- Email: [email protected]
The Effect of Haemoglobin on the Detection of Immunoglobulins in Calf Sera
Br. vet.]. ( 1969),
125>
458
THE EFFECT OF HAEMOGLOBIN ON THE DETECTION OF IMMUNOGLOBULINS IN CALF SERA By M.
GITTER* AND S. S. STONEt
East Mrican Veterinary Research Organization, Muguga, P.O. Kabete, Kenya SUMMARY
When haemolysed blood was added to homologous calf sera in different concentrations and the mixtures tested for immunoglobulins, a precipitate was always formed with the standard zinc sulphate test but not with the sodium sulphite test. Immunoelectrophoretic and spectrophotometric analyses of the precipitates identified the presence of haemoglobin. The authors suggest that the zinc sulphate test should not be used for the detection of immunoglobulins in haemolysed calf sera. INTRODU CTION
The test for the detection of immunoglobulins (Ig) in the serum of young calves is often of considerable diagnostic value. Unfortunately, blood from live animals is not always available and a dead calf sent to the laboratory is often the first indication that all is not well on the farm. The value of such specimens in establishing diagnosis varies, and post-mortem changes can often mask even pathognomonic lesions. The tests commonly used for the detection of Ig in calf sera are the sodium sulphite turbidity test (SSTT) and the zinc sulphate test (ZST) (Aschaffenburg, 1949; Patterson, 1967). In the course of our investigations we noted that when serum samples from dead calves were tested for Ig, marked turbidity was often recorded when the ZST was used, whereas the same samples might remain clear with the SSTT (Gitter & Stone, 1969). This discrepancy occurred in samples in which haemolysis was evident. It was particularly marked in haemolysed pre-colostral sera where the SSTT was negative and turbidity was ( )served in the ZST. Since the immunochemical validity of the SSTT had been established (Stone & Gitter, 1969) it was postulated that the ZST precipitated other proteins in addition to Ig. This would agree with the observation of Penhale et al. (1967) that the ZST precipitate "is not entirely globulin but also contains other serum proteins" . The purpose of this investigation was to ascertain the effect of haemolysis on the validity of the SSTT and the ZST for the detection of Ig in calf sera. MATERIALS AND METHODS
Two lots of blood samples were collected from each of four calves, two newly * On secondment from the M.A.F.F. Veterinary Investigation Centre, Weybridge, Surrey. t Permanent address: U .S. D epartment of Agriculture, Plum Island Animal Disease Laboratory, P.O. Box 848, Greenport, Long Island, New York 11944, U.S.A.
PLATE I
Fig. I . Comparative sodium sulphite turbidity and zinc sulpha te tes ts on pre-colostral serum a nd haemolysed blood . Left to right : ( I ) serum and haemolysed blood mixture after SSTT; (2) haemolysed blood a fte r SSTT ; (3, 4) the sam e samples after ZnS0 4 test.
Gitter and Stone, Br. vet. J. ( 1969), 125, 9
PLAT E II
Fig. 2. Immunoelectrophore tic precipita te patte rns of pre-colostral calf serum/ haemolysed blood mixtures. L eft to right : well I , normal bovine serum ; well 2, pre-colostral calf serum ; well 3, zinc sulpha te precipitate of haemolysed precolos tral calf serum ; well 4, whole haemolysed pre-colostra l blood. Cha nnels I and 2, ra bbit anti-bovine serum; cha nnels 3 and 4, rabbit a nti-haemoglobin serum . The a nod e is at the top .
Giller and Stone, B/". vet.
