Removal Of Excess Reagent Research Articles

Agarose-Droplet-Based Digital LAMP Assay for Counting Virus DNA in Single-Particle ICP-MS.

Inductively coupled plasma mass spectrometry (ICP-MS) has emerged as a promising analytical platform for the quantification of biomolecules using elemental tags; however, absolute quantification at extremely low concentrations by ICP-MS without a calibration curve remains challenging. Here, we developed a digital loop-mediated isothermal amplification (LAMP) assay for counting hepatitis B virus (HBV) DNA using single-particle (sp) ICP-MS. The sample and LAMP reagents were mixed and encapsulated in agarose droplets, which were generated by homemade centrifugal droplet generators. The agarose droplets were incubated at 65 °C for amplifying the virus DNA with LAMP primers and then cooled to 4 °C for generating "gel" particles during the temperature-dependent "sol-gel" transition. The LAMP amplicons were intercalated into the agarose particles using polyacrylamide-modified LAMP primers, enabling the labeling of dsDNA with [Ru(bpy)2dppz]2+ and the removal of excess reagents. Only those agarose particles, containing virus DNA, could be labeled with 101Ru and detected in spICP-MS. We also embedded the 153Eu-containing polystyrene microspheres into agarose droplets as the internal standard for counting the total number of agarose droplets. The copy number of virus DNA could be counted from the 101Ru/153Eu pulse numbers in spICP-MS. We achieved the lowest quantification of 25 copy μL-1 virus DNA in one analysis without the need for a calibration curve. The developed assay can be easily tuned for counting multiple types of nucleic acid targets and extended for new possibilities of the spICP-MS-based digital assay.

Insights into siRNA Transfection in Suspension: Efficient Gene Silencing in Human Mesenchymal Stem Cells Encapsulated in Hyaluronic Acid Hydrogel.

Small interfering RNAs (siRNAs) are powerful tools for post-transcriptional gene silencing, which offers enormous opportunities for tissue engineering applications. However, poor serum stability, inefficient intracellular delivery, and inevitable toxicity of transfection reagents are the key barriers for their clinical translation. Thus, innovative strategies that allow safe and efficient intracellular delivery of the nucleic acid drugs at the desired site is urgently needed for a smooth clinical translation of therapeutically appealing siRNA-based technology. In this regard, we have developed an innovative siRNA transfection protocol that employs a short incubation time of just 5 min. This allows easy transfection in suspension followed by transplantation of the cells in a hyaluronic acid (HA) hydrogel system. We also report here the unique ability of siRNA to bind HA that was quantified by siRNA release and rheological characterization of the HA-hydrogel. Such interactions also showed promising results to deliver functional siRNA in suspension transfection conditions within 30 min using native HA, although removal of excess HA by centrifugation seem to be essential. In the 2D experiments, suspension transfection of hMSCs with RNAiMAX resulted in ≈90% gene silencing (with or without removal of the excess reagent by centrifugation), while HA demonstrated a modest ≈40% gene silencing after removal of excess reagent after 30 min. Transplantation of such transfected cells in the HA-hydrogel system demonstrated an improved knockdown (≈90% and ≈60% with RNAiMAX and HA respectively after 48 h), with lower cytotoxicity (up to 5-days) as determined by PrestoBlue assay. The gene silencing efficiency in the 2D and 3D conditions were also confirmed at the protein levels by Western blot analysis. We postulate this novel transfection method could be applied for in vivo applications as it allows minimal manipulation of cells that are to be transplanted and reduce toxicity.

Salting-out assisted liquid–liquid extraction for the determination of biogenic amines in fruit juices and alcoholic beverages after derivatization with 1-naphthylisothiocyanate and high performance liquid chromatography

