Single Radial Immunodiffusion Analysis for Quantitation of Colostral Immunoglobulin Concentration1

Single Radial Immunodiffusion Analysisfor Quantitation of Colostral Immunoglobulin Concentration' W. A. FLEENOR and G. H. STOTT Department of Animal Sciences The University of Arizona Tucson 85721 ABSTRACT

have been standardized to produce uniform results. However, Ouchterlony (21) reported extraneous substances in the reagent layer may affect diffusion of antigen. He also pointed out that increased saline or protein content of the medium has enhanced diffusion. No attempt has been made to determine whether properties of colostrum as a solvent differ significantly from either saline or serum that commonly has been used to dissolve reference proteins during sRID standardization. A difference in diffusion potential due to solvent may cause incorrect ring diameters and subsequently incorrect concentrations. The purpose of this experiment was to determine the best procedure for colostral preparation in sRID analysis.

Relative accuracy of the single radial immunodiffusion technique to measure immunoglobulin concentration of colostral preparations (whey, whole, or fat-free) has been assessed. Fresh colostrum samples were analyzed for major constituents. Gammaglobulin as a standard was compared to total immunoglobulin concentration derived from single radial immundiffusion analysis of colostral preparations with no differences except between standard and whey. Differences were in part from either enhancement or interference of immunoglobulin diffusion by colostral constituents. Removal of casein and fat during whey preparation caused a concentrating effect upon immunoglobulin constituents resulting in exaggerated precipitin rings. Whey has produced unreliable results; therefore, whole colostrum is recommended for single radial immunodiffusion analysis.

M A T E R I A L S A N D METHODS Collection of Colostral Samples

INTRODUCTION

Single radial immunodiffusion (sRID) has been used extensively for quantitation of immunoglobulins (Ig) in blood serum (3, 24, 31) and to evaluate Ig in colostrum (15, 18, 22). Various methods have been described for preparation of colostrum for sRID analysis. Table 1 lists various techniques of cotostral preparation and lg obtained by several researchers. Differences in lg are large and may be attributed to several factors, one of which may be technique of colostral preparation. Various studies have determined additional factors that affect diffusion-in-gel methods (7, 21) and sRID assays (2, 8, 11, 16). Most of these factors

Received May 8, 1980. I Journal paper 3324 of the Arizona Agricultural Experiment Station. 1981 J Dairy Sci 64:740-747

740

Colostrum samples were collected from nursed and unnursed Holstein-Friesian cows within 24 h postpartum. Specific gravity was measured for each sample immediately after collection by a hydrometer calibrated in increments of .002 with a range of 1.000 to 1.200. Several readings were obtained for each colostrum and averaged. Each sample then was aliquoted immediately into separate analysis tubes and stored at 4°C. Analysis was on samples within 1 wk of collection. Quantitative Analysis

Total solids were measured by the method outlined in AOAC (1). The sample (2.5 to 3.0 g whole colostrum) first was heated on a steambath for 10 to 15 rain, oven-dried at 100°C + 2°C for 3 h, cooled to room temperature in a desiccator, and weighed to the nearest .1 mg. Lactose was determined by the method of Teles, et al. (32). Colormetric determination was based upon the combined action of phenol, sodium hydroxide, picric acid, and sodium bisulfite with lactose.

T A B L E 1. Comparison o f bovine colostral preparation techniques and resulting i m m u n o g l o b u l i n concentrations. Researcher

Colostral preparation technique

Klaus et al. (12)

1. 2. 3. 4. 1. 2. 1. 2. 3.

IgG 1

IgG 2

lgM

IgA

Total

(mg/ml)

Penhale and Christie (23) Mach and Pahud (15)

Butler (4) Brandon, Watson, and Lascelles (3)

Penhale et al. (25) MeGuire et al.a (18) ~7

Porter (26)

Wilson et al. (33)

centrifugation fat removal casein precipitated with acid reneutralization high speed centrifugation fat removal centrifugation fat removal casein precipitated with acid or rennin n o t indicated 1. centrifugation 2. fat removal 3. casein precipitated with rennet 1. high speed centrifugation 2. fat removal 1. centrifugation 2. fat removal 1. casein precipitated using ultra-centrifugation 2. fat removal 1. casein precipitated using ultra-centrifugation 2. fat removal

