Method Article

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

DOI:

10.3791/63001

⸱

December 10th, 2021

 ,  ,  ,  ,  ,  ,  ,  ,  ,  , 
1Department of Molecular Biology and Genetics, Çanakkale Onsekiz Mart University, 2Uluova Dairy Farm, 3Department of Nutrition, University of Nevada, Reno, 4Department of Food Science and Technology, University of Nebraska, Lincoln, 5Evolve BioSystems Inc.

In This Article

Summary

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Bovine colostrum is both a primary source of nutrients and immunological support for the newborn calf. The understanding of the level of therapeutic proteins (lactoferrin and IgG) is important for the bovine colostrum dosing and standardization for human consumption.

Abstract

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Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a good source of macro- and micronutrients, bioactive peptides, and growth factors. Bovine colostrum is also a potential source of nutrition and bioactive because of its rich protein content that includes immunoglobulin G (IgG) and lactoferrin. However, the level of lactoferrin and IgG in bovine colostrum changes markedly during the lactation period. Therefore, monitoring the concentration of IgG and lactoferrin for the use of bovine colostrum as a protein source is an important question to study. Methods in this article describe how to determine protein content, as well as specific concentrations of lactoferrin and IgG. These methods include the following steps: Isolation of bovine colostrum proteins, Determination of protein concentration via Bicinchoninic acid assay (BCA), Visualization of proteins via SDS-PAGE, Determination of lactoferrin, and IgG concentration using an ELISA Assay.

Introduction

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Colostrum is the initial secretion of the mammary gland produced by mammals shortly after parturition. Colostrum is rich in macro- and micronutrients, antimicrobial peptides, and growth factors1,2,3,4. The composition varies gradually over time through the transition to mature milk5,6,7 but most significantly within 24 h after parturition8. The composition of colostrum is also influenced by maternal factors, including age, parity, breed, health, and nutritional status, as well as extrinsic factors, including season, premature parturition, premature lactation, colostral handling factors (pooling colostrum and storage temperature), and induction of parturition9,10,11. Compared with mature milk, colostrum contains less lactose and more fat, protein, peptides, non-protein nitrogen, ash, hormones, growth factors, cytokines, nucleotides, vitamins, and minerals12. Bovine colostrum contains a wide range of proteins, including immunoglobulins, lactoferrin, α-lactalbumin (α-LA), β-lactoglobulin (β-Lg), lactoperoxidase, and several growth factors13. The total protein concentration of bovine colostrum ranges between 11.26 mg/mL and 169.55 mg/mL14. The protein content comprises whey and casein at an average concentration of 124.00 mg/mL and 26.00 mg/mL, respectively15. The whey portion contains three major types of immunoglobulins (Igs) as IgG (85%-90%), IgM (7%), and IgA (5%)16. The major Ig in bovine colostrum is IgG, which provides passive immunity and modulates the adaptive and innate immune systems in the calf17. The initial Ig concentration of the first milking bovine colostrum can range from 20 to 200 mg/mL and decrease to around 0.4-1.0 mg/mL18. The mean IgG concentration is approximately 60 mg/mL and declines steadily to the levels below 1 mg/mL throughout the transition to mature milk19.

Another important bioactive protein in colostrum is lactoferrin, an iron-binding glycoprotein with a concentration of 1.5-5 mg/mL. Properties of lactoferrin include enhancing iron absorption as well as possessing antimicrobial activity20,21, binding lipopolysaccharide, immune-modulation, and stimulating the growth of intestinal epithelial cells and fibroblasts22. Bovine colostrum also contains α-lactalbumin and β-lactoglobulin. These proteins are sources of essential amino acids and also have bactericidal activity23,24,25. The mean α-LA and β-Lg concentrations in colostrum average 2.77 mg/mL2, and 11.5 mg/mL26, respectively. Thereafter, these concentrations decrease to 1-1.5 mg/mL27, and 4.8 mg/mL26 in mature milk. Colostrum also contains a significant amount of lactoperoxidase (mean 22.8 µg/mL) and lysozyme (mean 0.40 µg/mL)26. Lactoperoxidase is a glycoprotein that possesses antimicrobial activity against Gram-positive and negative bacteria28 by producing reactive oxygen species. Lysozyme functions as an antimicrobial agent by cleaving the peptidoglycan component of bacterial cell walls, thereby leading to celldeath29,30.

