HYBRIDOMAVolume 7, Number 5, 1988Mary Ann Liebert, Inc., Pubiishers

A Screening Technique for Monoclonal AntibodyProduction: Application of an Indium

Slide ImmunoassayROBERT REJ,1 CHARLES R. KEESE,2 and IVAR GIAEVER2

'Wadsworth Centerfor Laboratoriesand Research, New York Stale Department of Health, Albany, NY 12201-05092Research and Development Center, General Electric Co., Schenectady, NY 12301

ABSTRACT

A simple technique is described that is suitable for rapid screening of hybridoma microculturefluids for monoclonal antibody producing hybrids. The procedure measures increase in light scatter dueto the antigen-antibody reaction on a surface of indium metal coated upon glass and does not requireuse of a labeled second antibody. Techniques minimizing nonspecific binding in such assays arepresented. The procedure was used to screen hybridoma microculture fluids containing mousemonoclonal antibodies directed against human mitochondrial isoenzyme of aspartate aminotransferase(EC 2.6.1.1). The technique was compared to an enzyme-linked immunosorbent assay, and similar semi-quantitative results were found for sample cultures tested in the two procedures. The new screeningprocedure affords a simple screening assay without sacrificing sensitivity or specificity of standardmethods.

INTRODUCTION

The production of monoclonal antibodies by standard techniques is now well established.Screening for positive clones represents an important and time-consuming portion of the overall effortin this process. Procedures utilizing adaptations of various enzyme-linked, fluorescence, and radiometricimmunoassay methods have been applied for this purpose (1). Each of these techniques requires as areagent, an antibody or antigen with detectable label.

The indium-slide assay (2) allows detection of the antigen-antibody reaction without the need forsuch labeled reagent and has been applied to several analytical applications (3-5). We used thistechnique and rabbit polyclonal antibodies to determine various fractions of the isoenzymes of aspartateaminotransferase (AspAT) in human serum and speculated at that time that the technique might alsobe suitable for detection of monoclonal antibodies at the screening stage (4). We here systematicallyexplored variables related to this adaptation of the technique and compare results to an establishedmicro-ELISA procedure.

Abbreviations used: AspAT, aspartate aminotransferase (EC 2.6.1.1, L-aspartate:2-oxoglutarateaminotransferase); m-AspAT, mitochondrial (cathodal) isoenzyme of aspartate aminotransferase;s-AspAT, soluble (anodal) isoenzyme of aspartate aminotransferase; Tris, tris(hydroxmethyl)-aminomethane; Bis-Tris, [bis(2-hydroxyethyl)amino]-2(hydroxymethyl)-l,3-propanediol; ELISA,enzyme-linked immunosorbent assay; SIA, slide immunoenzymatic assay.

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FIGURE 1. Polyacrylamide-gel electrophoresis of purified m-AspAT. Electrophoresis was carried outin the presence of SDS under dissociating conditions. Lane A

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purified m-AspAT (212 U/mg),Lane B

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molecular weight markers (from top, a-lactalbumin [14.4 Kilodaltons], soybean trypsininhibitor [20.1], carbonic anhydrase [30], ovalbumin [43], bovine serum albumin [67], phosphorylase B[94]), Lane C

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semipurified m-AspAT, before final chromatography on G-I00 Sephadex (158 U/mg).

MATERIALS AND METHODS

Antigen

Chemicals used in the preparation and assay of enzyme activity were of the highest purity availablefrom either Boehringer Mannheim Biochemicals (Indianapolis, IN) or Sigma Chemical Corp. (St. Louis,MO). Ion-exchange resins were from Pharmacia (Piscataway, NJ). AspAT activity was determined bycoupling oxalacetate formation with NADH and malate dehydrogenase at 30 °C (6). MitochondrialAspAT used for production of antibodies was prepared from human liver. Tissue, obtained at autopsywithin 24 hr post mortem from accident cases, was divested of visible fat, cut into portions of ca. 10 g,and washed with cold 0.15 M NaCl solution. Liver was homogenized in cold 20 mM phosphate buffer,pH 7.4, containing 2-oxoglutarate (2.5 mM), 2-mercaptoethanol (14 mM), and pyridoxal phosphate (0.1mM). The homogenate was heated to 52 °C for 20 min, cooled to 4 °C, centrifuged, and the precipitatediscarded. To the clear supernatant, solid (NH4)jS04 was added to 47% saturation at 4 °C, and theprecipitate was discarded. The enzyme was precipitated from the supernatant by adding (NH4),S04 to82% saturation at 4 °C and collected by centrifugation. The precipitate was suspended in 10 mMphosphate buffer, pH 7.5, containing 0.1 mM pyridoxal phosphate and was dialyzed against this bufferwith frequent changes.

