Characterization and application of a radioimmunoassay for beta-endorphin using an antiserum with negligible cross-reactivity against beta-lipotropin

High avidity antisera against β-endorphin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}\text{)}$ were obtained in two of five rabbits immunized with unconjugated synthetic human $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. One of these antisera (K-7762) cross-reacted 1.5% on a molar basis with β-lipotropin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}\text{)}$ and did not recognize leucine-enkephalin in a concentration as high as 0.2 mmol/l. The cross-reaction with methionine-enkephalin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}\text{61–65)}$ was 9%, while that with α-endorphin ($\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}$ 61–76) was 69%. This implied that the specific recognition site was in the amino-terminal region of $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. Although this sequence is present in $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}$ it was poorly recognized by the antiserum, suggesting that the free amino-terminal is essential. This interpretation was supported by the finding that $\text{α-N-}\text{acetyl}\text{-β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was equally poorly recognized by the antiserum. The sensitivity of the radioimmunoassay was 1.9 pmol/l. $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was not detectable (< 3 pmol/l) in 26 of 27 extracted plasma samples in healthy blood donors, in one it was 5 pmol/l. In five of six patients with an enlarged sella turcica, but without clinical and laboratory evidence of pituitary dysfunction, $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was detectable ($\text{5 ± 3}\text{pmol/l}$; mean ± S.D.) after metyrapone. $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was elevated in Addison's disease (23, 54 and 76 pmol/l), Nelson's syndrome (37, 39 and 109 pmol/l), ectopic ACTH production (27, 59 and 76 pmol/l), but only detectable in one of three samples from patients with Cushing's disease (7 pmol/l). Gel chromatography of extracts of porcine pituitary revealed only one immunoreactive peak co-eluting with synthetic human $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. The specificity of the antiserum K-7762 was such that the $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ concentration in plasma extracts could be reliably estimated by radioimmunoassay without prior chromatography.     

A new radioimmunoassay for serum 16 alpha-hydroxyandrost-4-ene-3,17-dione with specific antiserum

A radioimmunoassay system for serum 16 alpha-hydroxyandrost-4-ene-3,17-dione was developed with the use of rabbit antiserum against 16 alpha-hydroxyandrost-4-ene-3,17-dione-3-(O-carboxymethyl)oxime which was conjugated with bovine serum albumin. The antiserum was highly specific for 16 alpha-hydroxyandrost-4-ene-3,17-dione, with cross reactions to other steroids being less than 0.8% except for androst-4-ene-3,17-dione(3.4% cross reaction). Use of LH-20 column chromatography, however, clearly separated these two steroids. Pregnancy sera were measured with this assay system after an addition of labelled internal standard, extraction and separation by column chromatography. The lower limit of detection for 16 alpha-hydroxyandrost-4-ene-3,17-dione was 2 pg/tube. The mean recovery rate of the added standard was 98.3 +/- 8.8% (mean +/- SE). Intra- and inter-assay coefficients of variation were 8.6% (n = 6) and 12.1% (n = 7), respectively.     

A new antiserum with conformational specificity for LHRH: usefulness for radioimmunoassay and immunocytochemistry

We have produced and characterized a new high titer, highly specific antiserum for luteinizing hormone-releasing hormone (LHRH), and demonstrated its usefulness for radioimmunoassay (RIA) and immunocytochemistry. The antiserum can be used at a final dilution of 1:500,000 to 1:600,000 for RIAs with a sensitivity of 0.2 pg/tube. Both the amino and carboxy terminal ends of the LHRH molecule are required for antibody recognition, and the antigenic determinant appears to be part of a three-dimensional structure of LHRH. Fragments of LHRH and other brain peptides are not recognized by the antiserum. Using immunocytochemical techniques, we have localized LHRH-containing neurons in the medial basal hypothalamus of the rhesus monkey, guinea pig, and rat. Staining of LHRH fibers and cell bodies was eliminated by preabsorbtion of the immune serum with synthetic LHRH. This antiserum should be useful in studies that require quantification of very low amounts of LHRH and in studies that require correlation between immunocytochemical localization and tissue content or secretion of LHRH.     

