Immunoaffinity Chromatography Procedure Research Articles

A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor

The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant proteins. Up to 20 mg of intact fusion protein were expressed by stably transfected Drosophila S2 cells per liter of culture using this strategy. Purification of these secreted fusion proteins from the conditioned serum free medium of S2 cells was accompanied by an efficient one-step immunoaffinity chromatography procedure using the immobilized anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between the various recombinant proteins and the linker region of the tag, which enables generation of highly pure preparations of tag-free recombinant proteins. Using this system we successfully produced soluble and intact recombinant forms of extracellular proteins such as CD59, C4.4A and vitronectin, as well as a number of truncated domain constructs of these proteins. In conclusion, the present tagging system offers a convenient general method for the robust expression and efficient purification of a variety of recombinant proteins.

Antigen-binding properties of monoclonal antibodies reactive with human TATA-binding protein and use in immunoaffinity chromatography

The TATA-binding protein (TBP) plays a central role in the assembly of most eukaryotic transcription initiation complexes. We have characterized 3 monoclonal antibodies (mAbs) that react in the far amino-terminal (N-terminal) domain of the human TBP molecule (residues 1–99). One of these mAbs (designated 1TBP22) is a polyol-responsive monoclonal antibody (PR-mAb) and was adapted to an immunoaffinity chromatography procedure for purifying bacterially expressed, recombinant human TBP. The epitope for mAb 1TBP22 maps to residues 55–99, which includes the polyglutamine region. However, mAb 1TBP22 does not react with poly- l-glutamine. Human TBP, contained on the pET11a plasmid, was expressed in Escherichia coli Rosetta (DE3)pLysS. The cell lysate from 330 ml of induced culture was treated with polyethyleneimine (PEI) at 0.5 M NaCl to precipitate the nucleic acids. After centrifugation, the supernatant fluid was applied to an immunoadsorbent containing mAb 1TBP22. After extensive washing, the TBP was eluted with buffer containing 0.75 M ammonium sulfate and 40% propylene glycol. Human TPB purified by the immunoaffinity chromatography method was found to be active in gel-shift assays and transcription assays. Preliminary data indicate that this mAb might be useful for purifying protein complexes containing TBP from HeLa cell extracts.

An epitope tag derived from human transcription factor IIB that reacts with a polyol-responsive monoclonal antibody

Polyol-responsive monoclonal antibodies (PR-mAbs) provide a strategy to purify active, nondenatured proteins by a single-step immunoaffinity chromatography procedure. The high affinity interaction between these antibodies and the antigen can be dissociated in the presence of a nonchaotropic salt and a low molecular weight polyhydroxylated compound (polyol). The epitope for PR-mAb IIB8 is located near the N-terminus of the human transcription factor IIB (TFIIB). The epitope is an eight amino acid sequence, TKDPSRVG, that can be fused to a desired protein for use as a purification tag. This epitope tag (termed hIIB) was fused to the C-terminus of green fluorescent protein (GFP). An additional GFP fusion protein utilized another version of hIIB containing a point mutation at position two. These fusion proteins, expressed in Escherichia coli, allowed successful separation of the desired protein in a single chromatographic step. This strategy extends PR-mAb gentle-release purification to numerous expressed proteins.

Development of an epitope tag for the gentle purification of proteins by immunoaffinity chromatography: application to epitope-tagged green fluorescent protein

Polyol-responsive monoclonal antibodies (mAbs) are useful tools for the gentle purification of proteins and protein complexes. These are high-affinity mAbs that release the antigen in the presence of a nonchaotropic salt and a low-molecular-weight polyhydroxylated compound (polyol). The epitope for the polyol-responsive mAb NT73, which reacts with Escherichia coli RNA polymerase, was located at the C terminus of the β ′ subunit. Using recombinant DNA techniques, we have identified the epitope to be within the 13-amino-acid sequence SLAELLNAGLGGS and have developed an epitope tag that can be fused to a protein of interest for use as a purification tag. This epitope tag (designated Softag1) was fused to either the N or the C terminus of the green fluorescent protein. These tagged proteins were expressed in E. coli, and the tagged proteins were purified from the soluble fraction by a single-step immunoaffinity chromatography procedure. This approach extends the powerful technique of gentle-release immunoaffinity chromatography to many expressed proteins.

