Corticotropin Releasing Factor (CRF): Immunocytochemical localization and Radioimmunoassay (RIA)

Recent isolation, structural identification, and synthesis of ovine CRF has made possible the generation of specific antibodies against this hypothalamic peptide. Two fragments of the amino acid sequence corresponding to ovine CRF (CRF 37-41 and CRF 22-41), as well as the full sequence of 41 residues (CRF 1-41), synthesized in our laboratories by solid-phase methods, were coupled to bovine serum albumin (BSA) with glutaraldehyde. New Zealand white rabbits were immunized with the emulsified mixtures of peptide-BSA conjugates and Freund's adjuvant as immunogens. The specificity of the generated antibodies was studied by agar-gel diffusion, absorption tests in the immunohistochemical system, and with the aid of displacement curves in RIA. ¹²⁵I-Tyr(35)-CRF 36-41 and ¹²⁵I-Tyr(0)-CRF 1-41 were used as radioligands in the RIA. The minimum detectable dose was 20 pg. The linearity observed in RIA for immunoreactive CRF in extracts of rat hypothalami, together with the immunocytochemical findings in the rat brain, indicate the presence of substance(s) immunologically indistinguishable from CRF. Immunohistochemistry with the peroxidase-antiperoxidase (PAP) technique detected the following CRF-immunoreactive structures in vibratome sections of hypothalami of colchicine-treated rats: CRF-containing cell bodies were observed mainly in smaller neurons of the paraventricular nucleus. CRF-positive nerve fibers and/or terminals were present in the external zone of the median eminence, with some immunoreactive CRF also present in the internal zone. The CRF-positive terminals were localized in the central regions of the median eminence. These morphological data reinforce the view that this polypeptide plays a physiological role in the control of ACTH release.     

The photoactive yellow protein from Ectothiorhodospira halophila as studied with a highly specific polyclonal antiserum: (intra)cellular localization, regulation of expression, and taxonomic distribution of cross-reacting proteins.

A rabbit antiserum was raised against the photoactive yellow protein (PYP) from Ectothiorhodospira halophila and purified by adsorption experiments to obtain a highly specific polyclonal antiserum. This antiserum was used to obtain the following results. (i) In E. halophila, PYP can be isolated from the fraction of soluble proteins. In the intact cell, however, PYP appeared to be associated with (intra)cytoplasmic membranes, as was concluded from analysis of immunogold-labelled thin sections of the organism. (ii) The regulation of expression of PYP was studied by using dot blot assays, Western blotting (immunoblotting), and rocket immunoelectrophoresis. Under all conditions investigated (light color, salt concentration, and growth phase), PYP was expressed constitutively in E. halophila. However, when Rhodospirillum salexigens was grown aerobically, the expression of PYP was suppressed. (iii) A large number of prokaryotic microorganisms contained a single protein, with an apparent size of approximately 15 kDa, that cross-reacted with the antiserum. Among the positively reacting organisms were both phototrophic and chemotrophic, as well as motile and nonmotile, organisms. After separation of cellular proteins into a membrane fraction and soluble proteins, it was established that organisms adapted to growth at higher salt concentrations tended to have the cross-reacting protein in the soluble fraction. In the cases of R. salexigens and Chromatium salexigens, we have shown that the cross-reacting protein involved is strongly homologous to PYP from E. halophila.     

Determination of immunoreactivity of doxorubicin antibody immunoconjugates by a Le(y) competitive RIA.

