Immunochtmistry. Vol ~Pergamon Press Ltd.
IS. pp. 331-333. 197f1. Prinkd in Grear Britun
ool?-?791/78/0.(01-0331~0?
lwn
CHEMILUMINESCENCE IMMUNOASSAY; A NEW SENSITIVE METHOD FOR DETERMINATION OF ANTIGENS RARUCH Department of Biochemistry.
VELAN
and
MIRJAM
HALMANN
Israel Institute for Biological Research. Ness-Ziona. Israel
(First receilvd 13 July 1977: in revised form 25 October 1977) Abstract-A chemiiuminescent immunoassay was developed using peroxidase labeled antigen. The high sensitivity obtained permitted the determination of I ng staphylococcal enterotoxin B used as test substance.
INTRODL’CTION
Radioimmunoassay (RIA)andenzyme linked immunosorbent assay (ELISA)are the best methodsavailable today for the determination of small amounts of antigens and antibodies. Although RIA is more sensitive, and picogram to nanogram quantities can be determined, ELISA offers the advantage of greater stability of the reagents used and safety in handling as no radioactive elements are involved. It is usually based on calorimetric determinations, and for peroxidase. which is one of the enzymes most commonly used, there is a series of substrates which produce colored products (van Weemen & Schuurs. 1971; Avrameas & Guilbert, 1971). Puget et al. (1977) described a method for determination of antibody linked enzymes based on light emission techniques. The present paper describes a new immunoassay based on chemiluminescence. The chemiluminescence results from the oxidation of pyrogallol by H202 which is catalyzed by peroxidase labeled antigen. Using competition techniques, unlabeled. unknown antigen can be determined by measuring the decrease in light emission.
MATEKIALS
AND
methods
Determination of protein-Lowry el a/. (1951). Colorimetricdeterminationof peroxidase-Worthington Enzymes Manual. Preparation of antibody loxin f? (SEB)
against
of Sepharose
linked 10 ASEB
To I g (dry weight) of Sepharose CNBr activated in 0. I M NaHCOn were added IO mg of globulin made up of the indicated percentage of ASEB in normal rabbit globulin (NRG). Binding conditions were as indicated by the manufacturer. The coupled Sepharose was suspended in 25 ml PBS. Peroxidase
labelling
of SEB
Conjugation of 10 mg peroxidase to 5 mg SEB was performed according to Avrameas and Ternynck (1971). The conjugate was completely separated from free SEB by filtration through a 100 x I .6 cm column of Sephadex G-100 in saline containing 0.05 M Tris-HCI. pH 7.6 (running upwards). The fractions were tested for SEB (Weirether et al.. 1%) and for peroxidase with o-dianisidine (Worthington. 1972). Conjugate containing both SEB antigenicity and peroxidase enzymatic activity was eluted after 50 ml in a volume of 7 ml. There was some overlapping of free peroxidase on theconjugatefraction. but the free enzyme did not interfere with the immunoassay. The conjugation did not impair strongly the immunoreactivity of SEB as 5 ng unbound antigen caused a 70% decrease in the binding of 5 ng of conjugated SEB in a competition test with ASEB-Sepharose (standard immunoassay procedure). Standard
Pyrogallol. Perhydrol. NaCI. Na,SO, and phosphates were obtained from Merck, glutaraldehyde (Grade I), o-dianisidine and peroxidase (from horse radish, Rz = 2.6) from Sigma, bovine serum albumin (BSA) (Fr. V) from Mann;Sephadex G-IOOandSepharose4B-CNBractivated from Pharmacia.
slaphylococcal
enfero-
SEB was prepared and purified by the method of Schantz et al. (1%5). Serum against SEB was raised in rabbits (Silverman, 1963). and containe\l 8 mg specific antibody per ml determined by precipitation test. The globulin fraction of the serum was separated by precipitation with 17% sodium sulfate at room temperature and dissolved in IYU. 1) 5- E
Preparulion
METHODS
Chemicals
Analytical
saline containing 0.02 M phosphate, pH 7.6 (PBS). to a concentration of 20 mg protein per ml. This globulin was called ASEB.
