Introduction

IgG is an antibody probe that has an affinity for binding specifically to the IgG protein antigen. The research goal was to identify how closely the IgG of the tested species are related. This was accomplished by examining how goat IgG, specific for goat protein, responded to the same protein from cows, pigs, rats, and humans, and it was determined which animals have the most similar antigen binding reactions. The variability in each animal’s IgG protein was revealed by the level of affinity that the anti-goat IgG probe had for the species tested. Since the variability in each animal’s proteins is due to their genetic coding sequence this study clarified which species have the most similar genetic code. Immunoglobulins are the antigen-recognition molecules of B cells and are used as the main effector function in adaptive immunity. (Janeways, 2001) Their are five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM and all five Ig classes are present in mammals and are produced from B lymphocytes as part of the immune response system. (Urich, 1994) IgG antibodies are large molecules, having a total molecular weight of 150kDa, composed of two heavy (H) peptide chains weighing approximately 50kDa and two light (L) peptide chains approximately 25kDa. (Janeways, 2001). The region of Light and Heavy chains connected by a disulfide bond makes up the Fragment antigen binding (Fab) structure, while the remaining Heavy chain region is referred to as the Fragment crystallizable (Fc) fragment. IgG has a “Y” shaped structure due to the disulfide bond between the H chains that forms a hinge. Both types have a constant region (C) and a variable region (V). The amino-terminal variable of the H and L chains together make up the V region of the antibody and confer on the ability to bind specific antigen. (Janeways, 2001) This diversity of immunoglobulins is thought to have come from duplication, which is considered to be a rare event enabling IgG to transform in complexity overtime. (Urich, 1994) Gene segments are coded for during lymphocyte development which controls the structure of the V region binding site any rearrangements to these coded segments can alter the composition of the binding site and create isotypes of Ig with diverse specificity. These genetic modifications in the experimental goat IgG make it adapt to binding specifically to goat’s IgG protein. So the closer the resemblance of the species IgG protein to that of goat, the greater affinity it has for goat IgG. This relationship between protein homology and evolutionary relatedness has also been examined in the following current research. Animals IgG Fc fragment’s ability to bind to the Human cytomegalovirus (HCMV) categorized animals into relational groups with similar IgG properties (Antonsonn, 2001). The taxonomic classification system, which is determined by combinations of methods such as that listed above, was utilized to categorize the tested species in order of their similarity to goats (Table 1).

Goat
Cow
Pig
Human
Rat
Kingdom:AnimaliaAnimaliaAnimaliaAnimaliaAnimaliaPhylum:ChordataChordataChordataChordataChordataClass:MammaliaMammaliaMammaliaMammaliaMammaliaOrder:ArtiodactylaArtiodactylaArtiodactylaPrimatesRodentiaFamily:BovidaeBovidaeSuidaeHominidaeMuroideaSubfamily:
Caprinae
Bovinae
Suinae
Homo
Muridae
Genus:
Capra
Bos
Sus
H. sapiens
Rattus
Species:
C. aegagrus
B. taurus
S. domestica
H. s. sapiens
R. rattus

Utilizing these resources the experimental hypothesis is that the polycolonal antibody specific for goat IgG will bind best to species with the most homologous IgG protein, which are predicted to be in the following order: goat, cow, pig, rat, human. The affinity that goat IgG has for goat IgG protein will gradually decrease in these species at the Fc binding domain. Binding to the light chain will have a low affinity to the tested species.