Due to their properties, IgG and lactoferrin are processed into different food products to fortify infant formulas, food supplements, high-protein preparations for convalescents and sportsmen as well as in pharmacology and cosmetology31,32,33. Bovine colostrum represents an important source of IgG and lactoferrin. However, the composition of these bioactive proteins in bovine colostrum changes markedly during the lactation period. Therefore, monitoring changes in the concentration of these bioactive proteins in colostrum samples used for research and food processing is critical. This study aims to describe the methods for monitoring the concentration and compositions of the total protein, lactoferrin, and IgG in bovine colostrum during the 6 days after calving.

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Protocol

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Colostrum samples were collected for 6 days after calving in the noon over the period July-August, from 28 Holstein dairy cows from Uluova Milk Trading Company in Çanakkale, Turkey, and deep-frozen. The samples collected on the same day were pooled according to the day of each sample and analyzed for their total protein, lactoferrin, and IgG concentrations. All samples were assayed in duplicate.

1. Sample preparation

  1. Mix 200 µL of bovine colostrum with 400 µL of dH2O to obtain a diluted sample for the analysis. Dilute all the samples accordingly.
  2. Centrifuge diluted and undiluted samples at 4 °C, 1000 x g for 30 min.
  3. Separate the middle phase to an appropriately labeled new tube. Repeat step 1.2. to obtain clear middle phase. Store the whole middle phase and diluted samples at -20 °C, if not used immediately.
  4. Use the middle phase obtained from diluted samples for BCA, SDS-PAGE, and Lactoferrin assays. Collect the middle phase from undiluted samples for IgG assay.
  5. Prepare sample dilutions for each sample to ensure that the readings are within the standard curve range. Dilute each middle phase obtained from the diluted samples to 1:300 for BCA assay, and 1:30,000 for Lactoferrin assay, and dilute each middle phase obtained from undiluted samples to 1:400,000 for IgG assay.
    NOTE: Dilution factors are determined based on the absorbance value and standard curve.

2. Determine the protein concentration using BCA protein assay kit

  1. Preparation of standards and reagents
    1. Use the reagents provided in the commercially available kit (see Table of Materials) to be used for the assay: BCA reagent A, containing sodium carbonate, sodium bicarbonate, bicinchoninic acid, and sodium tartrate in 0.1 M sodium hydroxide. BCA reagent B contains 4% cupric sulfate. Albumin (BSA) standards, contains bovine serum albumin at 2.0 mg/mL in 0.9% saline and 0.05% sodium azide.
    2. Equilibrate all samples and protein standards to room temperature (RT).
    3. Prepare sufficient volumes of working reagent (WR) by mixing 50:1 ratio of reagent A:B. 200 µL of WR is required for each sample and standard.
    4. Prepare diluted albumin (BSA) standards according to the following dilution scheme (Table 1), that presents a working range between 20-2,000 µg/mL final BSA concentration. Use dH2O as diluent.

Table 1: Dilution scheme of BSA standards.

VialVolume of Diluent (µL)Volume and Source of BSA (µL)Final BSA Concentration (µg/mL)
A0300 µl of stock2000
B125375 µL of stock1500
C325325 µL of stock1000
D175175 of vial B diluton750
E325325 of vial C dilution500
F325325 of vial E dilution250
G325325 of vial F dilution125
H400100 of vial G dilution25
I40000 = Blank
  1. BCA assay procedure
    1. Transfer 25 µL of each BCA standard or sample into a 96-well plate. Add 200 µL of the WR to each well-containing standard or sample. Mix the plate thoroughly on a plate shaker for 30 s.
    2. Cover the plate with a plate sealer and incubate at 37 °C for 30 min. After incubation, allow the reactions to equilibrate to RT for around 10 min. Read each plate at 562 nm using a microplate reader with its associated software.
  2. Generating standard curve and determining results
    1. Record the absorbance values for the standards and samples. Please see Table 1 for the dilution series. Subtract absorbance values of the standard blank from each absorbance value of standards and sample. Average the duplicate readings for each standard and sample to estimate total protein concentration.
    2. Construct a standard curve by plotting the corrected mean absorbance for each standard on the x-axis and concentration on the y-axis. Draw a linear curve with appropriate software capable of the four-parameter curve fit.
    3. Use the standard curve to determine the concentration of each sample by interpolating its response to the concentration. Multiply with the dilution factor (step 1.5) to obtain the actual concentration of the sample.

