Prior Purification Research Articles

Monoclonal Antibodies to Complement Components Without the Need of Their Prior Purification. I. Antibodies to Mouse C1q

Rat monoclonal antibodies (MAbs) reactive with mouse complement subcomponent C1q were raised applying a principle that requires minute amounts of serum and circumvents purification of serum-derived C1q. The principle involves a) using the high affinity of certain cell-bound antibody isotypes for intercalating C1q from serum of various species, b) selecting such antibodies as are syngeneic to the immunized animal species, thus avoiding the production of antibodies against the intercalating antibodies and c) screening for the anti-C1q MAb in microtiter plates coated with C1q-intercalating MAb isotypes that are heterogeneic to the immunized animal species. We could establish 3 MAbs of IgM subclass, whose reactivity to mouse C1q was shown by ELISA techniques. One of them (RmC13C9) crossreacted with human C1q and was shown by SDS-PAGE and subsequent immunoblotting to recognize the C-chain of mouse and human C1q. The other two MAbs are directed against SDS-sensitive epitopes on mouse C1q and were, therefore, further characterized by native agarose gel electrophoresis and immunohistochemical studies.

A new membrane permeabilization method for the detection of intracellular antigens by flow cytometry

This article describes a new cell membrane permeabilization method for the detection of intracellular antigens by immunofluorescence staining and flow cytometry. The number of cells remained unaltered and no cell aggregation or loss of intracellular antigenicity was observed after this permeabilization treatment. A mixed leukocyte population from human peripheral blood was used in this study and the leukocytes were fixed and permeabilized, which permitted monoclonal anti-vimentin antibodies to reach intracellular antigens. The stabilization of cell membranes and preservation of intracellular antigenicity was achieved with paraformaldehyde fixation. This pretreatment prevents cell destruction and subsequent treatment with the detergent n- octyl-β- D-glucopyranoside results in permeabilization of the cell membrane. The procedure does not alter the expression of cell surface antigens, which is of importance if phenotypic characterization of intracellularly stained cells is to be undertaken. Furthermore, this simple, rapid and reproducible technique makes it possible to detect and distinguish between different human peripheral blood leukocytes without prior purification steps. The leukocyte subpopulations remain well-separated and easily detectable by flow cytometry.

Nucleic acid hybridization assays employing dA-tailed capture probes: II. Advanced multiple capture methods

A fourth capture is added to the reversible target capture procedure of the preceding paper. This results in an improved radioisotopic detection limit of 7.3 × 10 −21 mol of target. In addition, the standard triple capture method is converted into a nonradioactive format with a detection limit of under 1 amol of target. The principal advantage of nonradioactive detection is that the entire assay can be performed in about 1 h. Nucleic acids are released from cells in the presence of the (‘capture probe’) which contains a 3′-poly(dA) sequence and the (‘labeled probe’) which contains a detectable nonradioactive moiety such as biotin. After a brief hybridization in solution, the target is captured on oligo(dT) magnetic particles. The target is further purified from sample impurities and excess labeled probe by recapture either once or twice more on fresh magnetic particles. The highly purified target is then concentrated to 200 nl by recapture onto a poly(dT) nitrocellulose filter and rapidly detected with streptavidin-alkaline phosphatase using bromochloroindolyl phosphate and nitroblue tetrazolium. Using this procedure, as little as 0.25 amol of a target plasmid has been detected nonradioactively in crude samples in just 1 h without prior purification of the DNA and RNA. Finally, a new procedure called background capture is introduced to complement the background-reducing power of RTC.

A rapid separation procedure to analyse phosphorylation of two putative protein kinase C substrates in intact lymphocytes.

