A 1-Hour Enzyme-Linked Immunosorbent Assay for Quantitation of Acrolein- and Hydroxynonenal-Modified Proteins by Epitope-Bound Casein Matrix Method

A simple and rapid enzyme-linked immunosorbent assay (ELISA) method for quantitation of acrolein and 4-hydroxy-2-nonenal (HNE)-modified proteins was developed. Microtiter plate wells were precoated and blocked simultaneously with epitope-bound bovine caseins as matrix proteins, and aldehyde-modified proteins were quantitated by a competition assay with a monoclonal antibody specific for acrolein-modified lysine or HNE-modified histidine epitopes. Minimal reaction times required for the coating/blocking; first monoclonal antibody and the peroxidase-conjugated second antibody binding steps were 3, 3, and 7 min, respectively, the former two steps being found to be or akin to diffusion-rate-limiting reactions. The convenient ELISA should find an application for analyses of the intricate processes involved in oxidative stress and carcinogenic insult. The epitope-attachment methodology may also be advantageous for the quantitation of various other biologically important haptenic molecules.     

Characterization and use in ELISA of a new monoclonal antibody for quantitation of abscisic acid in senescing rice leaves

A new monoclonal antibody (mAb) was generated against abscisic acid (ABA), and an indirect enzyme-linked immunosorbent assay (ELISA) using this mAb was developed for convenient quantitative analysis of ABA levels in rice leaf extracts. The mAb, raised against (+-)-ABA conjugated to bovine serum albumin (BSA) through its carboxyl group (C₁), reacted preferentially with the (+)-ABA enantiomer, and equally well with both free and methyl-ester (+-)-ABA. Cross-reactivity with several ABA-related compounds was negligible. Linearity was obtained between 3 and 1000 pmo1 of (+)-ABA. The ABA-mAb was further used to quantitate pmol quantities of (+)-ABA in attached and detached rice leaves. Results obtained with such ELISA quantitation showed an increase in the free ABA content of detached rice leaves at progressive stages of senescence, which was regarded as a senescence-related response. This quantitation compared favorably with other presently used techniques for ABA determination, with regard to their detection limits, cost and assay time. The results suggest that the combination of a specific mAb with a sensitive ELISA technique is quite promising for quantitation of ABA.     

Polyclonal antibodies to domoic acid, and their use in immunoassays for domoic acid in sea water and shellfish

Ovine antibodies raised against conjugates linked through the secondary amino group of domoic acid (1) were used, together with activated-ester-derived conjugates of domoic acid (DA) as the plate coater, to develop a robust indirect competitive enzyme-linked immunosorbent assay (cELISA) for DA in shellfish and seawater. The ELISA was used to analyze shellfish samples for DA, and was compatible with several extraction procedures. The ELISA had a detection limit below 0.01 ng ml(-1), a limit of quantitation (LOQ) of 0.15 ng ml(-1) and a working range of 0.15-15 ng ml(-1) DA. The LOQ is equivalent to 38 ng g(-1) DA in shellfish flesh, assuming a 250-fold dilution during extraction. This is more than 500 times lower than the maximum permitted level (20 microg g(-1) flesh). The ELISA is designed for use alongside regulatory analyses, and, following formal validation, should be available for pre-screening of regulatory shellfish flesh samples. The ELISA was also shown to be appropriate for analysis of DA in algal cultures and in samples of seawater, and thus has the potential to provide early warning of developing algal blooms.     

Development and optimization of an enzyme-linked immunosorbent assay employing two murine monoclonal antibodies for absolute quantitation of human beta-glucuronidase.

We have developed and optimized an enzyme-linked immunosorbent assay (ELISA) for absolute quantitation of human beta-glucuronidase. This is a double antibody sandwich system employing two murine monoclonal antibodies specific for human beta-glucuronidase developed in our laboratories. The method involves (a) coating of the high binding polystyrene microtitration plate with the first antibody (7B6 IgG), (b) blocking of remaining active sites with 3% bovine serum albumin in phosphate-buffered saline, (c) application of samples, (d) addition of the biotinylated second antibody (6D2 IgG), (e) addition of streptavidin-horseradish peroxidase, and (f) development of color with o-phenylenediamine dihydrochloride-H2O2 and reading in a microplate reader at a wavelength of 490 nm. The method is highly sensitive with an optimal range of 10 to 100 ng/ml of the enzyme and is reproducible with intraday and interday precisions of 3.2 and 4.1%, respectively. The enzyme contents of 20 urine and 20 bile samples quantitated by this ELISA method were, respectively, 148 +/- 101 and 6380 +/- 3780 ng/ml (means +/- SD) which correlated well with their enzyme activities. Such a method for absolute quantitation of human beta-glucuronidase is essential for studying its pathophysiologic roles in cholelithiasis and carcinogenesis and can also be used clinically as an indicator for tissue damage or malignancy.     