3. Visualization of protein using SDS-PAGE assay

  1. Preparation of sample and solutions
    1. Prepare stock solutions
      1. Prepare 10% (w/v) SDS, 1.5 M Tris-HCl pH 8.3, 0.5 M Tris-HCl pH 6.8, 10% (w/v) ammonium persulfate (APS) solution (prepare fresh).
      2. (w/v) SDS: Weigh 1 g of SDS and add 10 mL of dH2O.
      3. M Tris-HCl pH 8.8: Weigh 18.15 g of Tris and dissolve with ~60 mL of dH2O. Adjust to pH 8.8 with HCl. Add dH2O to bring the volume to 100 mL.
      4. M Tris-HCl pH 6.8: Weigh 6.00 g of Tris and dissolve with ~60 mL of dH2O. Adjust to pH 6.8 with HCl. Add dH2O to bring the volume to 100 mL.
      5. APS: Weigh 15 mg of APS and add 150 µL of dH2O.
        CAUTION: Acrylamide and SDS are toxic and harmful. Wear protective gloves and work under a hood.
    2. Prepare the sample buffer by adding 50 µL of β-Mercaptoethanol into 950 µL of 2x SDS-PAGE sample buffer.
      CAUTION: β-mercaptoethanol is toxic if inhaled. Wear protective gloves and work under a hood.
    3. Prepare the running buffer by mixing 100 mL of 10x Tris-glycine SDS Running Buffer with 900 mL of dH2O.
    4. Prepare the staining solution (45% dH2O, 45% Methanol, 10% glacial acetic acid, 2 g of Coomassie Brilliant Blue R).
    5. Prepare the destaining solution (50% dH2O, 40% Methanol, 10% glacial acetic acid).
  2. Preparation of gel
    1. Prepare the electrophoresis unit equipment, including gel cassette, power supplies, electrodes, and cables for the assay. Clean the glass plates with ethanol and assemble the sandwich. Ensure that the lower edges of glass plates and spacers are well aligned.
    2. Prepare the separating gel mixture containing 3.5 mL of dH2O, 2.4 mL of 40% Acrylamide/Methylene bis Acrylamide, 2 mL of 1.5M Tris-HCl, 100 µL of 10% (w/v) SDS, 80 µL of 10% APS, 8 µL of N,N,N′,N′-Tetramethyl ethylenediamine (TEMED).
      CAUTION: TEMED is toxic and/or an irritant. Wear protective gloves and work under a hood.
    3. Pour the separating gel mixture into the gel plates to a level approximately 1-1.5 cm below the top of the shorter plate.
    4. Layer the top of the separating gel with isopropanol to remove bubbles at the top of the gel and keep the polymerized gel from drying out.
    5. Pour isopropanol on top of the separating gel after the separating gel has polymerized for at least 15 min.
    6. Prepare the stacking gel mixture containing 1.92 mL of dH2O, 300 µL of 40% Acrylamide/Methylene bis Acrylamide, 750 µL of 0.5M Tris-HCl, 100 µL of 10% (w/v) SDS, 30 µL of 10% APS, and 3 µL of TEMED.
    7. Pour the stacking gel solution on top of the separating gel so that gel plates are filled. Insert the comb to the top of the spacers.
    8. Let the stacking gel polymerize at room temperature for approximately 15 min.
  3. Running the gel
    1. Attach the gel to the electrode assembly. Add freshly prepared 1x Tris-glycine SDS Running Buffer to both the chambers of the apparatus.
    2. Remove the comb.
    3. Load 5 µL of the ladder (10-250 kDa) and 8 µL of the middle phase of diluted samples into the wells of the gel. Run the gel at 80 V until the dye migrates into the separating gel and increase to 120 V until the dye reaches the bottom of the gel. Turn off the applied power after the dye reaches the bottom of the gel.
  4. Staining and destaining the gel
    1. Remove the gel from the apparatus once the run is complete and remove the spacers and glass plates. Place the gel into a small tray.
    2. Stain the gel by adding staining solution (step 3.1.4) for 30 min with gentle shaking at 55 rpm.
    3. Pour off the staining solution from the gel. Rinse the gel with a little amount of destaining solution and discard the dye.
    4. Add a sufficient volume of destaining solution to cover the gel and destain with gentle shaking for ~1 h until the bands are visible.