A highly specific radioimmunoassay for progesterone using antibodies covalently linked to arylamine-glass particles

Antibody was produced in sheep following the administration of the antigen, progesterone-11α-succinyl-BSA. After treatment with Rivanol, the protein of the albumin-free antiserum was covalently bound to arylamine glass particles using glutar-aldehyde as the coupling agent. This antibody-glass preparation was used for measurement of plasma progesterone by radioimmunoassay. Progesterone was extracted from human plasma with petroleum ether. Of those Steroids which are petroleum ether extractable, only pregnenolone exhibited minimal cross-reactivity. Immobilization of the antibody by attachment to an insoluble support allows the separation of free from antibody-bound steroid without the use of absorbents, such as florisil and charcoal.     

Elimination of cross-reactivity to some component of erythrocytes in an antiserum to parathyroid hormone by absorption with fixed erythrocytes

Cross-reactivity to some component of rat erythrocytes with an antiserum to parathyroid hormone (anti-PTH) was detected in fixed demineralized sections of bone using the peroxidase-antiperoxidase method. The cross-reactivity was eliminated by the preabsorption of anti-PTH with fixed, washed rat erythrocytes. This technique provides an easy and rapid method with which to eliminate cross-reactivity to erythrocytes whenever such a situation is encountered in immunohistochemical procedures.     

Parameters in the binding of 16 alpha, 17 beta estriol with ewe antiestriol immune serums highly specific to rabbits

The binding of 16 α, 17 β-estriol to highly specific rabbit and ewe anti-esteriol antisera is studied comparatively, with an equilibrium dialysis technique. The affinity indices, association constants and concentrations of binding sites are measured. These parameters are discussed in relation to some immunological characteristics of the sera, previously determined by radioimmunoassay.     

Rapid and efficient isolation of highly specific antibodies from an antiserum against a pool of proteins

Antibodies with desired specificity to proteins of interest provide important and versatile tools for detecting and localizing specific proteins in organisms. With the rapidly increasing number of genes cloned, the demand for antibodies to the gene products is increasing greatly. We developed a procedure to isolate highly specific antibodies to an insect intestinal mucin (IIM) from a polyclonal antiserum, which served as a “library of antibodies,” by using an E. coli lysate of the IIM cDNA clone. This procedure allows rapid and efficient isolation of target protein specific antibodies from a polyclonal antiserum made against a pool of antigens.     

Rapid and efficient isolation of highly specific antibodies from an antiserum against a pool of proteins.

Antibodies with desired specificity to proteins of interest provide important and versatile tools for detecting and localizing specific proteins in organisms. With the rapidly increasing number of genes cloned, the demand for antibodies to the gene products is increasing greatly. We developed a procedure to isolate highly specific antibodies to an insect intestinal mucin (IIM) from a polyclonal antiserum, which served as a "library of antibodies," by using an E. coli lysate of the IIM cDNA clone. This procedure allows rapid and efficient isolation of target protein specific antibodies from a polyclonal antiserum made against a pool of antigens.     

A new method for preparation of an antiserum to penicillin and its application for novel enzyme immunoassay of penicillin.

A new method for the preparation of ampicillin-BSA conjugate by a three step procedure was developed. The first step is the introduction of a maleimide residue to ampicillin by a cross-linking reagent, MBS. The second step is reductive cleavage of disulfide bonds in BSA. The third step is thioether formation between the introduced maleimide residues and the reduced thiol groups. The obtained ampicillin-BSA conjugated raised an anti-ampicillin serum in rabbits. A new reagent, MPGS, was used for enzyme labelling of ampicillin to avoid immunological cross reaction. Using the enzyme labelled ampicillin and anti-ampicillin serum, enzyme immunoassay of ampicillin was successful in detecting 4 ng to 1 mug. Cross reactivities of anti-ampicillin to ampicillin analogs were studied by the enzyme immunoassay to find that the antiserum is specific to penicillin especially to ampicillin but hardly reacts with cephalosporins or the penicilloic acid derivative of ampicillin.     

Elimination of cross-reactivity by addition of an excess of cross-reactant for radioimmunoassay of 17alpha-hydroxypregnenolone.