Many monoclonal antibody-drug immunoconjugates have been evaluated for their ability to deliver cytotoxic drugs to tumors. It is essential to establish that the ability of the conjugates to bind antigen, i.e. their immunoreactivity, is not adversely affected by the drug conjugation procedure. We have described herein a measurement of the immunoreactivity of BR96-DOX, a conjugate comprised of BR96, a chimeric monoclonal antibody specific for the Le(y) tetrasaccharide commonly expressed on human carcinomas, and doxorubicin, an anticancer agent in widespread clinical use. We have employed a competitive RIA, in which microtiter wells were coated with synthetic Le(y) conjugated to human serum albumin and then incubated with 125I-labeled antibody BR96 in the presence of test conjugate or intact BR96 mAb. The test conjugates were found to compete as effectively as unconjugated BR96. This assay is highly applicable to QC processes with the intra-assay CV = 2.0% and the interassay CV = 4.3%.     

Affinity in radioimmunoassay of antibody cross-reactive with carcinoembryonic antigen (CEA) and colon carcinoma antigen-III (CCA-III).

Several antisera raised against purified carcinoembryonic antigen (CEA) were evaluated for their content of antibody cross-reactive with colon carcinoma antigen-III (CCA-III). All antisera gave a reaction of partial identity between CEA and CCA-III and demonstrated a high titer in CEA radioimmunoassay (RIA). Between 50 and 70% of the CEA RIA activity was removed, however, by absorption with soluble CCA-III or adsorption onto CCA-III-containing immunoadsorbents. Immunoadsorbent retained antibody gave a line of complete identity between CEA and CCA-III. Purified CCA-III (2 mug) only partially depressed CEA binding by this common site antibody, whereas nanogram quantities of CCA-III inhibited the reaction between specific CCA-III antibody and radioiodinated CCA-III. In addition, low levels of CEA were equally effective in depressing CEA binding by the common site or CEA-specific antibody. The higher affinity in RIA of the common site antibody for CEA over CCA-III suggests that the common determinant expressed on CEA is stereochemically different from that on CCA-III. The results further demonstrate that interference by plasma CCA-III is not a significant factor in the measurement of CEA by RIA.     

Mouse pituitary tumor mRNA directed cell-free synthesis of polypeptides that are cross-reactive with adrenocorticotropic hormone antiserum.

A wheat germ extract was used to translate mRNA isolated from the mouse pituitary tumor AtT-20. This RNA directed the synthesis of a product which was precipitated with antiserum specific for the synthetic adrenocorticotropic hormone polypeptide β(1–24)(Synacthen). Analysis of the products by the radioimmunoassay technique also indicated the presence of an immunoreactive adrenocorticotropic hormone-like material. The molecular weight of this product was 31,000 as determined by electrophoresis in sodium dodecylsulphate-polyacrylamide gels. These findings suggest that the 31,000 species may be the primary gene product in adrenocorticotropic hormone biosynthesis.     

Assessment of plasma profile of pregnancy-associated glycoprotein (PAG) in sheep with a heterologous (anti-caPAG55+59) RIA and its potential for diagnosing pregnancy

The purpose of the present investigation was to generate pregnancy associated glycoprotein (PAG)-profiles throughout pregnancy in a heterogenous sample of sheep using a radioimmunoassay with a heterologous antibody (anti-caPAG(55+59), #708) and utilize them for the purpose of pregnancy detection. From 2 weeks after the introduction of males into the breeding herd until 4 weeks after parturition, weekly blood samples were collected from 66 pregnant and 25 non-pregnant ewes of various breeds. Between 3 and 5 weeks after conception, plasma PAG levels increased, remained almost stable until week 17, then continued to increase, culminating in a drastic surge during the last 2 weeks of pregnancy. By 4 weeks of gestation, the plasma PAG level exceeded the level typical for non-pregnant ewes by five standard deviations, permitting a reliable pregnancy diagnosis. Plasma PAG levels were higher in twin-bearing ewes than in ewes carrying a single lamb, differences getting more evident as pregnancy proceeded. Neither breed and parity of the mother nor sex and weight of lambs borne exerted a significant effect. The heterologous assay system utilizing a caprine antibody proved to deliver results that are more consistent and less depending on various variables than those used in other studies. It may be concluded that, at the present state of development, the assay provides a reliable means of diagnosing pregnancy in sheep from the 4th week after they have been bred onward.     