4. Lactoferrin concentration using a Bovine Lactoferrin ELISA

  1. Preparation of standards and reagents
    1. Use the commercially available Bovine LF/LTF/Lactoferrin ELISA Kit for this assay.
    2. Equilibrate all the samples and standards to RT.
    3. Prepare sufficient volumes of detection reagent A and B Working Solution that are responsible for binding to the captured antigen.
    4. Dilute the detection reagents A and B to a ratio of 1:100 using assay diluents A and B, respectively.
    5. Prepare a 1x working wash buffer by diluting the 30x wash buffer concentrate with dH2O.
    6. Put a sufficient amount of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution into a sterile microtube.
    7. Resuspend one tube of lyophilized standard (100 ng/mL) with 0.5 mL of the sample diluent and incubate at RT for 10 min with gentle agitation. Spin the vial to ensure that all of the lyophilized sanple is collected at the bottom.
    8. Prepare a standard dilution series according to the following dilution scheme (Table 2).

Table 2: Dilution scheme of bovine lactoferrin standards.

VialVolume of Diluent (µL)Volume and Source of Lf (µL)Final Lf Concentration (ng/mL)
D10500 µL of stock100
D2250250 of vial D1 dilution50
D3250250 of vial D2 dilution25
D4250250 of vial D3 dilution12.5
D5250250 of vial D4 dilution6.25
D6250250 of vial D5 dilution3,125
D7250250 of vial D6 dilution1,563
D825000 = Blank
  1. Measuring the concentration of Bovine Lactoferrin
    1. Pipette 100 µL of each lactoferrin standard or sample into the coated 96-well strip plate. Cover the plate with a plate sealer to avoid evaporation. Incubate at 37 °C for 1 h.
    2. Aspirate the liquid of each well. Add 100 µL of the detection reagent A working solution to each well. Cover with a plate sealer and gently agitate to ensure thorough mixing. Incubate at 37 °C for 1 h.
    3. Wash three times by adding approximately 350 µL of 1x wash buffer after aspirating the liquid from each well. Allow each wash to sit for 1-2 min before completely aspirating. After the last wash, aspirate to remove any remaining wash buffer, then invert the plate and tap against a clean absorbent paper.
    4. Add 100 µL of the detection reagent B working solution to each well. Cover with a new plate sealer. Incubate at 37 °C for 30 min. Aspirate the liquid from each well and wash it five times as described in step 4.2.3. Put 90 µL of TMB substrate solution into each well, and cover with a new plate sealer.
    5. Incubate at 37 °C for 10-20 min away from light. Check the optimal color by monitoring periodically. Observe that the intense blue color in the well includes high concentrate lactoferrin.
    6. Add 50 µL of stop solution to each well. The color will change from blue to yellow. Measure the absorbance of each well immediately at 450 nm using a microplate reader with its associated software.
      NOTE: Tap the plate gently to ensure thorough mixing until the color change is uniform.
  2. Generating standard curve and determining results
    1. Follow step 2.3.1 for data generation to estimate lactoferrin concentration.
    2. Construct a standard curve by plotting the corrected mean absorbance for each standard as described in step 2.3.2 but drawing a polynomial curve with appropriate software capable of the four-parameter curve fit.
    3. Calculate the lactoferrin content of each sample by interpolating the absorption value onto the generated equation as described in step 2.3.3.

5. IgG concentration determination of samples using Bovine IgG ELISA

  1. Preparation of standards and reagents
    1. Use the items required from the ones provided in the Bovine IgG ELISA Kit.
    2. Equilibrate all the samples and standards to RT.
    3. Prepare sufficient volumes of working Enzyme-Antibody Conjugate solution by diluting 10 µL of horseradish peroxidase (HRP)-avidin concentrate (100x) with 990 µL of Enzyme-Antibody Conjugate diluent.
    4. Prepare sufficient volumes of 1x wash buffer by diluting 20x wash buffer concentrate with dH2O.
    5. Prepare sufficient volumes of the 1x diluent solution by diluting 20x diluent concentrate with dH2O.
    6. Add 1.0 mL of dH2O to the bovine IgG calibrator and mix gently until dissolved. The final concentration of the calibrator is 123.000 ng/mL.
    7. Prepare standard dilution series according to the dilution scheme described in Table 3.

Table 3: Dilution scheme of bovine IgG standards.