immunoassay
procedure
Sepharose-ASEB (0. I ml). SEB-peroxidase conjugate (as indicated). SEB solution to be tested (0.2 ml) and 0. I M phosphate. pH 7.6, containing 2.5 mg/ml BSA (0.05 ml) were mixed in a reaction cuvette (DuPont). Total volume was 0.4 ml. After 2 hr incubation at room temperature with reciprocal shaking in slanted position. the Sepharose was separated by centrifugation. washed twice with 0.18 M phosphate. pH 6.5. and the bound peroxidase was determined by the chemiluminescent reaction. The reaction can also be performed with a 0.85 ml sample in a final volume of I ml. with incubation at room temperature for 2 hr and overnight at 4°C. lmmunoassays in I ml volume were performed in Eppendorff centrifuge microtubes and at the end the washed Sepharose samples were transferred lo the Biometer cuvettes for the determination of peroxidase content. Chemiluminescence
determination
of peroxidase
The Sepharose containing peroxidase was suspended in 50 ~10.2% pyrogallol in 0.18 M phosphate, pH 6.5 and the cuvette introduced into a 760 luminescence biometer 331
332
BARUCH
VELAN
and MIRJAM HALMANN
This Sepharose-ASEB,% was used for immunoassays. When smaller concentrations of ASEB were needed Sepharose-ASEB,% was diluted with inactive Sepharose, linked to NRGonly (SepharoseASEBJ. Chemiluminescence determined after reaction of SEB-peroxidase conjugate with increasing dilutions of ASEB,, (Fig. 1) shows that with 5 ng RESULTS conjugate, the best ASEB concentration for inhibiIn our previous work (Halmann et al., 1977) it was tion tests, 30% saturation, is obtained with 0.1 ml shown that there is a second order relationship of a ten-fold dilution of Sepharose-ASEB,x in between the concentration of the peroxidase in the Sepharose-ASEB,. Under these conditions besolution and the amount of light emitted-double tween 1 and 10 ng SEB can be determined (Fig. 2a). concentration of enzyme induces a four-fold emisThis is also the limit reached by RIA test with Y sion of light. (Johnson et al., 1971; Collins et al., 1972). Similarly, The final concentration of peroxidase on the 20-200 ng SEB can be determined with undiluted Sepharose beads after immunoassay is afunctionof Sepharose-ASEB,% and 50 ng SEB-peroxidase conthe concentration of the homologous antibody on the jugate (Fig. 2b). To determine higher amounts of gel. In order to determine the optimal conditions for SEB with Sepharose-ASEB,, we would need larger light emission in the immunoassay, Sepharose was volumes of Sepharose resulting in higher blank linked to NRG containing increasing concentrations values due to carry-over. Therefore, for the l-30 pg of ASEB. Aliquots of 0.1 ml of the gels obtained SEB range we used Sepharose linked to ASEB were saturated with SEB-peroxidase conjugate and only-Sepharose-ASEBloon, and 0.4 pg SEBtested by the chemiluminescent reaction. The re- peroxidase conjugate. In this high range the amount sults, SummarizedinTable 1, show that maximallight of’peioxidase is sufficient for determination also by emission per molecule of peroxidase is obtained the usual colorimeteric method with o-dianisidine with Sepharose linked to globulin containing 5% (Worthington, 1972). The similarity of the results obtained with the chemiluminescent~nd the colorimetric methods of pg amounts of antigen is shown Table I. Influence of the concentration of ASEB linked to Sepharose on the chemiluminescent reaction in Fig. 3. The time needed for the immunoreaction depends on the concentrations of SEB and SEB conjugate. ASEB linked to Light emission Light emission X 100 For the high concentration Sepharose ranges it is sufficient to (maximal reading) (Q) % SEB incubate for 30 min at room temperature. To detect I-10 ng SEB it is necessary to incubate for 2 hr. (DuPont Instruments) set at sensitivity 8 and minimal exponent 3. The reaction was started by the injection of 50 pl of 0.37% H,O, in 0.18 Mphosphate, pH 6.5. The light emitted was determined by repeated measurements at 6 set intervals until maximal value was reached.
100 20 5 I 0.2 0
3x IO”
I .5 x IOfi 5x 4X 4x 3x
lo” 104 10:’ III
3 x 10” 7.5 x IO” 10’ 4x IO” 2x 106 -
Description in text-immuno and chemiluminescent reactions as described in Methods.
ASEB.
DISCUSSION
The chemiluminescence obtained by the enzyme attached to the gel in the immunoassay is much higher than the decrease in the light emission obtained when the supernatant is tested, as the number of photons produced depends not only on the
J
x
Q
2
8-
4-
AMOUNT
Fig.