J. ( 196 9),
12 5,
9
DETECTION OF IMMUNOGLOBULINS IN CALF SERA
459
born and two 3-4 days old. One lot was allowed to clot and the serum was separated; the other was collected in bottles containing heparin. Preparation of haemoglobin. The whole blood samples were haemolysed by repeated freezing at - 20 ° C and thawing in a water bath at ambient temperatures, until 0·05 ml. diluted in 5 ml. of saline produced a crystal-clear solution. The haemoglobin concentration in the lysed samples was determined (as described by Hawk, Oser & Sommerson, 1955) and samples of the sera were mixed with equal volumes of haemolysed whole blood from the same animal. Three-fold dilutions of each of the mixtures were made until haemolysis could not be visually detected. Detection of Ig. The serum and the serum and haemolysed blood mixtures were subjected to the SSTT and ZST. Turbidity in the ZST was measured, using a I cm. i.d. cell, on the log scale of an EEL colorimeter; a filter was not employed. Blank control readings for each dilution were obtained using normal 0·85 per cent saline in place of zinc sulphate. No turbido-metric measurements were carried out with the SSTT which was scored "negative" for no precipitate; ± for a slight cloudiness, and + for a definite precipitate forming immediately. The immunoelectrophoretic technique, as described by Scheidegger (1955) was used, employing 5 rnA of current for 90 minutes in barbital-acetate buffer, pH 8·6, ionic strength 0.025. The precipitin patterns were developed using rabbit anti-bovine and anti-haemoglobin sera and photographed 24 hours after incubation in a humid chamber. The anti-bovine haemoglobin serum was prepared in rabbits. The haemoglobin had been prepared by lysing thoroughly washed bovine erythrocytes in distilled water. The stroma was separated by centrifugation. RESULTS
Tables I and II show the increase in the zinc sulphate turbidometric readings which correspond with the increased concentration of haemoglobin in the samples. Haemolysis was visually evident in all dilutions tested. No precipitate could be seen when 0·85 per cent saline was used instead of zinc sulphate. TABLE I T HE
EFFE C T
OF
HAEMOGLOBIN IN PRE-COLOSTRAL CALF SERA ON TURBIDITY TEST AND ZINC SULPHATE TEST
Calf A Dilutions of serumJHb mixture
SSTT 1:2 1:6 1:18 1:54 1:162 Serum only
Initial haemoglobin
All negative
THE
SODIUM
SULPHIT E
CalfB
:{,nSO. turbidometric reading 3 ' 15 1'45 0 '55 0 '3 0'1 0 '00 [3'00 g, %
SSTT
All negative
:{,nSO. turbidometric reading 6 .40 2'00 0'95 0 '55 0' 1I O 'II
[6'05 g, %
BRITISH VETERINARY JOURNAL, 125, 9
TABLE II THE EFFECT OF HAEMOGLOBIN ON THE SODIUM SULPHITE TURBIDITY TEST AND ZINC SULPHATE TEST IN THE SERA OF CALVES 3-4 DAYS OLD
Galf D
GalfG Dilutions of serum/Hb mixture
SSTT
± ± ± ± ± ±
1:2 1:6 1:18 1:54 1:162
Serum only Initial haemoglobin
ZnSO. turbidometric reading 3.65 0'95 0·80 0'3 0 0'15 0'15 12·4g· %
SSTT
+ + + + + +
ZnSO. turbidometric reading 4'45 3'20 1'20 0'7 0 0.625 0·60 IO·Sg.%
In Fig. I the difference between the two tests is evident: while the sample of serum with haemolysed blood and whole blood remained clear with SSTT, the same samples showed marked turbidity with zinc sulphate. Figure 2 shows that pre-colostral calf serum in well 2 was devoid of Ig. The zinc sulphate precipitate of haemolysed pre-colostral calf serum in well 3 contains haemoglobin and other serum proteins of slow anodic migration. 10·0
'Wave Length
Fig. 3. Spectrophotometric profile of pre-colostral haemolysed calf serum reacting to the zinc sulphate test. Curve I (solid line), calf serum haemolysed blood mixture control; curve 2 (broken line), supernatant fraction serum, haemolysed blood and zinc sulphate mixture; curve 3 (dotted line), redissolved ZnS04 precipitate.