VialVolume of Diluent (µL)Volume and Source of IgG (µL)Final IgG Concentration (ng/mL)
D1900100 µL of stock12300
D2900100 of vial D1 dilution1230
D3178122 of vial D2 dilution500
D415050 of vial D3 dilution250
D5150150 of vial D4 dilution125
D6100100 of vial D5 dilution62.5
D7100100 of vial D6 dilution31.25
D8100100 of vial D7 dilution15,625
D9100100 of vial D8 dilution7,813
D1010000 = Blank
  1. Bovine IgG ELISA assay procedure
    1. Pipette 100 µL of each IgG standard or sample into the coated 96-well strip plate. Cover the plate with a plate sealer and incubate at RT for 30 min. Aspirate the liquid from each well.
    2. Wash four times by filling wells with 1x wash buffer and aspirate. After the last wash, aspirate to remove any residual wash buffer, then invert the plate and tap against clean absorbent paper. Add 100 µL of appropriately diluted Enzyme-Antibody Conjugate to each well. Cover with a plate sealer and gently agitate to ensure thorough mixing.
    3. Incubate at RT for 10 min. Wash and remove residual wash buffer from the wells as described in step 5.2.2. Add 100 µL of TMB substrate to each well; cover with a new plate sealer.
    4. Incubate at RT for precisely 10 min away from light. Stop the reaction by adding 100 µL of stop solution to each well. Read each plate at 450 nm using a microplate reader with its associated software.
  2. Generating standard curve and determining results
    1. Follow step 2.3.1 for the data edition to estimate IgG concentration.
    2. Construct a standard curve by plotting the concentration on the x-axis and the corrected mean absorbance for each standard on the y-axis. Draw a polynomial curve with appropriate software capable of the four-parameter curve fit.
    3. Calculate the IgG content of each sample by interpolating the absorption value onto the generated equation as described in step 2.3.3.

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Results

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Following the protocol, the bovine colostrum samples were analyzed to determine protein, lactoferrin, and IgG concentration. The results of protein, lactoferrin, and IgG analyses of bovine colostrum are shown in Table 4.

Table 4: Concentration of protein, lactoferrin, and IgG of bovine colostrum.

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Discussion

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This study provides information about considerable changes in the protein, lactoferrin, and IgG concentrations in colostrum throughout the transition to mature milk. Detection of changes in the lactoferrin and IgG concentration was carried out by sandwich ELISA, and total protein concentration was analyzed by the BCA assay. Results indicate that early colostrum has the highest protein, lactoferrin, and IgG concentration, that subsequently decreased over the next 3 days. Accurate measurements of these proteins are relevan...

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This study is supported by Uluova Süt Ticaret A.Ş (Uluova Milk Trading Co.). RMD and BMH are employees of Evolve BioSystems, a company focused on restoring the infant microbiome.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
10X Running Buffer (Tris-Glycine-SDS)ClearBandTGS10SDS-Page analysis
2-mercaptoethanolgibco31350-010SDS-Page analysis
Acetic Acid GLACIALIsolab901,013,2500SDS-Page analysis
Bovine IgG ELISA KitAviva Systems BiologyOKIA00005Determination of IgG concentration
Bovine LF / LTF / Lactoferrin ELISA KitLSBio Lifespan BiosciencesLS-F4884Determinaton of lactoferrin concentration
Coomassie Brillant Blue R 250amresco0472-25GSDS-Page analysis
Hydrochloric Acid Fuming 37%Isolab932,103,2501SDS-Page analysis
IsopropanolIsolab961,023,2500SDS-Page analysis
Laemmli Sample Buffer (2X)ClearBandLSB-2xSDS-Page analysis
MethanolIsolab947,046,2500SDS-Page analysis
PageRuler Plus Prestained Protein Ladder 10 to 250Thermo Scientific26619SDS-Page analysis
Pierce BCA Protein Assay KitThermo Scientific23225Determination of protein concentration
Sodium dodecyl sulfate (SDS)BioShopSDS001.500SDS-Page analysis
SureCast Acrylamide Solution 40% (w/v)InvitrogenHC2040SDS-Page analysis
SureCast Ammonium persulfate (APS)Thermo Scientific17874SDS-Page analysis
SureCast Tetramethylethylenediamine (TEMED)InvitrogenHC2006SDS-Page analysis
TECAN Infinite M200 Plate ReaderTecan30035094Measurement of absorbance
Tris baseBioShopTRS001.1SDS-Page analysis

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