The dialysate was applied to a column of DEAE-Sephadex A-50 equilibrated with 10 mMphosphate buffer, pH 7.5, under which conditions the enzyme does not bind. The eluate containingm-AspAT was then applied to a column of SE-Sephadex C-50 under identical conditions and washedwith buffer until the eluate protein concentration was < 5 /ig/ml. The enzyme was then eluted with alinear gradient of NaCl (0-100 mM) in 10 mM phosphate, pH 7.5. The final specific activity of thepreparation was > 180 U/mg, and the enzyme was homogeneous by two polyacrylamide gelelectrophoresis systems (Fig 1). Human s-AspAT was prepared as described previously (7), and mono-clonal antibodies against this isoenzyme, prepared essentially as described below, were used as a control.

Antibodies

Antibodies obtained from commercial sources were: peroxidase-labeled goat immunoglobulinspecific for mouse IgG (heavy and light chains specific) was from Cappel (Cooper Biomédical, Malvern,

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PA) and was used as second antibody for the micro-SIA; second antibody for the indium slide assay wasgoat immunoglobulin specific for mouse IgG (Miles Laboratories, Elkhart, IN).

For antibodies against human m-AspAT, male BALB/cJ mice (Jackson Laboratories, Bar Harbor,ME) 8 weeks old were used for immunization to obtain antisera and spleen cells. Animals received aprimary inter-peritoneal injection of 150 fig of m-AspAT in 75 p\ (ca. 350 U/ml of active enzyme)containing 10 mM Tris and 0.1 mM pyridoxal phosphate emulsified with 0.75 ml of complete Freund'sadjuvant containing 1.5 mg of Mycobacterium tuberculosis H37RA (Difco Laboratories, Detroit, MI).One booster injection was given without adjuvant 11 weeks after the primary dose. Four mice wereimmunized, of which one was selected for fusion on the basis of serum antibody activity. The sera fromall immunized animals exhibited antibodies to m-AspAT as detected by both slide immunoassays usedhere and by immunoprecipitation of active enzyme (7).

The spleen was excised four days after the booster dose, and spleen cells (1 x 10 ) were fused withX63-Ag8.653 myeloma cells (2.5 x 10 ) using procedures and reagents described by Rudofsky et al.(8). RPMI 1640 medium (Gibco, Grand Island, NY) containing 20% (by volume) fetal bovine serum,gentamicin, amino acid supplement, hypoxanthine, aminopterin, and thymidine was used. Hybrid-cells were cloned by the method of limiting dilution in micro-culture plates. Supernatant liquid fromthese plates were screened by both the indium slide and micro-SIA techniques.Slide micro-irnmunoenzvmatic assay (micro-SIA)

The micro-SIA was performed essentially as described (9) using slides obtained from Cel-LineAssociates (Newfield, NJ). Slides were washed with ethanolwater (95:5 by volume) and dried just beforeuse. Antigen (m-AspAT) was adsorbed to each circle on the slide by adding 5 p\ of a solutioncontaining approximately 250 U/ml (about 1.5 mg of active enzyme per ml) in 10 mM Tris buffer.Slides were dried in a 40 °C oven for a period of 1 to 2 hr. Prepared slides were washed with runningdistilled water and dried at 40 °C.

Neat culture media to be screened for presence of monoclonal antibodies were added (10 p\ each)to respective slide circles and incubated at room temperature in a humid chamber for 30 min. Slideswere washed with running distilled water and dried at 40 °C. Peroxidase-labeled second antibody (5/il of a 1:1000 dilution prepared in culture medium) was added to each circle, incubated for 30 min. atroom temperature, washed and dried as above. Presence of labeled second antibody was determinedby adding 10 ^1 of freshly prepared peroxidase substrate (o-phenylenediamine [0.09 mM], H202 [1.76mM] in citrate buffer [pH 4.5, 100 mM]) and allowing the reaction to proceed for approximately 5 min.Results were classified into three groups ranging from negative (no or faint color), to strongly positiveor (++) with an intense yellow color. A negative control (tissue culture medium) and strongly positivecontrol (immunized mouse serum diluted 500-fold with tissue culture medium) were included on eachslide. Dulbecco's modified Eagle medium containing 5% (v:v) calf serum and 10% (v:v) y-globulin-free horse serum was used for these dilutions.