Polyclonal antiserum against 3beta,17alpha-dihydroxypregn-5-en-20-one-19-O-(carboxymethyl )-oxime bovine serum albumin (17alpha-hydroxypregnenolone-19-CMO:BSA), was raised in rabbits. Its main structural determinants were the substituents on D-ring as demonstrated by its 107% cross-reaction with 17alpha-hydroxyprogesterone. This unspecificity was almost completely eliminated by addition of the excess of the cross-reactant directly to the analytical system. The contribution of the cross-reactant from the sample in such a system became negligible due to saturation of the populations of polyclonal antibodies recognizing the analyte as well as the cross-reactant. The possible interference of 17alpha-hydroxypregnenolone-3-sulfate was avoided by inserting ether extraction. The analytical system appeared to be stable to differences in cross-reactant concentrations even in samples from patients with pathologically elevated serum levels of 17alpha-hydroxyprogesterone. The radioimmunoassay was compared with the system using the unspecific antiserum alone, but after separation of the cross-reactants by HPLC. As demonstrated by parallel measurement of 125 samples of human plasma from both sexes and various ages either before and/or after adrenocorticotropin stimulation and 17 samples with elevated basal of human plasma 17alpha-hydroxyprogesterone levels, an excellent correlation was achieved between both methods. The method, based on a simple addition of the cross-reactant, avoids the time-consuming chromatographic separation and, in comparison with the other approaches for improving the specificity of polyclonal antisera, is efficient and rapid. Mathematical analysis of the relations in equilibrium demonstrates that such a simple approach is an efficient way for improvement of immunoassay specificity using some polyclonal antisera.     

A new approach to apple proliferation detection: a highly sensitive real-time PCR assay

The present paper describes a new approach for diagnosis of apple proliferation (AP) phytoplasma in plant material using a multiplex real-time PCR assay simultaneously amplifying a fragment of the pathogen 16S rRNA gene and the host, Malus domestica, chloroplast gene coding for tRNA leucine. For the first time, such an approach, with an internal analytical control, is described in a diagnostic procedure for plant pathogenic phytoplasmas enabling distinction between uninfected plant material and false-negative results caused by PCR inhibition. Pathogen detection is based on the highly conserved 16S rRNA gene to ensure amplification of different AP phytoplasma strains. The newly designed primer/probe set allows specific detection of all examined AP strains, without amplifying other fruit tree phytoplasmas or more distantly related phytoplasma strains. Apart from its specificity, real-time PCR with serial dilutions of initial template DNA ranging over almost five orders of magnitude (undiluted to 80,000-fold diluted) demonstrated linear amplification over the whole range, while conventional PCR showed a reliable detection only up to 500-fold or 10,000-fold dilutions, respectively. Compared to existing analytical diagnostic procedures for phytoplasmas, a rapid, highly specific and highly sensitive diagnostic method becomes now available.     

Phosphate-starvation-induced outer membrane proteins of members of the families Enterobacteriaceae and Pseudomonodaceae: demonstration of immunological cross-reactivity with an antiserum specific for porin protein P of Pseudomonas aeruginosa

Bacteria from members of the families Enterobacteriaceae and Pseudomonadaceae were grown under phosphate-deficient (0.1 to 0.2 mM Pi) conditions and examined for the production of novel membrane proteins. Of the 17 strains examined, 12 expressed a phosphate-starvation-induced outer membrane protein which was heat modifiable in that after solubilization in sodium dodecyl sulfate at low temperature the protein ran on gels as a diffuse band of higher apparent molecular weight, presumably an oligomer form, which shifted to an apparent monomer form after solubilization at high temperature. These proteins fell into two classes based on their monomer molecular weights and the detergent conditions required to release the proteins from the peptidoglycan. The first class, expressed by species of the Pseudomonas fluorescens branch of the family Pseudomonadaceae, was similar to the phosphate-starvation-inducible, channel-forming protein P of Pseudomonas aeruginosa. The second class resembled the major enterobacterial porin proteins and the phosphate-regulated PhoE protein of Escherichia coli. Using a protein P-trimer-specific polyclonal antiserum, we were able to demonstrate cross-reactivity of the oligomeric forms of both classes of these proteins on Western blots. However, this antiserum did not react with the monomeric forms of any of these proteins, including protein P monomers. With a protein P-monomer-specific antiserum, no reactivity was seen with any of the phosphate-starvation-inducible membrane proteins (in either oligomeric or monomeric form), with the exception of protein P monomers. These results suggest the presence of conserved antigenic determinants only in the native, functional proteins.     