Monoclonal antibodies to DNA modified by 8-methoxypsoralen and ultraviolet A light.

A panel of monoclonal antibodies have been developed which specifically recognize DNA modified by 8-methoxypsoralen (8-MOP) and ultraviolet A light (320-400 nm) (UVA). These antibodies have been characterized as to sensitivity and specificity by an enzyme linked immunosorbent assay (ELISA). In a competitive ELISA with the most sensitive antibody, 50% inhibition of antibody binding occurred at 17 fmole 8-MOP-DNA photo adducts. One adduct per 10(7) bases could be reliably detected. There was also some antibody cross-reactivity with DNAs modified by 4' aminomethyl-4, 5, 8-trimethylpsoralen and 4', 5-dimethylangelicin as well as DNA isolated from cells treated with 8-MOP and UVA. The primary specificity of one of the antibodies was shown to be the 4', 5' thymine monoadduct by competitive inhibition studies using HPLC fractions of an enzymatic digest of 8-MOP poly(dA-dT) . poly(dA-dT). These antibodies should allow the quantitation of adduct levels in various in vitro systems as well as humans exposed clinically to 8-MOP and UVA.     

Comparison between inhibitory indirect ELISA and HPLC methods to quantify free and adducted aflatoxins in human urine

HPLC and Inhibitory Indirect ELISA (I.I. ELISA) methods for quantitation of aflatoxins (AF) in human urine were compared in terms of specificity, sensitivity, easiness and cost. I.I. ELISA was optimized in kind of antibody in use, type of plastic plate, adduct synthesis technique, peroxidase and antibody dilutions, etc. Both polyclonal (Cuban) and monoclonal (British) anti-AF antibodies were statistically studied and the process was standardized. HPLC and electrophoresis were performed while synthetizing AFB(1)-DNA and AFB(1)-Cl-Ovalbumin (AFB(1)-Cl-Ov) adducts. Costar polystyrene plate had the best adherence. Optimum coating dilution was 10 ng of AFB(1)-Cl-Ov per well. Dilutions of 1:1000 of monoclonal antibody from purified culture or 1:300 from monoclonal antibody from tissue culture and 1:1000 of peroxidase anti-mouse conjugate were the best. Optimum separation with HPLC was obtained isocratically with 60% MeOH and 40% distilled water mobile phase. ELISA had a sensitivity of 1 pg mL(-1) AFB(1) and HPLC sensitivity was 0.1 ng mL(-1) AFB(1) with fluorescence detector and 4.5 ng mL(-1) with UV detector. Monoclonal antibody gave more accurate results for determination of free and adducted AFB(1) in urine analysis.     

Determination of 8-hydroxydeoxyguanosine by an immunoaffinity chromatography-monoclonal antibody-based ELISA

The postulated importance of oxidative damage to DNA in aging and age-related degenerative pathologies such as cancer has prompted efforts to develop sensitive quantitation methods. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is a widely used marker for oxidative damage to DNA. To develop an immunoassay for quantitation of 8-OHdG, two monoclonal antibodies have been developed and characterized by competitive enzyme-linked immunosorbent assay (ELISA). Antibody 1F7 has 50% inhibition at 5 pmol 8-OHdG and 1 × 105 pmol dG, while antibody IF11 has 50% inhibition at 2.5 pmol 8-OHdG and 2000 pmol dG. Both antisera crossreact with guanosine and several structurally related derivatives, including 6-and 8-mercaptoguanosine, 8-bromoguanosine, 8-methylguanine, and 7-methylguanosine. Immunoaffinity columns were prepared with antibody 1F7, which exhibits higher selectivity than 1F11, to isolate 8-OHdG from DNA hydrolyzates followed by ELISA quantitation with antibody 1F11. This method allows the analysis of approximately one 8-OHdG/105 dG using 100μg DNA. To validate the assay, DNA extracted from human placental tissues were assayed by both ELISA and HPLC with electrochemical detection. Values by both methods correlated well (r = 0.87, p < 0.001), but the levels determined by ELISA were approximately sixfold higher than those determined by HPLC. This may be due to oligonucleotides detected by the ELISA but not the HPLC method or crossreactivity with other damaged bases present in the immunoaffinity purified material. Placental samples from current smokers had significantly higher 8-OHdG by ELISA than those from nonsmokers (p < 0.05). The method of immunoaffinity purification combined with ELISA quantitation has sufficient sensitivity for detecting 8-OHdG in human DNA samples. Although absolute values are higher than those determined by HPLC, the method provides a good alternative to the HPLC-EC method for monitoring relative oxidative damage in molecular epidemiological studies.     