Activation of protein kinase C (PKC) has been implicated in the regulation of many biological processes including the regulation of T and B lymphocyte responses. A direct means of studying activation of this kinase is to analyse specific phosphorylation events of cellular substrates. In the present report we describe a fractionation method that allows quantitative analysis of phosphorylation of two distinct PKC substrates using normal human T cells and T-cell tumour lines. This method, which selectively visualizes PKC-dependent phosphorylation of both an 80- and a 19-kDa substrate, involves three simple fractionation steps and allows a large number of samples to be analysed simultaneously. Since specific antibodies to these cellular substrates are not commonly available, the present method provides an alternative approach which makes it feasible to use phosphorylation of the 19- and 80-kDa proteins as a sensitive marker for PKC activation. Finally, in a cellular system where PKC-mediated phosphorylation of these substrates can be studied without prior purification, the present method results in greatly improved resolution of these phosphorylation events.

T200 alternate exon use in murine lymphoid cells determined by reverse transcription-polymerase chain reaction.

T200 glycoproteins of lymphoid and myeloid cells exhibit cell lineage-specific structural heterogeneity. Peptide heterogeneity appears to arise from alternate 5'-exon use (Ex-4, 5, and 6), potentially giving rise to eight distinct forms of T200 mRNA containing 0 to 3 of these alternate exons. A method is described for determining the number and identity of the three alternate T200 exons expressed in cells by using the polymerase chain reaction (PCR) and the reverse transcription-polymerase chain reaction (RT-PCR) without prior purification of RNA. Synthetic primers flanking the alternate exon region of T200 were designed to yield products for each possible exon combination having unique size and restriction enzyme sites. PCR amplification of plasmids containing T200 cDNA with none (pLy-5-68) or all three (p70Z/3-3) known alternate exons resulted in the amplification of 186 and 603 bp products, respectively. That amplified products were derived from T200 cDNA was verified by restriction enzyme mapping of each PCR product. T200 cDNA prepared from cell lines utilizing no alternate exons (BW5147) or all three exons (70Z/3.12) were analyzed by RT-PCR and contained amplified products of 186 bp (zero alternate exons) and 603 bp (containing Ex-4+5+6), respectively. RT-PCR of EL4 cells revealed approximately 186 and 330 bp products suggestive of zero and one alternate exon forms. Restriction mapping confirmed that EL4 cells contained a zero-exon form and a one-exon form containing Ex-5. Analysis of the 3B3 pre-B cell line yielded 186, 330, 460, and 603 bp products; restriction mapping revealed T200 mRNA for a zero alternate exon form, two distinct one- and two-exon forms (Ex-4; Ex-5; Ex-4+5; Ex-5+6), and a three-exon form (Ex-4+5+6). Other lymphoid cell lines were heterogeneous in T200 alternate exon use, with distinct patterns distinguishing B and T cells. RT-PCR can facilitate the analysis of variations in T200 alternate exon use among developmentally and functionally distinct lymphoid and myeloid cells.

Synthèse du 5-bromométhyl- et du 5-chlorométhyl-2-furannecarboxaldéhyde

The action of hydrogen halides, sulfur and phosphorus halides, and halotrimethylsilanes on 5-(hydroxymethyl)-2-furancarboxaldehyde ( 1) led to the corresponding 5-(chloromethyl)- ( 2) or 5-(bromomethyl)-2-furancarboxaldehyde ( 3). Thus, treatment of 1 in diethyl ether solution with gaseous dry hydrogen chloride under very mild experimental conditions led to high yields of 2. The selective and quantitative conversion of 1 into 3 was achieved with bromotrimethylsilane. In the same manner, chlorotrimethylsilane gave high yields of 2 from 1. The latter compound was obtained by acid-catalyzed dehydration of d-fructose in dimethyl sulfoxide, and then converted into 2 without prior purification.

Analysis of complement Factor B activation by PAGE-immunoblotting

Initiation of complement activation via the alternative pathway results in cleavage of Factor B into two fragments Ba and Bb. Employing PAGE and immunoblotting analysis we have demonstrated Ba and Bb activation fragments following in vitro and in vivo activation in a range of biological fluids including cerebrospinal fluid and synovial fluid. Quantitative linearity was obtained over the range 5–20 μg/ml of Factor B and its fragments in body fluids. Using Davis-PAGE a previously unrecognised triple component heterogeneity of the Ba fragment was observed. This study demonstrates that immunoblotting may be used as a sensitive and quantitative tool for the detection and characterisation of the individual cleavage products of Factor B in complex biological fluids without prior purification.