43.3 -+ 14.0

3.2 + 1.7

NA b

46.5

34,1 -+ 2.1

4.9 -+ .4

NA

39.0 85.2

75.0

1.9

4.9

4.4

33.8 79.2 ± 6.5

3.6 5.2 + .8

NA 10.7 + 1.3

2.0 6.5 + 1.1

39.4 102.0

~

O 'n C3 O v" "J t~"

36.0

5.1

139.5 -+ 53.7

6.9

48.0

8.1 +- 3.3

NA

147.6

Z O

103.3

O ¢¢

88.2

2.5

9.2

3.4

34.0 ± 11.4

3.5 +- 1.2

3.9 -+ 1.1

1.5 + .6

r' 42.9

aBeef cattle. bNot available.

4~

742

FLEENOR AND STOTT

Fat content was measured gravimetricaUy by extraction of fat from an ammoniacal alcohol solution of colostrum with diethyl ether and light petroleum ether as outlined in AOAC (1). The solvents were evaporated, and residue was weighed to the nearest. 1 mg. Total nitrogen, noncasein nitrogen, nonprotein nitrogen, and globulin nitrogen were measured by Roland's method (28, 29). Total nitrogen, globulin, and nonprotein nitrogen were determined directly, and casein, total protein, and albumin were obtained by difference. The macro-Kjeldahl procedure was utilized rather than the micro-Kjeldahl procedure recommended by Roland (29). We employed purified bovine gammaglobulin (19) to assess accuracy of magnesium sulfate precipitation and Kjeldahl procedures as outlined by Roland (29). Bovine gammaglobulin was added to a colostrum sample in .5 g increments beginning at 6.853 g/100 g colostrum and ending at a concentration of 10.785 g/100 g. Colostrum samples then were analyzed according to the procedure outlined above and results compared to theoretical gammaglobulin. Percent error increased from a minimum of 2.6 to a maximum of 9.3 at the greatest concentration. Further trials demonstrated that during initial acid precipitation of casein, small amounts of gammaglobulin were coprecipitated. The amount of gammaglobulin precipitated during acid precipitation of casein followed approximately the same percent error pattern for the overall gammaglobulin assay procedure. Many of the colostrum samples assayed were less than the starting concentration, and thus, error due to acid precipitation of gammaglobulin was considered negligible. Whole colostrum, fat-free colostrum, and whey were assayed for IgG and IgM by the radial immunodiffusion procedure of Fayhey and McKelvey (6). This technique has been used extensively in past research (10, 12, 22, 31, 33). Reasons for its popularity are: rapid results( and conservation of antisera. We felt that reported advantages of the Mancini et al. (16) procedure are obviated by additional incubation time required, distortion of precipitin rings with increased time, and increased antisera requirements. That the accuracy of the Fahey technique at times may be semiquantitative is acknowledged. Analysis for IgA utilized microdiffusion /

Journal of Dairy Science Vol. 64, No. 5, 1981

discs as described by Masseyeff and Zisswiller (17). Antisera to bovine lgG, IgM, and IgA were prepared in rabbits by methods similar to those described by Campbell et al. (5). Antisera were rendered monospecific by gluteraldehyde insolubilization immunoadsorption techniques. The specificity of all antisera was determined by Ouchterlony plates and immunoelectrophoresis. Standards for sRID analysis were quantitated by Lowry protein determinations (14) and verified by quantitation kits available from Miles Laboratories (19). Purified bovine gammaglobulin was the primary protein standard for Lowry protein determination. Colostral Preparation Procedures

Whey was prepared by incubating 12.5 mg rennin with 5 ml whole colostrum for 30 rain at 37°C and centrifuging at 8000 x g at 5°C for 1 h. The clear supernatant then was separated carefully from the casein-fat layer and stored at -10°C. Fat-free colostrum was prepared by centrifugation at 1650 x g at 5vC for 10 rain. The tubes then were chilled at - 1 0 ° C for 10 min to solidify the fat rim. The fat free supernatant was pipetted carefully from underneath the fat layer and stored at - 10°C. Statistical Analysis