Competitive binding of dynorphin-(1-13) and beta-endorphin to cerebroside sulfate in solution

Circular dichroism was used as a probe for competitive binding of two opioid peptides, dynorphin-(1-13) and beta-endorphin, with cerebroside sulfate, a membrane lipid thought to be part of the morphine receptor complex. The rationale was that bound beta-endorphin is partially helical but bound dynorphin-(1-13) remains unordered, thus making it possible to detect the degree of binding of beta-endorphin. The addition of dynorphin-(1-13) to a cerebroside sulfate solution of beta-endorphin invariably displaced beta-endorphin from the peptide-lipid complex, but the addition of beta-endorphin had little effect on dynorphin-(1-13) bound to the lipid. Similar results were obtained for competitive binding of the two peptides with two other amphiphiles, sodium dodecyl and decyl sulfate. The maximum number of binding sites on dynorphin-(1-13) and beta-endorphin was between five and six, which coincides with the five positively charged side chains plus an alpha NH+3 group at the NH2 terminus on both peptide molecules. The results support our working hypothesis that dynorphin-(1-13) may displace beta-endorphin bound to the receptor, which in turn can account for the inhibition of beta-endorphin-induced analgesia by dynorphin-(1-13).     

Comparative quantitation of abscisic acid in plant extracts by gas-liquid chromatography and an enzyme-linked immunosorbent assay using the avidin-biotin system

In this report we describe an enzyme-linked immunosorbent assay (ELISA) for the quantitation of abscisic acid (ABA) in plant extracts. A microtitration plate is coated with an ABA-protein complex. The ABA, standard or sample, is then added to each well with a limiting quantity of rabbit anti-ABA antibodies. During the following incubation period, antibodies bind either to free or to bound ABA on the plates. After washing, bound antibodies are indirectly labelled in two steps by the means of biotinylated goat antirabbit immunoglobulin-G antibodies which act as a link between rabbit anti-ABA antibodies and an avidin-alkaline phosphatase complex. The relative enzyme activity bound is measured spectrophotometrically. The detection limit of this method is 5 pg ABA and the measuring range extends to 10 ng. Gas-liquid-chromatography controls, with an electron capture detector, show a good correlation with ELISA results obtained using extracts of Lycopersicon esculentum, Nicotiana tabacum and Pseudotsuga menziesii samples purified by high-performance liquid chromatography. This provides a good argument for the accuracy of the immunoenzymatic method. The indirect labelling of antibodies, with the avidin-biotin amplifying system, should make this technique suitable for the quantitation of other plant growth substances against which specific antibodies are available.Abbreviations ABA abscisic acid - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - GLC gas liquid chromatography - HPLC high-performance liquid chromatography - IgG Immunoglobulin G - PBS phosphate-buffered saline     

Colorimetric competitive inhibition method for the quantification of avidin, streptavidin and biotin.

A colorimetric competitive inhibition assay for avidin, streptavidin and biotin was developed. The method for avidin or streptavidin was based on the competitive binding between avidin or streptavidin and a streptavidin-enzyme conjugate for biotinylated dextrin immobilized on the surface of a microtitre plate. For biotin quantitation the competition is between free biotin and the immobilized biotin for the streptavidin-enzyme conjugate. The limits of detection which was determined as the concentration of competitor required to give 90% of maximal absorbency (100% inhibition) was approximately 20 ng/100 microl per assay for avidin and streptavidin and 0.4 pg/100 microl per assay for biotin. The methods are simple, rapid, highly sensitive and adaptable to high throughput analysis.     

