Detection of several strains of the bacterium-like organism of citrus greening disease by DNA probes

Greening disease of citrus is caused by a phloem-restricted, bacterium-like organism (BLO). DNA was purified from phloem tissue of periwinkle plants infected with an indian strain of the greening BLO, restricted withHindIII endonuclease, and cloned in the replicative form of bacteriophage M13mp18.By differential hybrizations involving DNA from healthy and infected periwinkle plants, we have selected three recombinant phages containing BLO DNA. The BLO DNA inserts (In-2.6, In-1.9, and In-0.6) have been purified from the viral replicative forms and used as probes. Southern and dot hybridizations have shown that In-2.6 and In-1.9 recognized all asian strains tested (strains from India, Thailand, the Philippines, Indonesia, China, and Taiwan), but not a South African strain. In-0.6 reacted only with the indian BLO strain.     

Purification of recombinant proteins by immunoaffinity chromatography with preselected antibodies

Summary Nuclease produced by recombinant Escherichia coli was successfully purified by immunoaffinity chromatography using an antibody preselected for the purpose on the basis of an elution assay. The elution assay was also used to optimize elution conditions. One step recovery of nuclease from crude periplasmic extract was 66%.     

Purification of antigenic protein of sparganum by immunoaffinity chromatography using a monoclonal antibody

The quality improvement of antigen (crude saline extract) of Spirometra mansoni pleroceroid (sparganum) was investigated by protein purification. The crude extract was fractionated by gel filtration through Sephacryl S-300 Superfine. Its third fraction was purified by affinity chromatography using a monoclonal antibody as ligand. When observed by SDS-PAGE, the purified protein was composed of 2 bands of 36 kDa and 29 kDa which were found already as the most sensitive components in the crude extract by immunoblots with patients sera. The quality of the purified antigen was evaluated in comparison with the crude extract by enzyme-linked immunosorbent assay (ELISA) for the specific (IgG) antibody in sera of human sparganosis, other parasitic and neurologic diseases, and normal control. When the purified antigen was used, the sensitivity was not altered but remained high (96.4%) while the specificity was increased from 86.8% to 96.9%.     

Monoclonal antibodies to avian lipoprotein lipase. Purification of the enzyme by immunoaffinity chromatography

Three monoclonal antibodies to avian lipoprotein lipase have been isolated by fusing spleen cells from immunized BALB/c mice with myeloma P3X-63 Ag 8. The antibodies were detected by their ability to bind immobilized lipoprotein lipase in enzyme-linked immunosorbent assay (ELISA) and by immunoprecipitation of purified enzyme in the presence of second (rabbit anti-mouse) antibodies. Two of these antibodies, CAL1-7 and CAL1-11, inhibited catalytic activity, whereas with CAL1-2 interaction with lipoprotein lipase could be demonstrated only in ELISA and in Western blot assays following denaturation of the enzyme with sodium dodecyl sulfate. An immunoadsorbent column was prepared by coupling immunopurified CAL1-11 to Sepharose-4B. When acetone powder extracts of adipose tissue were applied on the column, 70% of the catalytic activity bound to the matrix. Effective elution was achieved with 1.8 M NaCl, 40% glycerol, 5% acetone, 20 mM Chaps (3[(3-cholamidopropyl)dimethylammonio]propanesulfonate), 0.5 mM EDTA, 1 mM phosphate (pH 6.5). After concentration of the active fractions on a heparin-Sepharose 4B column, the purified enzyme was obtained with an overall recovery of 25%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrates that the preparation is homogeneous with a major band at Mr 60900. Thus, avian adipose lipoprotein lipase has been purified by a one-step immunoaffinity followed by a concentrating step on heparin-Sepharose 4B.     

Purification of human adult and foetal intestinal alkaline phosphatases by monoclonal antibody immunoaffinity chromatography.

We have used the technique of monoclonal antibody immunoaffinity chromatography to purify adult and foetal intestinal alkaline phosphatases. Pure adult intestinal enzyme was obtained from a crude tissue extract with a single immunoaffinity chromatographic step in yields exceeding 95%. An additional ion-exchange chromatographic step was necessary for purification of the foetal enzyme, but yields still exceeded 70%. Experiments to optimize the efficiency of the monoclonal antibody immunoaffinity chromatography procedure suggest that the relative strength of binding of an antibody to its antigen is the most important factor to consider when constructing such columns. A column made from an antibody of too low an avidity will not retain the enzyme, while one of too high an avidity will make elution of enzyme in the active state difficult. A scheme is suggested for the application of this technique to a general approach to enzyme purification.     