Experimental Design

Testing of the anti-Goat probe required an experimental plan to prepare the samples before they could be probed; using the list provided in the lab manual techniques were ordered into a testing sequence based on the type of information they provided. Serial dilutions were performed first so that a standard curve for the IgG species concentrations could be generated. 150μl of a 10 mg/ml bovine serum albumin (BSA) stock solution was diluted by adding 150μl of phosphate buffered saline (PBS) to make 300μl at 1mg/ml. Serial dilutions were continued making concentrations at 0.5mg/ml, 0.25mg/ml, 0.125mg/ml and 0.0625mg/ml. The dilution values were calculated by applying the equation C1V1=C2V2. These BSA concentrations were then used to create a standard curve. A Bradford assay was conducted to identify where the species samples fell on the standard curve, so that their concentrations could be determined. The prepared solutions, one blank of pure PBS, and each of the IgG samples were transferred into separate cuvettes. Then combined all samples with Bradford Reagent to determine their absorbance at 595nm in the UV/VIS spectrophotometer. The starting concentrations for each type of IgG were calculated using the equation for the slope of the standard curve (y = 1.392x + 0.0142). The IgG samples were then corrected by a dilution ratio 1:10; Initial volumes of samples were then calculated by applying the equation C1V1=C2V2, and diluting all samples to 1.6 mg/ml by the addition of PBS in order to keep sample concentrations equal as possible. The absorbance values of each BSA standard concentration was used to generate a standard curve and determine the concentration of each IgG species sample using their absorbance. The PBS blank in the Bradford Assay, and the standard concentration curve, were also used as controls to determine each species IgG protein concentration.
Sodium Dodecyl Sulfate 12% polyacrylamide gel electrophoresis (SDS-PAGE) and coomassie blue staining were performed to separate the proteins within the samples. The IgG protein was coated with negatively charged SDS and applied to the top of a 12% polyacrylamide gel matrix, along with a molecular weight standard. An electric field was connected to the gel causing the smaller negatively charged light chains to separate from the heavy chains as they migrated away from the negative charged electrode. The gel was then removed and placed in coomassie blue stain approximately 30 minutes and allowed to de-stain overnight. Rf values were calculated on the stained gel by measuring the distance traveled by the band divided by the distance traveled by the dye front. These values were then compared to the standard curve, using this data the molecular weight of the protein was determined. A control was the standard marker used in SDS-PAGE that showed polypeptide separation while running the gel
A Western blot was then performed to view the affinity for anti-Goat in each species. This was performed by electroblotting, which transported the separated IgG species chains from the SDS-PAGE gel and permanently fixed them to a nitrocellulose membrane. The nitrocellulose membrane was then blocked preventing the probe from adding to open sites, antibody binding, and washing followed the blocking step. The IgG proteins were then incubated in alkaline phosphatase (BCIP/NBT) and a developer was added. The reaction caused by the developer was observed by the antibody-labeled bands on the pig IgG turning purple and the intensity of the bands designated which species IgG had the greatest homology with goat’s IgG antibody. Noted that the pig IgG began to appear first, then cow and goat all three showing both light and heavy chain.
There was also the experimental control of other groups performing this same tests and using the same samples each group’s results were considered a comparative control. This was very helpful when certain equipment would not work properly.

Data and Calculations

TABLE 2. Absorbance Values for BSA Standards. The absorbance values for the five bovine serum albumin (BSA) standards were determined by utilizing a UV/VIS spectrophotometer set at 595nm.

Cuvette
BSA (mg/ml)
BSA Absorbance
1
0
0
2
0.50
0.70
3
0.25
0.38
4
0.125
0.192
5
0.0625
0.104

TABLE 3. Species IgG Concentrations and Adjustments. The starting concentrations for each type of IgG were calculated using the equation for the slope of the standard curve (y = 1.392x + 0.0142). The equation was rearranged in order to solve for “x”
(x = [y -0.0142]/1.392), where the absorbance of each IgG type was used as the value for “y.” The concentrations were then corrected by multiplying by the dilution factor.

IgG Sample Data and Calculations
Species
Absorbance
Initial Concentration (mg/mL)
Corrected Initial Concentration (mg/ml)
Final Concentration (mg/mL) Final Volume (μL)
Amount of Sample Used (μL)
PBS Added (μL)
Goat
0.404
0.28
2.8
1.6
100
57.4
42.6
Cow
0.52
0.36
3.6
1.6
100
44.4
55.6
Pig
0.23
0.16
1.6
1.6
100
100
0
Rat
0.72
0.51
5.1
1.6
100
31.4
68.6
Human
0.36
0.25
2.5
1.6
100
64.4
35.6

Concentration Calculation: x = [y - 0.0142] / 1.392 = [0.404 - 0.0142] / 1.392 = 0.28mg/ml

Corrected Concentration for Dilution Calculation:
0.28mg/ml x 10 = 2.8mg/ml

Adjustment Calculation:

Ci = starting concentration, Vi = initial volume, Cf = desired concentration, Vf = 100ml

CiVi = CfVf

Vi = [1.6mg/ml (100μl)] / 2.8= 57.4μl IgG Goat

57.4μl IgG Goat - 100μl = 42.6μl PBS added

FIGURE 2. Coomassie Blue Stain. Depicts similar band pattern alignment, but different concentrations.