In Fig. 3 the spectrophotometric profile of the zinc sulphate precipitate is essentially similar to that of the control haemolysed blood-serum mixture in which saline was used instead of zinc sulphate. These two curves and that of
DETECTION OF IMMUNOGLOBULINS IN CALF SERA
461
the supernatant fraction indicate that haemoglobin was precipitated in the ZST (Keilin & Hartree, 1941; White, Handler & Smith, 1959). Using o.d. values at the 350 mp, plateau region and the 410 mp, peak, 58 per cent of haemoglobin was precipitated; of the precipitate only 56 per cent was soluble in 0·85 per cent saline. DISCUSSION
There is little doubt that haemoblogin is precipitated by zinc sulphate and that in addition to Ig the precipitate contains other serum proteins as demonstrated in the immunoelectrophoretic precipitin patterns, thus confirming the observation made by Penhale et at. (1967) . The samples of calf serum mixed with haemolysed blood to which zinc sulphate was added became increasingly turbid in proportion to the amount of haemoglobin. This turbidity could easily be observed and confirmed by turbidometric readings. The presence of haemoglobin in the zinc sulphate precipitate was demonstrated by immunoelectrophoretic and spectrophotometric techmques. The spectrophotometric curves in Fig. 3 are essentially similar to those reported by Keilin & H artree (1941) and White et at. (1959). The slight deviation in the sharpness of the peaks is probably due to difference in degrees of purity of the samples used. In contrast to the ZST, no precipitate was formed with the sodium sulphite, irrespective of the amount of haemoglobin added to the calf serum. It would therefore appear that the ZST for the detection of Ig in haemolysed calf sera is not reliable, since false positive results may be obtained. In such circumstances the very simple SSTT, which is immunochemically valid (Stone & Gitter, 1969) even in the presence of haemoglobin, would be the test of choice. ACKNOWLEDGMENT
We are grateful to Mr T. F. Brand for valuable technical assjstance. REFEREN CES
ASCHAFFENBURG, R. (1949) . Br. ] . Nutr., 3, 200. GITTER, M. & STONE, S. S. (1969). Vet. Rec. , 84> 72 . HAWK, P. B., OSER, B. L. & SOMMERSON, W. H . ( 1955). Practical Physiological Chemistry, 13th edn, pp. 616, 617. London: Churchill. KElLIN, D. & HARTREE, E. F. ( 1941). Nature, Lond. , 148,75,77. PATTERSON, D. S. P . ( 1967) . Vet. R ec., 80, 260. PENHALE, W. ] ., CHRISTIE, G., McEWAN, A. D. , SELMAN, I. E . & FISHER, E. W. (1967) . Vet. Rec. , 81, 416. SCHEIDEGGER, ]. (1955) . Int. Archs Allergy, 7, 103. STONE, S. S. & GITTER, M. (1969) . Br. vet. ] ., us, 68. WHITE, A., HANDLER, P. & SMITH, E. L. ( 1959). Principles 0/ Biochemistry, 3rd edn, p. 199. New York: McGraw-Hill. (Accepted/or publication 14 February 1969)
BR ITISH VETERINARY JOUR NAL,
12 5,
9
Action de l'hem.oglobine sur la m.ise en evidence d'im.m.uno-globulines dans Ie serum. de veau (Gitter et Stone) Resum.e . La recherche d'immuno-globulines apres adjonction de sang hemolyse a differentes concentrations de serum de veau a ete marquee par la formation d'un precipite lors de l'epreuve au sulfate de zinc, mais non lors de celle au sulfite de soude. De l'avis des auteurs, l'epreuve au sulfate de zinc ne devrait pas etre utilisee pour la mise en evidence d'immunoglobulines dans Ie serum d e veau. Der Einfiu8 des Hiim.oglobins auf den Nachweis der Im.m.unoglobuline bei Kiilberseren (Gitter und Stone) Zusam.m.enfassung. Wenn hamolysiertes Blut homologen Kalberseren in verschiedenen Konzentrationen zugefligt und das Gemisch auf Immunoglobuline geprtift wurde, ergab sich bei der Standard-Zinksalfatprobe immer ein Prazipitat, jedoch nicht bei der Natriumsulfitprobe. Anhand immunoelektrophoretischer und spektrophotometrischer Analysen des Prazipitats wurde das Vorhandensein von Hamoglobin nachgewiesen. Die Verfasser weisen darauf hin, daB die Zinksulfatprobe flir den Immunoglobulinnachweis bei hamolysierten Kalberseren nieht verwendet werden darf. El efecto de la hem.oglobina en el descubrim.iento de inm.unoglobinas en los sueros de ternero (Gitter y Stone) Resum.en. Cuando se aiiadia sangre hemolizada a sueros homalogos de terneros en diferentes concentraciones y se ensayaron las mezclas para investigar la presencia de inmunoglobinas, se forma siempre un precipitado con la prueba normal de sulfato de cinc pero no con la prueba del sulfuro sadico. El amHisis inmunoelectroforetico y espectrofotometrico de los precipitados identificaron la presencia de la hemoglobina. Los autores sugieren que Ie prueba del sulfa to de cinc no debe usarse para el descubrimiento de inmunoglobulinas en los su eros hemolizados de ternero.
125>
458
THE EFFECT OF HAEMOGLOBIN ON THE DETECTION OF IMMUNOGLOBULINS IN CALF SERA By M.
GITTER* AND S. S. STONEt
East Mrican Veterinary Research Organization, Muguga, P.O. Kabete, Kenya SUMMARY
When haemolysed blood was added to homologous calf sera in different concentrations and the mixtures tested for immunoglobulins, a precipitate was always formed with the standard zinc sulphate test but not with the sodium sulphite test. Immunoelectrophoretic and spectrophotometric analyses of the precipitates identified the presence of haemoglobin. The authors suggest that the zinc sulphate test should not be used for the detection of immunoglobulins in haemolysed calf sera. INTRODU CTION
The test for the detection of immunoglobulins (Ig) in the serum of young calves is often of considerable diagnostic value. Unfortunately, blood from live animals is not always available and a dead calf sent to the laboratory is often the first indication that all is not well on the farm. The value of such specimens in establishing diagnosis varies, and post-mortem changes can often mask even pathognomonic lesions. The tests commonly used for the detection of Ig in calf sera are the sodium sulphite turbidity test (SSTT) and the zinc sulphate test (ZST) (Aschaffenburg, 1949; Patterson, 1967). In the course of our investigations we noted that when serum samples from dead calves were tested for Ig, marked turbidity was often recorded when the ZST was used, whereas the same samples might remain clear with the SSTT (Gitter & Stone, 1969). This discrepancy occurred in samples in which haemolysis was evident. It was particularly marked in haemolysed pre-colostral sera where the SSTT was negative and turbidity was ( )served in the ZST. Since the immunochemical validity of the SSTT had been established (Stone & Gitter, 1969) it was postulated that the ZST precipitated other proteins in addition to Ig. This would agree with the observation of Penhale et al. (1967) that the ZST precipitate "is not entirely globulin but also contains other serum proteins" . The purpose of this investigation was to ascertain the effect of haemolysis on the validity of the SSTT and the ZST for the detection of Ig in calf sera. MATERIALS AND METHODS
Two lots of blood samples were collected from each of four calves, two newly * On secondment from the M.A.F.F. Veterinary Investigation Centre, Weybridge, Surrey. t Permanent address: U .S. D epartment of Agriculture, Plum Island Animal Disease Laboratory, P.O. Box 848, Greenport, Long Island, New York 11944, U.S.A.
PLATE I
Fig. I . Comparative sodium sulphite turbidity and zinc sulpha te tes ts on pre-colostral serum a nd haemolysed blood . Left to right : ( I ) serum and haemolysed blood mixture after SSTT; (2) haemolysed blood a fte r SSTT ; (3, 4) the sam e samples after ZnS0 4 test.
Gitter and Stone, Br. vet. J. ( 1969), 125, 9
PLAT E II
Fig. 2. Immunoelectrophore tic precipita te patte rns of pre-colostral calf serum/ haemolysed blood mixtures. L eft to right : well I , normal bovine serum ; well 2, pre-colostral calf serum ; well 3, zinc sulpha te precipitate of haemolysed precolos tral calf serum ; well 4, whole haemolysed pre-colostra l blood. Cha nnels I and 2, ra bbit anti-bovine serum; cha nnels 3 and 4, rabbit a nti-haemoglobin serum . The a nod e is at the top .
Giller and Stone, B/". vet.