Determinations along with a control were duplicate for each analysis with a maximum of 5% error allowed. Data were analyzed in part by a statistical computer package (20). Means were compared by the least significant difference test (LSD) described by Steel and Torrie (30). RESULTS

Major constituents of 14 colostrums are tabulated in Table 2. Single radial immunodiffusion analysis for IgG, IgM, and IgA was on whey, whole and fat-free colostrum for each respective sample, and results are compiled in Table 3. Total Ig was computed for three colostral preparations (whey, whole, and fat-free) of each sample and compared to standard magnesium sulfate gammaglobulin (standard globulin) for that sample. Gammaglobulin has been accepted to represent total Ig concentration in colostrum (13, 27). The percent errors for the three colostral preparations of each sample in Table 3 then were

TABLE 2. Quantitative analysis of bovine colostrum.

Sample number

Specific gravity

Total solids

Total N Lactose

Fat

(I)

NoncaseinN (II)

Nonprotein N (I11)

Globulin (IV)

Casein (1-II)

Albumin (lI-lllqV)

Total protein (I-III)

Z ,.-}

(g/lO0 g secretion)

e.

P-

o,,

O0

r,.

g

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1.046 1.050 1.062 1.046 1.058 1.050 1.052 1.032 1.04.4 1.036 1.045 1.066 1.061 1.072

12.796 15.377 24.811 15.215 21.449 17.601 19.024 12.459 13.554 12.880 17.988 21.711 20.925 36.920

3.934 2.793 2.379 2.933 3.577 2.748 2.834 3.896 3.852 4.034 3.129 2.520 2.754 1.343

1.245 .707 3.653 .392 2.403 .903 1.676 1.903 .213 1.585 2.765 .841 1.017 8.395

6.626 10.243 17.952 11.139 14.698 13.135 13.387 5.996 8.661 6.594 10.880 16.597 15.584 21.825

3.113 5.981 13.416 7.801 6.596 9.057 7.985 2.323 3.864 3.462 5.004 10.187 10.712 16.708

.402 .354 .342 .383 .366 .365 .351 .271 .269 .362 .326 .318 .387 .420

1.876 4.833 11.532 5.890 4.413 6.819 5.561 .949 2.487 2.156 3.258 7.703 8.288 13.658

3.543 4.262 4.536 3.338 8.102 4.078 5.402 3.673 4.797 3.132 5.876 6.410 4.872 5.117

.835 . .794 1.542 1.527 1.816 1.873 2.073 1.103 1.108 .945 1.420 2.166 2.037 2.630

6.224 9.888 17.679 10.756 14.332 12.770 13.037 5.725 8.392 6.232 10.554 16.279 15.198 21.404

.X % CV

1.051 1.074

18.765 34.663

3.052 24.625

1.978 105.090

12.380 37.807

7.586 54.825

.351 12.503

5.673 65.190

4.796 28.197

1.562 35.920

12.033 38.804

Z Q fb 0 tQ f/3 ,--I t-

Z O ¢3 tO t-

.< o

2: o ..q

4~ 4~

t~

a

T A B L E 3. Radial i m m u n o d i f f u s i o n analysis of whey, whole and fat-free colostrum.

o<

Whole colostrum Sample number

Standard globulin a

IgG

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1.962 5.075 12.247 6.161 4.669 7,160 5.851 .980 2.596 2.233 3.404 8.212 8,794 14.642

1.65 2.80 7.60 5.00 2.12 4,30 3.10 .77 1.60 1.50 1.60 3.60 6.80 8.80

~, % CV

5.999 66.10

Whey

IgA

Total lg

% Error b

IgG

.18 .45 .78 .75 .39 .41 .45 .19 .29 .28 .35 .54 .45 1.50

.15 1.08 2.80 1.60 .46 1.48 1.28 .12 .32 .40 .40 .42 .92 2.20

1.98 4.33 11.18 7.25 2.97 6.19 4.83 1.08 2.21 2.18 2.35 4.56 8.17 12.50

.65 -14.67 -8.71 19.29 -36.29 -13.54 -17.44 10.24 -14.88 -2.61 -30.97 -44.47 -7.09 -14.63