A new method for determining biogenic amines in fruit juices and alcoholic beverages is described involving reaction of biogenic amines with 1-naphthylisothiocyanate followed by extraction of 1-naphthylthiourea derivatives with water-miscible organic solvent acetonitrile when solvents phase separation occurred using ammonium sulphate, a process called salting-out assisted liquid–liquid extraction. The extract was analyzed by high-performance liquid chromatography with UV detection at 254nm. The new reagent avoided many of the inconveniences as observed with existing derivatizing agents, such as dansyl chloride and benzoyl chloride, in regard to their inselectivity, instability, adverse effect of excess reagent, and necessity to remove excess reagent. The procedure has been optimized with respect to reaction time and temperature, water-miscible extraction solvent, and salt for solvents phase separation. Use of reagent as dispersed phase in aqueous medium produced derivatives in high yield. A linear calibration was obtained between the amount of biogenic amines in range 1–1000μgL−1 and peak areas of corresponding thioureas formed; the correlation coefficient was 0.9965, and the limit of detection and limit of quantification found were 1.1μgL−1 and 3.2μgL−1, respectively. The pre-concentration method gave an average enrichment factor of 94. The application of the method has been demonstrated in the determination of biogenic amines in commercial samples of fruit juices and alcoholic beverages. In spiking experiments to real samples, the average recovery found by the present method was 94.5% that agreed well with 95.8% obtained by established comparison methods.

Underivatized oxysterols and nanoLC–ESI-MS: A mismatch

Due to their non-charged character, liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) measurements of oxysterols are often performed after derivatization with e.g. charged Girard reagents. However, derivatization reactions are time-consuming and may require numerous steps to remove excess reagent. In addition, extensive sample handling can be associated with cholesterol autoxidation, resulting in analyte artifacts and hence false positives.Nano scale liquid chromatography in combination with electrospray-mass spectrometry (nanoLC–ESI-MS) is a powerful tool for analyzing limited samples, due to substantially increased sensitivity compared to conventional LC–ESI-MS. The signal enhancement may compensate for the poor ionization of the oxysterols; hence we have explored the possibility to quantify oxysterols without derivatization using nanoLC–ESI-MS.Non-derivatized oxysterols and nanoLC were however not compatible, due to persistent and large carry-over. This was attributed to the extended contribution of surface to volume ratio in such miniaturized systems and interactions with the materials of the nanoLC instrumentation (e.g. adsorption to the fused silica tubing).Two contemporary MS instruments (Q-Exactive™ hybrid quadrupole-Orbitrap and TSQ Quantiva™ triple quadrupole) were used. However, both the MS and MS/MS spectra of non-derivatized oxysterols were ambiguous and/or unrepeatable for both of the instruments employed.Derivatizing oxysterols is more cumbersome, but provides more selective and reliable results, and Girard derivatization+nanoLC–ESI-MS continues to be our recommended choice for measuring oxysterols in very limited samples.These investigations also indicate that extra care should be taken to remove lipids prior to nanoLC of other analytes, as adsorbed oxysterols, etc. can compromise analysis.

3-(Dimethylamino)-1-propylamine: a cheap and versatile reagent for removal of byproducts in carbohydrate chemistry.

Inexpensive 3-(dimethylamino)-1-propylamine (DMAPA) was found to be effective in anomeric deacylation reactions giving 1-O deprotected sugars in high yield as precursors for the formation of imidate glycosyl donors. DMAPA was also found to be useful for removing excess reagents such as benzoyl chloride, tosyl chloride, and 2,2,2-trifluoro-N-phenylacetimidoyl chloride. The deacylation reaction could be conducted in moist THF and did not require chromatographic purification since an acidic wash was sufficient to remove excess reagent and the formed byproduct.

A novel condition for capillary electrophoretic analysis of reductively aminated saccharides without removal of excess reagents

We have identified novel CE conditions for the separation of 7-amino-4-methylcoumarin-labeled monosaccharides and oligosaccharides from glycoproteins. Using a neutrally coated capillary and alkaline borate buffer containing hydroxypropylcellulose and ACN, saccharide derivatives form anionic borate complexes, which move from the cathode to the anode in an electric field and are detected near the anodic end. Excess labeling reagents and other fluorescent products remain at the cathodic end. Fluorimetric detection using an LED as a light source enables determination of monosaccharide derivatives with good linearity between at least 0.4 and 400 μM, may correspond to 140 amol to 140 fmol. The lower LOD (S/N = 5) is only 80 nM in the sample solution (ca. 28 amol). The results were comparable to reported values using fluorometric detection LC. The method was also applied to the analysis of oligosaccharides that were enzymatically released from glycoproteins. Fine resolution enables profiling of glycans in glycoproteins. The applicability of the method was examined by applying it to other derivatives labeled with nonacidic tags such as ethyl p-aminobenzoate- and 2-aminoacridone-labeled saccharides.