PRAS40(Ser183)磷酸化多克隆抗体的制备及检测

PRAS40为富含脯氨酸、分子量为40kD的Akt底物蛋白,能够与雷怕霉素哺乳动物细胞靶点复合物1(mTORC1)结合,其苏氨酸183位点(Ser183)可被mTORC1磷酸化。为了制备PRAS40(Ser183)磷酸化多克隆抗体,本实验通过蛋白疏水性抗原性分析设计多肽抗原,用其免疫家兔获得抗血清,ELISA检测其效价为1:10000;Western blotting法检测发现,通过rProtein A Sepharose亲和层析纯化并经非磷酸化的抗原条吸附处理后的抗体可以明显提高磷酸化抗体的特异性;用PRAS40抗体及PRAS40(Ser183)磷酸化抗体对正常细胞HL7702、HEK293及肿瘤细胞HepG2、A549、S180的检测显示:磷酸化的Ser183在不同细胞中表达差异不显著,而在经细胞饥饿处理的HEK293细胞中却明显观察到了S183磷酸化水平随氨基酸含量降低而减弱的现象。因此,本实验所制备的抗体可用于PRAS40(Ser183)磷酸化位点的功能研究。     

Identification of the epitope for a highly cross-reactive monoclonal antibody on the major sigma factor of bacterial RNA polymerase.

A highly cross-reactive monoclonal antibody (MAb), 2G10, was found to react in a conserved region of Escherichia coli RNA polymerase sigma70. The epitope was localized to amino acids 470 to 486, which included part of conserved region 3.1. The epitope for MAb 3D3, a MAb which maps close to the 2G10 epitope, was also determined.     

Radioimmunoassay for the detection of hepatitis e antigen (HBeAG) and antibody (anti-HBe).

A solid phase micro-immunoradiometric assay (micro-SPIRA) for the detection of hepatitis e antigen (HBeAg) and antibody has been developed. Chimpanzee anti-HBe/2 was developed by repeated immunizations with purified antigen containing HBeAg/1 and HBeAg/2. An anti-HBe/2 titer of 1:4 was determined by immunodiffusion (ID) analysis. Anti-HBe/1 was not detected. The anti-HBe IgG used in the assay was purified from plasma by a combination of DEAE-cellulose and affinity chromatography. The sensitivity of the micro-SPIRA for antigen and antibody was 193 ng/ml and 65 ng/ml, respectively. By comparing relative endpoint titers obtained by ID to micro-SPIRA, it was determined that micro-SPIRA for antigen and antibody is 320 and greater than 1300 times more sensitive, respectively, than ID. The specificity of the assay was ascertained by the examination of various non-B specimens. The application of the assay to a panel of 50 hepatitis B surface antigen (HBsAg)-positive specimens resulted in an increase in positivity of 18% for antigen and 22% for antibody.     

Apparent cleavage of poly(ADP-ribose) polymerase in non-apoptotic mouse LTA cells: An artifact of cross-reactive secondary antibody

Proteolytic cleavage of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) to fragments of 89 kD and 24 kD is widely observed during apoptotic cell death. In the present study, labelling of a M_r 89000 polypeptide was demonstrated in untreated mouse LTA cells during probing of immunoblots with C-2-10 monoclonal anti-PARP antibody. The source of the labeling was traced to the secondary antibody preparation, which labeled a M_r ~89000 polypeptide in murine LTA cells but not in human cells. These observations indicate that assessment of PARP cleavage must be (1) performed with appropriate controls when new cell lines are investigated and (2) carefully interpreted in light of additional biochemical or morphological data demonstrating apoptotic changes.     