1.90 4.65 12.00 5.05 5.30 6.00 6.60 1.00 2.35 2.05 2.90 8.00 10.50 13.60

.39 1.14 1.53 ,86 1.32 .86 1.14 .38 .59 .55 1.16 1.80 1,08 2.80

.19 1.48 5.20 2.08 .64 1.60 1.36 ,15 .48 .48 .46 .92 1.40 3.50

3.66 .50 69.03 67.69

.97 83.83

5.13 69.04

-12.5 136,89

5.85 67.32

1.11 57.46

1.42 98,84

IgM

IgM

IgA

Fat-free colostrum Total Ig

% Error

IgG

lgM

lgA

Total lg

% Error

2.48 7.27 18.73 7.99 7.26 8.46 9.10 1.53 3,42 3,08 4,52 10.72 12.98 19.90

26.40 43.26 52.93 29.68 55.49 18.16 55,54 55.66 31.73 37.92 32.77 30.54 47.60 35.91

1.45 3.05 8.20 4.35 2.25 4.70 3.30 .78 1.80 1.75 2.00 4.40 6.80 8.80

.20 .75 1.00 .75 .50 .50 .50 .21 .32 .33 .46 .54 .50 1.50

.15 1.08 2.80 1.92 .46 1.48 1.08 .12 .32 .40 ,35 .42 .98 2.40

1.81 4.88 12.00 7.02 3.21 6.68 4.88 1.11 2,44 2.48 2.81 5.36 8.28 12.70

-8.00 -3.84 -2.02 13.94 -31.25 -6.70 -16.59 13.30 -6.21 10,83 -17.46 -34.73 -5.84 -13.26

8.39 67.68

39.5 31.05

3.83 66.40

.58 59.72

1.00 86,45

5.40 66.95

-7.7 190.41

(g/lO0 ml secretion)

a s t a n d a r d globulin concentration was converted from m a s s / m a s s to m a s s / v o l u m e by using colostrum specific gravity and globulin from Table 2. bpercent error was c o m p u t e d by subtracting standard globulin from total lg, dividing by standard globulin and multiplying by 100.

"n t~

Z Q =

> Z ©

QUANTITATION OF COLOSTRAL IMMUNOGLOBULINS regressed linearly against the major constituents in each respective colostrum sample. Casein was the only constituent related (P<.001) to percent errors for whole and fat-free colostral preparations. No relationships were found between percent error for whey and any colostral constituent. In whole and fat-free colostrum preparations, an increase in casein resulted in an underprediction of total Ig as indicated by negative percent errors in Table 3. During whey preparation, immunoglobulins are concentrated by removal of casein and fat from the sample. Concentrating of immunoglobulins in whey would cause an overprediction of total Ig as indicated by positive percent errors in Table 3. A correction equation (Table 4) was formulated based upon the volume of fat and casein removed from colostrum during whey preparation. Correction equations also were computed for whole and fat-free colostrum based upon regression coefficients obtained when percent error was regressed against casein. Table 4 summarizes means and percent errors for three colostral fractions before and after correction equations. As indicated, percent error and respective standard deviation were reduced by the appropriate correction equation. The total Ig and percent error means for each colostral fraction were compared by least

745

significant difference test (Table 5). Statistical differences were found between total Ig for whey and globulin standard and whole or fat-free colostrums. Statistical differences also were found between percent error for whey and whole or fat-free colostrums.

DISCUSSION

Effect of sample type, e.g., whey, whole, or fat-free colostrum, on sRID analysis has not been investigated thoroughly to determine sources of potential error. Colostral Ig concentrations have been evaluated by sRID under the assumption that whey does not affect diffusion characteristics of Ig through agarose gel. Results from this experiment indicate that this assumption is not valid. In addition, different methodologies of colostral preparation may contribute to variability between laboratories (Table 1). Colostral whey has been utilized most frequently by researchers evaluating colostral Ig concentrations. Primary justification for using whey was based upon removal of interfering substances (i.e., casein) that may plug gel pores. However, during whey preparation immunoglobulins are concentrated in the remaining solvent as a consequence of removal of casein