Indium slide assay

The indium-coated substrate used in the assay was prepared by evaporating indium metal upon aglass surface under reduced pressure (0.13 mPa). Under these conditions, the indium condenses on theglass slide forming small metallic islands whose average size is determined by the amount of indiumevaporated (10). When the average diameter of these islands is a few hundred nanometers, a singlemonomolecular layer of protein causes a change in the optical density of the slide that can be easilydetected by visual observation in transmitted light. This phenomenon relies on the fact that the amountof light scattered by the metallic islands depends strongly on whether these areas are coated with adielectric layer. Adsorbed protein (or other macromolecules) act as the dielectric layer and, in general,the more protein present, the more the scattering increases and the darker the appearance of the coatedarea. Thus, one can readily distinguish between single- and double-layers of protein. In the workpresented here, we estimated the extent of antibody binding by simple visual inspection; wherespecified, we also employed a densitometer (4,5) to quantitatively measure the amount of darkening.

To screen hybridoma cultures, an indium-coated slide (#2, 25 mm square coverslip) was coveredwith a solution of m-AspAT (50 ¿ig/ml in 150 mM NaCl) and incubated for 15 min, allowing adsorptionof a complete monolayer of antigen. The slide was then rinsed and dried in a jet of air. Culture mediato be screened for the presence of specific monoclonal antibodies were mixed with one tenth volume ofa 1.0 M solution of Bis-Tris (pH 7.5) and then spotted on the antigen-coated slide. Each spot wasapproximately 5 p\ in volume, and 24 spots could be conveniently placed in a grid pattern on a singleindium-coated slide. Culture medium not exposed to growing hybridomas, and dilutions of specificmouse antiserum with culture medium, were spotted on each slide as a negative and positive control,respectively.

The spotted slide was placed in a wet box for 1 hr and then rinsed in running tap water. At thispoint the slide could be blown dry in a jet of air and observed for antibody binding. However, toincrease the sensitivity of the method, the slide was taken through a development step to form a doubleantibody layer and, hence, increase the darkness of the positive spots. To accomplish this, the slideswere placed in a wet box and their surface was flooded with about 200 n\ of a 50-fold dilution of goat

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FIGURE 2. Effect of Bis-Tris on nonspecific binding of culture medium. An indium slide wasprecoated with m-AspAT as described. The dried slide was spotted with (A) fresh culture medium or(B) fresh culture medium containing 0.1 M Bis-Tris. Following an incubation of 30 min, the slide wasrinsed in running water and forcefully blown dry in a jet of nitrogen.

anti-mouse IgG in 100 mM Tris (pH 7.5), 50 mM NaCl. Following a 30 min incubation, the cover slipswere rinsed in running water and blown dry in a jet of air.

The slides were observed on a light box and each area corresponding to a specific culture mediumspot was evaluated relative to the background darkness of the slide. The spots were assigned a value of(++) or (+) depending on the relative contrast of the spot. A negative value was assigned to a spot if itwas not visible or only visible upon very careful scrutiny.Method comparison

Screening was performed in a blind manner in two different laboratories by different investigators.Only after all screening was performed were results compared. Method comparison was performed byusing Spearman rank correlation of categorical observations.

RESULTS AND DISCUSSION

In initial experiments, the undiluted media from hybridoma cultures were spotted directly onantigen-coated indium slides and incubated in a wet box. Under these circumstances, however, therewas a deposition of material on the slide causing considerable darkening that could not be attributed tobinding of specific antibody (Fig. 2A). A series of experiments suggested that the deposits wereinsoluble salts of calcium and phosphate. These spots were not observed when either of these ions wereabsent from the medium or the pH was maintained at about neutrality. Apparently as droplets of culturemedia are spotted and exposed to the air, the level of dissolved carbon dioxide decreases from therelatively high levels obtained during incubation in the 5% CO, environment of the incubator. Theconcomitant rise in pH causes calcium phosphate salts to precipitate from solution and deposit on thesurface of the slide in a thin layer. This layer is not washed from the slide during prolonged rinsing butremains tightly bound and results in enhanced darkening of the slide in the region of the spot. To avoidthis undesirable situation, each culture medium was mixed with Bis-Tris buffer prior to spotting. Weselected Bis-Tris since it has the dual properties of a buffer at neutral pH (pKa = 6.46) as well as achelator of divalent metal ions (11). A final concentration of Bis-Tris at 100 mM was sufficient for thispurpose. This step was essential in carrying out the incubation in an air environment, and it eliminatedthe problem of false positive results arising from insoluble salt deposition (Fig. 2B). Alternatively,although less convenient, we could also avoid such precipitates by carrying out the incubation in a tissueculture incubator.

The sensitivity of the indium slide assay in detecting polyclonal antibody has been well documented(10). In general, the test is capable of detecting less than 1 /ig/ml of specific antibody following a 15min incubation. Using a second antibody development as described in Materials and Methods, thesensitivity of the assay is enhanced by a factor of as much as tenfold. Lengthening the incubation time

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C D

FIGURE 3. Indium slide assay of supernatant solution from post-fusional cultures. An indium slidewas precoated with m-AspAT as described. The dried slide was spotted with: (A) polyclonal mouseanti-human m-AspAT antiserum diluted 100-fold in culture medium. All other applications were withspent tissue culture medium from: (B) a hybridoma clone positive for m-AspAT (C) a hybridoma clonenegative for m-AspAT and (D) a hybridoma clone positive for s-AspAT. Bis-Tris buffer was addedto all solutions as described in Materials and Methods. Following a one hour incubation, the slides weredeveloped with anti-mouse IgG antiserum as described.

also raises the level of sensitivity. In general, our experience showed that using a 60 minute incubationtime and a second antibody development step resulted in unambiguous detection of concentrations lessthan 50 ng/ml of specific polyclonal antibody. Still longer incubation times and further developmentsteps with additional antibodies can be employed to push the assay to its limit, but such manipulationstend to compromise the inherent simplicity of the assay. As the levels of monoclonal antibodies inhybridoma culture medium generally fall in the ¿tg/ml level, the sensitivity of the indium slide assaydescribed is well suited for screening media from hybridoma cultures.

Typical slides obtained from the indium slide assay used to screen post-fusional cultures are shownin Fig. 3. These representative slides show binding of a monoclonal antibody from medium used toculture a positive hybridoma clone (Fig. 3B) and a negative hybridoma clone (Fig. 3C). As controls, Fig3D demonstrates the lack of binding of monoclonal antibodies directed against s-AspAT and as apositive control, polyclonal mouse anti-human m-AspAT antiserum diluted in culture medium (Fig. 3A).The positive clone (Fig. 3B) would have been classified as (+) by its relative contrast. Densitometerreadings from the spot shown in Fig. 3A (polyclonal antibodies) were approximately twofold greater thanthat found for Fig. 3B (monoclonal antibody). Densitometer readings for regions of the slide spottedwith no, or nonbinding, antibody (shown in Figs 3C and 3D) could not be distinguished frombackground absorbance. Supernatant solutions from cloned hybrids were assayed by the indium slideassay and, as a comparative procedure, the micro-SIA. Results (Table 1) showed identical semi-quantitative classification of 86% of the samples and >90% were classified as either positive or negativeby both techniques. Of the 94 specimens tested by both procedures, only 3 were found to be positiveby the micro-SIA technique yet were negative by the indium-slide assay. Spearman rank correlationcoefficient for these data was 0.431.

An essential prerequisite for production of monoclonal antibodies is the availability of a rapidscreening assay used to identify cultures that synthesize antibody that reacts with the antigen of interest.The indium slide procedure has proven suitable to this application. Several hundred supernatants couldeasily be examined within a single working day. Once indium coated slides are prepared, no specializedequipment is required and the antibody-antigen reaction can be detected visually without the need foran enzyme or radioactive label.

The application to detection of human m-AspAT antibodies is appropriate since monoclonalantibodies to this enzyme have not been described, and there is considerable interest in this enzyme as

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TABLE 1. COMPARISON OF INDIUM SLIDE AND MICRO-SIA PROCEDURES FORSCREENING MONOCLONAL ANTIBODIES

Indium Slide Result

++ + 0

Micro-SIA Result

++ 2 0 0

+ 4 3 3

0 0 6 76

a marker in alcoholic liver damage (12-14). We have previously shown a discrepancy in the amount ofcatalytically active and immunologically active enzyme in circulation in human serum (4); an observationrecently confirmed by others (15). The availability of monoclonal antibodies for this antigen would beuseful in further examining this phenomenon. This antigen also presents an analytical challenge inthat it has a relatively basic pi (> 9.5) a property that increases nonspecific binding, a potential drawbackthat could interfere with application as a screening technique.

The problem we observed involving surface deposition of calcium phosphate salts (Fig. 2) may alsointerfere with other screening assays for monoclonal antibodies. If culture medium is allowed toequilibrate with the ambient CO, level in an assay, precipitation of the insoluble salts will occur.Although we have only studied the indium/glass surface, where such precipitation can be detectedvisually, we suspect that most other surfaces used in solid state assays will also become coated in anessentially irreversible manner with these deposits. Presumably these layers would interfere to somedegree with the sensitivity of the assay. Since it is easy to avoid the precipitation, it would be arelatively simple matter for investigators using other surface detection schemes to examine this possibleinterference.

The results (Table 1 ) show that no strongly positive cultures were found by the indium slide assaythat were not also detectible by the micro-SIA, indicating that the methods are of comparablespecificity. Although Spearman rank correlation coefficient was unimpressive, only 9 of the specimenswere found to contain detectable antibody by one procedure but not the other assay. Six of 94 sampleswere considered positive by the indium slide method while negative by the micro-SIA. Under theconditions that we used, it is possible that the indium slide assay is more sensitive at distinguishingweakly positive clones, since longer incubation times lead to an increased blank in enzymeimmunoassays. This could also explain why four clones were classified as (++) in the indium slidemethod and were rated a (+) in the micro-SIA. Since the measurements were carried out in differentlaboratories, the subjective nature of such readings could also not be excluded. This discrepancy islikely not due to non-specific binding of antibody, since blocking antigen coated slides using a solutionof bovine serum albumin (1 mg/ml) did not result in significant changes in the outcome of the indiumslide assay.

We are uncertain why the indium slide assay failed to detect antibodies demonstrated to be presentin three of the culture fluid by the micro-SIA. It is doubtful that this is due to relative insensitivity ofthe indium slide assay as replicate analysis using longer incubation times and higher concentrations ofantibody did not substantially change results. It could be the result of differences in the accessibilityof antigenic sites of the antigen adsorbed onto a metal surface in the indium coated slide assay and onthe glass surface used in the micro-SIA. Differences in the methods employed to coat the surface withantigen may also be important. In the indium slide assay, the antigen layer was adsorbed from solutionfollowed by thorough rinsing whereas in the micro-SIA, the antigen solution was allowed to dry on theglass surface before rinsing. Differences between screening techniques in solution and solid phase havebeen described (16) and it is possible that differences between the two solid-phase techniques used inthis study may account for the three discrepant results. In addition to the methods of antigen deposition,there were also significant differences between the amount of antigen used; 50 Mg/ml in the indiumslide assay while 1.5 mg/ml was required for the SIA. However, in studies not presented here, we foundthat increasing the concentration of antigen in solution used to coat the indium slides, did notsignificantly alter the amount of antigen typically bound at the surface (approximately 0.5 jjg/cm2 isbound). An alternative explanation is that there may have been differences in the binding specificitiesof the second antibodies used in the development steps in each assay.

Although the method is clearly useful for hybridoma screening, the spot contrast obtained for thepositive hybridoma culture fluid was, in general, not as great as that commonly observed for the indium

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slide detecting polyclonal antibodies (Fig. 3; references 4 and 5). For high spot contrast, the specificallybound layer of antibodies must consist of many antibody molecules. With polyclonal sera, there will bea large number of available binding sites on each antigen molecule making up the layer. Hence, even

though some sites are hidden or destroyed in the adsorption process, each antigen molecule would beexpected to bind at least one and possibly more antibody molecules. With monoclonal antibodies,however, each antigen molecule presumably contains only one determinant that will be recognized byeach antibody. If this site is lost in the adsorption process, the maximum number of antibodies moleculethat can specifically bind to the antigen layer could be significantly reduced.

We conclude that the indium slide assay is readily adaptable as a screening procedure fordetermining the presence of monoclonal antibodies. Unlike enzyme-linked or radio-labeled assays, theindium slide immunoassay does not require a tagged second antibody. This eliminates the extra steps,expense, or problems involved in measuring enzymatic reactions or in handling radioisotopes. Thesensitivity and specificity of the new method are similar to an ELISA technique and it is inherentlysimpler as it also allows use of simple dilutions of antiserum, rather than purified conjugated antibody,for the development step.

ACKNOWLEDGEMENTS

The authors are grateful to Drs. Everly Conway de Macario and Alberto J. L. Macario for theirhelp and assistance with the Micro-SIA procedure and their comments on this paper. We thank UlrichRudofsky and Brenda Evans for their assistance in preparing monoclonal antibodies against m-AspAT,Joanne Martinez and Ray Ciaglowski for their assistance in preparing monoclonal antibody against s-

AspAT, Larry Turner for preparation of the indium slides, and Carol Norton for her excellent technicalassistance.

REFERENCES

1. Langone, J.J. and Van Vunakis, H., Eds. (1986). Immunochemical Techniques. I. HybridomaTechnology and Monoclonal Antibodies, Meth. Enzymol. 121, 1-947.

2. Giaever, I. (1973) The antibody-antigen reaction: a visual observation. J. Immunol. 110, 1424-1426.

3. Giaever, I., and Laffin, R.J. (1974). Visual detection of hepatitis B antigen. Proc. Nail. Acad. Sei.USA 71, 4533-4535.

4. Rej, R., Keese, C. R., and Giaever, I. (1981). Direct immunological determination of aspartateaminotransferase isoenzymes. Clin. Chem. 27, 1597-1601.

5. Giaever, I., Keese, C.R., and Rynes, R.I. (1984). A new assay for rheumatoid factor. Clin. Chem.30, 880-883.

6. Rej, R., and Harder, M. (1983). Aspartate aminotransferase (Glutamate oxaloacetate transaminase).In: Methods of Enzymatic Analysis (H. U. Bergmeyer, Ed.), Verlag Chemie, Weinheim, Vol. 3, 3rded., pp. 416-433.

7. Rej, R. (1980). An immunochemical procedure for determination of mitochondrial aspartateaminotransferase in human serum. Clin. Chem. 26, 1694-1700.

8. Rudofsky, U.H., Dilwith, R.L., Lynes, M., and Flaherty, L. (1982). Murine monoclonal antikidneyautoantibodies. I. Anti-renal proximal tubular basement membrane autoantibodies. Clin. Immunol.Immunopathol. 25, 165-178.

9. Conway de Macario, E., Macario, A.J.L., and Jovell, R.J. (1983). Quantitative slide micro-immunoenzymatic assay (micro-SIA) for antibodies to particulate and non-particulate antigens.J. Immunol. Methods 59, 39-47.

10. Giaever, I. (1976). Visual detection of carcinoembryonic antigen on surfaces. J. Immunol. 116,766-771.

11. Serieller, K.H., Abel, T.H.J., Polanyi, P.E., Wenk, P.K., Fischer, B.E., and Sigel, H. (1980). Metalion/buffer interactions. Stability of binary and ternary complexes containing 2-[Bis(2-hydroxy-ethyl)amino]-2(hydroxymethyl)-l,3-propanediol (Bistris) and adenosine 5'-triphosphate (ATP).Eur. J. Biochem. 107, 455-466.

12. Nalpas, B., Vassault, A., Le Guillou, A., Lesgourgues, B., Ferry, N., Lacour, B., and Berthelot, P.

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(1984). Serum activity of mitochondrial aspartate aminotransferase: a sensitive marker ofalcoholism with or without alcoholic hepatitis. Hepatology 4, 893-896.

13. Nalpas, B., Vassault, A., Charpin, S., Lacour, B., and Berthelot, P. (1986). Serum mitochondrialaspartate aminotransferase as a marker of chronic alcoholism: diagnostic value and interpretationin a liver unit. Hepatology 6, 608-614.

14. Lumeng, L. (1986). New diagnostic markers of alcohol abuse. Hepatology 6, 742-745.

15. Hirano, K., Matsuda, K., Adachi, T., Ito, Y., Hayashi, K., Okuno, F., and Muto, Y. (1986).Determination of mitochondrial aspartate aminotransferase in serum. Clin. Chim. Acta 155, 251-262.

16. Vaidya, H.C., Dietzler, D.N., and Ladenson, J.H. (1985). Inadequacy of traditional ELISA forscreening hybridoma supernatants for murine monoclonal antibodies. Hybridoma 4, 271-276.

Address reprint requests to:

Robert Rej, Ph.D.Wadsworth Center for Laboratories and Research

New York State Department of HealthAlbany, NY 12201-0509

Received for publication June 6, 1988Accepted after revisions July 20, 1988

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