Polyomavirus small t antigen: overproduction in bacteria, purification, and utilization for monoclonal and polyclonal antibody production

Polyomavirus small t antigen was purified from genetically engineered Escherichia coli and used as the immunogen for the production of polyclonal and monoclonal antibodies. A new series of plasmids for increased expression of polyomavirus T antigens or a T antigen-beta-galactosidase fusion protein was constructed by replacing sequences coding for the ribosome-binding site of previously published plasmids with a chemically synthesized sequence that has a higher degree of complementarity to the 3' end of the 16S rRNA. Cells expressing the fusion protein from the plasmid with the synthetic sequence contained 5- to 10-fold more fusion protein after a 3-h induction than did control cells. Pulse-labeling of cells bearing the new plasmids revealed that the T antigens were synthesized at high levels after induction: 10% of total synthesis for small t; 15% for Py-1387T middle T, a truncated mutant of middle T; and probably 1 to 5% for middle T. Small t and Py-1387T middle T, but not wild-type middle T, were seen as minor bands in total cell protein analyzed on sodium dodecyl sulfate-polyacrylamide gels stained with Coomassie blue. A simple, rapid procedure for purification of bacterial small t from the pellet of sonicated bacteria yielded 1 to 2 mg of small t per liter of bacterial culture at 80 to 90% homogeneity. High-titer polyclonal rabbit antisera raised against purified small t recognized all three T antigens and were suitable for immunoaffinity purification of middle T. Mouse monoclonal antibodies raised against bacterial small t were of four classes, immunoprecipitating either all three polyomavirus T antigens, small t and middle T only, primarily small t, or middle T and large T in preference to small t. One of the latter monoclonal antibodies also immunoprecipitated large T but not small t of simian virus 40, suggesting that the site recognized by this antibody may be functionally important. None of the monoclonal antibodies yielded an immunoprecipitate active in phosphorylating middle T in vitro.     

Production and characterization of a monoclonal antibody cross-reactive with most group A trichothecenes.

A monoclonal antibody cross-reactive with most group A trichothecenes was produced by fusion of P3/NS-1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse that had been immunized with 3-acetyl-neosolaniol-hemisuccinate conjugated to bovine serum albumin. One stable clone, H159B1D5, which produced monoclonal antibody that bound with both T-2 toxin and diacetoxyscirpenol (DAS) was obtained after subcloning. Enzyme-linked immunosorbent assay (ELISA) revealed that the antibody belongs to the immunoglobulin G1 (kappa chain) isotype and had binding constants of 2.81 x 10(9), 1.05 x 10(9), and 1.57 x 10(8) liters per mole for T-2 tetraol tetraacetate, T-2 toxin, and DAS, respectively. The relative cross-reactivities of the antibody with T-2 tetraol tetraacetate, T-2 toxin, and DAS were 200, 100, and 20, respectively, with tritiated T-2 toxin as the marker ligand. The relative cross-reactivities for the above toxins were 667, 100, and 73, respectively, with tritiated DAS as the marker ligand. No cross-reaction with HT-2 and deoxynivalenol triacetate was observed in either system. By using this monoclonal antibody, an indirect ELISA for analysis of T-2 toxin was also developed. The linear portion of the standard curve for analysis of T-2 toxin in each analysis by radioimmunoassay and ELISA was in the range of 0.1 to 2 ng and 0.05 to 1.0 ng, respectively.     

Characterization of a polyclonal anti-peptide antibody to the angiotensin II type-1 (AT1) receptor.

A polyclonal antibody has been prepared against a synthetic peptide corresponding to amino acids 14-23 of the angiotensin II type-1 (AT1) receptor. The antibody is of high titer and mono-specific. Western blot analysis of membranes from rat liver, kidney, and adrenal gland showed that the antibody specifically recognizes a protein band of MW 70,000 whose amounts are highest in the liver, followed by kidney and adrenals. In addition, a relatively less prominent band of MW 95,000 was also detected. The relative distribution of this protein correlates well with the values obtained for [3H]-DuP753 binding and AT1 receptor mRNA.     