TABLE 4. Summary table for total Ig and percent error means, standard deviations, and correction equations based upon the results of radial immunodiffusion analysis of whey, and whole and fat-free colostrum. % Error

% Error SD

Correction equations b

........ -12.5 .2

.171 .114

Total Ig whole colostrum

Fraction

.~a

Standard globulin Whole colostrum Corrected whole colostrum

5.999 5.13

Fat-free colostrum Corrected fat-free colostrum

5.40 5.96

-7.7

.1

.147 .078

Whey Corrected whey

8.39 7.28

39.5 23.9

.123 .115

5.99

1.395 - (.096 X casein) Total Ig fat-free colostrum 1.385 - (.096 × casein) Total Ig Whey (1-.022 Fat-.014 casein)

aGrams per 100 ml. bcorrection equations for whole and fat-free colostrum were computed by first regressing, percent error (y) against casein (x). Percent error is defined as total Ig whole or fat-free colostrum (T) minus standard globulin (S) and divided by standard globulin (y=(T--S)/S). The resulting regression equation, y=mx+b, where re=slope and b=y-intercept, is rearranged to (T-S)/S=mx+b. Solving for S the final correction equation is obtained: S=T/[(b+l)+mx]. Correction equation for whey was determined by estimating the relative volume of fat and casein per unit volume of whole colostrum. Journal of Dairy Science Vol. 64, No. 5, 1981

746

FLEENOR AND STOTT

TABLE 5. The least square means (g/lO0 ml) for total Ig and percent error from the radial immunodiffnsion analysis of whey, and whole and fat-free colostrum. Fraction

Total Ig % Error

Standard globulin

Whole colostrum

Whey

Fat-free colostrum

5.999b ....

5.13 b --12.5 d

8.39a 39.5c

-7.7 d

5.4b

a'bMeans with different superscripts are different. LSD = 1.10 (P = .01). C'dMeans with different superscripts are different. LSD = 6.87 (P = .01).

and fat. Subsequently, results of sRID analysis with whey are consistently high as indicated by positive percent errors in Table 3. Some researchers have recognized the need to correct lg concentration based upon proportion of whey in the colostrum sample (9). Due to high variability of the casein-fat matrix during whey preparation, volume of whey exuded is not always constant. Between inconsistent whey volume and concentrating effect upon Ig from removal of casein and fat, highly unpredictable and inaccurate Ig may result. Correction equations based upon volume of casein and fat removed tend to decrease variability (Table 4) b u t do n o t account for all variability. Whole and fat-free colostrum have produced statistically reliable results as compared to the standard globulin. Whey produced inaccurate results that were statistically different from the standard globulin, whole and fat-free colostrum. In whole and fat-free colostrum sRID analysis, casein interfered with Ig diffusion through agarose gel and subsequently produce lower Ig (Table 3). The effect from casein virtually can be eliminated by a casein correction equation (Table 4). Based upon the above discussion, a standardized method of colostrum analysis may be derived. Whole colostrum, having no preparation requirement and exhibiting no statistical difference from standard globulin, is recommended for sRID analysis. The whole colostrum sample should be used undiluted, if possible, to minimize effects from solventdilution interaction (16). Reference proteins, working standard, and u n k n o w n samples should be diluted in identical volumes of solvent (16). Further research is required Journal of Dairy Science Vol. 64, No. 5, 1981

to assess specific effects that solvent type, e.g., saline, serum, or whey, has on diffusion characteristics of Ig in either Mancini et al. (16) or Fahey and McKelvey (6) sRID techniques. Until such work, reference proteins should be dissolved in a solvent of similar composition to the sample to be analzed. In whole colostrum analysis, reference proteins may be dissolved in a milk sample virtually devoid of gammaglobulins. In blood serum analysis, reference proteins should be dissolved in an agammaglobulinemic serum, e.g., precolostral neonatal calf serum. ACKNOWLEDGMENTS

The authors are grateful to Shamrock Dairies, Inc., Tucson, AZ, for animals and facilities that kindly were supplied for this project and to the United Dairymen of Arizona for financial assistance.

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Journal o f Dairy Science Vol. 64, No. 5, 1981