1. Dilution
OF ASEB- SEPHAROSEy/,, .ml/ assay
curve for Sepharose-ASEB,,
conjugate. SEB-peroxidase theindicatedamountof Sepharose-ASEBb,.
with SEB-peroxidase
conjugate (5 ng) wasreactedunderstandardconditionswith
Volume of Sepharose was made up to a minimum of 0.1 ml with Sepharose-ASEB,. was determined by the chemiluminscent reaction (Methods).
Bound peroxidase
Chemiluminescence
%
100
0
ae
03
I
3
IO
SEB , ng / ossoy
c
%
80
t\
l
I
2 too
3
h
w 60 P
333
h
-
01
Immunoassay
’
‘;a ’ .
60
9 3 om 40
j: 3
IO
30
100
300
SE3 jog /cssoy
Fig. 2. Determination of SEi by competition. Assay conditions as described in Methods. Reagents used: A-5 ng SEB-peroxidase conjugate and 0.01 ml SepharoseASEB, in 0.1 ml Sepharose ASEBB; B-50 ng SEBperoxidase conjugate and 0.1 ml Sepharose-ASEB,,; 100% values(withoutaddedSEB)(A) 1.8 x IO”,(B)9 x 10’. B-9 x IP.
of enzyme present but also on its concentrations. The determination of antigen by the method described takes advantage of both the amount and the density of distributions of the enzyme. First, there is the competitive decrease in the number of enzyme molecules attached to the Sepharose-as in RIA and ELISA. Second, the distance between the molecules of enzyme increases with increasing amounts of the free antigen tested, resulting in a greater decrease in the Iight emission than the simple arithmetical proportion. The net result is a steeper calibration curve giving higher accuracy. In the test of high concentrations of SEB (I-30 pg) performed under conditions where light emission is proportional to the number of mofecules of enzyme only, the results with both the chemiluminescent and the calorimetric methods show the usual shahow curve (Fig. 3). As the maximal light emission per molecule of peroxidase is a function of the proximity between the molecules of the enzyme the optimal ratio of antibody linked to Sepharose or any other support used wili vary with the specific activity of the antibody and the characteristics of the gel.
Fig. 3. Comparison of determinations of SEB by chemiluminescent (A) and coiorimetric (B) methods. Conditions as described in Methods with 0.4 pg SEB-peroxidase conjugate and Sepharose linked to ASEB only. 100% valueschemihtminescent reaction-2 x 106. calorimetric reaction in 1 ml-AO.D.~min = 0.2 (corresponding to 0.018 units peroxidasei.
In conclusion, the chemihrminescent method here described has all the advantages of ELISA and a sensitivity similar to RIA using iz51. Acknowledgement-We wish to thank Tamar Sery for excellent technical assistance.
amount
REFERENCES Avrameas S. & Guilbert B. (1971). C.r. hebd. sdanc. Acad. Sci., Paris (Serie D) 273, 2705.
Avrameas S. & Ternynck T. (1971) immunochemjstry
117.5. Collins W. S., II, Metzger J. F. &Johnson _KImmun. loS, 852.
8,
A. D. (1972)
Halmann M., Velan B. % Sery T. (1977) Appl. environm. Microbial.
34, 473.
Johnson H.M., Bukovic J. A.. Kauffman P. E. & Peeler
J. T. (1971) Appl. Microbial.
22, 837.
Kalmakof J., Parkinson A. J., Crawford A. M.&Williams B. R. G. (1977) .I. immunol. Meth. 14, 73. Lowry 0. H.. Rosebrough N. J., Farr- A. L. & Randall R. J. (1951) J. biol. Chem. 193, 265. Puget K., Michelson A. M. & Avrameas S. (19773 Analgt. Biochem. 79,447.
Schantz E. J., Roessler W. G., Wagman J., Spero L., Dunnery D. A. & Bergdoll M. S. (1965) Biochemist? 4, 1011. Silverman S. J. (1963) J. Bact. 85, 955. Van Weemen B. K. & Schuurs A. H. W. M. (1971) FEB.9 Lett. 15, 232. Weirether F. J., Lewis E. E.. Rosenwald A. J. & Lincoln R. E. (1966)Appl. ~icrobiol. 14, 284. Worthington Enzyme Manual (1972). p. 43.