Development of a highly sensitive ELISA for the determination of PBAN and its application to the analysis of hemolymph in Spodoptera littoralis

A highly sensitive enzyme linked immunosorbent assay (ELISA) for the determination of the pheromone biosynthesis activating neuropeptide (PBAN) has been developed. Six antisera have been obtained that recognize the carboxyl terminal side of this peptide. Two immunogens have been rationally designed and synthesized in order to direct antibody specificity, using as haptens PBAN or PBAN(20-33) with a Cys residue attached to their amino-terminal side. The Cys thiol group has been used to covalently bind the peptide to keyhole limpet hemocyanin (KLH) by using N-succinimidyl-4-(maleidimidomethyl) cyclohexane carboxylate (SMCC) as a convenient heterobifunctional cross-linker. Several usable competitive immunoassays have been obtained by synthesizing eight different coating antigens and screening the sera against all of them. The best assay was obtained with antibody 4 using Cys-Hez-PBAN(20-33) coupled to bovine serum albumin (BSA) through the Lys groups by using the homobifunctional cross-linker dimethylpimelidate dihydrochloride (DMP) as the coating antigen. The optimized assay allows to detect PBAN at concentrations as low as 1 fmol/well (l₅₀ = 2.5 fmol/well). An extraction procedure for the hemolymph has been developed that allows to perform PBAN measurements in this tissue even after a tenfold dilution. In these conditions matrix effect is negligible. Preliminary results on the presence of PBAN like immunoreactivity (PBAN-IR) in the hemolymph of Spodoptera littoralis females are reported.© 1995 Wiley-Liss, Inc.     

Mechanism of inhibition of RNA polymerase II and poly(adenylic acid) polymerase by the O-n-octyloxime of 3-formylrifamycin SV.

Factors affecting the inhibition of RNA polymerase II from rat liver by the O-n-octyloxime of 3-formylrifamycin SV (AF/013) were investigated. Using either native or denatured calf-thymus DNA as template, almost complete inhibition of RNA polymerase II was observed when AF/013 was added directly to the enzyme. Considerable resistance to AF/013 was observed when RNA polymerase II was preincubated with denatured DNA at either 0 or 37 degrees. However, under similar conditions, no resistance was observed when enzyme was preincubated with native DNA. Only when AF/013 was added to the ongoing reaction using native DNA did a resistance to AF/013 occur. The inhibition of RNA polymerase II by AF/013 was competitive with respect to all four nucleoside triphosphate substrates. The inhibition by AF/013 remaining after enzyme-DNA complex formation also appeared competitive with nucleoside triphosphate levels. The effect of exogenous protein (bovine serum albumin, BSA) on the inhibition of RNA polymerase II was also investigated. BSA reduced the extent of inhibition by AF/013, but did not alter the competitive nature of inhibition. Concurrently, the inhibition of highly purified nuclear poly(A) polymerase from rat liver, a template independent enzyme which incorporates AMP in a chain elongation reaction, was examined. As in the case of RNA polymerase, poly(A) polymerase was inhibited by AF/013 in a manner competitive with the nucleoside triphosphate substrate. The competitive nature of inhibition of RNA polymerase by AF/013 with respect to all four nucleoside triphosphate substrates, before and after enzyme-DNA complex formation, as well as the competitive nature of inhibition of poly(A) polymerase with respect to ATP tend to indicate that the major effect of AF/013 on RNA polymerase II is at the level of the substrate binding as opposed to a specific inhibition of initiation.     

Development of enzyme-linked immunosorbent assay for determination of polybrominated diphenyl ether BDE-121

Our interests are in the development of immunoassay-based fast scanning methods for persistent organic pollutants. To develop the immunoassay method of polybrominated diphenyl ether (PBDE), a model compound of PBDE, 2,3′,4,5′,6-pentabromodiphenylether (BDE-121), has been chosen to develop its antibody and the competitive indirect enzyme-linked immunosorbent assay (ELISA) is developed. The hapten of BDE-121 containing reactive carboxylic acid was synthesized and conjugated to carrier proteins (bovine serum albumin [BSA] and ovalbumin [OVA]). Anti-BDE-121 polyclonal antibody was then developed in rabbits as a result of immunization with the BDE-121–BSA conjugate. The optimal amount of coating antigen BDE-121–OVA conjugate and the dilution of antiserum needed in the ELISA were determined with the checkerboard method, and the effects of the properties of PBST (phosphate-buffered saline and Tween 20) buffer (pH and salt concentration) and chemical solvent (types and concentrations) on the ELISA were investigated to achieve a rapid robust assay with high sensitivity. Under the optimized conditions, the developed indirect ELISA shows a linear detection range from 1.74 to 84.1 ng/ml, with an IC₅₀ value of 8.07 ng/ml and a detection limit of 0.644 ng/ml. In total, 11 kinds of compounds were tested for calculating the cross-reactivity, which was less than 8% for nearly all of them. Real samples were analyzed by the proposed immunoassay and gas chromatography/mass spectrometry (GC/MS).     

