an N-terminal biotin in the peptide enabled binding to streptavidin-coated ELISA plates as monitored using Helix pomatia agglutinin or anti-Tn monoclonal antibody. We also found that the biotinylated IgA-Tn peptide is a functional acceptor for β1-3-galactosylation using recombinant T-synthase (β1-3-galactosyltransferase). Besides its immunochemical functionality as a possible diagnostic tool for IgA nephropathy, the peptide is an excellent substrate for glycan elongation and represents a novel template applicable for glycan-antigen-associated diseases.     

Characterization of a somatomedin (insulin-like growth factor) synthesized by fetal rat liver organ cultures.

Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.     

Identification and purification of a stress associated nuclear carbohydrate binding protein (Mr 33000) from rat liver by application of a new photoreactive carbohydrate probe

A photoreactive -D-glucose probe has been designed for the specific detection of carbohydrate binding proteins (CBPs). The probe consists of four parts: (i) an -D-glucose moiety; (ii) the digoxigenin tag; (iii) the photoreactive cross-linker; and (iv) the lysyl-lysine backbone. After incubation with lectins in the dark, the probe is activated and cross-linked to the CBPs after being treated by several flashes.Using this method we have identified a new -D-glucose CBP ofM _r=33000, termed CBP33, in the nuclei of rats exposed to transient immobilization stress. Monoclonal antibodies were raised against the partially purified protein and subsequently used to enrich CBP33. It was purified (>2400-fold) to apparent homogeneity from a 0.6M nuclear salt extract by two subsequent affinity chromatography steps (antibody-affinity as well as -D-glucose affinity column).Abbreviations BSA bovine serum albumin - CBP carbohydrate binding protein - DIG digoxigenin - Gal galactose - Glc glucose - Lys lysine - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate.