IN VITRO CELLULAR ~[ DEVELOPMENTAL BIOLOGY Volume 25, Number 11, November 1989 �9 1989 Tissue Culture Association, Inc.

LOSS OF SOYBEAN T R Y P S I N I N H I B I T O R IN CALLUS AS M O N I T O R E D BY I N H I B I T I O N ENZYME IMMUNOASSAY

D. M. WRIGLEY, M. B. LINDSAY, AND R. J. LEBOWITZ

Department of Biological Sciences. Box 34, Mankato State University, Mankato. Minnesota 56001

IReceived 4 January 1989; accepted 30 July 19891

SUMMARY

A monoclonal antibody was produced against Kunitz soybean trypsin inhibitor iKSBTI) and used in an inhibition enzyme immunoassay (EIA). The inhibition EIA was as sensitive as competetive EIAs and was easily modified for other protein-antibody interactions. The KSBTI assay described detected KSBTI in complex mixtures from 100 ~g/ml to 50 ng/ml and did not react with the Bowman-Birk trypsin inhibitor. The assay was used to examine levels of KSBTI in Glycine max hypocotyl-derived callus tissue. The developing hypocotyis contained 0.21 /~g KSBTI per mg of fresh tissue. This level of KSBTI rapidly decreased when placed in culture and was undetectable 6 days later. The decrease in KSBTI correlated with the development of callus.

Key words: inhibition enzyme immunoassay; Glycine max; trypsin inhibitor; hypocotyl-derived callus.

[NTRODUCTION

The Kunitz soybean trypsin inhibitor (KSBTI) is the major trypsin inhibitor found in the seed and seedlings of Glycine ms~x. Along with the Bowman-Birk trypsin inhibitor, it may comprise up to 6% of the seed's protein Ill). In addition to its presence in the seed, the KSBTI has also been detected in other parts of the seedling. While the negative effects of KSBTI on animals, including interference with digestive serine proteinases and chronic toxicity, are well known (6,7,10), the role of the KSBTI in the plant has not been delineated. It has been suggested that KSBTI acts as a sulfur-containing amino acid storage protein in the seed and as an inhibitor of endogenous proteinases (14). To help determine the role of KSBTI in the plant it would be important to detect small quantities of the KSBTI in complex mixtures of plant protein. The following study describes an inhibi- tion enzyme-linked immunosorhent assay (EIA) to detect nanogram levels of KSBTI using a monoclonal antibody against KSBTI and a commercial kit manufactured by Hyclone to detect the monoclonal antibody. The assay was tested on complex protein mixtures derived from soybean hypocotyl and hypocotyl-derived callus tissues.

Various sensitive immunoassays have been developed to detect KSBTI. These include radial immunodiffusion (3), immunoblotting (3,13), sandwich EIA (4) and competetive EIA IlL Each of these has the advantage of specificity and good sensitivity. However, the disadvan- tages of the procedures include the need for specialized reagents and procedures. An inhibition EIA would provide a simple system that could be easily modified for

any protein antibody system without requiring extensive labelling or purification schemes (1). In a KSBTI inhibition EIA, the anti-KSBTI is mixed with and binds to free KSBTI and then transferred to KSBTI coated wells. Only the remaining unbound antibody will adhere to the KSBTI on the well surfaces. After the wells are washed to remove unbound material, enzyme-labelled anti-mouse immunoglobulin and the enzyme's substrate are added to the wells to test for the presence of anti-KSBTI. To make the assay quantitative, known concentrations of KSBTI are assayed simultaneously. One advantage of this assay is that all the reagents, except for the anti-KSBTI, are commercially available.

MATERIALS AND METHODS

.4nti-KSBTI monoclonal antibody. The monoclonal antibody against KSBTI used in this study was produced by the method of Strahli et al. (12L Briefly, Balb/c mice were hyperimmunized over 5 weeks by i.p. inoculation with 500/~g KSBTI (US Biochemical #21735) suspended in a 0.5 ml phosphate buffered saline (pH 7). One mouse was reinoeulated four days prior to the fusion process. The spleen cells from the mouse were fused with the mouse myeloma line NS-1 using polyethylene glycol (9). The hybrid cells were selected by culture in RPMI 1640 with hypoxanthine, aminopterin and thymidine iSigma #R3505L Following selection, the surviving cell populations were expanded and screened for antibody production using

t h e Hyclone EIA screening kit for mouse antibodies (E-5010-KL Positive cultures were cloned and one hybrid

1075

1076 WRIGLEY ET AL.

0.12

0.10

0.O8 t5 d 0.06

0.04

0.02 I I I I

-2 -1 0 1 2

Log KSBTI FI~3. 1. Inhibition EIA for KSBTI. Known concentration of

KSBTI were incubated with monoclonal anti-KSBTI and transferred to KSBTI coated plates as described in text. Optical density was read of 405 nm.

line producing ant i-KSBTI was selected and used for this study. Anti-KSBTI was produced by culturing the cells in RPMI 1640 (Sigma #R7755) supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, 50 units /ml penicillin and 50 ~g/ml streptomycin. Culture supernatants were collected from 48 hour-old cultures and stored at 4 ~ C until used. The hybridoma could be stored at --80 ~ C and thawed with little loss of activity. The monoclonal ant i -KSBTI showed no cross reactivity with other trypsin inhibitors.

Inhibition EIA. The efficacy and sensitivity of the inhibition EIA using the Hyclone EIA hybridoma screening kit reagents to detect known KSBTI concentra- tions were determined as follows. The assay plates were prepared by adding 100 ~1 of a 20 ~g/ml KSBTI in Hyclone carbonate buffered plate coating solution to each well of a Falcon ProBind assay plate and incubating at 4 ~ C for 20 hr to permit the KSBTI to bind to the polystyrene. The plates were then washed as described in the kit and used immediately or washed, patted dry and stored for up to a week at 4 ~ C.

To begin the assay, 0.2 ml of known concentrations of KSBTI were mixed with an equal volume of the monoclonal ant i -KSBTI supernatant and incubated at room temperature for 30 min. After the incubation, 0.2 ml of PBS-surfactant solution was added to the KSBTI /an t i - KSBTI mixture. Then 150 ~1 of the mixture was transferred to wells on the KSBTI-coated EIA plate. All samples were tested in triplicate. The plate was incubated at 37 ~ C for 30 min during which time any unbound antibodies from the first incubation could attach to the KSBTI in the wells. The wells were thoroughly washed with distilled water. Then 100 ~1 enzyme-labelled anti-mouse immunoglobulin from the Hyclone hybrido- ma screening kit was added to each well, and the plate incubated at room temperature for 90 min. Finally, the wells were washed with distilled water and the substrate from the Hyclone hybridoma screening kit added. The eolorimetric reaction was measured using a Bio-tek EIA

reader set at 405 nm. To test whether the KSBTI could be detected in complex mixtures, an aqueous extract consisting of 0.7 g of course ground seed mixed with 4 ml of phosphate buffered saline was prepared and assayed as described above.

Hypocotybderived callus cultures. To obtain the hypocotyls for the cultures, seeds of Glycine max L. Merr. cv. Hardin were surface disinfested in a 10% Chlorox solution containing 0.1% Triton X100 for 10-15 min, then rinsed three times in sterile distilled water. The disinfested seeds were cultured in 6 ounce baby food jars covered with Magenta B-caps on Murashige and Shooge (MS) medium (8) containing no phytohormones, 0.2% sucrose and solidified with 0.6% bactoagar. The cultured seed were maintained in the dark at 25 ~ C for 5-7 days until they germinated and the hypocotyls had elongated.

One cm long hypoeotyl sections were transferred to a callusing medium consisting of MS medium (pH 5.7) supplemented with 2% sucrose, 0.5 mg/L 2,4-D, and 0.8% bactoagar (2,5). Callus cultures were established under low intensity cool white fluorescent light at 25 ~ C. These were maintained on this medium for 6 weeks, then the live, green-colored friable calluses were subcultured to fresh medium.

Newly established callus cultures were collected and analyzed for KSBTI content every three days over a 21 day period. The tissues were weighed, washed in 0.16M mannitol, suspended by vortexing for 30 sec, then pelleted by centrifugation at 400 Xg for 10 min. The pelleted cells were resuspended in 2 ml of an extraction medium (1 M sucrose, 5.6 mM 2-mercaptoethanol, 0.2 M Tris-HCl buffer) at pH 8.5, frozen, thawed and homogenized for 5 min in a glass homogenizer (13). Non-callused hypocotyl tissues were treated in a similar manner except they were not centrifuged. The thawed cell suspensions were tested for KSBTI by the inhibition EIA. To quantitate the assay, known KSBTI concentra- tions suspended in the extraction buffer were assessed simultaneously. Soluble extracted protein from each tissue was determined using the BioRad protein assay with bovine serum albumin as the standard.

TABLE 1

EFFECT OF UNRELATED PROTEIN ON THE INHIBITION EIA FOR KSBTI

Optical Density with BSA at: KSBTI ~g/ml 100 og/ml 20t~g/ml 0~g/m]

100.00 0 .008(0 .012) 0.005(0.002} 0.006(0.003) 2 0 . 0 0 0.032t0.0021 0.033(0.007) 0.040(0�9 4.00 0 .165(0 .023) 0.178(0.024) 0.182(0.026) 0.80 0 .271(0 .018) 0.238(0.020) 0.234(0.023) 0.16 0 .287(0 .037) 0.263(0.016) 0.270(0.039) 0.03 0.30210.036} 0.29710.036) 0.290(0.027) 0 0�9 0.31010.028) 0.322(0.036)

Known concentrations of KSBTI were added to buffer containing bovine serum albumin�9 These solutions were tested for reactivity in the inhibition EIA as described in the methods. Standard deviation is given in parentheses�9

TRYPSIN INHIBITOR IN SOYBEAN CALLUS 1077

5

4

3

2

1

0

30O

250

2OO

150

1 O0

50,

I I' �9 !

6 12 18 24

Day

K58TI

0 o ~ i 0 6 12 18 24

Day

FIG. 2. KSBTI and protein levels in soy callus tissue. Hypocotyl sections were placed in culture (day 0) and total tissue examined for KSBTI and protein concentrations at three day intervals. Results are expressed as a ratio of/~g o1 KSBTI to mg of tissne based on an average of three cultures.

RESULTS AND DISCUSSION

The efficacy of the inhibition EIA is shown in Fig. 1. The optical density is inversely proportional to the concentration of the KSBTI present. The detection limits were between 10 /~g/ml KSBTI and 100 ng/ml. Accuracy was greatest between 8/~g/mi and 200 ng/ml.

W h e n KSBTI was added in known concentrations to an unrelated protein, bovine serum albumin, the resultant optical densities remained consistent. As shown in Table 1, the variation between the optical densities for a given amount of KSBTI in the different serum concentrations did not vary by more than ten percent.

The assay was also used to measure KSBTI concentra- tions in complex protein mixtures, such as aqueous extracts from ground soybeans and callus tissue. 'Hardin' seeds contained an average of 0.34/~g fresh weight and 12.4 ~g KSBTI /mg protein. KSBTI levels in developing hypocotyl explants as determined by inhibition EIA are summarized in Fig. 1, KSBT! levels decreased by two-thirds within a six day period. Within 15 days, bright green, friable calluses had developed on the ends of the hypocotyl segments and KSBTI was no longer detectable. The decrease in KSBTI was more rapid than the decrease in total soluble protein per mg fresh tissue weight.

In eleven week-old callus cultures, protein levels were 0.58 _ 0.12 /~g/mg tissue. When subcultured on fresh medium, the callus protein levels increased rapidly, then declined to background levels (on day three of subculture 1.63 4- 0.41/~g/mg, day six 1.09 -t- 0.33 ~g/mg, and day nine 0.67 + 0.26/~g/mg). During this time KSBTI was never detected. Tan-Wilson et al. (13) reported a similar lack of KSBTI in established callus cultures. The data in- dicate that KSBTI levels are not correlated with total protein levels.

In conclusion, the inhibition EIA described in this study is highly sensitive for analyzing KSBTI in small tissue samples. Unpublished studies of work done in our laboratory with bacterial proteins and their specific antibodies indicate that the same assay can be easily modified to detect any protein, not just KSBTI. The availability of commercially-prepared enzyme-labelled antibody against mouse immunoglobulins simplifies the protocol and there is no requirement of extensive purifications and chemical labelling.

REFERENCES

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2. Gamborg, O. L.; Miller, R. A.; Ojima, K. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell. Res. 50:148-151; 1968.

3. Hughes, M. A.; Dunn, M. A. The use of immunochemical techniques to study plant proteins. Plant nol. Biol. Rep. 3:17-23; 1985.

4. Hitchcock, C. H. S.; Bailey, F. J.; Crimes, A. A., et al. Deter- mination of soya proteins in food using an enzyme-linked immunosorbent assay procedure. J. Sci. Food Agric. 32:157-165; 1981.

5. Kerns, H. R.; Barwale, U. B.; Meyer, M. M., et al. Correlation of cotyledonary node shoot proliferation and somatic embryoid development in suspension cultures of soybean (Glycine max L. MerrA. Plant Cell Rep. 5:140-143; 1986.

6. Liener, I. E. Trypsin inhibitors: concern for human nutrition or not? Nutrition 116:920-924; 1973.

7. Liener, I. E. Legume toxins in relation to protein digestibility: A review. J. Food Sci. 41:I076-1081; 1976.

8. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tabacco tissue cultures. Physiol. Plant 15:473-497; 1962.

9. Oi, V. T.; Herzenberg, L. A. Imunoglobulin-producing hybrid cell lines. Mishell, B. B.; Shiigi, S. M. eds. Selected methods in cellular immunology. San Francisco: W. H. Freeman and Co.; 1980:351-372.

10. Rackis, J. J.; Anderson, R. L. Isolation of four soybean trypsin inhibitors by DEAE-cellulose chromatography. Biochem. Biophys. Res. Commun. 15:230-235; 1964.

11. Ryan, C. A. Proteolytic enzymes and their inhibitors in plants. Ann. Rev. Plant Physiol. 24:173-196; 1973.

1078 WRIGLEY ET AL.

12. Stahli, C.; Staehelin, T.; Miggiano, V., et al. High frequencies of antigen-specific hybridomas: dependence on immunization parameters and prediction by spleen cell analysis. J. Immunol. Meth. 32:297-304; 1980.

13. Tan-Wilson, A. L.; Hartl, P. M.; Delfel, N. E., et al. Differential expression of Kunitz and Bowman-Birk soybean proteinase

inhibitors in plant and callus tissue. Plant Physiol. 78:310-314; 1985.

14. Wilson, K. A. The structure, function and evolution of legume proteinase inhibitors. In: Ory, R. L., ed. Antinutrients and natural toxicants in food. Westport, Connecticut: Food and Nutrition Press; 1981:187-202.