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A turbidity test for the estimation of immune globulin levels in neonatal calf serum
CLINICA CHIMICA ACTA
155
A T U R B I D I T Y TEST FOR T H E ESTIMATION OF IMMUNE GLOBULIN LEVELS IN NEONATAL CALF SERUM
A. D. McE~,VAN, E. W. F I S H E R , I. E. SELMAN a x o W. J. P E N H A L E *
Department of Veterinary Medicine, University Veterinary Hospital, Bearsden Road, Bearsden, Glasgow ( U.K.) * Department of Veterinary Pathology, Royal (Die,{) School of Veterinary Studies, Edinburgh (U.K.) (Received August 5, t969)
SUMMARY
A simple and rapid method for the determination of immune globulin concentration in neonatal calf serum is described. The method uses a turbidity reaction and investigations into some factors which influence this reaction and into the correlation between the reaction and specific inmmne globulin concentrations have been performed.
INTRODUCTION
Calves are hvoog!obulinaewdc at birth and are dependent i:pon the absorption of immune globulins from colostrum for passive immunity to neonatzl diseases. Since the quantity of immune globulin absorbed has been shown to bear a relationship with susceptibility to neonatal disease t, the estimation of serum imnmne globulin concentratiot:s in early life is of clinical importance. For practical purposes, the technique of estimation should be cheap, rapid and simple, so that the immune globulin concentration o, large numbers may be determined as easily as that of the individual calf. A method of detecting whether or not colostrum has been fed to newborn ca'. es of a serum turhiditv was described by Aschaffenburg'. This involved *lie Inoulncatlon ~"" " test originally described and designed by Kunkel s to detect alterations in the serum gamma globulin concentrations of humans suffering from liver diseases. The purpose of this paper is to describe some factors which affect the turbidity reaction when performed under the conditions described by Aschaffenburg, and to correlate the intensity of the reaction when performed under standard conditions with the concentration of specific immune globulin fractions. MATERIALS AND METHODS
Serum. Serum was obtained from bull calves less than one ~ 'eek of age, largely of the Ayrshire breed, which had been bought in local markets. Clin. Chim. Acta, 27 (I97 o) i 5 5 - I 6 3
I56
MCEWAN et al.
Zinc sulphate solution. A solution of zinc sulphate (208 mg/l ZnSO4"7 H20) was prepared in a volumetric flask using distilled water which had been boiled for lO-15 rain in order to remove dissolved carbon dioxide. This solution was then kept in an aspirator bottle and was further protected against the uptake of carbon dioxide by the insertion of a soda lime tube into the stopper. The aspirator bottle was connected by means of polythene tubing to an automatically dispensing pipette* set to deliver 6 ml. An identical arrangement was used toldeliver a similar volume of distilled water for control samples. Measurement of turbidity. The test was performed in sets of matched Io-ml E E L colorimeter tubes. For each serum sample tested, a "control" tube containing 6 ml of distilled water and a "test" tube containing 6 ml of zinc sulphate solution were measured out. To each of these, o.I ml of serum was delivered from an Autozero high precision pipette**. The solution was then gently shaken in order to ensure mixing of reagents, and the tubes were allowed to stand for 6o min at room temperature, which was 20 °. After this time had elapsed, the tubes were shaken again to ensure an even redistribution of the precipitate and inserted into a photoelectric absorptiometer***, which had been previously zeroed using a colorimeter tube containing distilled water. A blue-green filter (Ilford No. 623) was used in the absorption in place of the red one (Ilford No. 6o8) recommended by both Kunkel a and Aschaffenburg 2 as this provided greater sensitivity to measurement of turbidity. The increased sensitivity which this filter exhibits to haemoglobin and hence to the effects of haemolysis, is compensated by the use of control samples. The turbidity reading was found by subtracting the reading obtained from the control tube to that of the test tube and by multiplying the difference by a factor of IO. In order to eliminate variations in the sensitivity of the absorptiometer and to facilitate comparison of results obtained in this laboratory with results possibly obtained in the future in other laboratories, the barium sulphate standard recommended by Shank and Hoagland 4 for use in the thymol turbidity test was adopted. Three millilitres of a barium chloride solution containing 1.15 g barium chloride (BaCI2.2 H~O) per IOO ml were made up to IOO ml in a volumetric flask with o.2 N sulphuric acid. When