Quantitative determination of acetylcholinesterase by enzyme antigen immunoassay: Methodological aspects and clinical use

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<ul><li><p>Scand. J . clin Lab. Invest. 44. 717-724. 1Y84. </p><p>Quantitative determination of acetylcholinesterase by enzyme antigen immunoassay: methodological aspects and clinical use </p><p>J . H A N G A A R D . K E L D S B R E N S E N . U R S B R O D B E C K . &amp; B E N T N 8 R G A A R D - P E D E R S E N : I Department of Clinical Chemistry, Sonderborg Hospital, Denmark, Medizinisch-Chemisches Institut, University of Bern, Switzerland, and The Hormone Department, Statens Seruminstitut, Copenhagen, Denmark </p><p>Hangaard. J. . Sorensen. K . , Brodbeck, U. &amp; Norgaard-Pedersen, B. Quantitative determination of acetylcholinesterase by enzyme antigen im- munoassay: methodological aspects and clinical use. Scund. J. clin. Lub. Invest. 44, 717-724, 1984. </p><p>We here describe the optimization of an immunochemical measuring method for acetylcholinesterase (AChE) found in different human body fluids. The principle is the binding of a polyclonal antibody to a solid support (microtitre plate), followed by quantitation of the enzymatic active antigen. by its own enzymatic activity. The test is mainly thought as a diagnostic tool for the prenatal detection of neural tube defects (NTD). The assay is optimized with respect to antibodies and a number of physical parameters. The test is accurate and reliable. and it yields quantitative results. Further we show i t to give neither false positive nor false negative values in the prediction of NTD. The assay also performs well on other sample-materials. We suggest that the test will be routinely used alongside amniotic a-fetoprotein (AFP) determinations to ensure the correct prenatal diagnosis of NTD. </p><p>Key-words: acetylcholinesterase: amniotic fluid: neural tube defects; prenatal diagnosis </p><p>Bent NQrguard-Pedersen, The Hormone Department, Statens Seritrninstitirt, Amuger Boulevard 80. D K-2300 Copenhagen, Denmurk </p><p>Measurements of amniotic a-fetoprotein (AFP) is the main prenatal diagnostic test for open neural tube defects (NTD) [12. 13, 17, 181. However, the test may yield both false positive and false negative results. In order to corrobor- ate the findings of the AFP test it is imperative to have a confirmatory test. Several studies </p><p>have recently confirmed the significance of qualitative determination of acetylcholinesterase (AChE) (E.C. in amniotic fluid by polyacrylamide gel electrophoresis (PAGE) [4, 6, 19-21] for this purpose. As a supple- mentary and confirmatory test for NTD, the (AChE) (E.C. in amniotic fluid by </p><p>717 </p><p>Scan</p><p>d J </p><p>Clin</p><p> Lab</p><p> Inv</p><p>est D</p><p>ownl</p><p>oade</p><p>d fr</p><p>om in</p><p>form</p><p>ahea</p><p>lthca</p><p>re.c</p><p>om b</p><p>y O</p><p>saka</p><p> Uni</p><p>vers</p><p>ity o</p><p>n 12</p><p>/08/</p><p>14Fo</p><p>r pe</p><p>rson</p><p>al u</p><p>se o</p><p>nly.</p></li><li><p>718 J . Hangaard et al. </p><p>applied to amniotic fluid samples with positive AFPresults [14,16,17]. The spectrophotometric method for AChE determination has not improved the discrimination provided by AFP [17, 191 due to the low content of the enzyme in the sample material and the lack of absolute specificity of the inhibitors used. </p><p>Recently, a new quantitative enzyme antigen immunoassay (EAIA) for AChE has shortly been described by Norgaard-Pedersen et al. [ 151 This assay utilizes an antibody to AChE which forms an enzymatically active irnmuno-complex with AChE. The EAIA gives comparable results to the PAGE technique, when applied to normal and pathological amniotic fluid sam- ples [15], and has the advantage of a higher sensitivity and sample throughput, the potential of being automatized, not requiring toxic sub- stances, yielding quantitative results and being relative insensitive to the reagents used. </p><p>The present study describes the methodolo- gical aspects of EAIA in further details, i.e. the optimization