TABLE 4. Rf Value of Standard Markers Western Blot Membrane. Molecular weights were obtained from the Sigma-Aldrich Handout

Rf Value
Color
MW (kDa)
Logged MW
0.0714
Violet
220
2.34
0.096774
Pink
100
2.00
0.16129
Blue
60
1.78
0.258065
Pink
45
1.65
0.354839
Orange
30
1.48
0.451613
Blue
20
1.30
0.79677
Pink
12
1.08
0.870968
Blue
8
0.90

Logged Molecular Weight: Log (220) = 2.34

TABLE 5. Species IgG Rf Values and Calculated MW for Heavy and Light Chains. Molecular Weights of the species light (Rf value 1) and heavy chains (Rf value 2) was determined by using the linear regression equation from Figure 3.

Lane #
Species
Rf Value 1
MW 1 (kDa)
Rf Value 2
MW 2 (kDa)
1
Standard
*
*
*
*
2
Goat
0.1929
71
0.4464
28
3
Cow
0.1786
74
0.4286
30
4
Pig
0.1893
72
0.4107
32
5
Rat
0.1929
71
0.45
28
6
Human
0.1821
73
0.4286
30
7
Goat
0.1929
71
0.4464
28

Rf Values Formula
(Distance Protein Traveled in Gel in cm) / (Distance Solute Traveled in cm)

The Rf Values plugged into standard curve line equation to determine logged molecular weight:

y = -0.6368x + 1.3702
0.1893 = -0.6368x + 1.3702
0.1893 – 1.3702 = -0.6368x
-1.1809 = -0.6368x x = -1.1809 / -0.6368 = 1.85413723

The inverse of the logged molecular weight was taken to calculate the actual molecular weight of each IgG species:

10 ^ 1.85413723 = 71 kDa

Results

The coomassie blue stained gel was done to verify that the sample concentrations of IgG distinctly separated into heavy and light chains and that the samples were all of the same concentration. The coomassie stain however did not appear to show equal concentration. This was seen by the thickness of some bands width compared to others. Fortunately, there was similarity in the band patterns revealing that both heavy and light chains are present and by comparing the bands to their approximate weight corresponding to the marker their molecular weights were calculated. The upper band was determined to be the heavy chain by comparing it to the standard the weight was approximated to be 50 kDa and the lower band was determined to be roughly 25kDa. Since, IgG has two of each of these chains as stated in the introduction, their total weight is 150kda, which is the standard molecular weight for IgG. There are other visible bands showing faintly on the gel, which may be due to contamination of proteins other than IgG during the transfer and possibly human contact if gloves were not worn. The samples may have had altered protein structures if not properly handled or if not kept in the proper refrigerator while not in use. All of these possibilities may have been determined had a Ponceau stain been performed, this stain would have shown the band separation and concentration after being transblotted to the nitrocellulose membrane. The stain depicts clear heavy and light chain separation. There may have been evidenced that the samples needed to run at higher concentrations or that there was too much variance in the concentration used. Band alignment could have been supported showing that all of the IgG proteins have similar structures and that the samples were not contaminated. The Western blot revealed the anti-goat probe’s affinity to bind to each species IgG by the concentration of color change caused by the reaction with alkaline phosphatase (AP) that was conjugated to the antibody. The initial appearance and greater the intensity of the purple color caused by AP signified a greater binding affinity. Every lane in the Western blot containing a sample of IgG was labeled by an AP-conjugated antibody. Both heavy and light chains were labeled by the probe, which was expected because Fab binding region is contained on both the heavy and light chains. Additional bands are faintly visible, which may be smaller components of IgG or other protein components that have some ability to bind to the antibody. The blocking solution should have prevented the probe from binding to any unoccupied binding sites on the nitrocellulose paper. The order of band labeling from most strongly labeled to least was pig, cow, goat, rat, and human; the heavy chain of each animal’s IgG was evident. The light chain was only evidently bound in goat, cow and pig species. The differences observed in antibody labeling intensity were due to differences in IgG protein homology and differences in IgG sample concentrations. The Ponceau red stain would have revealed that samples were in different concentrations, so when the western blot was performed samples of higher concentration would appear as a thicker band. However, differences in each specie’s IgG homology to the antibody appeared as bands with a darker colored intensity, since antibody’s were most concentrated on these bands.