measured under the same conditions as used in the zinc sulphate turbidity test, this solution gave a turbidity reading of 20 units. Investigation of factors influencing the zinc sulphate turbidity reaction r. Time and temperature. To study the effects of time and temperature on the turbidity reaction, serum samples were obtained from ten neonatal calves. The turbidity test was performed as described above except that the turbidities were measured 15, 3o, 6o and 12o min after the addition of the serum sample to the zinc sulphate solution. This procedure was performed using the same sera at the following temperatures: 6 °, zo °, 25 °, 3 l°, 37 °. The results of this experiment are shown in Table I, in which tile average turbidity of the ten samples is shown in relation to time and temperature. From this, it can be seen that the reaction continues to develop throughout the 12o-nlill period and is influenced greatly by temperature. 2. pH. In the original description of the zinc sulphate turbidity test, Kunkel * W.G. Flaig & Sons Ltd., Excelo Works, Margate Road, Broadstairs, Kent. ** H . J . Elliot Ltd., E-Mil Works, Treforest, Glamorgan. *** Evans Electroselenium Ltd., St. Andrew's Works, Halstead, Essex. Clin. Chim. Acta, 27 (x97o) x55-t63
IMMUNE GLOBULIN IN NEONATAL CALF SERUM TABLE
I~7
I
THE EFFECT OF TIME AND TEMPERATURE ZINC SULPHATE TURBIDITY OF IO SERA
Temp. °C 6 20 25 3I 37
ON
THE
MEAN
Time (rain) "5 30
60
z2o
I2.8 i4. 7 I6.8 30.3 41.6
15.8 18.6 24. 9 38.4 44.0
16.6 zI.2 27.6 4°.6 46.7
15.x i7. 4 22.o 34.6 42.8
maintained tile pH of the reagent at 7.5 by use of a weak barbiturate buffer. Subsequent workers 6,' have continued this practice, although Reinhold 7 had cause to alter the relative concentrations of the constituents of the buffer as the original concent:ations provided an alkaline pH in his laboratory. Aschaffenburg ~ found it unnecessary to use buffer and used only distilled water. Where necessary, the pH was adjusted to neutrality by the addition of dilute acid or alkali. The following experiment was performed to find out what effect pH had on the zinc sulphate turbidity reaction. Twenty calf sera with turbidity readings between o and 4° were selected. These sera were then tested using (x) unbuffered zinc sulphate solution, (z) zinc sulphate solution to which had been added barbiturate buffer in the proportions described by KunkeP, i.e. 28o mg barbituric acid and 2xo mg sodium barbitone per litre, (3) a zinc sulphate solution to which had been added barbiturate buffer in the proportions described by Reinhold 7, i.e. 3o2 mg barbituric acid and xoo mg sodium barbitone per litre. The measured pH of these solutions were respectively, 4.7, 7.53 and 7.6. The turbidity test was then performed under the conditions described previously. When compared with unbuffered reagents, it was found that both buffers depressed the development of the turbidity reaction. The depression produced bv the buffer originally described by Kunkel 3 was so great that only one sample gave a reading of greater than 2.5 (Fig. x). 3. Albumin. In human patients with hver disease, KunkeP produced a slight fall in the intensity of the turbidity reaction by administenng intravenously large amounts of human albumin. He also demonstrated that by adding albumin to positively reading serum taken from patients suffering from hepatitis that there was a decrease in the amount of globulin precipitated. The method used to determine the influence of albumin on the zinc sulphate reaction was as follows. A range of concentrations of bovine albumin solution was prepared by dissolving crystallized bovine albumin in distilled water and making taLt~, w a s serial dilutions. A more concentrated solution of zinc sulphate, z5o mg per ~='-^ prepared in order to allow for the dilution which occurs when the albumin solution is added. Five serum samples with turbidity readings ranging between 3o and 50 were selected and the test performed in the following manner. For each concentration TABLE
II
THE EFFECT
ON THE TU~°TDITY
A d d e d a l b u m i n (mg/mi) Average turbidity Difference
REACTION
b 43-7 o
OF THE ADDITION
2.5 42.~ --o.8
5.0 43 .2 --o.5
OF ALBUMIN TO 5 SERA
io.o 43.8 +o.r
20.0 43.4 --o.3
30.0 -~4.° +o.3
40.0 42"5 -- 1.2
Clin. Chim. Acta, 27 (x97 o) I 5 5 - 1 6 3
McEWA~ a al.
x58 40, o.-o ZnS04 •
~,--.xZnS04+Reinhold buffer' /
35 ~"~ZnS04+Kunkel buffer
30.
~)0"
~
P~ J
25'
E~40. o
E
.__c
~20-
30. .Q
O
I-
E E
-b ~5-
x 10' m
10 5 .,
o
g
Ib
~g
:/o
Calf no.
,
,
,
. . . . . .
,
.
.
10 20 30 40 Zinc sulphate turbidity units
.
.
,
50
Fig. t. The effect of pH on the zinc sulphate turbidity reaction. Fig. e. The effect on the sulphate turbidity reaction of adding a bovine gamma globulin preparation to hypogammaglobulinaemic calf serum.