Determination of the primary structure of paim II, an α-amylase inhibitor from Streptomyces coruchorushii, by high-performance tandem mass spectrometry

This study indicates one of the advantages of tandem mass spectrometry; the primary structures of proteins with little structural difference can be determined by using tandem mass spectrometry without prior purification of each component. The primary structure of Paim II, a protein alpha-amylase inhibitor from Streptomyces coruchorushii, was determined by using tandem mass spectrometry. Paim II consists of two component proteins with ragged N-terminus, and was sequenced on the basis of the structure of Paim I, an analogous alpha-amylase inhibitor from the same natural origin.

Detection and use of recombinant staphylococcal protein A fusion proteins to localize nucleic-acid-binding domains of proteins

We developed a rapid method to visualize recombinant staphylococcal protein A fused to segments of another protein and detect DNA-binding proteins simultaneously. Fusion genes were expressed in Escherichia coli to produce fusion proteins of protein A and segments of c-myc oncoprotein. Polypeptides from total E. coli lysates were separated in sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes. Without the use of an antigen-specific antibody, protein A/c-myc fusion proteins were visualized by incubation of nitrocellulose membranes with horseradish-peroxidase (HRP)-conjugated immunoglobulin, the Fc region of which directly binds protein A. The sensitivity of detection of the protein A/c-myc fusion proteins was greatly enhanced by incubation of nitrocellulose membranes with rabbit immunoglobulin before incubation with HRP-conjugated goat anti-rabbit antibody. Prior to incubation of nitrocellulose membranes with immunoglobulin, the membranes were incubated with radiolabeled DNA to visualize DNA-binding proteins by autoradiography. Purified staphylococcal protein A does not bind DNA, whereas a segment of c-myc oncoprotein fused to protein A binds DNA in vitro. These methods may be applied to identify nucleic-acid-binding domains of other proteins without prior purification of the fusion proteins.

A Comparison of Two Extraction Procedures For The Recovery of Cytokinin Nucleotides

While it is contended in the literature that alcoholic extraction of plant material could result in excessive breakdown of cytokinin nucleotides no support for this could be obtained in this investigation. The recovery rate of DHZMP was similar when using Bieleski's solution and 80 % ethanol for the extraction of this molecule when added to soybean callus. The latter procedure is more rapid and allows material to be quickly processed by HPLC with a minimum of prior purification.

Possibilities of application of some new algorithms for standardisation purposes; standardisation of sodium hydroxide solution against commercial potassium hydrogen phthalate

A method for the standardisation of sodium hydroxide solution against commercial potassium hydrogen phthalate without its prior purification is described. Application of the curve-fitting method to the corresponding regression functions, denoted PADE W, PADE 1/W and SIMMS 1/W, gives the possibility of evaluating the parameters related to the system tested, i.e., concentrations of the species, activity coefficient of hydrogen ions and equilibrium constants.

Analysis by high-performance gel permeation chromatography of hyaluronic acid in animal skins and rabbit synovial fluid

The simultaneous analysis of the molecular weight and concentration of hyaluronic acid in biological samples using high-performance liquid chromatography with two gel permeation columns is described. The elution volumes of various molecular weights of hyaluronic acids were linearily related to the logarithms of their molecular weights up to 600 000. The concentration of hyaluronic acid could be determined in the range from 20 to 100 μg/ml, i.e., from 4 to 20 μg per 200 μl injected. The method was applied to the analysis of several animal skin extracts and rabbit synovial fluid. Skin extracts from house, rat, guinea-pit and rabbit could be chromatographed without prior isolation and purification. Hyaluronic acids in skin were separated clearly from chondroitin sulphates and their concentrations were determined. The molecular weights were estimated simultaneously to be more than 10 6. Rabbit synovial fluids from intact joints and saline- and carrageenin-treated joints could be chromatographed directly. The chromatograms showed that the concentration of hyaluronic acid in carrageenin-treated synovial fluid is lower than that in saline-treated fluid and the molecular weight distribution is broader. This technique enabled the rapid analysis of hyaluronic acid present at low levels in biological samples.