IS. pp. 331-333. 197f1. Prinkd in Grear Britun
ool?-?791/78/0.(01-0331~0?
lwn
CHEMILUMINESCENCE IMMUNOASSAY; A NEW SENSITIVE METHOD FOR DETERMINATION OF ANTIGENS RARUCH Department of Biochemistry.
VELAN
and
MIRJAM
HALMANN
Israel Institute for Biological Research. Ness-Ziona. Israel
(First receilvd 13 July 1977: in revised form 25 October 1977) Abstract-A chemiiuminescent immunoassay was developed using peroxidase labeled antigen. The high sensitivity obtained permitted the determination of I ng staphylococcal enterotoxin B used as test substance.
INTRODL’CTION
Radioimmunoassay (RIA)andenzyme linked immunosorbent assay (ELISA)are the best methodsavailable today for the determination of small amounts of antigens and antibodies. Although RIA is more sensitive, and picogram to nanogram quantities can be determined, ELISA offers the advantage of greater stability of the reagents used and safety in handling as no radioactive elements are involved. It is usually based on calorimetric determinations, and for peroxidase. which is one of the enzymes most commonly used, there is a series of substrates which produce colored products (van Weemen & Schuurs. 1971; Avrameas & Guilbert, 1971). Puget et al. (1977) described a method for determination of antibody linked enzymes based on light emission techniques. The present paper describes a new immunoassay based on chemiluminescence. The chemiluminescence results from the oxidation of pyrogallol by H202 which is catalyzed by peroxidase labeled antigen. Using competition techniques, unlabeled. unknown antigen can be determined by measuring the decrease in light emission.
MATEKIALS
AND
methods
Determination of protein-Lowry el a/. (1951). Colorimetricdeterminationof peroxidase-Worthington Enzymes Manual. Preparation of antibody loxin f? (SEB)
against
of Sepharose
linked 10 ASEB
To I g (dry weight) of Sepharose CNBr activated in 0. I M NaHCOn were added IO mg of globulin made up of the indicated percentage of ASEB in normal rabbit globulin (NRG). Binding conditions were as indicated by the manufacturer. The coupled Sepharose was suspended in 25 ml PBS. Peroxidase
labelling
of SEB
Conjugation of 10 mg peroxidase to 5 mg SEB was performed according to Avrameas and Ternynck (1971). The conjugate was completely separated from free SEB by filtration through a 100 x I .6 cm column of Sephadex G-100 in saline containing 0.05 M Tris-HCI. pH 7.6 (running upwards). The fractions were tested for SEB (Weirether et al.. 1%) and for peroxidase with o-dianisidine (Worthington. 1972). Conjugate containing both SEB antigenicity and peroxidase enzymatic activity was eluted after 50 ml in a volume of 7 ml. There was some overlapping of free peroxidase on theconjugatefraction. but the free enzyme did not interfere with the immunoassay. The conjugation did not impair strongly the immunoreactivity of SEB as 5 ng unbound antigen caused a 70% decrease in the binding of 5 ng of conjugated SEB in a competition test with ASEB-Sepharose (standard immunoassay procedure). Standard
Pyrogallol. Perhydrol. NaCI. Na,SO, and phosphates were obtained from Merck, glutaraldehyde (Grade I), o-dianisidine and peroxidase (from horse radish, Rz = 2.6) from Sigma, bovine serum albumin (BSA) (Fr. V) from Mann;Sephadex G-IOOandSepharose4B-CNBractivated from Pharmacia.
slaphylococcal
enfero-
SEB was prepared and purified by the method of Schantz et al. (1%5). Serum against SEB was raised in rabbits (Silverman, 1963). and containe\l 8 mg specific antibody per ml determined by precipitation test. The globulin fraction of the serum was separated by precipitation with 17% sodium sulfate at room temperature and dissolved in IYU. 1) 5- E
Preparulion
METHODS
Chemicals
Analytical
saline containing 0.02 M phosphate, pH 7.6 (PBS). to a concentration of 20 mg protein per ml. This globulin was called ASEB.