A new approach to detect a particular DNA sequence by UV-immobilization of its hybridization complex with a highly specific probe resulting from ligation of a tandem of short oligonucleotides in solution

A new approach was proposed for detecting amplified DNA fragments by hybridization with a highly selective oligonucleotide probe obtained by ligation of a tandem of three short oligonucleotides (pN8 + pN4 + pN8' Bio) in solution, with subsequent UV-immobilization of the hybridization product on a nylon membrane and its colorimetric detection with the streptavidin-alkaline phosphatase technique. Owing to the high selectivity of ligation, the 20-mer ligation product was detected on a membrane only when it was completely complementary to a template fragment. The results showed that any single-nucleotide substitution in the tetramer-binding site can be localized and identified with the use of all 12 possible tetramers.     

Development of a radioimmunoassay for prostaglandin D2 using an antiserum against 11-methoxime prostaglandin D2

A sensitive and specific radioimmunoassay for prostaglandin D2 has been developed using its stabilized 11-methoxime derivative, which was obtained after treatment of prostaglandin D2 with methoxamine-HCl. The antiserum was obtained after injection of prostaglandin D2-methoxamine coupled to bovine serum albumin. A (125I)-Histamide prostaglandin D2-methoxamine tracer was prepared by iodination of the corresponding histamide, followed by thin layer chromatography purification. The sensitivity of the assay was 280 femtomoles per ml at 50% displacement. The cross reactivities were 15% with prostaglandin D1-methoxamine and less than 0.20% with other prostaglandins. Determination of the half-life of prostaglandin D2 in a solution containing albumin was also carried out, since it has been shown to catalyze prostaglandin D2 destruction. The unstability of this prostaglandin is due to the presence of a beta-hydroxy ketone group, and all prostaglandins possessing this labile moiety could be stabilized by such a derivatization before developing a radioimmunoassay.     

Further purification of human pituitary-derived chondrocyte growth factor: heparin-binding and cross-reactivity with antiserum to basic FGF

The previously described human pituitary-derived chondrocyte growth factor (CGF), mitogenic for rabbit fetal chondrocytes, was found to bind to heparin-Sepharose and was eluted with 1.5M NaCl. Further characterization of CGF demonstrated a molecular weight of 18-20 kD and cross-reactivity with antiserum to synthetic bovine basic fibroblast growth factor (FGF1-24). When human pituitaries were homogenized in 0.15 ammonium sulfate (pH 5.5) and the extract chromatographed on heparin-Sepharose, 98% of the mitogenic activity was adsorbed to heparin and eluted with 3M NaCl. These findings indicate that CGF is closely related or identical to basic FGF and that the bulk of mitogenic activity in the human pituitary extracts binds to heparin.     

Functional characteristics of a highly specific integrase encoded by an LTR-retrotransposon

Background

The retroviral Integrase protein catalyzes the insertion of linear viral DNA into host cell DNA. Although different retroviruses have been shown to target distinctive chromosomal regions, few of them display a site-specific integration. ZAM, a retroelement from Drosophila melanogaster very similar in structure and replication cycle to mammalian retroviruses is highly site-specific. Indeed, ZAM copies target the genomic 5′-CGCGCg-3′ consensus-sequences. To enlighten the determinants of this high integration specificity, we investigated the functional properties of its integrase protein denoted ZAM-IN.

Principal Findings

Here we show that ZAM-IN displays the property to nick DNA molecules in vitro. This endonuclease activity targets specific sequences that are present in a 388 bp fragment taken from the white locus and known to be a genomic ZAM integration site in vivo. Furthermore, ZAM-IN displays the unusual property to directly bind specific genomic DNA sequences. Two specific and independent sites are recognized within the 388 bp fragment of the white locus: the CGCGCg sequence and a closely apposed site different in sequence.

Conclusion

This study strongly argues that the intrinsic properties of ZAM-IN, ie its binding properties and its endonuclease activity, play an important part in ZAM integration specificity. Its ability to select two binding sites and to nick the DNA molecule reminds the strategy used by some site-specific recombination enzymes and forms the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.