Albumin's Influence on Carprofen Enantiomers–Hymecromone Interaction

Hymecromone is an important coumarin drug, and carprofen is one of the most important nonsteroidal antiinflammatory drugs (NSAIDs). The present study aims to determine the influence of bovine serum albumin (BSA) on the carprofen–hymecromone interaction. The inhibition of carprofen enantiomers on the UDP‐glucuronosyltransferase (UGT) 2B7‐catalyzed glucuronidation of hymecromone was investigated in the UGTs incubation system with and without BSA. The inhibition capability of increased by 20% (P < 0.001) of (R)‐carprofen after the addition of 0.5% BSA in the incubation mixture. In contrast, no significant difference was observed for the inhibition of (S)‐carprofen on UGT2B7 activity in the absence or presence of 0.5% BSA in the incubation system. The Lineweaver‐Burk plot showed that the intersection point was located in the vertical axis, indicating the competitive inhibition of (R)‐carprofen on UGT2B7 in the incubation system with BSA, which is consistent with the inhibition kinetic type of (R)‐carprofen on UGT2B7 in the incubation system without BSA. Furthermore, the second plot using the slopes from the Lineweaver‐Burk versus the concentrations of (R)‐carprofen showed that the fitting equation was y=39.997x+50. Using this equation, the inhibition kinetic parameter was calculated to be 1.3 μM. For (S)‐carprofen, the intersection point was located in the horizontal axis in the Lineweaver‐Burk plot for the incubation system with BSA, indicating the noncompetitive inhibition of (S)‐carprofen on the activity of UGT2B7. The fitting plot of the second plot was y=24.6x+180, and the inhibition kinetic parameter was 7.3 μM. In conclusion, the present study gives a short summary of BSA's influence on the carprofen enantiomers–hymecromone interaction, which will guide the clinical application of carprofen and hymecromone. Chirality 28:226–229, 2016. © 2015 Wiley Periodicals, Inc.     

Quantitation of the endopeptidase activity generating gamma-endorphin from beta-endorphin in rat brain synaptic membranes by a radiometric assay

gamma-Endorphin is a naturally occurring biologically active peptide that is produced by an endopeptidase activity cleaving its precursor beta-endorphin. This enzyme was termed gamma-endorphin generating enzyme (gamma-EGE). In order to quantitate gamma-EGE activity by means of a simple and sensitive assay two synthetic peptides derived from the sequence surrounding the gamma-EGE cleavage site in beta-endorphin were tested as substrates. One of these peptides Ac-Val-Thr-Leu-Phe-Lys-NHCH3 fulfilled all criteria for a suitable gamma-EGE substrate. The peptide was exclusively cleaved at the correct bond for gamma-EGE upon incubation with brain synaptic membranes, and this cleavage was inhibited by the naturally occurring substrate beta-endorphin. The peptide was insensitive to cleavage by exopeptidases and cathepsin D. Addition of a 14C-labeled methyl group at the lysine residue of this peptide by reductive methylation did not alter its properties as a substrate for gamma-EGE activity. The use of the 14C-labeled peptide allowed sensitive quantitation of its radioactive products after simple separation by hydrophobic chromatography on minicolumns containing polystyrene beads. gamma-EGE activity increased linearly with a protein concentration and incubation time. This assay can be used for reliable quantitation of gamma-EGE activity and permits investigations on the regulation of gamma-endorphin production.     

Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface

Glutathione S-transferase genetically fused with an affinity peptide tag, PS19 (RAFIASRRIKRP) having a specific affinity for a hydrophilic polystyrene (PS) surface, was preferentially immobilized on a hydrophilic PS (phi-PS) plate without suffering from interference by coexisting protein molecules. Furthermore, rabbit IgG chemically conjugated with a peptide, KPS19R10, in which (10)Lys in PS19 was replaced with Arg and one Lys residue was added at the N-terminus as a coupling site for glutaraldehyde, showed a higher immobilization affinity to the phi-PS plate than that conjugated with the PS19 peptide. On the basis of these findings, the use of a phi-PS plate and peptide tag-linked ligand proteins permitted a one-step or two-step enzyme-linked immunosorbent assay (ELISA) to be achieved, resulting in a substantial reduction in operational time compared with the conventional ELISA method using a hydrophobic PS (pho-PS) plate, while maintaining a high sensitivity. Furthermore, the sensitivity was increased to a greater extent compared to the conventional ELISA meihod when the one-step ELISA was applied to the detection of bovine insulin in a sandwich mode, due to the reduced number of washing and incubation steps. The method proposed here would be a versatile method for use in various ELISA techniques such as sandwich and competitive ELISAs using an antigen, an antibody and streptavidin that are genetically fused or chemically conjugated with the PS-specific affinity peptide as the ligand protein.     