Conclusion

The experimental results rejected the hypothesis, which predicted the homology of species to goat IgG to be in the following order from greatest to least: goat, cow, pig, rat, human. The predicted order was most greatly disrupted by pig appearing to be more homologous than cow and goat itself. An explanation for this is that goat and rat might have been run at too low of a concentration, in which the amount of IgG was too small to detect. Therefore, if all sample concentrations were the same, the hypothesized order may still be correct. The fact that sample concentrations were revealed to be different was the greatest weakness in the research because it severely decreased the ability to definitively conclude which species IgG was most homologous to goat’s IgG.
Running at these concentrations of cow and pig IgG affinity for the anti-goat probe turned out to be easily differentiated. The comparison revealed that more of the anti-goat probe bound to pig IgG and that they were the only species that showed the both the light and heavy band labeling, which supported the hypothesized close relatedness.
The next observable band concentration comparison was difficult to determine between rat and human especially because human was not visible until looked at under the dot cam. The low concentration seemed to identify that rat IgG caused a slightly darker band to appear than human IgG. These results were supported by the research used to form the hypothesis and, therefore, rat was concluded to be the next most related IgG to goat. To be more certain of this conclusion a comparison of rat and human should be made by running them both at an equal higher concentration.
If this study were repeated it would be critical to not make calculation errors when correcting for the dilution factor. An additional adjustment may be to dilute the standards for each specie’s IgG a little more. The bands were thick and difficult to measure the Rf values properly. But with this we also need to be careful in not lowering the dilution factor too much causing it to be difficult for proper identification in the western blot. If a less diluted initial sample is used than the final concentration should be higher when all samples are made the equivalent and enable clearer probe affinity. Once again there should be no steps skipped such as the Ponceau stain. Had this step been performed the clarity of our results may have supported our hypothesis better.

References

Prasad, Arjun. Allard, Marc. Green, Eric. (2008) Confirming the Phylogeny of Mammals by Use of Large Comparative Sequence Data Sets. Molecular Biology Evolution. 25(9):1795–1808.

Gellin, Joel. Brown, Steve. Graves, Jennifer. Rothschild, Max. Schook, Lawrence. Womack, Jim. Yerle, Martine. (2000) Comparitive gene mapping workship: progress in agriculturally important animals. Mammalian Genome. 11 140-144.

Antonsson, Annika. Johansson, Hugo. (2001) Binding of human and animal immunoglobulins to the IgG Fc receptor induced by human cytomegalovirus. Journal of General Virology. 82.1137-1145.

Urich, Klaus. (1994) Comparative Animal Biochemistry. Spinger-Verlog. New York. 222-227.

Weaver, D. (2009) Biochem Lab Manual. Campbell University. 50-71.

Janeways, Charles A. Shlomchik, Mark J. Travers, Paul. Walport, Mark. (2001) Part II Chapter 3. The Recognition of Antigen. Immunobiology Ed. 5. Garland Publishing. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=imm. 23 October 2008.

Hickman, Cleveland. Roberts, Larry. Larson, Allan. I’Anson, Helen. Eisenhour, David. (2005). Integrated Principles of Zoology. McGraw-Hill Science. 741-743.

FIGURE 1 Bradford Assay Standard Curve. The absorbance values of each BSA standard concentration was used to generate a standard curve and determine the concentration of each IgG species sample using their absorbance.

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7
MW Standards Goat Cow Pig Rat Human Goat II

FIGURE 3. Standard Curve of Marker Rf Values and their Logged Molecular Weight. Rf values were measured on the nitrocellulose membrane and plugged in the y variable of the standard curve’s line equation to calculate the logged molecular weight.