of albumin solution, five colorimeter tubes were set up. Into each was added 5 ml of the zinc sulphate solution, I ml of the appropriate albumin solution, and o.I ml of each serum sample. Thereafter, the performance of the test was as described previously. The results of this experiment are ~hown in Table II. From the results obtained in the present work, it would appear that there is very little depression of turbidity below concentrations of 3o mg of albumin per ml. 4. Bovine gamma globulin. In view of the fact that the serum of newborn calves is markedly hypogammaglobulinaemic until immune globulins have been absorbed from colostrum, the addition of gamma globulin to such sera would allow a method of quantitating the zinc sulphate turbidity reaction through the range of levels encountered naturally. A preparation of bovine gamma globulin is available commercially* which has been prepared by the Cohn fractionation technique and is the equivalent of fraction I I. This has been used in the following experiment. By means of serial dilutions, solutions of bovine gamma globulin of concentrations ranging from 1.5 to 30 mg per ml of serum were set up. Serum samples were obtained from newly born co!ostrum-deprived calves, split into two groups, each containing five samples, and submitted to the zinc sulphate turbidity test, using the standard technique. The turbidity test on the samples containing bovine gamma globulin was performed as follows. Each concentration of bovine gamma globulin in a series of dilutions was tested against five of the serum samples. Therefore, for each * Armour Pharmaceutical Company Ltd., Eastbourne, England. Clin. Chim. Acta, 27 (x97o) x55-x63
159
IMMUNE GLOBULIN IN NEONATAL CALF SERUM
concentration, five co!ofimeter tubes were set up, and to each was added 5 ml zinc sulphate solution. This solution was made up at a strength of 250 mg zinc sulphate per litre in order to allow for the dilution which occurs when the globulin solutions were added. To each part of the five tubes, I ml of the appropriate solution was added. Finally, o.I ml of the serum was taken from each serum sample and added to one tube, Thereafter, the performance of the test was as described in the MATERIALS AND METHODS section. The average turbidity obtained from each concentration of bovine gamma globulin used was calculated, and the results are shown in Fig. 2. It would appear that the relationship existing between the turbidity devel.)ped by the -~inc sulphate test and the concentration of bovine gamma globulin added to the serum is linear only up to levels of about 30 mg bovine gamma globulin per ml. Thereafter, the turbidity would appear to increase in an exponential manner. 5. The comparative turbidities developed by serum and plasma. Serum and plasma 40-
35-
30"
.=_ 2510 0 L
.~ 20.
/i/
g i,-
15-
/
d/ /
10-
x~x
/
/
Plasma
(>-----<)Serum
--_
I
s
Io
15
20
Calf number
Fig. 3. A c o m p a r i s o n of t h e zinc s u l p h a t e t u r b i d i t i e s developed b y p l a s m a a n d serum. TABLE THE
II I
REPRODUCIBILITY
PERFORMED
ON 6
Serum
Mean
O F THI~ Z I N C S U L P H A T E
DIFFERENT
TURBIDITY" RE~tCTION
SERA
Standard deviation
I
9.0
~
2 3 4 5 6
x9.o 25. I 33.0 3 8.t 4o.9
± ~: ± 5: 4-
i.l 0.8 0.9 x.4 I.t 0.8
Coeff. of variahon
(%)
i2.2 4.2 3 .6 4 .2 2.9 1.9 Clin. Chim. Acta, 27 (x97 o) I 5 5 - I 6 3
x6o
MCEWAN et
al.
samples were taken from a group of 2o neonatal calves. Heparin was used as the anticoagulant for plasma samples. These samples were then submitted to the zinc sulphate turbidity test, and the results obtained are shown in Fig. 3. It would appear that plasma gives a greater turbidity reaction than serum. 6. The reproducibility of the zinc sulphate turbidity reaction. To ascertain the reproducibility of the method, 5 determinations were performed on 6 different serum samples. The means, standard deviations and coefficients of variation are shown in Table I I I. From these results it can be seen that the reproducibility of this method of measuring the turbidity reaction is high. 50y = 0.97x - 1.95 r = 0.959
v
40-
/
/
30-
v sv ~ v ~ v v Vq
Ill 2o-
~'~v v
v
./
o
.v
,1~
0
Z"
.
.
.
.
.
.
.
20 30 40 sulphote turbidity units
10 Zinc
50
Fig. 4. The correlation between the serum IgG concentration and the turbidity developed by the zinc sulphate turbidity reaction.
y=
0.1X -
024
r = 0.780 v
E
2.
/
v ~
v
v
~V
10 Zinc
,~,
V
V
V
. . . . .
20 30 sulphate turbidity units
40
50
Fig. 5. The correlation between the serum IgM concentration arid the turbidity developed by the zinc sulphate turbidity reaction. Clin. Ckim. Aaa, 27 (I97 o) I55-I63
IMMUNE GLOBULIN IN NEONATAL CALF SERUM
I6I
50y ==1 0 7 x - 2.17 r =, 0.96,2 40.
/
E3O-
v
E OI
';' 20. (9 0 i,,,,11
10"
0-~
10
50
Zinc sulpl~ote t u r b i d i t y units
Fig. 6. The correlation between the serum I g G + igM concentration and the turbidity developed by the zinc sulphate turbidity reaction.
7. Correlation of the zinc sulphate turbidity test with serum immune globulin concentrations. Determinations of IgG and IgM concentrations were made without the knowledge of the zinc sulphate turbidity of a series of 53 neonatal calf sera. The immune globulin concentrations were determined by a quantitative immunodiffusion technique 8. The correlations existing between the zinc sulphate turbidity test and the concentration of IgG and IgM and IgG plus lgM are shown in Figs. 4, 5 and 6. These results show that there is a highly significant correlation between the specific immune globulin fractions when measured both singly and in combination. The turbidity reaction would appear to be influenced mainly by the concentration of IgG and to a lesser extent by the IgM concentration. DISCUSSION
In the investigation of the effects of time and temperature, several points of importance arise when considering the development of a stand~xd technique for the performance of this turbidity test. At a given temperature, the rate of development of turbidity would appear to be fairly uniform during the :2o-min period studied. The observation of Aschaffenburg that the reaction was not quite complete after I h was confirmed. The study of the effect of temperature indicates that the reaction is very sensitive to temperatures above 2o °. This finding is in agreement with that of Niemann-Sorensen el al. ° who also noted that the reaction was very sensitive to temperatures over 2o °. In the light of these findings, it is suggested that in the performance of the zinc sulphate turbidity test, the temperature of the reagents should be 2o ° and that the intensity of the reaction should be read 60 rain after the start. The depression of turbidity which was found by alteration of the pH of the zinc sulphate reagent suggests that unbuffered reagent should be used. Serious changes Clin. Chim. Acta, 27 (x97o) 155-163
x62
,~CEWAN et al.
in the pH of the reagent were found to occur if the solution was allowed to absorb carbon dioxide t°. In order to overcome this source of error, Discombe '° kept the zinc sulphate reagent in an automatic burette protected by a soda lime tube, and Reinhold 7 recommended that the distilled water used for making up the reagent should be boiled for Io min in order to drive off dissolved carbon dioxide. Both of these precautions have been adopted in the preparation of the zinc sulphate reagent. The effect of adding albumin to the turbidity reaction of neonatal calf serum has been found to be negligible at concentrations below 3o mg per ml. By increasing the amount of albumin added beyond the range described, a progressive fall in the turbidity reaction has been detected. The depressant effect of added albumin on the turbidity reaction in human serum has been described 3,1~,~, at concentrations in excess of lO-15 mg per ml. The concentration of albumin in the serum ef newborn colostrumdeprived calves has been measured by several workers13-~L It would appear, therefore, that the normal variations encountered in the serum albumin concentration are unlikely to affect the turbidity reaction. The investigation into the addition of quantities of bovine gamma globulin to hypogammaglobulinaemic calf serum has revealed interesting results. In this present work a deviation from the linear relationship between zinc sulphate turbidity and the amount of added gamma globulin was found at concentrations greater than 30 mg per ml. In contradistinction, Adner ~a noted that at low gamma globulin concentrations, the relationship was not linear. However, only one estimation was made at a concentration greater than 30 mg per ml, and ~q the relationship at higher concentrations is still undetermined for human serum. It has generally been accepted by all workers who have used the zinc sulphate turbidity test that it should be performed on serum rather than plasma. The reasons for this are several. The effect of whatever anticoagulant is used on the turbidity reaction must be ascertained. Heparin is reported to cause a decrease in zinc sulphate turbidity ~6. Further, reaction of some anticoagulants is not long, and this may allow tile development of fibre clots if samples are stored for some time. Finally, the effect of variations in the fibrinogen concentration may cause different turbidity readings from samples with the same gamma globulin concentration. In this study, a difference in turbidity between serum and plasma is most marked in the samples which have lowest turbidities, but becomes less so in samples in the higher ranges. Therefore, the use of plasma in place of serum is not recommended. In the correlation which has been shown between the zinc sulphate turbidity reaction and the concentration of IgG and IgM, it should be stressed that the closest correlation exists with the total immune globulin concentration rather than with that of a specific immune globulin class. The reason why it appears that there is a correlation with the individual classes is that it so happens that in neonatal calf serum samples there is a direct relationship between lgG and IgM concentrations tT. ACKNOWLEDGEMENTS
This work was financed by a grant from the Agricultural Research Council.
Clin. Chim. Aaa, 27 (I97 o) x55-I63
IMMUNE GLOBULIN IN NEONATAL CALF SEI~UM
16 3
REFERENCES C. C. GAY, N. ANDERSON, E. XV. FISHER AND A. D. McEwA,~, Vet. Rec., 77 (I965) 148R. ASCHAFFE,~BtmG, Brit. J. Nutr., 3 (I949) 200. H. G. KtJNKEL, Proc. Soc. Exptl. Biol. Med., 66 (I947) 217. R. E. SHANK A,~D C. L. HOAGLA~D, J. Biol. Chem., i62 (I946) 133. G. DmCOMBE, R. F. Jo,~Es AND D. P. WI.~STa,~LEV, J. Clin. Pathol., 7 (1954) io6. V. L. YO~AN AXD J. G. REINHOLD, Clin. Chem., 3 (I957) 685J. G. REInhOLD, Advan. Clin. Chem., 3 (t96o) 83. W. J. Pv-~HALE AND G. CrmlsTm, Res. Vet Sci., in press (t969). A. NIEMANN-SonENSEN, S. P. KONGGAARD .~ND V. Ki~trSE, LandoKon Aarb., (1966) 347. G. DISCOMBZ, Lancet, i (t959) 6t9. J. DE LA HUERGA A.~D H. POPPER, J. Lab. Clin. Med., 35 (x95 o) 459P. L. ADNER, Acta Soc. Med. Upsalien., 62, Suppl. 6 (t957). H. E. ROBERTS, A. N. WORDEN AND E. T. REES-EVANS, J. Comp. Pathol. Therap., 64 (1954) 283. 14 A. E. PIERCE, J. Hyg., 53 (1955) 247. r ¢ B. TENNANT, D. HARROLD, M. REL~A GUERRA AND R. C. LABEN, Am. J. I'et. Res., 30 (19o9)
I 2 3 4 5 6 7 8 9 to Ix t2 x3
345. 16 Z. HORN AND E. KOVACKS, Acta ~lled. Scand., 164 (I959) 143. 17 G. G. B. KLAUS, A. BENNETT AND E. W. Jo,~Es, Immunology, 16 (t969) 293.
Clin. Chim. Acta, 2 7 (t97 o) 155-163
155
A T U R B I D I T Y TEST FOR T H E ESTIMATION OF IMMUNE GLOBULIN LEVELS IN NEONATAL CALF SERUM
A. D. McE~,VAN, E. W. F I S H E R , I. E. SELMAN a x o W. J. P E N H A L E *
Department of Veterinary Medicine, University Veterinary Hospital, Bearsden Road, Bearsden, Glasgow ( U.K.) * Department of Veterinary Pathology, Royal (Die,{) School of Veterinary Studies, Edinburgh (U.K.) (Received August 5, t969)
SUMMARY
A simple and rapid method for the determination of immune globulin concentration in neonatal calf serum is described. The method uses a turbidity reaction and investigations into some factors which influence this reaction and into the correlation between the reaction and specific inmmne globulin concentrations have been performed.
INTRODUCTION
Calves are hvoog!obulinaewdc at birth and are dependent i:pon the absorption of immune globulins from colostrum for passive immunity to neonatzl diseases. Since the quantity of immune globulin absorbed has been shown to bear a relationship with susceptibility to neonatal disease t, the estimation of serum imnmne globulin concentratiot:s in early life is of clinical importance. For practical purposes, the technique of estimation should be cheap, rapid and simple, so that the immune globulin concentration o, large numbers may be determined as easily as that of the individual calf. A method of detecting whether or not colostrum has been fed to newborn ca'. es of a serum turhiditv was described by Aschaffenburg'. This involved *lie Inoulncatlon ~"" " test originally described and designed by Kunkel s to detect alterations in the serum gamma globulin concentrations of humans suffering from liver diseases. The purpose of this paper is to describe some factors which affect the turbidity reaction when performed under the conditions described by Aschaffenburg, and to correlate the intensity of the reaction when performed under standard conditions with the concentration of specific immune globulin fractions. MATERIALS AND METHODS
Serum. Serum was obtained from bull calves less than one ~ 'eek of age, largely of the Ayrshire breed, which had been bought in local markets. Clin. Chim. Acta, 27 (I97 o) i 5 5 - I 6 3
I56
MCEWAN et al.
Zinc sulphate solution. A solution of zinc sulphate (208 mg/l ZnSO4"7 H20) was prepared in a volumetric flask using distilled water which had been boiled for lO-15 rain in order to remove dissolved carbon dioxide. This solution was then kept in an aspirator bottle and was further protected against the uptake of carbon dioxide by the insertion of a soda lime tube into the stopper. The aspirator bottle was connected by means of polythene tubing to an automatically dispensing pipette* set to deliver 6 ml. An identical arrangement was used toldeliver a similar volume of distilled water for control samples. Measurement of turbidity. The test was performed in sets of matched Io-ml E E L colorimeter tubes. For each serum sample tested, a "control" tube containing 6 ml of distilled water and a "test" tube containing 6 ml of zinc sulphate solution were measured out. To each of these, o.I ml of serum was delivered from an Autozero high precision pipette**. The solution was then gently shaken in order to ensure mixing of reagents, and the tubes were allowed to stand for 6o min at room temperature, which was 20 °. After this time had elapsed, the tubes were shaken again to ensure an even redistribution of the precipitate and inserted into a photoelectric absorptiometer***, which had been previously zeroed using a colorimeter tube containing distilled water. A blue-green filter (Ilford No. 623) was used in the absorption in place of the red one (Ilford No. 6o8) recommended by both Kunkel a and Aschaffenburg 2 as this provided greater sensitivity to measurement of turbidity. The increased sensitivity which this filter exhibits to haemoglobin and hence to the effects of haemolysis, is compensated by the use of control samples. The turbidity reading was found by subtracting the reading obtained from the control tube to that of the test tube and by multiplying the difference by a factor of IO. In order to eliminate variations in the sensitivity of the absorptiometer and to facilitate comparison of results obtained in this laboratory with results possibly obtained in the future in other laboratories, the barium sulphate standard recommended by Shank and Hoagland 4 for use in the thymol turbidity test was adopted. Three millilitres of a barium chloride solution containing 1.15 g barium chloride (BaCI2.2 H~O) per IOO ml were made up to IOO ml in a volumetric flask with o.2 N sulphuric acid. When measured under the same conditions as used in the zinc sulphate turbidity test, this solution gave a turbidity reading of 20 units. Investigation of factors influencing the zinc sulphate turbidity reaction r. Time and temperature. To study the effects of time and temperature on the turbidity reaction, serum samples were obtained from ten neonatal calves. The turbidity test was performed as described above except that the turbidities were measured 15, 3o, 6o and 12o min after the addition of the serum sample to the zinc sulphate solution. This procedure was performed using the same sera at the following temperatures: 6 °, zo °, 25 °, 3 l°, 37 °. The results of this experiment are shown in Table I, in which tile average turbidity of the ten samples is shown in relation to time and temperature. From this, it can be seen that the reaction continues to develop throughout the 12o-nlill period and is influenced greatly by temperature. 2. pH. In the original description of the zinc sulphate turbidity test, Kunkel * W.G. Flaig & Sons Ltd., Excelo Works, Margate Road, Broadstairs, Kent. ** H . J . Elliot Ltd., E-Mil Works, Treforest, Glamorgan. *** Evans Electroselenium Ltd., St. Andrew's Works, Halstead, Essex. Clin. Chim. Acta, 27 (x97o) x55-t63
IMMUNE GLOBULIN IN NEONATAL CALF SERUM TABLE
I~7
I
THE EFFECT OF TIME AND TEMPERATURE ZINC SULPHATE TURBIDITY OF IO SERA
Temp. °C 6 20 25 3I 37
ON
THE
MEAN
Time (rain) "5 30
60
z2o
I2.8 i4. 7 I6.8 30.3 41.6
15.8 18.6 24. 9 38.4 44.0
16.6 zI.2 27.6 4°.6 46.7
15.x i7. 4 22.o 34.6 42.8
maintained tile pH of the reagent at 7.5 by use of a weak barbiturate buffer. Subsequent workers 6,' have continued this practice, although Reinhold 7 had cause to alter the relative concentrations of the constituents of the buffer as the original concent:ations provided an alkaline pH in his laboratory. Aschaffenburg ~ found it unnecessary to use buffer and used only distilled water. Where necessary, the pH was adjusted to neutrality by the addition of dilute acid or alkali. The following experiment was performed to find out what effect pH had on the zinc sulphate turbidity reaction. Twenty calf sera with turbidity readings between o and 4° were selected. These sera were then tested using (x) unbuffered zinc sulphate solution, (z) zinc sulphate solution to which had been added barbiturate buffer in the proportions described by KunkeP, i.e. 28o mg barbituric acid and 2xo mg sodium barbitone per litre, (3) a zinc sulphate solution to which had been added barbiturate buffer in the proportions described by Reinhold 7, i.e. 3o2 mg barbituric acid and xoo mg sodium barbitone per litre. The measured pH of these solutions were respectively, 4.7, 7.53 and 7.6. The turbidity test was then performed under the conditions described previously. When compared with unbuffered reagents, it was found that both buffers depressed the development of the turbidity reaction. The depression produced bv the buffer originally described by Kunkel 3 was so great that only one sample gave a reading of greater than 2.5 (Fig. x). 3. Albumin. In human patients with hver disease, KunkeP produced a slight fall in the intensity of the turbidity reaction by administenng intravenously large amounts of human albumin. He also demonstrated that by adding albumin to positively reading serum taken from patients suffering from hepatitis that there was a decrease in the amount of globulin precipitated. The method used to determine the influence of albumin on the zinc sulphate reaction was as follows. A range of concentrations of bovine albumin solution was prepared by dissolving crystallized bovine albumin in distilled water and making taLt~, w a s serial dilutions. A more concentrated solution of zinc sulphate, z5o mg per ~='-^ prepared in order to allow for the dilution which occurs when the albumin solution is added. Five serum samples with turbidity readings ranging between 3o and 50 were selected and the test performed in the following manner. For each concentration TABLE
II
THE EFFECT
ON THE TU~°TDITY
A d d e d a l b u m i n (mg/mi) Average turbidity Difference
REACTION
b 43-7 o
OF THE ADDITION
2.5 42.~ --o.8
5.0 43 .2 --o.5
OF ALBUMIN TO 5 SERA
io.o 43.8 +o.r
20.0 43.4 --o.3
30.0 -~4.° +o.3
40.0 42"5 -- 1.2
Clin. Chim. Acta, 27 (x97 o) I 5 5 - 1 6 3
McEWA~ a al.
x58 40, o.-o ZnS04 •
~,--.xZnS04+Reinhold buffer' /
35 ~"~ZnS04+Kunkel buffer
30.
~)0"
~
P~ J
25'
E~40. o
E
.__c
~20-
30. .Q
O
I-
E E
-b ~5-
x 10' m
10 5 .,
o
g
Ib
~g
:/o
Calf no.
,
,
,
. . . . . .
,
.
.
10 20 30 40 Zinc sulphate turbidity units
.
.
