Determination of Immunoglobulin G Content in Bovine Colostrum
Colostrum is the first milk produced by mammals following the delivery of a newborn. Containing a high number of bioactive compounds, it serves a number of functions including "jump starting" the newborn's immune system. Although its role in the health of newborns is well understood, research has suggested that bovine colostrum, which is quite similar to human colostrum in makeup, can be consumed to provide health benefits long after infancy.
Immunoglobulins are a class of proteins that play a role in numerous biological functions, which includes but is not limited to regulation of physiological functions and boosting of the immune system. They are widely present in serum, breast milk, and colostrum. As our understanding of immunoglobulins has developed, so too has our understanding of its potential applications. Bovine Immunoglobulin G (IgG) isolated from cow milk has become an important additive in infant milk powder formula and other health products. The content of immunoglobulin G directly determines the quality of the product, as well as its price. As such, accurate determination of IgG content in such a product is of paramount importance to those performing research, or to those involved at the product quality assurance level.
Immunoglobulin G Assay
The following equipment were used in the determination of IgG content in bovine colostrum experiment: A 5-piece Dionamix HPLC system (Consisting of an L-3120 Solvent Organizer, an L-3220 Binary High-Pressure Gradient Pump, an L-3400 Column Thermostat, an L-3300 Autosampler, and an L-3500 UV/Visible Detector), a 1mL HiTrap Protein G HP column, a Millipore Milli-Q ultrapure water purification system, a Sartorius Germany BT 124S scale, a set of Eppendorf 10-100µL and 10-1000µL pipettes, a Changsha Xiangyi H1650 centrifuge, a Tianjin Autoscience AP-01P Vacuum Pump, and a German MEMBRANA Company 0.20µm filter membrane.
The experiment ran for 30 minutes, and was carried out under the following parameters: A flow rate of 0.4mL/min, an injection volume of 20µL, a detection wavelength of 280nm, a column temperature of 40°C.
Mobile Phase
Two mobile phases were prepared, A and B. Mobile Phase A was produced by mixing 5.6765g of disodium hydrogen phosphate 4.6478g of potassium dihydrogen phosphate, diluted to 1000ml with water, to obtain a solution with a pH of 6.5 and a concentration of 0.05mol/L. Mobile Phase B was produced by weighing 3.7535g of glycine and adding water to reach a volume of 1000mL, adjusting the pH to 2.5 with concentrated hydrochloric acid to prepare a 0.05mol/L glycine hydrochloric acid buffer solution.
Sample Preparation
Standard Curve Solutions
0.025mg of Immunoglobulin G reference substance was added to a 25mL volumetric flask and mixed with mobile phase A to produce a solution with a concentration of 1.0000mg/mL (Standard Solution (5)). 5mL of that solution was extracted and added to a 10mL volumetric flask and mixed with mobile phase A to produce a solution with a concentration of 0.5000mg/mL (4). This process was repeated three more times each using the subsequently produced solution to obtain solutions with the concentrations of 0.2500mg/mL (3), 0.1250mg/mL (2), and 0.0625mg/mL (1).
Sample Solutions
0.1000g of bovine colostrum powder was added to a 10mL volumetric flask and diluted to the mark with mobile phase A. After sonication for 10 minutes, it was centrifuged at 4000rpm for 10 minutes and then filtered with a 0.20μm filter. 0.5mL of the continuous filtrate was added to a 10mL volumetric flask and diluted to the mark with mobile phase A (Test Solution (1)), and another 0.5mL was added to a 10mL volumetric flask and mixed with 8mL of Standard Solution (5), before being diluted to the mark with mobile phase A (2).
System Suitability
Standard Solution (4) was injected. It was found to have a retention time of 15.863 seconds and a tailing factor of 2.829 with a column efficiency of 10757. The Chromatogram of the IgG Standard Solution can be seen on the right.
System Suitability Repeatability
Standard Solution (4) was injected 6 times. The qualitative and quantitative chromatogram of the IgG standard solution can be seen on the left. A qualitative repeatability RSD% (n=6) of 0.206 and a quantitative repeatability RSD% (n=6) of 0.186 were calculated from the values below.
| Name | Retention Time | Peak Area |
| IgG-1 | 15.830 | 691.260 |
| IgG-2 | 15.867 | 694.066 |
| IgG-3 | 15.803 | 690.451 |
| IgG-4 | 15.770 | 692.287 |
| IgG-5 | 15.800 | 692.909 |
| IgG-6 | 15.810 | 692.845 |
Linear Range of the Standard Curve
Standard Solutions (1), (2), (3), (4), and (5) were successively injected once each, the chromatogram of which can be seen on the right.
The standard curve of the IgG reference substance can be seen on the left. The concentration range was found to be 0.0625mg/mL - 1.0000mg/mL, the regression equation Y = 1244.4625 * X + 50.9864, and the correlation coefficient r=0.9999.
Sample Measurement Results
Test Solution (1) was injected, the chromatogram of which can be seen on the right. With a peak area of 278.643, a peak height of of 11.869, and a column efficiency of 13751, the IgG content was calculated to be 36.58%, using the content calculation formula below.
IgG Content (%) =
(AS - 50.9864) / 1244.4625 * 10 * 20 / 1000 / 0.1 * 100%
Where AS is the IgG peak of test solution (1)
Sample Measurement Repeatability
Test Solution (1) was injected 6 times. The qualitative and quantitative chromatogram of the IgG test solution can be seen on the left. A qualitative repeatability RSD% (n=6) of 0.107 and a quantitative repeatability RSD% (n=6) of 0.401 were calculated from the values below.
| IgG Sample Name | Retention Time | Peak Area |
| Repeatability 1-1 | 15.787 | 279.979 |
| Repeatability 1-2 | 15.757 | 279.854 |
| Repeatability 1-3 | 15.787 | 277.335 |
| Repeatability 1-4 | 15.793 | 277.761 |
| Repeatability 1-5 | 15.753 | 279.515 |
| Repeatability 1-6 | 15.777 | 278.643 |
Rate of Recovery
Test Solution (2) was injected 3 times, the chromatogram of which can be seen on the right. Recovery rates were calculated from the formula below.
Rate of Recovery (%) =
[(AR - 50.9864) / 1244.4625 - (AS - 50.9864) / 1244.4625] / CA * 100%
Where
AR is the peak area of the IgG in bovine colostrum powder, after adding the IgG standard substance,
AS is the peak area of the IgG in bovine colostrum powder, and
CA is the Concentration of IgG standard solution added to bovine colostrum powder (mg/mL)
Of the three injections, the AR were given at 1242.841, 1237.100, 1259.821. All three shared an AS of 278.643, and a CA of 0.8, giving the calculated rate of recoveries as 96.93%, 96.35%, and 98.63%, with an average rate of recovery of 97.30%.
Conclusion
The L-3000 HPLC is capable of quickly and efficiently carrying out the bovine colostrum immunoglobulin G content experiment to a high degree of accuracy, and with good qualitative and quantitative repeatability.
This makes the Dionamix L-3000 HPLC system and method well suited to the needs of research scientists, quality control managers, industry regulators, and other similar roles that require a precise understanding of the nutrient content in infant and/or adult food supplements.
Applicable to research and quality assurance processes, the HPLC system and method also delivers more accurate results than other methods, and is additionally quite versatile.