Determination of Irgarol 1051 in Western Mediterranean sediments. Development and application of supercritical fluid extraction–immunoaffinity chromatography procedure

A supercritical fluid extraction (SFE) procedure for Irgarol 1051 (i.e. 2-(tert-butylamino)-4-(cyclopropylamino)-6-(methylthio)-1,3,5-triazine) determination in marine sediments, which minimises the solvent usage, is developed and compared to a conventional extraction technique (i.e. sonication). First, the use of methanol (MeOH) in the presence of trifluoroacetic acid (TFA) as secondary modifier of supercritical carbon dioxide was evaluated. Extraction efficiency was strongly dependent on the modifier content but lesser on pressure (100–410 bar) and temperature (60–200°C). In the selected extraction conditions (20% MeOH/TFA 0.65 M, 370 bar, 150°C) recoveries higher than 87% were obtained and the limit of detection was 3 ng g −1 and the relative standard deviation of 10% ( N=3) by GC coupled to mass spectrometry (GC-MS) in the electron impact mode. The developed SFE procedure is more convenient to extract Irgarol 1051 than the agitation plus sonication methods concerning on solvent usage (1.5 vs . 20 mL) being compatible with immunochemical procedures avoiding any solvent transfer step. The developed SFE combined with immunoaffinity chromatography (IAC) is highly selective allowing the determination of Irgarol by gas chromatography with nitrogen–phosphorus detection or in sediments at low ng g −1 level (11–35 ng g −1) from Mediterranean marina and harbour sediments.

Characterization of plasma inhibin forms in fertile and infertile men.

The reciprocal relationship between plasma FSH and inhibin B generally reflects the state of spermatogenesis but data in some settings indicate further complexity in their relationship. Inhibin circulates as a range of higher molecular weight (mol wt) forms of varying bioactivity such that the serum profile of inhibin forms may differ between normal men and those with varying types of spermatogenic failure. The aim of this study was to establish if the inhibin B mol wt distribution was altered in men with infertility. The mol wt profiles of inhibin B and free alpha-subunit were determined in plasma of fertile (n = 11) and infertile (n = 17) men using a combined immunoaffinity chromatography, preparative SDS-PAGE and electro-elution procedure and fractions assayed using ELISAs for inhibin B, total inhibin (all forms containing the alpha-subunit) and free alpha-subunit. Inhibin B was identified as precursor (60-65 k) and mature (26-30 k) forms in plasma in similar proportions (29%) in fertile men and oligospermic men (25%), but was undetectable in azoospermic men. The free alpha-subunit detected by the pro-alphaC ELISA was identified as both the precursor and processed (pro-alphaC) forms with similar proportions in fertile (8%) and all infertile (4-14%) men. The pro-alphaC ELISA did not detect the precursor forms of inhibin B in plasma while the inhibin B ELISA detected all total inhibin forms following removal of pro-containing forms by immunoabsorption. (i) the proportions of precursor inhibin B and alpha-subunit forms in the circulation are unchanged in men with spermatogenic disorders indicating there is no alteration of the Sertoli cell inhibin secretory pattern; (ii) these fractionation studies indicate that pro-alphaC and inhibin B ELISAs specifically detect the free alpha-subunit and inhibin B forms present in male plasma.

Placental, Maternal Serum and Amniotic Fluid Molecular Weight Forms of Inhibin A and Pro-αC

Previous studies have identified the presence of unidentified small molecular weight (mol wt) forms of inhibin and the pro-αC region of the inhibin α subunit in serum from women during late pregnancy. The aim of this study was to investigate if these gestational-related changes in mol wt forms arose from changing placental production. Pooled placental extracts, derived from normal healthy singleton pregnancies in the 1st, 2nd and 3rd trimesters of pregnancy, were fractionated by a combined immunoaffinity chromatography, preparative PAGE and electroelution procedure. Inhibin A, inhibin B and the pro-αC region of the inhibin α subunit were determined in the eluted fractions by specific ELISAs, with the profiles of immunoactivity characterized in terms of molecular size and percentage recovery. Inhibin B was undetectable in all samples. Mol wt peaks of 36k, 75K and 97K for inhibin A and 29k, 55K and 97K for pro-αC were detected in placental extracts across all three trimesters. The relative abundancy of small mol wt inhibin A forms (<30K) present in the placenta increased significantly in the third trimester placenta, increasing from 0.3 per cent in the first trimesters to 6 per cent in the third trimester (P=0.01, chi-squared test). The relative abundances of various mol wt forms of pro-αC was similar at all three gestations (P=0.67). In serum, small mol wt inhibin A and pro-αC forms accounted for 23.4 per cent and 37.4 per cent of inhibins, respectively, in the third trimester.These data suggest that the presence of small mol wt forms of both inhibin A and pro-αC in maternal serum is only partially attributed to placental production and/or secretion. We conclude that inhibin A and pro-αC inhibins in maternal serum are processed in late pregnancy by more than one mechanism to form low mol wt circulating forms of, as yet, undetermined structure.

Characterization of Inhibin Forms and Their Measurement by an Inhibin α-Subunit ELISA in Serum from Postmenopausal Women with Ovarian Cancer

The aim of this study was to characterize the molecular wt forms of inhibins A and B and its free alpha-subunit present in serum from women with ovarian cancer as a basis for developing improved monoclonal antibody-based inhibin assays for monitoring ovarian cancer. Three new inhibin alpha-subunit (alphaC) ELISAs were developed using monoclonal antibodies directed to three nonoverlapping peptide regions of the alphaC region of the inhibin alpha-subunit. To characterize serum inhibin molecular wt forms present in women with ovarian cancer, existing inhibin immunoassays (inhibin A, inhibin B, and pro-alphaC) and the new alphaC ELISAs were applied to sera from women with granulosa cell tumors and mucinous carcinomas previously fractionated using a combined immunoaffinity chromatography, preparative SDS-PAGE, and electroelution procedure. The distribution and molecular size of dimeric inhibins and alpha-subunit detected were consistent with known mol wt forms of inhibins A and B and inhibin alpha-subunit and their precursor forms present in serum and follicular fluid from healthy women. The alphaC ELISAs recognized all known forms of inhibin and the free inhibin alpha-subunit, although differences between alphaC ELISAs were observed in their ability to detect high mol wt forms. To assess which of the alphaC ELISAs was preferred in application to ovarian cancer, the alphaC ELISAs were applied to serum from a range of normal postmenopausal women (n = 61) and postmenopausal women (n = 152) with ovarian (serous, mucinous, endometrioid, clear cell carcinomas, and granulosa cell tumors) and nonovarian (breast and colon) cancers. Despite differences in their ability to detect high mol wt forms of inhibin, the alphaC ELISAs showed similar sensitivity (i.e. proportion of cancer patients correctly detected) and specificity (proportion of controls correctly detected) indexes in the detection of mucinous carcinomas (84% and 95%) and granulosa cell tumors (100% and 95%) compared with earlier inhibin RIA or polyclonal antibody-based immunofluorometric assays. A combination of the alphaC ELISAs with the CA125 assay, an ovarian tumor marker that has a high sensitivity and specificity for other ovarian cancers (serous, clear cell, and endometrioid), resulted in an increase in sensitivity/specificity indexes (95% and 95%) for the all ovarian cancer group. These new monoclonal antibody-based inhibin alphaC ELISAs now provide practical and sensitive assays suitable for evaluation as diagnostic tests for monitoring ovarian cancers.

Changes in molecular weight forms of inhibin A and pro-alpha C in maternal serum during human pregnancy.

Maternal serum pools obtained from healthy women throughout normal pregnancy were fractionated by a combined immunoaffinity chromatography, preparative PAGE, and electroelution procedure. Inhibin A and the pro-alpha C region of the inhibin alpha-subunit were determined in the eluted fractions by specific ELISAs, and the profiles of immunoactivity characterized in terms of molecular weight and percent recovery. The molecular weight patterns of inhibin A and pro-alpha C in serum during early pregnancy (<19 wk gestation) showed peaks between 25-40K and approximately 60K, consistent with the presence of known mature and larger precursor inhibin forms. However, during late pregnancy (>19 wk gestation), an increase in the proportion of smaller molecular weight forms (from 2% to approximately 25%) of inhibin A and pro-alpha C of unknown structure were observed in the less than 30K and less than 25K regions, respectively. To assess whether this change in molecular weight distribution in late pregnancy was related to the method of serum collection, serum and plasma from women during early and late pregnancy were collected and snap-frozen. Three pools [one from early pregnancy (12-15 wk), two from late pregnancy (28-39 wk)] of serum and plasma were then fractionated as described above. No differences in molecular weight patterns of inhibin A and pro-alpha C were observed between serum and plasma pools obtained in early pregnancy. However, in late pregnancy there was a reduction in the proportion of low molecular weight forms between serum (25% inhibin A, 35% pro-alpha C) and plasma (12% and 17%, respectively), but not to the low levels seen in early pregnancy. Incubation of iodinated 30K human inhibin A with serum or plasma obtained from early or late pregnancy showed no evidence of cleavage, suggesting that 30K inhibin A is not the cleavage precursor. It is speculated that the formation of small molecular weight forms of both inhibin A and pro-alpha C is attributed to proteolytic changes, in part induced in the circulation during late gestation and in part by the placenta before secretion. It is concluded that inhibin A and pro-alpha C are processed in late pregnancy by more than one mechanism to form low molecular weight circulating forms of unknown structure.

Development and application of immunoaffinity chromatography for the determination of the triazinic biocides in seawater

The development of an immunoaffinity chromatography (IAC) procedure for the selective extraction of the anti-fouling agent Irgarol 1051 [2-( tert.-butylamino)-4-(cyclopropylamino)-6-(methylthio)-1,3,5-triazine] from seawater is described. The anti-Irgarol 1051 antibodies were covalently bound to agarose-based beads support. IAC column capacities were higher than 400 ng and ethanol–water (70:30) was selected as eluting mixture. After percolation of 250 ml of water sample containing Irgarol 1051 at environmental levels (ng l −1), the breakthrough volume was still not achieved. Other triazine herbicides percolated through the IAC column showed good recoveries. Thus, this IAC procedure may be useful to extract related compounds. The developed IAC column was applied to real seawater samples and compared with RP-C 18 cartridges. The limit of detection (LOD) reached by using the IAC procedure was twenty times lower than the LOD achieved by the RP-C 18 cartridges using the same detection system. Irgarol 1051 was detected at ng l −1 levels in the Barcelona marina (northwestern Mediterranean Sea). An acceptable correlation between enzyme-linked immunosorbent assay and gas chromatography with nitrogen–phosphorus detection was observed, thus analysis of Irgarol 1051 can be performed by either one of the methods. In this work, further confirmation of the analyte identity for real samples was accomplished by gas chromatography–electron impact mass spectrometry.

Multiresidue analysis of β 2-agonists in human and calf urine using multimodal solid-phase extraction and high-performance liquid chromatography with electrochemical detection

Various β 2-agonists are used as illegal growth promoters in man and in animals. We developed a multiresidue procedure for the analysis of four β-agonists in human and calf urine. The sample was pre-extracted with an Extrelut column at alkaline pH. The β-agonists were eluted with a mixture of tert.-butylmethyl ether and hexane. Then the extract was further cleaned with a mixed mode SPE column, or with a combination of immunoaffinity chromatography (IAC) and the mixed mode SPE column. The IAC column contained antibodies against salbutamol, which were suitable for multiresidue extractions. The extract was then brought onto a mixed mode SPE column at an acidic pH. The column was washed with 70% methanol in water. Thereafter, the β-agonists were eluted with ammoniated ethanol–hexane. The extract was analysed with an HPLC method with electrochemical detection. The β-agonists were separated on a reversed-phase column using a mobile phase buffered at pH 5.5 and containing an ion-pair reagent. Recoveries were higher when the IAC procedure was not performed (90–105% vs. 65–75%), but the extracts were cleaner when the latter step was included. Detection limits in human and calf urine were in the low ng/ml range. The study indicated that β 2-agonists can be analysed in human and calf urine without the selectivity of a mass spectrometer, but that comprehensive clean-up is required to avoid the interference of urine matrix components.

Immunopurification of human β2-glycoprotein I with a monoclonal antibody selected for its binding kinetics using a surface plasmon resonance biosensor

The β 2-glycoprotein I ( β 2GPI)-binding properties of five murine monoclonal antibodies immobilized as capture antibodies were studied using surface plasmon resonance detection. The monoclonal antibody with the fastest dissociation kinetics (6F3) was selected for the development of an immunoaffinity chromatography procedure, assuming that its behaviour would be similar in both systems since the covalent coupling chemistries involved amino groups in both cases. Under our experimental conditions of a fast one-step procedure, β 2GPI was purified to homogeneity from human plasma with a yield of about 50%. β 2GPI was eluted under fairly mild conditions, either at low pH or at high pH. The immunoadsorbent was used five times without any apparent loss of binding capacity. The immunopurified protein showed similar binding to cardiolipin-coated polystyrene wells as β 2GPI purified by conventional methods. However, differences in the pattern of immunoreactivity in relation to the purification procedure were observed by surface plasmon resonance using the monoclonal antibody with the highest association kinetics (9G1) immobilized on the sensor surface.

Development of monoclonal antibodies for the selective isolation of human plasma apolipoprotein A-containing particles.

Monoclonal antibodies (MAbs) to human plasma apolipoprotein A-I (apo A-I), denoted FF9 and 6B9, and apolipoprotein A-II (apo A-II), 3F5, were developed to be used in an immunoaffinity chromatography procedure to isolate lipoprotein particles Lp A-I and Lp A-I:A-II. MAb FF9 and MAb 6B9 reacted with apo A-I and high-density lipoprotein (HDL) while MAb 3F5 was directed to apo A-II and HDL. The apparent affinity constant (Kapp) for apo A-I of the MAb FF9 was higher (2 x 10(7) M-1) than that of 6B9 (5 x 10(6) M-1). MAb 3F5 recognized the apo A-II with a Kapp value of 1 x 10(9) M-1. The isolated lipoparticles Lp A-I and Lp A-I:A-II will be used to standardize an immunoassay for the measurement of these apo A-I-containing lipoprotein particles in human plasma.

Prevention of in vitro low-density lipoprotein oxidation by an albumin-containing Lp A-I subfraction

High-density lipoprotein (HDL) and two lipoprotein particles, those containing apo A-I and apo A-II (LpA-I:A-II) and those containing only apo A-I (LpA-I) were examined for their effect on Cu 2+-catalyzed oxidation of human low-density lipoprotein (LDL). Lipoproteins and lipoprotein particles were prepared from plasma samples of five healthy subjects. LDL and HDL were purified by ultracentrifugation, LpA-I and LpA-I:A-II were isolated by an immunoaffinity chromatography procedure. The contaminating albumin often linked to the LpA-I affinity purified particles was eliminated by selected affinity immunosorption. The presence of HDL, LpA-I or LpA-I:A-II, at an apo A-I-containing lipoproteins/LDL ratio of 1, did not prevent LDL oxidation when assessed by oxidation kinetics, electrophoretic mobility, amounts of thiobarbituric acid-reactive products and fragmentation of apo B-100. On the other hand, when the albumin removing step was omitted, the subfraction of albumin-containing LpA-I particles impeded and even inhibited the oxidation of LDL in an albumin dose-dependent manner.

Different NH2-Terminal Form with 12 Additional Residues of α2-Plasmin Inhibitor from Human Plasma and Culture Media of HEP G2 Cells

α2-Plasmin inhibitor (α2PI) was purified from plasma or from the culture media of Hep G2 cells by one-step immunoaffinity chromatography procedure. Majority of α2PI purified from plasma was the previously recognized plasma α2PI with NH2-terminal Asn (Asn-α2PI), whereas majority of α2PI purified from the culture media was retaining the "pro" peptide of 12 amino acids with NH2-terminal Met (Met-α2PI). When Hep G2 cells were cultured in serum-free media, the α2PI secreted to the media was totally in a form of Met-α2PI. Incubation of Met-α2PI with human plasma induced the complete conversion of Met-α2PI to Asn-α2PI. The results indicate that α2PI is synthesized and secreted from liver cells as Met-α2PI and Met-α2PI is converted to Asn-α2PI by proteolytic cleavage in plasma during the circulation.

Expression of a gene encoding a scorpion insectotoxin peptide in yeast, bacteria and plants

The nucleotide sequence encoding the scorpion insectotoxin I 5A was chemically synthesized and expressed in yeast, bacteria and tobacco. The I 5A peptides produced in these organisms were purified using an immunoaffinity chromatography procedure. I 5A produced using the bacteria secretion system was efficiently secreted and released into the culture medium. In contrast, only a trace amount of I 5A was detected in bacterial cytosols when expressed from a direct expression vector, suggesting that I 5A was unstable in bacterial cells. I 5A secreted from yeast using an α-factor signal sequence was shown to have an N-terminal (Glu-Ala) 2 extension, indicating incomplete processing of the secreted peptide by dipeptidyl aminopeptidase A. In tobacco, a nonsecreted form of the protein was produced. No measurable insect toxicity was observed when insect larvae were assayed, regardless of whether I 5A was produced in yeast, bacteria or tobacco. The lack of toxicity is almost certainly the result of improper folding due to incorrect disulfide bond formation. The inability to produce a biologically active peptide must be overcome before scorpion toxins might be used for the genetic engineering of plants for insect resistance. The yeast and bacterial expression systems described here may be useful for further studies on the problem of expressing a biologically active peptide.