Purification of human erythrocyte transglutaminase by immunoaffinity chromatography

Human erythrocyte transglutaminase was purified using a reusable immunoaffinity column prepared from a monoclonal antibody described previously (Birckbichler et al., Hybridoma, 4, 179-186, 1985). The purified TGase was catalytically active and exhibited a single band of apparent Mr = 85,000 on SDS-PAGE and Western blotting. The amino acid composition of the enzyme was determined. The amino terminus was blocked, and the carboxy-terminal residue appeared to be isoleucine.     

Purification of nucleotide-requiring enzymes by immunoaffinity chromatography.

Monospecific (affinity-purified) anti-(yeast glucose-6-phosphate dehydrogenase) IgG inhibits three different NADPH-requiring enzymes, chicken liver dihydrofolate reductase, pigeon liver fatty acid synthetase and chicken liver malic enzyme. The inhibition of all three enzymes was approx. 50% in a 2h incubation with 100 micrograms of IgG. Similarly, with several different NADH-requiring enzymes, an immunocrossreactivity was observed. Monospecific anti-(rabbit muscle glyceraldehyde-3-phosphate dehydrogenase) IgG inhibited yeast alcohol dehydrogenase and pig heart malate dehydrogenase by 39% and 55% respectively. The cross-reactivity observed was tested by affinity chromatography. Immunoaffinity columns made with each monospecific IgG were able to bind each of the enzymes it immunotitrated. Enzymes were eluted with a nondenaturing solvent with little loss of activity. The immunoaffinity column with monospecific anti-(glucose-6-phosphate dehydrogenase) IgG as the bound ligand was also used to purify partially (over 150-fold) both isocitrate dehydrogenase and dihydrofolate reductase from crude rat liver homogenate.     

Purification of an enterotoxin produced by Bacillus cereus by immunoaffinity chromatography using a monoclonal antibody.

A murine monoclonal antibody (MAb) with high reactivity to an enterotoxin produced by Bacillus cereus was used to prepare an immunoadsorbent for purification of the enterotoxin. By immunoaffinity chromatography using the immunoadsorbent, approximately 25% of crude enterotoxin applied was recovered in the eluate. The purified enterotoxin was found to be electrophoretically and antigenically homogeneous. It also showed vascular permeability activity and mouse lethality, and caused fluid accumulation in mouse ligated intestinal loops, whereas it did not show any hemolytic and lecithinase activities. Thus, immunoaffinity chromatography proved useful in the purification of enterotoxin produced by B. cereus in terms of recovery, purity, and relative ease of performing the purification.     

Selection of monoclonal antibodies for immunoaffinity chromatography: model studies with antibodies against soy bean trypsin inhibitor

To facilitate selection of monoclonal antibodies for immunoaffinity chromatography, an ELISA screening procedure was developed. The assay is based on the avidin-biotin system and provides a profile of the monoclonal antibody which is based on the binding characteristics of the antigen binding site when exposed to different elution reagents. The elution profiles of 5 monoclonal antibodies to soy bean trypsin inhibitor (SBTI) were determined and for 2 of the antibodies the results obtained in the ELISA were verified using column experiments. The affinity constants were determined for the same 5 monoclonal antibodies and no correlation was seen with the ease of elution. The elution profiles presented here are easily obtained and the results indicate that a general screening procedure for suitable combinations of antibodies and elution conditions can be carried out using an elution ELISA assay when modified as described herein.     