,
50
Fig. t. The effect of pH on the zinc sulphate turbidity reaction. Fig. e. The effect on the sulphate turbidity reaction of adding a bovine gamma globulin preparation to hypogammaglobulinaemic calf serum.
of albumin solution, five colorimeter tubes were set up. Into each was added 5 ml of the zinc sulphate solution, I ml of the appropriate albumin solution, and o.I ml of each serum sample. Thereafter, the performance of the test was as described previously. The results of this experiment are ~hown in Table II. From the results obtained in the present work, it would appear that there is very little depression of turbidity below concentrations of 3o mg of albumin per ml. 4. Bovine gamma globulin. In view of the fact that the serum of newborn calves is markedly hypogammaglobulinaemic until immune globulins have been absorbed from colostrum, the addition of gamma globulin to such sera would allow a method of quantitating the zinc sulphate turbidity reaction through the range of levels encountered naturally. A preparation of bovine gamma globulin is available commercially* which has been prepared by the Cohn fractionation technique and is the equivalent of fraction I I. This has been used in the following experiment. By means of serial dilutions, solutions of bovine gamma globulin of concentrations ranging from 1.5 to 30 mg per ml of serum were set up. Serum samples were obtained from newly born co!ostrum-deprived calves, split into two groups, each containing five samples, and submitted to the zinc sulphate turbidity test, using the standard technique. The turbidity test on the samples containing bovine gamma globulin was performed as follows. Each concentration of bovine gamma globulin in a series of dilutions was tested against five of the serum samples. Therefore, for each * Armour Pharmaceutical Company Ltd., Eastbourne, England. Clin. Chim. Acta, 27 (x97o) x55-x63
159
IMMUNE GLOBULIN IN NEONATAL CALF SERUM
concentration, five co!ofimeter tubes were set up, and to each was added 5 ml zinc sulphate solution. This solution was made up at a strength of 250 mg zinc sulphate per litre in order to allow for the dilution which occurs when the globulin solutions were added. To each part of the five tubes, I ml of the appropriate solution was added. Finally, o.I ml of the serum was taken from each serum sample and added to one tube, Thereafter, the performance of the test was as described in the MATERIALS AND METHODS section. The average turbidity obtained from each concentration of bovine gamma globulin used was calculated, and the results are shown in Fig. 2. It would appear that the relationship existing between the turbidity devel.)ped by the -~inc sulphate test and the concentration of bovine gamma globulin added to the serum is linear only up to levels of about 30 mg bovine gamma globulin per ml. Thereafter, the turbidity would appear to increase in an exponential manner. 5. The comparative turbidities developed by serum and plasma. Serum and plasma 40-
35-
30"
.=_ 2510 0 L
.~ 20.
/i/
g i,-
15-
/
d/ /
10-
x~x
/
/
Plasma
(>-----<)Serum
--_
I
s
Io
15
20
Calf number
Fig. 3. A c o m p a r i s o n of t h e zinc s u l p h a t e t u r b i d i t i e s developed b y p l a s m a a n d serum. TABLE THE
II I
REPRODUCIBILITY
PERFORMED
ON 6
Serum
Mean
O F THI~ Z I N C S U L P H A T E
DIFFERENT
TURBIDITY" RE~tCTION
SERA
Standard deviation
I
9.0
~
2 3 4 5 6
x9.o 25. I 33.0 3 8.t 4o.9
± ~: ± 5: 4-
i.l 0.8 0.9 x.4 I.t 0.8
Coeff. of variahon
(%)
i2.2 4.2 3 .6 4 .2 2.9 1.9 Clin. Chim. Acta, 27 (x97 o) I 5 5 - I 6 3
x6o
MCEWAN et
al.
samples were taken from a group of 2o neonatal calves. Heparin was used as the anticoagulant for plasma samples. These samples were then submitted to the zinc sulphate turbidity test, and the results obtained are shown in Fig. 3. It would appear that plasma gives a greater turbidity reaction than serum. 6. The reproducibility of the zinc sulphate turbidity reaction. To ascertain the reproducibility of the method, 5 determinations were performed on 6 different serum samples. The means, standard deviations and coefficients of variation are shown in Table I I I. From these results it can be seen that the reproducibility of this method of measuring the turbidity reaction is high. 50y = 0.97x - 1.95 r = 0.959
v
40-
/
/
30-
v sv ~ v ~ v v Vq
Ill 2o-
~'~v v
v
./
o
.v
,1~
0
Z"
.
.
.
.
.
.
.
20 30 40 sulphote turbidity units
10 Zinc
50
Fig. 4. The correlation between the serum IgG concentration and the turbidity developed by the zinc sulphate turbidity reaction.
y=
0.1X -
024
r = 0.780 v
E
2.
/
v ~
v
v
~V
10 Zinc
,~,
V
V
V
. . . . .
20 30 sulphate turbidity units
40
50
Fig. 5. The correlation between the serum IgM concentration arid the turbidity developed by the zinc sulphate turbidity reaction. Clin. Ckim. Aaa, 27 (I97 o) I55-I63
IMMUNE GLOBULIN IN NEONATAL CALF SERUM
I6I
50y ==1 0 7 x - 2.17 r =, 0.96,2 40.