A simple, rapid, and precise direct radioimmunoassay method for 17α,20β-dihydroxy-4-pregnen-3-one in salmon plasma

This report describes a radioimmunoassay method for 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-diOHP) in the blood plasma of Atlantic salmon ( Salmo salar). 3H-labeled 17α,20β-diOHP was synthesized from 17α-[ 3H]hydroxy-4-pregnen-3-one (17α-OHP) by enzymatic reduction of the C-20 oxo group and the simultaneous oxidation of coenzyme-reduced nicotinamide adenine dinucleotide. Antiserum to 17α,20β-diOHP-3-CMO-BSA exhibited high specificity; all steroids tested against the antiserum gave less than 1% cross-reactivity at 50% displacement. Crude dichloromethane extracts of small volumes (50 μl or less) of plasma can be assayed directly without prior purification. The working range is 5 to 300 pg. Interassay and intraassay coefficients of variation were less than 10%. Various parallelism tests showed high accuracy and sensitivity. The assay is simple and rapid and at least 75 samples can be easily assayed in duplicate by two analysts in 1 day.

Taxonomic Application of Crossed Immunoelectrophoresis

We used crossed immunoelectrophoresis, crossed-line immunoelectrophoresis, tandem-crossed immunoelectrophoresis, and crossed immunoelectrophoresis with an intermediate gel (containing antibodies) in studies of bacterial taxonomy. By these quantitative immunoelectrophoretic methods, as many as 90 antigens from a bacterial species can easily and reproducibly be analyzed for immunological cross-reactivity with antigens from other bacterial species without prior purification of antigens. The immunological cross-reactivity between various bacterial species is expressed as the matching coefficient (MC), i.e., the ratio between the number of cross-reactive antigens with another bacterial species and the total number of antigens of the reference bacterial species. The MC has been found to be significantly correlated with phenotypic similarities (biochemical characteristics) and nucleic acid hybridization results obtained with the taxa investigated so far. In general, closely related strains (serotypes within a species) have a high MC (≥0.90), while different species of the same genus have a lower MC (0.90 to 0.70 or lower in some cases); genera within a family have an intermediate MC (0.70 to 0.15), and distantly related taxa have a very low MC (≤0.15).

Molecular hybridization with RNA probes in concentrated solutions of guanidine thiocyanate

Hybridizations with RNA probes were performed in 3–6 m guanidine thiocyanate concentrations where cells can be solubilized. At these concentrations the melting temperature of hybrids and the optimum temperature of hybrid formation are greatly reduced. The rate of hybridization in GuSCN at room temperature was greater than that in 50% formamide at 42°C. Hybridizations were performed on DNA and RNA in cells dissolved in GuSCN without prior nucleic acid purification.

Simultaneous isolation of preparative amounts of RNA and DNA from Trichinella spiralis by cesium trifluoroacetate isopycnic centrifugation

A rapid method for simultaneously banding preparative amounts of RNA and DNA from Trichinella spiralis muscle larvae by isopycnic centrifugation in cesium trifluoroacetate (CsTFA) is described. Larvae were homogenized in guanidinium isothiocyanate and the DNA, RNA, glycogen, and denatured protein components were isopycnically separated without prior purification. This procedure resulted in the isolation of nucleic acids suitable for molecular biological application. Agarose gel electrophoresis of gradient fractions indicated the separation of undegraded RNA and DNA where total RNA was of sufficient purity to efficiently direct in vitro translation of parasite protein and total DNA was greater than 20 kb in size and sensitive to restriction endonuclease digestion. Oligo (dT)-purified poly(A) + mRNA was 3.6% of total RNA with greater than 18% conversion to cDNA.