J. ( 196 9),
12 5,
9
DETECTION OF IMMUNOGLOBULINS IN CALF SERA
459
born and two 3-4 days old. One lot was allowed to clot and the serum was separated; the other was collected in bottles containing heparin. Preparation of haemoglobin. The whole blood samples were haemolysed by repeated freezing at - 20 ° C and thawing in a water bath at ambient temperatures, until 0·05 ml. diluted in 5 ml. of saline produced a crystal-clear solution. The haemoglobin concentration in the lysed samples was determined (as described by Hawk, Oser & Sommerson, 1955) and samples of the sera were mixed with equal volumes of haemolysed whole blood from the same animal. Three-fold dilutions of each of the mixtures were made until haemolysis could not be visually detected. Detection of Ig. The serum and the serum and haemolysed blood mixtures were subjected to the SSTT and ZST. Turbidity in the ZST was measured, using a I cm. i.d. cell, on the log scale of an EEL colorimeter; a filter was not employed. Blank control readings for each dilution were obtained using normal 0·85 per cent saline in place of zinc sulphate. No turbido-metric measurements were carried out with the SSTT which was scored "negative" for no precipitate; ± for a slight cloudiness, and + for a definite precipitate forming immediately. The immunoelectrophoretic technique, as described by Scheidegger (1955) was used, employing 5 rnA of current for 90 minutes in barbital-acetate buffer, pH 8·6, ionic strength 0.025. The precipitin patterns were developed using rabbit anti-bovine and anti-haemoglobin sera and photographed 24 hours after incubation in a humid chamber. The anti-bovine haemoglobin serum was prepared in rabbits. The haemoglobin had been prepared by lysing thoroughly washed bovine erythrocytes in distilled water. The stroma was separated by centrifugation. RESULTS
Tables I and II show the increase in the zinc sulphate turbidometric readings which correspond with the increased concentration of haemoglobin in the samples. Haemolysis was visually evident in all dilutions tested. No precipitate could be seen when 0·85 per cent saline was used instead of zinc sulphate. TABLE I T HE
EFFE C T
OF
HAEMOGLOBIN IN PRE-COLOSTRAL CALF SERA ON TURBIDITY TEST AND ZINC SULPHATE TEST
Calf A Dilutions of serumJHb mixture
SSTT 1:2 1:6 1:18 1:54 1:162 Serum only
Initial haemoglobin
All negative
THE
SODIUM
SULPHIT E
CalfB
:{,nSO. turbidometric reading 3 ' 15 1'45 0 '55 0 '3 0'1 0 '00 [3'00 g, %
SSTT
All negative
:{,nSO. turbidometric reading 6 .40 2'00 0'95 0 '55 0' 1I O 'II
[6'05 g, %
BRITISH VETERINARY JOURNAL, 125, 9
TABLE II THE EFFECT OF HAEMOGLOBIN ON THE SODIUM SULPHITE TURBIDITY TEST AND ZINC SULPHATE TEST IN THE SERA OF CALVES 3-4 DAYS OLD
Galf D
GalfG Dilutions of serum/Hb mixture
SSTT
± ± ± ± ± ±
1:2 1:6 1:18 1:54 1:162
Serum only Initial haemoglobin
ZnSO. turbidometric reading 3.65 0'95 0·80 0'3 0 0'15 0'15 12·4g· %
SSTT
+ + + + + +
ZnSO. turbidometric reading 4'45 3'20 1'20 0'7 0 0.625 0·60 IO·Sg.%
In Fig. I the difference between the two tests is evident: while the sample of serum with haemolysed blood and whole blood remained clear with SSTT, the same samples showed marked turbidity with zinc sulphate. Figure 2 shows that pre-colostral calf serum in well 2 was devoid of Ig. The zinc sulphate precipitate of haemolysed pre-colostral calf serum in well 3 contains haemoglobin and other serum proteins of slow anodic migration. 10·0
'Wave Length
Fig. 3. Spectrophotometric profile of pre-colostral haemolysed calf serum reacting to the zinc sulphate test. Curve I (solid line), calf serum haemolysed blood mixture control; curve 2 (broken line), supernatant fraction serum, haemolysed blood and zinc sulphate mixture; curve 3 (dotted line), redissolved ZnS04 precipitate.