of the assay and antisera. Most important. the test is evaluated on a large number of amniotic fluid samples. both normal and pathological. </p><p>A further interest in the accurate determina- tion of AChE in body fluids has been stimu- lated by the reportings that a change in brain- AChE is found in patients suffering from Alzheimer's disease [ I ] . The presented test can be utilized for the determination of AChE in cerebrospinal fluid and post-mortem brain extract. </p><p>M A T E R I A L S A N D M E T H O D S </p><p>The material included supernatants (centrifuga- tion: 3000 g ; 10 min) of amniotic fluid samples obtained from diagnostic amniocentesis a t 12-26 weeks of gestation (1470 normal. 135 pathological and 40 blood contaminated). </p><p>The assay has been described by Norgaard- Pedersen et al. [15]. Briefly, the procedure consists of hydrophobic binding of the IgG to polystyrene microtitre plates followed by in- cubation with sample and subsequent demon- stration of AChE activity by the Ellman method. In this paper, the individual para- meters of the assay have been extensively varied in order to optimize the procedure. All reactions were carried out in flat bottom </p><p>polystyrene microtitre plates (Immuno plate 11, 2-42404 from NUNC, Denmark). The absor- bance was measured with a Titertek Multiscan spectrophotometer (Flow Laboratories) at 405 nm. Blanking was automatically performed on the first row of the microtitre plate. Polyac- rylmide gel electrophoresis used for compari- son, was carried out as described by Smith er ul. [221. </p><p>Reagents </p><p>Antigens. Detergent soluble AChE (DS- AChE) from human erythrocyte membranes and from human brain were purified by affinity chromatography according to the procedures described by Brodbeck et al. and Sorensen rt ,al., respectively [5, 231. This purification yielded pure preparations as judged by SDS- PAGE. </p><p>Antisera. The pure AChE's were used for immunization of rabbits following the recom- mendations given by Harboe &amp; Ingild [ 101. The IgG fraction was isolated by ammoniumsul- phate precipitation [ 121. </p><p>The antibodies were functional monospecific, i.e. they reacted only with one protein. namely the AChE in the original crude homogenate from which the enzyme is purified. The antigen from red blood cell reacted also with the antibody against the brain enzyme and vice versa. </p><p>Standardization. For general routine purpose an inexpensive, easily obtainable standard is of paramount importance. For this purpose we have used a serum pool from healthy blood donors. A serum pool from 40 donors was collected, and defined to contain 1000 arbitrary AChE units/l. Different pools were identical with respect to the AChE content. </p><p>For the determination of conversion factors between IU and arbitrary units, a well defined pure preparation of AChE had to be used. We used the pure enzyme from erythrocyte mem- brane with a specific activity of 4500 IU/mg protein. </p><p>Other materiuh </p><p>All substances were of the highest purity available. </p><p>Buffers. The following buffers were used: phosphate buffered saline (PBS) 10 mmol/l </p><p>Scan</p><p>d J </p><p>Clin</p><p> Lab</p><p> Inv</p><p>est D</p><p>ownl</p><p>oade</p><p>d fr</p><p>om in</p><p>form</p><p>ahea</p><p>lthca</p><p>re.c</p><p>om b</p><p>y O</p><p>saka</p><p> Uni</p><p>vers</p><p>ity o</p><p>n 12</p><p>/08/</p><p>14Fo</p><p>r pe</p><p>rson</p><p>al u</p><p>se o</p><p>nly.</p></li><li><p>Acetylcholinesteruse immunoussuy 719 </p><p>phosphate pH 7.2 with 145 mmol/l NaCI. For washing of the immuno-plate the buffer is supplemented with 0.05% Tween 20. Substrate for enzyme determination: 1 mmol/l acetyl- thiocholineiodide and 0.5 mmol/l5.5'dithio-bis- (2-nitro-benzoic acid) (3,3'-6) (DTNB) in 50 mmol/l phosphate, pH 7.0 [8]. </p><p>Inhibitors. Two inhibitors have been used, each specific for either AChE or ChE: (a) Lysivane (ChE inhibition) from May &amp; Baker Ltd. in a stock solution of 1.7 mmol/l (test solution 10 pmol/l) and (b) BW 284C59 (AChE inhibition) from Sigma, Mo., USA in a stock solution of 4.4 mmol/l (test solution 26 pmol/l). </p><p>Busic procedure (Fig. 1) </p><p>(a) Diluted IgG fraction (100 pl) from anti- serum was pipetted into the wells of the </p><p>1. ANTIBODY - COATING. antibody </p><p>k t Q -solut ion 4 </p><p>/incubate </p><p>2. ANTIGEN-ANTIBODY REACTION. </p><p>3. E N Z Y M E - REACTION. </p><p>FIG. I . Schematic analytical procedure for the enzyme antigen immunoassay (EAIA) . Note that the dem- onstration o f bound antigen is performed using the enzymatic activity of the antigen in the immunecom- plex. </p><p>polystyrene microtitre plate and incubated for at least 16 h at 37C. (b) The wells were washed three times with the PBS buffer containing 0.05% Tween 20. (c) A 100-pI sample was added to each of the wells, and incubated for 4 h at 37C. (d) As (b). (e) Substrate solution (100 pl) was added to each well and the reaction allowed to proceed for 2 h at 37C. (f) Recording of the absorbance of the well con- tent, at 405 nm (or 412 nm). </p><p>R E S U L T S Optimizing ussuy conditions </p><p>Different antibodies were tested for their usefulness in this assay. Antisera was raised to erythrocyte membrane AChE. using either the antigen in a Triton X-100 buffer, or in a detergent free buffer. The two different anti- sera showed exactly the same reactivity towards the different types of samples (normal amniotic fluid, pathological amniotic fluid, serum and cerebrospinal fluid). The antibody raised against DS-AChE from human caudate nucleus showed a slightly different reaction pattern; the values for pathological amniotic samples turned out to be higher, whereas the normal samples gave lower responses. </p><p>Within the range of antibody dilution 1500 to 1:2000 only minor differences were observed. In our routine assay we chose an antibody dilution of 1:1000. which gives a lower limit of detection of 4 arbitrary unitdl and linear response up to 500 arbitrary units/l. Furthermore, this antibody concentration yielded very satisfactory 'within' and 'between' assay precision (both below 6.5%; see Table I ) . </p><p>TABLE 1 . Precision studies </p><p>Mean AChE Within-assay Between-assay arb unitsll CV 'XI ( n ) CV '%) (tf) </p><p>.~ </p><p>Control 1 15 6 . I (10) 6.5 (10) Control 2 70 2.8 (10) 3.5 (10) Control 3 240 2.7 (10) 3.5 (10) </p><p>4.4 ( 1 0 ) 4.4 (10) Control 4 350 </p><p>Four different samples (control 1 to control 4) have been tested. The within-assay precision was dcter- mined in 10 experiments, and the values given are coefficient of variation ( C V ) within the individual plate. The between-assay CV was determined by measuring the samples on different days. with different lot-number of plates. In all cases an internal standard (normal human serum pool) was used. </p><p>Scan</p><p>d J </p><p>Clin</p><p> Lab</p><p> Inv</p><p>est D</p><p>ownl</p><p>oade</p><p>d fr</p><p>om in</p><p>form</p><p>ahea</p><p>lthca</p><p>re.c</p><p>om b</p><p>y O</p><p>saka</p><p> Uni</p><p>vers</p><p>ity o</p><p>n 12</p><p>/08/</p><p>14Fo</p><p>r pe</p><p>rson</p><p>al u</p><p>se o</p><p>nly.</p></li><li><p>720 J . Hangaurd et al. </p><p>Lower dilutions of the antisera did not increase the sensitivity, and the response to zero dose did not increase: i.e. the slope o f the standard curve remained the same. The antibody-coated plates can be stored at 4C for at least ii week. provided they are sealed airtight t o halt evaporation. </p><p>Effkct of tcwiperutirrr und time on untihody coating. Diluted antibody ( I 0 0 pl) was dis- pensed into the wells of the microtitre plates, covered and left overnight at 4, 22 and at 37C. After the normal washing procedure a number o f standards were run in the plates, according to the basic procedure. The fraction o f IgG bound was nearly identical in all cases, regardless of the incubation temperature. The highest hind- ing was found at 37C. Experiments with a prolonged incubation time, up to 72 h , did not increase the binding of the antibody to the solid support; consequently, as a standard proce- dure, we have chosen to incubate overnight at 37C. </p><p>The use o f a