Purification of monoclonal antibodies by hydrophobic interaction chromatography under no-salt conditions

Hydrophobic interaction chromatography (HIC) is commonly used as a polishing step in monoclonal antibody purification processes. HIC offers an orthogonal selectivity to ion exchange chromatography and can be an effective step for aggregate clearance and host cell protein reduction. HIC, however, suffers from the limitation of use of high concentrations of kosmotropic salts to achieve the desired separation. These salts often pose a disposal concern in manufacturing facilities and at times can cause precipitation of the product. Here, we report an unconventional way of operating HIC in the flowthrough (FT) mode with no kosmotropic salt in the mobile phase. A very hydrophobic resin is selected as the stationary phase and the pH of the mobile phase is modulated to achieve the required selectivity. Under the pH conditions tested (pH 6.0 and below), antibodies typically become positively charged, which has an effect on its polarity and overall surface hydrophobicity. Optimum pH conditions were chosen under which the antibody product of interest flowed through while impurities such as aggregates and host cell proteins bound to the column. This strategy was tested with a panel of antibodies with varying pI and surface hydrophobicity. Performance was comparable to that observed using conventional HIC conditions with high salt.     

Rapid purification of phospholamban by monoclonal antibody immunoaffinity chromatography

Monoclonal antibodies have been raised against canine phospholamban purified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). Four of twenty-four antibodies were purified to close to homogeneity from mouse ascites. All four antibodies could react with isolated bovine cardiac sarcoplasmic reticulum (SR) to result in the stimulation of ATP-dependent Ca2+ pump activity and blocking of phospholamban phosphorylation by cAMP-dependent protein kinase. Relative efficiencies of antibodies in Ca2+ pump stimulation and on phospholamban phosphorylation were not correlated. An immunoabsorbent prepared by conjugating antibody Al to Affi-Gel 10 was used for the purification of phospholamban. Isolated bovine cardiac SR was solubilized in a buffer containing deoxycholate and the soluble fraction was applied to the immunoaffinity column. After washing the column with a series of detergent-containing buffer solutions, the column-bound protein which contained essentially pure phospholamban was eluted by a buffer containing 2.8 M MgCl2. The phospholamban recovery from the immunoaffinity column was close to 100%; the overall yield of purification from SR vesicles was about 70%. SDS-PAGE analysis showed that purified phospholamban consisted of a 25 and 5 kilodalton (kDa) protein species. Upon brief boiling (20 s) of the sample in SDS-PAGE sample buffer, five molecular species ranging from 5 to 25 kDa could be detected by immunotransblotting following SDS-PAGE. This observation supports the notion that phospholamban is composed of five 5-kDa polypeptides. The pure phospholamban could be phosphorylated maximally by cAMP-dependent protein kinase to 1-1.5 mol phosphate/mol phospholamban (25,000 g). This stoichiometry of phosphorylation could be increased to about 5 upon addition of the immunoaffinity column flow through fraction.     

Purification of a brain-specific astroglial protein by immunoaffinity chromatography

An immunoaffinity chromatography procedure for the isolation of bovine glial fibrillary acidic (GFA) protein is described. Degraded GFA protein isolated by hydroxyapatite chromatography from human spinal cord was used to prepare the antiserum. The immunoglogulin G fraction of the antiserum was covalently linked to CNBr-activated Sepharose, and columns of the immuno-affinity gel were used to adsorb bovine GFA protein from brain extracts. Elution was accomplished with a solution of 1 m acetic acid, 5 m urea, 0.8 m sodium chloride, pH 2.5. The yield of about 0.5 mg of highly purified protein/g of cerebral white matter could be increased to 1.5 mg/g of tissue by lowering the ionic strength of the extracting buffer from 50 mm to 1 mm sodium phosphate. Isolation in the presence of EDTA prevented the formation of an oxidation product migrating as a dimer of the monomeric species on SDS-polyacrylamide gel electrophoresis.     

Purification of swine kidney alkaline phosphatase by immunoaffinity chromatography

A homogeneous alkaline phosphatase preparation was obtained from swine kidney cortex by a simple purification step of immunoaffinity chromatography. The enzyme was purified 426 times that of the initial acetone powder with a recovery of 69.6% and a specific activity of 1206 units/mg of protein. The sodium dodecyl sulfate-gel electrophoretic pattern showed a single 80,000-M_r protein band as the monomer of the purified enzyme.     