immunoassay
procedure
Sepharose-ASEB (0. I ml). SEB-peroxidase conjugate (as indicated). SEB solution to be tested (0.2 ml) and 0. I M phosphate. pH 7.6, containing 2.5 mg/ml BSA (0.05 ml) were mixed in a reaction cuvette (DuPont). Total volume was 0.4 ml. After 2 hr incubation at room temperature with reciprocal shaking in slanted position. the Sepharose was separated by centrifugation. washed twice with 0.18 M phosphate. pH 6.5. and the bound peroxidase was determined by the chemiluminescent reaction. The reaction can also be performed with a 0.85 ml sample in a final volume of I ml. with incubation at room temperature for 2 hr and overnight at 4°C. lmmunoassays in I ml volume were performed in Eppendorff centrifuge microtubes and at the end the washed Sepharose samples were transferred lo the Biometer cuvettes for the determination of peroxidase content. Chemiluminescence
determination
of peroxidase
The Sepharose containing peroxidase was suspended in 50 ~10.2% pyrogallol in 0.18 M phosphate, pH 6.5 and the cuvette introduced into a 760 luminescence biometer 331
332
BARUCH
VELAN
and MIRJAM HALMANN
This Sepharose-ASEB,% was used for immunoassays. When smaller concentrations of ASEB were needed Sepharose-ASEB,% was diluted with inactive Sepharose, linked to NRGonly (SepharoseASEBJ. Chemiluminescence determined after reaction of SEB-peroxidase conjugate with increasing dilutions of ASEB,, (Fig. 1) shows that with 5 ng RESULTS conjugate, the best ASEB concentration for inhibiIn our previous work (Halmann et al., 1977) it was tion tests, 30% saturation, is obtained with 0.1 ml shown that there is a second order relationship of a ten-fold dilution of Sepharose-ASEB,x in between the concentration of the peroxidase in the Sepharose-ASEB,. Under these conditions besolution and the amount of light emitted-double tween 1 and 10 ng SEB can be determined (Fig. 2a). concentration of enzyme induces a four-fold emisThis is also the limit reached by RIA test with Y sion of light. (Johnson et al., 1971; Collins et al., 1972). Similarly, The final concentration of peroxidase on the 20-200 ng SEB can be determined with undiluted Sepharose beads after immunoassay is afunctionof Sepharose-ASEB,% and 50 ng SEB-peroxidase conthe concentration of the homologous antibody on the jugate (Fig. 2b). To determine higher amounts of gel. In order to determine the optimal conditions for SEB with Sepharose-ASEB,, we would need larger light emission in the immunoassay, Sepharose was volumes of Sepharose resulting in higher blank linked to NRG containing increasing concentrations values due to carry-over. Therefore, for the l-30 pg of ASEB. Aliquots of 0.1 ml of the gels obtained SEB range we used Sepharose linked to ASEB were saturated with SEB-peroxidase conjugate and only-Sepharose-ASEBloon, and 0.4 pg SEBtested by the chemiluminescent reaction. The re- peroxidase conjugate. In this high range the amount sults, SummarizedinTable 1, show that maximallight of’peioxidase is sufficient for determination also by emission per molecule of peroxidase is obtained the usual colorimeteric method with o-dianisidine with Sepharose linked to globulin containing 5% (Worthington, 1972). The similarity of the results obtained with the chemiluminescent~nd the colorimetric methods of pg amounts of antigen is shown Table I. Influence of the concentration of ASEB linked to Sepharose on the chemiluminescent reaction in Fig. 3. The time needed for the immunoreaction depends on the concentrations of SEB and SEB conjugate. ASEB linked to Light emission Light emission X 100 For the high concentration Sepharose ranges it is sufficient to (maximal reading) (Q) % SEB incubate for 30 min at room temperature. To detect I-10 ng SEB it is necessary to incubate for 2 hr. (DuPont Instruments) set at sensitivity 8 and minimal exponent 3. The reaction was started by the injection of 50 pl of 0.37% H,O, in 0.18 Mphosphate, pH 6.5. The light emitted was determined by repeated measurements at 6 set intervals until maximal value was reached.
100 20 5 I 0.2 0
3x IO”
I .5 x IOfi 5x 4X 4x 3x
lo” 104 10:’ III
3 x 10” 7.5 x IO” 10’ 4x IO” 2x 106 -
Description in text-immuno and chemiluminescent reactions as described in Methods.
ASEB.
DISCUSSION
The chemiluminescence obtained by the enzyme attached to the gel in the immunoassay is much higher than the decrease in the light emission obtained when the supernatant is tested, as the number of photons produced depends not only on the
J
x
Q
2
8-
4-
AMOUNT
Fig.