Traceless Approach for the Synthesis of 3,5-Disubstituted Thiohydantoins on Functionalized Ionic-Liquid Support

A traceless approach for the synthesis of 3,5-disubstituted thiohydantoins on a novel functionalized ionic-liquid support, 5, is described. Acylation of benzylamine functionalized ionic-liquid support with amino acids yielded ionic-liquid-supported amino acids, which reacted with isothiocyanates to afford ionic-liquid-supported thioureas. Following intramolecular cyclization cleavage from the ionic-liquid support by trifluoroacetic acid (TFA), the desired 3,5-disubstituted thiohydantoins were obtained in good yields and purities. The efficiency of this ionic-liquid-phase strategy facilitated isolation and analysis of intermediates and removal of excess reagents and by-products during the reaction process.

Dess-Martin Periodinane Mediated Intramolecular Cyclization of Phenolic Azomethines: A Solution-Phase Strategy toward Benzoxazoles and Benzothiazoles

Dess-Martin periodinane (DMP), a highly versatile hypervalent iodine(V) reagent, was found to efficiently mediate the intramolecular cyclization of phenolic azomethines/Schiff bases at ambient temperature leading to the rapid and expeditious synthesis of substituted benzoxazoles and benzothiazoles. Furthermore, a solution-phase strategy has been developed by treating the reaction mixtures sequentially with Amberlyst A-26 thiosulfate resin and diisopropylaminomethyl resin (PS-DIEA), which remove excess reagent and byproducts, to give pure products.

Synthesis of Natural-Product-Like Molecules with Over Eighty Distinct Scaffolds

Synthesis of Natural-Product-Like Molecules with Over Eighty Distinct Scaffolds

Gas-Phase Pyridylamination of Saccharides: Development and Applications

Pyridylamination is a versatile method for fluorescence labeling of oligosaccharides. The technique affords sensitive detection of saccharides with reducing termini and high-resolution separation by high-performance liquid chromatography. The conventional method, based on a liquid-phase reaction, has been extensively used in various aspects of glycobiology and glycotechnology. Unfortunately, the necessity for removing excess 2-aminopyridine makes the technique both laborious and time-consuming. Furthermore, removal of excess reagent can result in a significant loss of short saccharide components. In the present paper, we report an alternative methodology based on a "gas-phase" reaction, in which dried saccharides are reacted with vaporized 2-aminopyridine. The resultant Schiff base was also reduced in the gas phase within the same reaction microtube using a purpose-built device. The newly developed procedure was applied to both monosaccharide (GlcNAc) and oligosaccharides (isomalto-oligosaccharides) at quantitative yields with no requirement to remove excess reagent. The acid-labile sialyl linkages of alpha2-6-disialobiantennary oligosaccharides proved to be fully stable during the procedure. The developed method was also successfully applied to profiling N-linked oligosaccharides liberated from glycoproteins by hydrazinolysis and, thus, should contribute to various fields of glycomics.

Efficient labelling of antibodies with horseradish peroxidase using cyanuric chloride

An efficient and mild method for labelling of immunoglobulin G (IgG) with horseradish peroxidase (HRP) using cyanuric chloride (2,4,6-trichloro-1,3,5-triazine, CC) as a bridging molecule is described. The enzyme was treated first with cyanuric chloride to introduce dichloro triazine and after removal of excess reagent, the activated enzyme was mixed with the IgG preparation and incubated to effect linkages with amine groups in the antibody protein. Various amounts of coupling reagent were tested to optimise the conjugation method using commercially available enzyme and affinity-purified sheep IgG antibody preparations to three different test haptens. The conjugates were assessed by solid phase Enzyme Linked Immunosorbent Assays (ELISA) and commonly used peroxidase substrate preparations. The binding activity of the conjugates rose with increasing coupling reagent added during the enzyme activation step. Use of the conjugates prepared by the new method gave comparable sensitivity in direct competitive ELISAs for the three test haptens to assays carried out using indirect ELISA with commercial anti-sheep-HRP conjugates. No deterioration of enzyme activity or hapten-binding activity in the conjugates was observed after storage in 50% glycerol at − 70 °C for up to 18 months. This study presents a relatively simple and efficient conjugating method for labelling antibodies with HRP and provides an additional and probably a better alternative to the periodate, glutaraldehyde and succinimide–maleimide procedures.