<Emphasis Type="Italic">Blastomyces Dermatitidis</Emphasis> Antigen Detection in Urine Specimens from Dogs with Blastomycosis Using a Competitive Binding Inhibition ELISA

A competitive binding inhibition enzyme linked immunosorbent assay (ELISA) was used to detect Blastomyces dermatitidis antigens in urine specimens from dogs with blastomycosis. Sera from rabbits immunized with B. dermatitidis killed whole yeast cells were used as the primary antibody in the competitive ELISA. This initial study was performed to determine if B. dermatitidis antigen detection was possible and to test the efficacy of the rabbit sera as a primary antibody. An indirect ELISA was also performed to compare antigen detection in urine to antibody detection in the sera of the infected dogs. The results indicate 100% (36/36 specimens) detection of both antigen and antibody. Cross reactivity with Histoplasma capsulatum, as well as non-specific binding with the normal urine specimens, was observed with the competitive binding inhibition ELISA.     

黄曲霉毒素B_1的免疫检测 Ⅱ.抗体的产生及应用

将黄曲霉毒素Ｂ１肟（ＡＦＢ１Ｏ）与牛血清白蛋白（ＢＳＡ）的连接物,通过多点、多次免疫法注射免疫兔子。分析了抗体的产生进程、效价以及特异性。注射抗原后的第６０天开始有较明显抗体产生,第１２０天达到高峰,维持１５天左右后开始下降;抗体的ＥＬＩＳＡ效价高达１：３００００;和黄曲霉毒素Ｂ１（ＡＦＢ１）的结构类似物的竞争ＥＬＩＳＡ表明,抗体有很好的特异性。运用该抗体,以ＥＬＩＳＡ分析检测了几种农产品及饲料中污染ＡＦＢ１的含量,并和薄层层析法的分析结构进行了比较,结果表明当ＡＦＢ１的含量大于等于５ｎｇ／ｍｌ时,两者间有很好的相关性。     

Characterization of 11-dehydro-thromboxane B2 recombinant antibody obtained by phage display technology

Recombinant monoclonal antibodies specific for 11-dehydro-thromboxane B(2) (11D-TX) were isolated from the combinatorial libraries on a pComb3 phage-display vector using a magnetic cell sorting (MACS) system. The libraries were constructed from repertories of light and heavy-chains derived from the total RNA of 11D-TX conjugated keyhole limpet haemocyanin-immunized mice. Biotinylation of 11D-TX conjugated bovine serum albumin (BSA) was performed through free thiol groups on BSA using 1-biotinamido-4-[4'-(maleimidomethyl) cyclohexanecarboxamido] butane (Biotin-BMCC). Affinity bio-panning was performed to enrich the phage display libraries against biotinylated 11D-TX conjugated BSA with the MACS system. Results indicated that the selected anti-11D-TX Fab fragments expressed by E. coli exhibited a five-fold higher affinity for BSA conjugated 11D-TX compared to BSA alone and little specificity to other related compounds as determined by the binding assay and inhibition enzyme-linked immunosorbent assay (ELISA). This is the first report of an antibody against prostaglandin produced by phage display technology and also determination of the DNA sequence of this antibody. The MACS system was shown to be a simpler and more efficient method of panning than the conventional ELISA procedure. According to our results, we concluded that the phage display technique combined with the MACS system allowed the selection of the antibody with high affinity and some specificity.     

Antibody to platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF)

Specific antibodies to platelet activating factor (PAF) were prepared by immunizing rabbits with a hapten-bovine serum albumin (BSA) conjugate. As the hapten we used the synthetic PAF derivative which is resistant against enzymatic inactivation by plasma or tissues and which can bind to BSA through covalent bonding. Antibody activity was determined by an enzyme-linked immunosorbent assay (ELISA). Anti-PAF IgG reacted strongly with PAF. By means of the ELISA inhibition assay, we found that the antibody did not cross-react with phosphocholine, glycerophosphocholine, dilaurylglycerophosphocholine or PAF analogues which have ethanolamine-type polar head groups instead of choline group.