Purification of human α2-plasmin inhibitor by a one-step immunoaffinity chromatography procedure

ala-plasmin inhibitor (cLzPI) has been purified from human plasma by conventional multistep methods (1, 2). Although the methods allow the recovery of high quality of aaP1, low overall yield and prolonged time required for purification make the methods unsuitable for routine use. In this communication, we describe a newly developed immunoaffinity purification method for claPI, which is simple, efficient and easily performed.

Purification of a multiprotein complex containing centrosomal proteins from the Drosophila embryo by chromatography with low-affinity polyclonal antibodies.

A 190-kDa centrosomal protein interacts with microtubules when Drosophila embryo extracts are passed over microtubule-affinity columns. We have obtained a partial cDNA clone that encodes this protein. Using a fusion protein produced from the clone, we have developed a novel immunoaffinity chromatography procedure that allows both the 190-kDa protein and a complex of proteins that associates with it to be isolated in in a single step. For this procedure, the fusion protein is used as an antigen to prepare rabbit polyclonal antibodies, and those antibodies that recognize the 190-kDa protein with low affinity are selectively purified on a column containing immobilized antigen. These low-affinity antibodies are then used to construct an immunoaffinity column. When Drosophila embryo extracts are passed over this column, the 190-kDa protein is quantitatively retained and can be eluted in nearly pure form under nondenaturing conditions with 1.5 M MgCl2, pH 7.6. The immunoaffinity column is washed with 1.0 M KCl just before the elution with 1.5 M MgCl2. This wash elutes 10 major proteins, as well as a number of minor ones. We present evidence that these KCl-eluted proteins represent additional centrosomal components that interact with the 190-kDa protein to form a multiprotein complex within the cell.

Isolation and characterization of human Lp-B lipoprotein containing apolipoprotein B as the sole apolipoprotein

A sequential immunoaffinity chromatography procedure was developed to isolate from whole normolipidemic human plasma a subpopulation of apoB containing particles (Lp-B) which is virtually free of non apoB protein. The absence of non apoB protein in Lp-B was assessed by enzyme immunoassay against apolipoproteins A-I, A-II, A-IV, E, C-III and (a). Electron microscopy and fractionation of the isolated particles by gel filtration demonstrated that these particles were heterogeneous in size. However, most of them had diameters between 18 and 26 nm. These particles were found to be rich in cholesterol (molar ratio cholesterol/apoB = 2246 ± 995) poor in triacylglycerol (molar ratio triacylglycerol/apoB = 555 ± 518) and had a phospholipids/apoB molar ratio of 713 ± 348. Most of the cholesterol was esterified (66% ± 5%). Lp-B particles bound to the apoB, E receptor of HeLa cells with a lower affinity than LDL prepared by ultracentrifugation (1.030 kg/l < d < 1.053 kg/l). ( K D = 18.9 vs 10.5 nmol/l).

Immunological properties of the fibrinolytic enzyme (fibrolase) from southern copperhead ( Agkistrodon contortrix contortrix) venom and its purification by immunoaffinity chromatography

H. M. Chen, A. L. Guan and F. S. Markland, Jr. Immunological properties of the fibrinolytic enzyme (fibrolase) from southern copperhead ( Agkistrodon contortrix contortrix) venom and its purification by immunoaffinity chromatography. Toxicon 29, 683–694.—An antibody to the fibrinolytic enzyme in southern copperhead venom was produced by immunizing rabbits with chromatographically purified enzyme. The antibody was purified from rabbit blood by ammonium sulfate fractionation and protein-A affinity chromatography. The purified antibody reacted only with the fibrinolytic enzyme in southern copperhead venom as demonstrated by immunodiffusion and immunoelectrophoresis. Western immunoblotting revealed that several snake venoms, including Agkistrodon piscivorus conanti, Crotalus atrox, Crotalus basiliscus basiliscus, and Bothrops asper, cross-reacted with the antibody to varying degrees. However, Deinagkistrodon acutus showed no cross-reaction. Immobilized antibody has been used, in combination with molecular sieve chromatography, to purify the fibrinolytic enzyme from southern copperhead venom. In this two-step purification procedure, the enzyme was purified in good yield within two days. The specific activity of the enzyme purified by the immunoaffinity chromatography procedure is comparable with that of enzyme purified by a four-step chromatographic procedure. The mol. wt of the purified enzyme is approximately 23,000–24,000 as determined by SDS-PAGE. Interestingly, the enzyme purified by this two-step immunoaffinity chromatography procedure possesses virtually no hemorrhagic activity.