Analysis of all protein amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid chromatography

A gas chromatographic method for the separation and quantitation of the 20 protein amino acids is described using N-methyl- N( tert.-butyldimethylsilyl)trifluoroacetamide, with 1% tert.-butyldimethylchlorosilane as catalyst, to prepare the tert.-butyldimethylsilyl amino acid derivatives. Alkylsilylation of amino acids proceeds at 140°C in 20 min. The derivatives formed in the one-step reaction are used directly for gas-liquid chromatographic analysis, using a flame-ionization detector, without prior isolation or purification. Complete separation and quantitation of all protein amino acids are readily achieved using a 15-m DB-5 capillary column. Strict linearity extends from less than 15 to about 100 ng for all amino acids except Arg, which has a linear range from 50 to 300 ng. The limits of detection, however, range from one to several hundred nanograms. The method was used to analyze the free amino acid pool in carnation petals.

Genetic polymorphism of haptoglobin subtypes in a Japanese population.

Haptoglobin (Hp) subtypes have been determined in the Japanese population by polyacrylamide gel isoelectric focusing followed by immunoblotting and by two-dimensional polyacrylamide gel electrophoresis. In the present study, neuraminidase-treated plasma samples were used for subtyping of Hp, without prior purification. These samples were obtained from 372 unrelated healthy donors. Allelic frequencies were: Hp*1F = 0.0014; Hp*1S+ = 0.2688; Hp*2FF = 0.0000; Hp*2FS = 0.7284, and Hp*2SS = 0.0014. The phenotypic distribution was in good accordance with the Hardy-Weinberg equilibrium.

Glycosyltransferases as probes for non‐reducing terminal monosaccharide residues on nitrocellulose immobilized glycoproteins: The β (1–4) galactosyltransferase model

AbstractThis paper reviews a technique for detecting non‐reducing terminal N‐acetyl‐glucosamine residues on glycoproteins in a mixture of proteins resolved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and immobilized on nitrocellulose by electrophoretic transfer. Detection is accomplished by fluorography after enzymatic modification with galactosyltransferase (UDP‐galactose: N‐acetyl‐D‐glucosaminyl‐glycopeptide 4‐β‐D‐galactosyltransferase, E.C. 2.4.1.38), which transfers radiolabeled galactose from its nucleotide sugar substrate, UDP‐[6‐3H]‐galactose, into any immobilized glycoprotein with a non‐reducing terminal 2‐acetamido‐2‐deoxy‐D‐glucose (GlcNAc) acceptor site. The kinetics of radiolabeling are independent of the amount of immobilized glycoprotein substrate. Half‐maximal incorporation of [6‐3H]galactose occurs by 5 h and saturation by 16 h. We have used enzyme blotting with galactosyltransferase: (i) to detect non‐reducing terminal GlcNAc residues on isolated glycoproteins, (ii) to detect, in combination with endo‐β‐N‐acetylglucosaminidase F digestion, Asn‐linked oligosaccharides on glycoproteins, and (iii) to identify a subset of glycoproteins with non‐reducing terminal N‐acetylglucosamine residues in both F9 and PYS murine teratocarcinoma cell extracts, two of which appear to be unique to PYS cells. Because the family of glycosyltransferases recognizes a large variety of non‐reducing terminal monosaccharide residues, this technique, termed enzyme blotting, could be applied to the analysis of any non‐reducing terminal monosaccharide. This method could be used to obtain initial information about the carbohydrate structures of a particular glycoprotein without any prior purification steps. This technique may also be potentially useful in elucidating the functional contribution of non‐reducing terminal monosaccharide residues in intercellular recognition and adhesion.

Comparison of a DNA-DNA dot-blot hybridisation assay with light microscopy and radioimmunoassay for the detection of a nuclear polyhedrosis virus

A dot-blot hybridisation assay was developed for the detection of a nuclear polyhedrosis virus (NPV) and was compared to light microscopy and radioimmunoassay (RIA). Using cloned NPV DNA labelled with 32P as a probe, a number of hybridisation assay procedures was examined. The assay was found to be more sensitive than differential staining, phase-contrast microscopy, or indirect solid-phase RIA with as few as 20 occlusion bodies (150 pg DNA) being detected. Samples do not require prior purification or DNA extraction. The assay was shown to be specific for NPV and has the potential to detect and discriminate between strains of the virus. With little modification the assay may be used to detect other insect viruses.