Analysis of cell membrane aminophospholipids as isotope-tagged derivatives

Glycerophosphoethanolamine (GPEtn) and glycerophosphoserine (GPSer) lipids were reacted with a multiplexed set of differentially isotopically enriched N-methylpiperazine acetic acid N-hydroxysuccinimide ester reagents, which place isobaric mass labels at a primary amino group. The resulting derivatized aminophospholipids were isobaric and chromatographically indistinguishable but yielded positive reporter ions (m/z 114 or 117) after collisional activation that could be used to identify and quantify individual members of the multiplex set. The chromatographic and mass spectrometric response of N-methylpiperazine amide-tagged aminophospholipids was probed using glycerophosphoethanolamine and glycerophosphoserine lipid standards. The [M+H]+ of each tagged aminophospholipid shifted 144 Da, and during collision-induced dissociation the major fragmentation ion was either m/z 114 or 117. This mode of detecting aminophospholipids was useful for an unbiased analysis of plasmalogen GPEtn lipids. Molecular species information on the esterified fatty acyl substituents was obtained by collisional activation of the [M-H]- ions. The isotope-tagged reagents were used to assess changes in the distribution of GPEtn lipids after exposure of liposomes made from phospholipids extracted from RAW 264.7 cells to Cu2+/H2O2 to illustrate the ability of these reagents to aid in the mass spectrometric identification of aminophospholipid changes that occur during biological stimuli.

Determination of amines as pentafluoropropionic acid anhydride derivatives in biological samples using liquid chromatography and tandem mass spectrometry.

Determination of amines in biological samples as markers of exposure to the amines or the corresponding isocyanates is an important tool for industrial exposure assessment. In this study, a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method for determination of amines in biological samples as perfluorofatty amides derivatives is presented. The method enables determination of diamines such as methylene diamine (MDA), toluene diamine (TDA), naphthalene diamine (NDA), hexamethylene diamine (HDA), isophorone diamine (IPDA), methylenedi(cyclohexylamine)(HMDA) and 4,4'-methylene-(2-chloroaniline)(MOCA) in human urine and plasma. The work-up procedure included hydrolysis of the biological samples with 3 M H(2)SO(4) at 100 degrees C for 16 h and extraction of the amines into toluene, where derivatisation of the amines with perfluorofatty acid anhydride was performed. Following removal of excess reagent and the acid formed and an exchange of solvent, the derivatives were analysed using gradient elution with an acetonitrile/water mobile phase composition and electrospray ionisation (ESI) with multiple reaction monitoring (MRM) of [M - H](-)-->[M - H - 120](-) or [119](-). Several perfluorofatty acid anhydrides were evaluated as derivatisation reagents, but the LC chromatographic properties of the pentafluoropropionic acid anhydride (PFPA) derivatives were favourable. Quantification of amine-PFPA derivatives was performed using deuterium labelled amine-PFPA derivatives as internals standards with good precision and linearity in the investigated range of 0-20 ng ml(-1) urine. The instrumental detection limits for the amine-PFPA derivatives were 0.2-3 fmol for MRM of [M - H](-)-->[119](-) and 0.3-8 fmol for [M - H](-)-->[M - H - 120](-). In 10 urine and 6 plasma samples from workers exposed to isocyanates, determination of TDA and MDA as PFPA derivatives was performed using LC-MS/MS and a reference GC-MS method. No significant difference between the two methods was observed.

Concentrations of low and high molecular weight thiols in wheat dough as affected by different concentrations of ascorbic acid.