/
E3O-
v
E OI
';' 20. (9 0 i,,,,11
10"
0-~
10
50
Zinc sulpl~ote t u r b i d i t y units
Fig. 6. The correlation between the serum I g G + igM concentration and the turbidity developed by the zinc sulphate turbidity reaction.
7. Correlation of the zinc sulphate turbidity test with serum immune globulin concentrations. Determinations of IgG and IgM concentrations were made without the knowledge of the zinc sulphate turbidity of a series of 53 neonatal calf sera. The immune globulin concentrations were determined by a quantitative immunodiffusion technique 8. The correlations existing between the zinc sulphate turbidity test and the concentration of IgG and IgM and IgG plus lgM are shown in Figs. 4, 5 and 6. These results show that there is a highly significant correlation between the specific immune globulin fractions when measured both singly and in combination. The turbidity reaction would appear to be influenced mainly by the concentration of IgG and to a lesser extent by the IgM concentration. DISCUSSION
In the investigation of the effects of time and temperature, several points of importance arise when considering the development of a stand~xd technique for the performance of this turbidity test. At a given temperature, the rate of development of turbidity would appear to be fairly uniform during the :2o-min period studied. The observation of Aschaffenburg that the reaction was not quite complete after I h was confirmed. The study of the effect of temperature indicates that the reaction is very sensitive to temperatures above 2o °. This finding is in agreement with that of Niemann-Sorensen el al. ° who also noted that the reaction was very sensitive to temperatures over 2o °. In the light of these findings, it is suggested that in the performance of the zinc sulphate turbidity test, the temperature of the reagents should be 2o ° and that the intensity of the reaction should be read 60 rain after the start. The depression of turbidity which was found by alteration of the pH of the zinc sulphate reagent suggests that unbuffered reagent should be used. Serious changes Clin. Chim. Acta, 27 (x97o) 155-163
x62
,~CEWAN et al.
in the pH of the reagent were found to occur if the solution was allowed to absorb carbon dioxide t°. In order to overcome this source of error, Discombe '° kept the zinc sulphate reagent in an automatic burette protected by a soda lime tube, and Reinhold 7 recommended that the distilled water used for making up the reagent should be boiled for Io min in order to drive off dissolved carbon dioxide. Both of these precautions have been adopted in the preparation of the zinc sulphate reagent. The effect of adding albumin to the turbidity reaction of neonatal calf serum has been found to be negligible at concentrations below 3o mg per ml. By increasing the amount of albumin added beyond the range described, a progressive fall in the turbidity reaction has been detected. The depressant effect of added albumin on the turbidity reaction in human serum has been described 3,1~,~, at concentrations in excess of lO-15 mg per ml. The concentration of albumin in the serum ef newborn colostrumdeprived calves has been measured by several workers13-~L It would appear, therefore, that the normal variations encountered in the serum albumin concentration are unlikely to affect the turbidity reaction. The investigation into the addition of quantities of bovine gamma globulin to hypogammaglobulinaemic calf serum has revealed interesting results. In this present work a deviation from the linear relationship between zinc sulphate turbidity and the amount of added gamma globulin was found at concentrations greater than 30 mg per ml. In contradistinction, Adner ~a noted that at low gamma globulin concentrations, the relationship was not linear. However, only one estimation was made at a concentration greater than 30 mg per ml, and ~q the relationship at higher concentrations is still undetermined for human serum. It has generally been accepted by all workers who have used the zinc sulphate turbidity test that it should be performed on serum rather than plasma. The reasons for this are several. The effect of whatever anticoagulant is used on the turbidity reaction must be ascertained. Heparin is reported to cause a decrease in zinc sulphate turbidity ~6. Further, reaction of some anticoagulants is not long, and this may allow tile development of fibre clots if samples are stored for some time. Finally, the effect of variations in the fibrinogen concentration may cause different turbidity readings from samples with the same gamma globulin concentration. In this study, a difference in turbidity between serum and plasma is most marked in the samples which have lowest turbidities, but becomes less so in samples in the higher ranges. Therefore, the use of plasma in place of serum is not recommended. In the correlation which has been shown between the zinc sulphate turbidity reaction and the concentration of IgG and IgM, it should be stressed that the closest correlation exists with the total immune globulin concentration rather than with that of a specific immune globulin class. The reason why it appears that there is a correlation with the individual classes is that it so happens that in neonatal calf serum samples there is a direct relationship between lgG and IgM concentrations tT. ACKNOWLEDGEMENTS
This work was financed by a grant from the Agricultural Research Council.
Clin. Chim. Aaa, 27 (I97 o) x55-I63
IMMUNE GLOBULIN IN NEONATAL CALF SEI~UM
16 3
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I 2 3 4 5 6 7 8 9 to Ix t2 x3
345. 16 Z. HORN AND E. KOVACKS, Acta ~lled. Scand., 164 (I959) 143. 17 G. G. B. KLAUS, A. BENNETT AND E. W. Jo,~Es, Immunology, 16 (t969) 293.
Clin. Chim. Acta, 2 7 (t97 o) 155-163