In Fig. 3 the spectrophotometric profile of the zinc sulphate precipitate is essentially similar to that of the control haemolysed blood-serum mixture in which saline was used instead of zinc sulphate. These two curves and that of
DETECTION OF IMMUNOGLOBULINS IN CALF SERA
461
the supernatant fraction indicate that haemoglobin was precipitated in the ZST (Keilin & Hartree, 1941; White, Handler & Smith, 1959). Using o.d. values at the 350 mp, plateau region and the 410 mp, peak, 58 per cent of haemoglobin was precipitated; of the precipitate only 56 per cent was soluble in 0·85 per cent saline. DISCUSSION
There is little doubt that haemoblogin is precipitated by zinc sulphate and that in addition to Ig the precipitate contains other serum proteins as demonstrated in the immunoelectrophoretic precipitin patterns, thus confirming the observation made by Penhale et at. (1967) . The samples of calf serum mixed with haemolysed blood to which zinc sulphate was added became increasingly turbid in proportion to the amount of haemoglobin. This turbidity could easily be observed and confirmed by turbidometric readings. The presence of haemoglobin in the zinc sulphate precipitate was demonstrated by immunoelectrophoretic and spectrophotometric techmques. The spectrophotometric curves in Fig. 3 are essentially similar to those reported by Keilin & H artree (1941) and White et at. (1959). The slight deviation in the sharpness of the peaks is probably due to difference in degrees of purity of the samples used. In contrast to the ZST, no precipitate was formed with the sodium sulphite, irrespective of the amount of haemoglobin added to the calf serum. It would therefore appear that the ZST for the detection of Ig in haemolysed calf sera is not reliable, since false positive results may be obtained. In such circumstances the very simple SSTT, which is immunochemically valid (Stone & Gitter, 1969) even in the presence of haemoglobin, would be the test of choice. ACKNOWLEDGMENT
We are grateful to Mr T. F. Brand for valuable technical assjstance. REFEREN CES
ASCHAFFENBURG, R. (1949) . Br. ] . Nutr., 3, 200. GITTER, M. & STONE, S. S. (1969). Vet. Rec. , 84> 72 . HAWK, P. B., OSER, B. L. & SOMMERSON, W. H . ( 1955). Practical Physiological Chemistry, 13th edn, pp. 616, 617. London: Churchill. KElLIN, D. & HARTREE, E. F. ( 1941). Nature, Lond. , 148,75,77. PATTERSON, D. S. P . ( 1967) . Vet. R ec., 80, 260. PENHALE, W. ] ., CHRISTIE, G., McEWAN, A. D. , SELMAN, I. E . & FISHER, E. W. (1967) . Vet. Rec. , 81, 416. SCHEIDEGGER, ]. (1955) . Int. Archs Allergy, 7, 103. STONE, S. S. & GITTER, M. (1969) . Br. vet. ] ., us, 68. WHITE, A., HANDLER, P. & SMITH, E. L. ( 1959). Principles 0/ Biochemistry, 3rd edn, p. 199. New York: McGraw-Hill. (Accepted/or publication 14 February 1969)
BR ITISH VETERINARY JOUR NAL,
12 5,
9
Action de l'hem.oglobine sur la m.ise en evidence d'im.m.uno-globulines dans Ie serum. de veau (Gitter et Stone) Resum.e . La recherche d'immuno-globulines apres adjonction de sang hemolyse a differentes concentrations de serum de veau a ete marquee par la formation d'un precipite lors de l'epreuve au sulfate de zinc, mais non lors de celle au sulfite de soude. De l'avis des auteurs, l'epreuve au sulfate de zinc ne devrait pas etre utilisee pour la mise en evidence d'immunoglobulines dans Ie serum d e veau. Der Einfiu8 des Hiim.oglobins auf den Nachweis der Im.m.unoglobuline bei Kiilberseren (Gitter und Stone) Zusam.m.enfassung. Wenn hamolysiertes Blut homologen Kalberseren in verschiedenen Konzentrationen zugefligt und das Gemisch auf Immunoglobuline geprtift wurde, ergab sich bei der Standard-Zinksalfatprobe immer ein Prazipitat, jedoch nicht bei der Natriumsulfitprobe. Anhand immunoelektrophoretischer und spektrophotometrischer Analysen des Prazipitats wurde das Vorhandensein von Hamoglobin nachgewiesen. Die Verfasser weisen darauf hin, daB die Zinksulfatprobe flir den Immunoglobulinnachweis bei hamolysierten Kalberseren nieht verwendet werden darf. El efecto de la hem.oglobina en el descubrim.iento de inm.unoglobinas en los sueros de ternero (Gitter y Stone) Resum.en. Cuando se aiiadia sangre hemolizada a sueros homalogos de terneros en diferentes concentraciones y se ensayaron las mezclas para investigar la presencia de inmunoglobinas, se forma siempre un precipitado con la prueba normal de sulfato de cinc pero no con la prueba del sulfuro sadico. El amHisis inmunoelectroforetico y espectrofotometrico de los precipitados identificaron la presencia de la hemoglobina. Los autores sugieren que Ie prueba del sulfa to de cinc no debe usarse para el descubrimiento de inmunoglobulinas en los su eros hemolizados de ternero.