Characterization of a polyclonal antibody to human pituitary tumor transforming gene 1 (PTTG1) protein.

Pituitary tumor transforming gene 1 (PTTG1), recently cloned from human testis, is a potent oncogene that is expressed in most tumors. However, assessment of its potential value as a prognostic marker is dependent on the development of a suitable antibody. We have developed a rabbit polyclonal antibody, SK601, that is highly specific for the PTTG1 gene product using recombinant PTTG1 protein (24 kD) containing an N-terminal His(6) tag as the immunogen. The antiserum is capable of detecting recombinant PTTG1 protein in ELISA assays at a titer of 1:100,000. Use of the antibody as the probe in Western blotting analyses revealed a single band with the anticipated relative molecular weights of 52 kD from E. coli expressing the GST-PTTG1 recombinant protein, and 56 kD from COS-7 cells transfected with the PTTG1-GFP chimeric construct. A single band with a relative molecular weight of 28 kD was observed in extract of COS-7 cells transfected with PTTG1 cDNA. The antiserum immunoprecipitated a protein of relative molecular weight of 56 kD from the extracts of COS-7 cells transfected with the PTTG1-GFP chimeric construct. Immunohistochemical analysis of COS-7 cells transfected with this construct confirmed that the antibody detected and was specific for expressing the PTTG1-GFP recombinant protein. Screening of various normal human tissues (testis, ovary, and breast) by immunohistochemistry indicated that these tissues did not exhibit staining with the exception of testis, a tissue that had previously been shown to express PTTG1 mRNA. In contrast all of the tumor tissues (testicular tumor, ovarian tumor, and breast tumor) that were assessed exhibited intense staining. The results suggest that antiserum SK601 is highly specific for the PTTG1 protein and therefore should prove useful in further analysis of the expression and interactions of this protein, including its potential application as an immunohistochemical marker of human tumors.     

Immunohistochemical detection of human epidermal growth factor in submandibular glands and their tumors using a polyclonal antiserum and a monoclonal antibody

Summary We applied immunohistochemical procedures to detect hEGF in salivary glands and pleomorphic adenomas of salivary-gland origin using a polyclonal hEGF antiserum and a monoclonal antibody against hEGF synthesized by applying the synthetic gene technique using Escherichia coli. In normal salivary glands, hEGF was mainly localized in the ductal system (i.e., intercalated, striated, and excretory ducts). The staining intensity and intracellular localization exhibited some variation depending on the fixative used. When a polyclonal hEGF antiserum was used for immunostaining, slight background staining was observed in sections prepared using the fixatives tested. Therefore, precise localization of hEGF was obtainable only in formalin-fixed sections using the monoclonal antibody against hEGF. In pleomorphic adenomas, positive hEGF staining was seen on the luminal side of tumors and in cells of ductal origin; no reactivity was present on the outer side of tumors or in cells of myoepithelial origin. Occasionally, long, spindleshaped tumor cells and chondroidally changed tumor cells also exhibited positive staining for hEGF.     

A monoclonal anti-type II collagen antibody with cross-reactive anti-Ig activity specific for the F(ab')2 fragment

An IgG2a hybridoma antibody (BC-10) was obtained by a myeloma fusion with lymphocytes from B10.RIII mice immunized against native bovine type II collagen. This anti-collagen monoclonal exhibited extensive cross-reactivity with several type II collagen species. BC-10 was found to have self-associating properties, but not the specificity of a typical IgG rheumatoid factor, inasmuch as this mAb bound to F(ab')2 fragments of itself and of normal mouse IgG. Self binding was inhibited by the association of BC-10 with type II collagen, and inhibition assays indicated that antibodies with the capacity to inhibit BC-10 binding to collagen were present in the sera from B10.RIII arthritic mice, but not from DBA/1 LacJ arthritic mice. Joint inflammation and histopathologic features consistent with arthritis were observed in mice injected with the BC-10 hybridoma.