1. Dilution
OF ASEB- SEPHAROSEy/,, .ml/ assay
curve for Sepharose-ASEB,,
conjugate. SEB-peroxidase theindicatedamountof Sepharose-ASEBb,.
with SEB-peroxidase
conjugate (5 ng) wasreactedunderstandardconditionswith
Volume of Sepharose was made up to a minimum of 0.1 ml with Sepharose-ASEB,. was determined by the chemiluminscent reaction (Methods).
Bound peroxidase
Chemiluminescence
%
100
0
ae
03
I
3
IO
SEB , ng / ossoy
c
%
80
t\
l
I
2 too
3
h
w 60 P
333
h
-
01
Immunoassay
’
‘;a ’ .
60
9 3 om 40
j: 3
IO
30
100
300
SE3 jog /cssoy
Fig. 2. Determination of SEi by competition. Assay conditions as described in Methods. Reagents used: A-5 ng SEB-peroxidase conjugate and 0.01 ml SepharoseASEB, in 0.1 ml Sepharose ASEBB; B-50 ng SEBperoxidase conjugate and 0.1 ml Sepharose-ASEB,,; 100% values(withoutaddedSEB)(A) 1.8 x IO”,(B)9 x 10’. B-9 x IP.
of enzyme present but also on its concentrations. The determination of antigen by the method described takes advantage of both the amount and the density of distributions of the enzyme. First, there is the competitive decrease in the number of enzyme molecules attached to the Sepharose-as in RIA and ELISA. Second, the distance between the molecules of enzyme increases with increasing amounts of the free antigen tested, resulting in a greater decrease in the Iight emission than the simple arithmetical proportion. The net result is a steeper calibration curve giving higher accuracy. In the test of high concentrations of SEB (I-30 pg) performed under conditions where light emission is proportional to the number of mofecules of enzyme only, the results with both the chemiluminescent and the calorimetric methods show the usual shahow curve (Fig. 3). As the maximal light emission per molecule of peroxidase is a function of the proximity between the molecules of the enzyme the optimal ratio of antibody linked to Sepharose or any other support used wili vary with the specific activity of the antibody and the characteristics of the gel.
Fig. 3. Comparison of determinations of SEB by chemiluminescent (A) and coiorimetric (B) methods. Conditions as described in Methods with 0.4 pg SEB-peroxidase conjugate and Sepharose linked to ASEB only. 100% valueschemihtminescent reaction-2 x 106. calorimetric reaction in 1 ml-AO.D.~min = 0.2 (corresponding to 0.018 units peroxidasei.
In conclusion, the chemihrminescent method here described has all the advantages of ELISA and a sensitivity similar to RIA using iz51. Acknowledgement-We wish to thank Tamar Sery for excellent technical assistance.
amount
REFERENCES Avrameas S. & Guilbert B. (1971). C.r. hebd. sdanc. Acad. Sci., Paris (Serie D) 273, 2705.
Avrameas S. & Ternynck T. (1971) immunochemjstry
117.5. Collins W. S., II, Metzger J. F. &Johnson _KImmun. loS, 852.
8,
A. D. (1972)
Halmann M., Velan B. % Sery T. (1977) Appl. environm. Microbial.
34, 473.
Johnson H.M., Bukovic J. A.. Kauffman P. E. & Peeler
J. T. (1971) Appl. Microbial.
22, 837.
Kalmakof J., Parkinson A. J., Crawford A. M.&Williams B. R. G. (1977) .I. immunol. Meth. 14, 73. Lowry 0. H.. Rosebrough N. J., Farr- A. L. & Randall R. J. (1951) J. biol. Chem. 193, 265. Puget K., Michelson A. M. & Avrameas S. (19773 Analgt. Biochem. 79,447.
Schantz E. J., Roessler W. G., Wagman J., Spero L., Dunnery D. A. & Bergdoll M. S. (1965) Biochemist? 4, 1011. Silverman S. J. (1963) J. Bact. 85, 955. Van Weemen B. K. & Schuurs A. H. W. M. (1971) FEB.9 Lett. 15, 232. Weirether F. J., Lewis E. E.. Rosenwald A. J. & Lincoln R. E. (1966)Appl. ~icrobiol. 14, 284. Worthington Enzyme Manual (1972). p. 43.