blocking agent. for excess binding capacity of the plate, was investigated. It appeared that the traditional coating with albumin could be omitted without affecting the non-specific binding, if the detergent Tween 20 was included 131. This yielded an efficient washing of the plates with the required low background as result and reduced the time needed for preparing the plates. </p><p>Effkct of time on untipw-utit iboiiy cornplex forniution (Fig. 2). AChE standards (100 pl. serum dilutions) was dispensed into the wells of microtitre plates. The incubation time was varied between 1 and 4 h, followed by the basic procedure as described in materials and methods. Figure 2 shows a significantly higher degree of antigen binding after 4 h compared to the shorter times. </p><p>Effect of temprruture on crntigen-untibody binding (Fig. 3 ) . Samples o f AChE were incubated at 37C. room temperature and 4C in the antibody-coated wells, and the extent of complex formation was monitored by the stan- dard procedure. Incubation at 37C gave the highest degree of antigen binding and a s a consequence this temperature was used f o r the assay (Fig. 3). </p><p>Effect of suhstrute und DTNB concentrution. AChE is known to display the phenomenon of substrate inhibition. We have investigated the influence o n enzyme activity o f substrate con- </p><p>1.4 </p><p>1.c </p><p>c 0 </p><p>U c </p><p>w </p><p>+ </p><p>- c </p><p>0.E </p><p>0.2 </p><p>Activi ty </p><p>FIG. 2. Effect ol time on antigen-antibody hinding. The abscissa is the activity i n arbitrary units/l 0 1 thc sample. The ordinate is the resulting extinction ;it 405 nm. read in the microtitre plate. Symbols: m, Ih: 0. 2 h; 0. 3 h; 0. 4 h . </p><p>1.L </p><p>1.C </p><p>c 0 </p><p>F + U - </p><p>0.E </p><p>0.2 </p><p>Act iv i t y </p><p>FIG. 3. Eft'cct of tcmpcraturc on antigen-antibody binding. Ordinate and abscissa a s in Fig. 2. Symbols: .. 4C: 0. 22C; 0. 37C. </p><p>Scan</p><p>d J </p><p>Clin</p><p> Lab</p><p> Inv</p><p>est D</p><p>ownl</p><p>oade</p><p>d fr</p><p>om in</p><p>form</p><p>ahea</p><p>lthca</p><p>re.c</p><p>om b</p><p>y O</p><p>saka</p><p> Uni</p><p>vers</p><p>ity o</p><p>n 12</p><p>/08/</p><p>14Fo</p><p>r pe</p><p>rson</p><p>al u</p><p>se o</p><p>nly.</p></li><li><p>Acetylcholinesterase immunoassay 721 </p><p>centrations in the range from 0.3 to 3.3 mmol/l. The results in Fig. 4 show that the optimal substrate concentration was 1 mmol/l. At higher substrate concentrations the typical sub- strate inhibition was observed and the effective- ness of the system was decreased. (Fig. 4). Although the DTNB and the thiocholine reacts in a 1 : l stochiometry, the staining solution contains 0.5 mmol/l DTNB and 1 mmol/l acetylthiocholiniodide. Concentrations of DTNB larger than 0.5 mmol/l resulted in increased background. At above 250 arbitrary units/l. the response is no longer linear at a DTNB concentration of less than 0.25 mmol/l. The optimal concentration was thus 0.5 mmol/l 16, 71. </p><p>The enzyme reaction was allowed to develop up to 4 h. The relation of colour development to time was linear up to 500 arbitrary unitdl within 2 h. Prolonged incubation time did result in higher extinctions values, but the response was non-linear. To obtain the highest discri- minatory power within normal working time, 2 h incubation was selected. </p><p>In order to verify the nature of the bound antigens, they were pre-incubated with the cholinesterase inhibitors BW284C51 (selective for AChE) and Lysivane (selective for ChE). The inhibitor BW284C51 at 26 pmol/l entirely inhibited enzyme activities up to at least 1000 arbitrar units/l; the highest enzyme concentra- tion tested. As expected. no inhibition was obtained with Lysivane in concentrations up to 10 pmol/l. </p><p>Determinations of AChE in normal and patho- logical amniotic fluids </p><p>With the described optimized assay we have examined 1470 clear amniotic fluids from pregancies with normal outcome. One- thousand-four-hundred of these were consecu- tively select...</p></li></ul>


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