Different amounts of ascorbic acid (AA) were added to flour, and the concentrations of low and high molecular weight thiols in the dough were determined. For the determination of the low molecular weight thiols, glutathione, cysteine, and the corresponding disulfides, an isotope dilution assay with a (14)C-labeled internal standard was used. For the determination of the high molecular weight thiols, a method was developed that involved derivatization of dough with Ellman's reagent, removal of excess reagent by dialysis, micro-Osborne fractionation, release of the label by reduction, and determination of reduced Ellman's reagent by reversed-phase high-performance liquid chromatography. Mixing of flour without AA led to a decrease of the glutathione and an increase of the cysteine concentration. Addition of AA reduced the concentration of both thiols to a minimum when 125 mg of AA/kg of flour was applied. Furthermore, the concentrations of high molecular weight thiols in the glutenins of flours from different wheat cultivars were determined. The values ranged from 5.6 to 8.2 micromol/kg of protein and showed a correlation between flour quality and SH concentration. On addition of AA and mixing of a dough, the concentrations of the protein thiols in the glutenins isolated from the dough increased to a maximum when 100 mg of AA/kg of flour was added. Higher concentrations of AA led to a decrease of the SH concentration. The last results are not in accordance with previously published data or with current hypotheses about the mechanism of the AA improver action.

Use of a small reporter molecule to determine cell-surface proteins by capillary electrophoresis and laser-induced fluorescence: use of 5-SAENTA-x8f for quantitation of the human equilibrative nucleoside transporter 1 protein.

The human equilibrative nucleoside transporter 1 protein (hENT1) is a major mediator of cellular entry of nucleosides and anticancer nucleoside drugs; its assay is important in understanding and diagnosing chemotherapy resistance. Here we present a novel assay for quantifying hENT1 using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A cellular population is treated with 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine-x8-fluorescein (5-SAENTA-x8f), which binds with high affinity and specificity to the hENT1 protein. The cells are washed to remove excess reagent, lysed, and centrifuged, and the supernatant is analyzed by CE-LIF with the use of an internal standard. Accuracy was evaluated by comparing the capillary electrophoresis results with those obtained by flow cytometry; the results were highly correlated, r = 0.96. The relative standard deviation of the hENT1 assay was 10%, determined from nine independent assays of the same cell line, which is 3 times superior to results obtained in a flow cytometry assay. The detection limit for 5-SAENTA-x8f was 4300 molecules injected into the capillary.

Assessment of ω-fatty-acid-supplemented human platelets for potential improvement in long-term storage

Uptake of omega (ω)−3 fatty acids can influence membrane stability and cell mobility. We investigated the effects of ω−3 and −6 fatty acids on the hemostatic efficacy of human platelets using an in vivo rabbit bleeding model. In vitro assays such as platelet aggregation, vWF bead-mediated ATP release and platelet adhesion to beads (measured by the residual platelet count [RPC] {free platelet count after reacting with the beads}/{baseline platelet count}×100=%RPC; a high %RPC indicates reduced platelet function) were conducted on platelets treated with 1% fish oil (ω−3); 2% fish oil emulsion or 1% soy oil (ω−6). Oil treatment of platelets reduced the vWF bead-induced ATP release insignificantly. Addition of ω−3 agents reduced physical reactivity (%RPC) with the vWF beads by a factor of 1.2 (oil) and 1.9 (emulsion). The ω−6 oil enhanced reactivity by a factor of 1.7. After washing to remove excess reagent, platelet resuspension was most efficient with the ω−3 emulsion. Platelet function was higher with the ω−3-treated platelets (%RPC=52.3%, ω−3 oil; 63.3%, ω−3 emulsion vs. 85%, ω−6 oil; 82% untreated platelets). Ethyl-palmitate-treated thrombocytopenic rabbits were infused with human platelets. Survival times of the treated platelets, as monitored by flow cytometry (6.2–8.2 h) were comparable to untreated platelets (8.6 h). In the rabbit kidney injury model, blood loss after infusion of the treated platelets was similar to that of saline-infused rabbits (75.3±3.4 g). However, platelets washed prior to infusion reduced blood loss to a value comparable to that of fresh platelets (48.3±5 g). Furthermore, the presence of the infused platelets at the injury site was clearly visualized using FITC-tagged anti CD42a antibody. Thus, the ω−3-based agents protect the platelets from damage during the washing procedure as demonstrated in vitro by improved platelet resuspension, low %RPC, high stimulus-responsive ATP secretion and a reduction in blood loss in vivo.

Inactivation of monomeric sarcosine oxidase by reaction with N-(cyclopropyl)glycine.