The genome of the non-cultured,bacterial-like organism associated with citrus greening disease contains thenusG-rplKAJL-rpoBC gene cluster and the gene for a bacteriophage type DNA polymerase

We have recently cloned three DNA fragments (In-2.6, In-1.0, and In-0.6) of the noncultured, bacterial-like organism (BLO) associated with citrus greening disease. Nucleotide sequence determination has shown that fragment In-2.6 is part of therplKAJL-rpoBC gene cluster, a well-known operon in eubacteria. The DNA fragment upstream of and partially overlapping with In-2.6 could be isolated and was shown to be thenusG gene. InEscherichia coli, nusG is also immediately upstream ofrplKAJL-rpoBC. Fragment In-1.0 carries the gene for a bacteriophage type DNA polymerase. Fragment In-0.6 could not be identified.When In-2.6 was used, at high stringency, as a probe to detect greening BLO strains in infected plants, hybridization was obtained with all Asian strains tested, but not with the African strain examined. At lower stringencies, In-2.6 was able to detect also the African strain. The implications of these reults in the taxonomical position of the greening BLO are discussed.     

Characteristics of monoclonal antibodies against human thyroid peroxidase for use in immunoaffinity chromatography and immunoassays

Two types of monoclonal antibodies (MABs) against human thyroid peroxidase (TPO) have been obtained, which interact with spatially separated conformational epitopes of the antigen (K _a values are in the range 10⁸–10⁹ M^?1). The binding site of MAB F8 is in the immunodominant region of the TPO molecule, in the vicinity of the autoantigenic determinants, whereas the epitope specific for MAB A1 lies outside this location. Both MABs retain the ability to form immune complexes after solid-phase immobilization and chemical modification with a biotin derivative. The above properties suggest that MABs A1 and F8 may be used in immunoaffinity chromatography (isolation and purification of TPO from natural sources) and immunoassays for determinations of TPO (in biological fluids) and TPO autoantibodies (in human blood serum).     

Characteristics of monoclonal antibodies against human thyroid peroxidase for use in immunoaffinity chromatography and immunoassays

Two types of monoclonal antibodies (MABs) against human thyroid peroxidase (TPO) have been obtained, which interact with spatially separated conformational epitopes of the antigen (Ka values are in the range 10(8)-10(9) M(-1)). The binding site of MAB F8 is in the immunodominant region of the TPO molecule, in the vicinity of the autoantigenic determinants, whereas the epitope specific for MAB A1 lies outside this location. Both MABs retain the ability to form immune complexes after solid-phase immobilization and chemical modification with a biotin derivative. The above properties suggest that MABs A1 and F8 may be used in immunoaffinity chromatography (isolation and purification of TPO from natural sources) and immunoassays for determinations of TPO (in biological fluids) and TPO autoantibodies (in human blood serum).     

Purification of A-subtype pancreatic cholecystokinin receptor by immunoaffinity chromatography.

So far, no efficient affinity chromatography for CCK receptor purification has been reported that prevented obtention of sequenceable amounts of purified receptor. In this work, 10% of plasma membrane receptor sites were specifically cross-linked with the photoreactive cleavable agonist 125I-ASD-[Thr28, Ahx31]-CCK-25-33, solubilized by NP-40, chromatographied on immobilized wheat germ agglutinin and further immunopurified using anti-CCK antibodies to an overall rate of 3000-3600-fold. Analysis of eluted material demonstrated a protein migrating at Mr 85,000-100,000 and the absence of 35S-labeled impurity. This single and efficient affinity chromatography should provide enough homogeneous receptor protein for microsequence determination and leads to consider immunoaffinity chromatography on immobilized anti-ligand antibodies as a potential tool for purification of membrane receptors.     

Component proteins in crude extract of adult Paragonimus westermani purified by immunoaffinity chromatography using monoclonal antibodies.

The nature of 2 component proteins in crude saline extract of adult Paragonimus westermani was investigated. By immunoaffinity chromatography using monoclonal antibodies (MAb) as ligands, the proteins were purified from the crude extract. Band 1 protein in disc-polyacrylamide gel electrophoresis (PAGE) was purified by PFCK-136 MAb. The protein, known to have molecular mass of 440 kDa, was composed of 23, 46 and 92 kDa subunits when observed by reducing SDS-PAGE and SDS-PAGE/immunoblot. This protein was originated from eggs of the worm as revealed by immunohistochemical staining with PFCK-136 Mab. Another affinity purified protein utilizing PFCK-44 MAb was the band 4 protein of 17 kDa in disc-PAGE. This was a monomer protein in reducing SDS-PAGE and SDS-PAGE/immunoblot. The protein was produced at intestinal epithelium of the worm.