Monomeric sarcosine oxidase (MSOX) catalyzes the oxidative demethylation of sarcosine (N-methylglycine) and contains covalently bound flavin adenine dinucleotide (FAD). The present study demonstrates that N-(cyclopropyl)glycine (CPG) is a mechanism-based inhibitor. CPG forms a charge transfer complex with MSOX that reacts under aerobic conditions to yield a covalently modified, reduced flavin (lambda(max) = 422 nm, epsilon(422) = 3.9 mM(-1) cm(-1)), accompanied by a loss of enzyme activity. The CPG-modified flavin is converted at an 8-fold slower rate to 1,5-dihydro-FAD (EFADH(2)), which reacts rapidly with oxygen to regenerate unmodified, oxidized enzyme. As a result, CPG-modified MSOX reaches a CPG-dependent steady-state concentration under aerobic conditions and reverts back to unmodified enzyme upon removal of excess reagent. No loss of activity is observed under anaerobic conditions where EFADH(2) is formed in a reaction that goes to completion at low CPG concentrations. Aerobic denaturation of CPG-modified enzyme yields unmodified, oxidized flavin at a rate similar to the anaerobic denaturation reaction, which yields 1,5-dihydro-FAD. The CPG-modified flavin can be reduced with borohydride, a reaction that blocks conversion to unmodified flavin upon removal of excess CPG or enzyme denaturation. The possible chemical mechanism of inactivation and structure of the CPG-modified flavin are discussed.

The Zymark BenchMate™. A compact, fully‐automated solution‐phase reaction work‐up facility for multiple parallel synthesis

The rapid growth of multiple parallel synthesis in our laboratories has created a demand for a robust, easily accessed automated system for solution-phase reaction work-up, since the manual work-up of large numbers of small-scale reactions is both time-consuming and tedious, and is a rate limiting step in the generation of large numbers of compounds for test. Work-up in chemical organic synthesis consists of a series of post-reaction operations designed using differential chemical properties to remove excess reagent or starting material, reagent products and, where possible reaction by-products. Careful consideration of post-reaction operations as a clean-up step can obviate the requirement for purification. Generally, work-up can be resolved into four operations: filtration, solvent addition (dilution, trituration), washing and separation (partition) and it is the selection and ordering of these four basic operations that constitutes a chemical work-up. Following the proven success of centralized Zymate robotic systems in the compilation, execution and work-up of complex reaction sequences, a centralized chemical work-up service has been in operation for over 12 months. It now seemed prudent that the needs of multiple parallel synthesis would be better served by the development of a compact, automated system, capable of operating in a standard chemistry laboratory fume-hood. A Zymark BenchMate platform has been configured to perform the four basic operations of chemical solution work-up. A custom-built filtration station, incorporating an integrated tipping facility for the sample tube has also been developed. Compilation of each work-up is through a set of Visual Basic procedure screens, each dedicated to a particular work-up scenario. Methods are compiled at the chemist's own PC and transferred to the BenchMate via a diskette.

Solution-phase synthesis of 2,6,9-trisubstituted purines

A simple three-step method for the solution-phase combinatorial synthesis of 2,6,9-trisubstituted purines from 2,6-dichloropurine is described. The synthesis exploits the use of resin capture to remove excess reagent used in the final step.

Bioengineering of silicon nitride

Selective functionalization of silicon nitride with biomolecules by light-dependent processes has been investigated, Aryldiazirin-based photoimmobilization procedures are used to achieve covalent biomolecule binding. Experimentally facile processes applied include the following steps: (i) adsorptive coating of the surface with photolabel-bearing reagents or photolabel-functionalized biomolecules; (ii) exposure of the coated surface to activating light (350 nm); and (iii) removal of excess reagent or functionalized biomolecule. The extent of photoreagent binding to silicon nitride depends on the time of light exposure as well as on the amount of photoreagent applied to the surface. Streptavidin is immobilized by photolinker polymer-mediated procedures, and antibody-derived F(ab') fragments are covalently immobilized on silicon nitride (45–50 fmol mm −2) with a low-molecular-weight crosslinker. Biomolecule binding is monitored by fluorescein-labelled ligand binding and by tracing radiolabelled proteins, respectively. Photoimmobilized streptavidin retains ligand binding activity, and immunoreagents remain biologically active. Mask-assisted photopatterning on silicon nitride is achieved and patterned structures are resolved by atomic force microscopic imaging of photobonded diazirin-derivatized bovine serum albumin.