Immunochemistry lab report

Immunochemistry lab report

INTRODUCTION:

In this lab Immunoglobulin G (IgG) was purified from sheep anti-IL-15R serum using a protein G column. The absorbance of this purified sample, as well as those of: pre-immunised serum, wash through and eluate are measured using a spectrophotometer and their protein contents calculated. This purified antibody was also used to create a biotinylated IgG sample.

Dilutions of both anti-IL-15R antibody and biotinylated anti-IL-15R antibody were made. A standard curve pre-immunised & immunised serum dilutions as well as eluate & biotinylated eluate dilutions were also made. These various dilutions were added to separate ELISA plates which had been prepared with a specific coating antigen for the relevant sample (biotinylated or non biotinylated). The ELISA was used to determine the amount of antibody in the various dilutions via an optical density measurement taken with an ELISA reader.

A sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) test was performed on the IgG & biotinylated IgG eluates in order to assess the weight in KDA of the heavy and light chains of the IgG antibody present in the serum.  Estimates of weight for these chains were attained by drawing a graph of distance [Rf] vs weight [KDA] using data from the SDS-PAGE standard proteins and taking a line of best fit.

A Western blot was also performed on the IgG & biotinylated IgG eluates. The Western blot could be used to ascertain the weight of the IgG samples; however it was more difficult to interpret than the SDS-PAGE for this purpose. The Western blot was of particular use in determining the purity of the sample being tested.

METHODS:

Experiments were carried out as described in the protocol outlined in the lab book.

Well 9-H of ELISA plate 1 was recorded as having an absorbance of “0.812”. This well should have been filled with dilution buffer in the same manner as row H columns 3 through 12. Dilution buffer should display a low absorbance, and it is likely that this well has been filled with a different sample such as 1:500 dilution eluate (which also has an absorbance of ~0.8).  There is an incongruity between the SDS-PAGE and Western blot measurement of Ig size. Using the SDS-PAGE and the accompanying graph of KDA vs Rf the light chain was worked out to be 30KDA and the heavy chain 60KDA for a total weight of 90KDA. The Western blot appeared to show that the light chain was 40KDA and the heavy chain 80KDA.

These measurements should be exactly the same as it is the same sample being measured in both cases. The likely reason for this discrepancy is that the Western blot is more difficult to use for assessing molecular weight than the SDS-PAGE. The difference in measurements amounts to one step higher on the ladder of standard proteins and thus the ladder may have been applied to the Western blot incorrectly.

 

 

RESULTS:

OBSERVATIONS

Upon adding the 50µl of substrate solution to each well the development of blue colouration occurred rapidly, within 3-4 minutes. This change occurred without the need to incubate the 96-well plate in the dark. Some wells (those with more IgG) were a darker, more azure blue colouration. The addition of the 25 µl H2SO4 stop solution immediately caused the blue colour to change to a light yellow. Within approximately 90 seconds the wells with a higher IgG concentration had changed to a deep, opaque yellow.

YIELD

The yield of the original serum (the total quantity of protein per ml) is given by the formula:

Protein mg/ml = OD280 x 1.4

The OD measurement for the samples is as follows:

  • Original Serum = 0.953
  • Flow Through    = 0.490
  • Eluate                    = 1.926

Hence the mg/ml protein content of these samples is as follows:

  • Original Serum = 1.3342 mg/ml
  • Flow Through    = 0.686     mg/ml
  • Eluate                    = 2.6964   mg/ml

This formula gives the value of all protein in the sample; it does not specifically give the amount of IgG protein in the sample.

 

 

 

 

 

 

 

 

PURITY, ANTIBODY TITRE, SPECIFICITY – TABULAR PRESENTATION OF ELISA DATA

ELISA 1 – IgG detection

1

2

3

4

5

6

7

8

9

10

11

12

A

0.561

0.86

0.757

0.7

0.607

0.415

0.74

0.803

0.615

0.655

0.842

0.843

B

0.365

0.462

0.753

0.779

0.445

0.486

0.81

0.837

0.727

0.765

0.899

0.917

C

0.248

0.229

0.618

0.894

0.357

0.245

0.813

0.729

0.87

0.731

0.977

0.921

D

0.205

0.205

0.688

0.771

0.21

0.206

0.827

0.684

0.701

0.729

1.085

0.962

E

0.127

0.155

0.644

0.706

0.201

0.171

0.761

0.731

0.476

0.57

0.192

0.416

F

0.12

0.2

0.18

0.817

0.147

0.129

0.843

0.943

0.379

0.391

0.186

0.185

G

0.181

0.163

0.825

0.849

0.204

0.165

0.888

0.839

0.29

0.308

0.211

0.194

H

0.154

0.21

0.395

0.187

0.193

0.232

0.294

0.201

0.812

0.301

0.434

0.227

 

ELISA 2 – biotinylated IgG detection

1

2

3

4

5

6

A

0.43

0.592

0.491

0.419

0.917

0.1

B

0.324

0.364

0.334

0.322

0.675

0.674

C

0.282

0.283

0.259

0.209

0.541

0.608

D

0.255

0.32

0.306

0.265

0.578

0.779

E

0.212

0.259

0.246

0.226

0.319

0.375

F

0.209

0.253

0.245

0.233

0.31

0.335

G

0.263

0.313

0.295

0.243

0.305

0.305

H

0.264

0.336

0.333

0.33

0.274

0.316

 

 

The means of each sample are as follows:

ELISA 1

  • Standard              = 0.278
  • Pre-serum          = 0.713
  • Anti-serum         = 0.285
  • Eluate                   = 0.803
  • Eluate biotin       = 0.586
  • Flow Through    = 0.825
  • Wash 1                  = 0.906
  • Wash 2                  = 0.949
  • Wash 3                  = 1.0235
  • Dilution Buffer  = 0.291

ELISA 2

  • Standard              = 0.301
  • Eluate                   = 0.292
  • Eluare biotin       = 0.487
  • Dilution Buffer = 0.313

 

 

 

DISCUSSION:

In this experiment IgG was purified using a protein G agarose spin column. Protein A (derived from Staphylococcus aureus rather than Streptococcus sp.) could also be used, however protein G binds a larger number of IgG subclasses and typically has a greater affinity.

This technique is known as protein G antibody affinity chromatography. The Fc region of Ig will bind specifically to protein G, other proteins will be removed through washing with binding buffer A. Complexes of protein G and Ig will be dissociated by inducing a low pH (2.5) with elution buffer after which antibodies can be separated from buffer and protein G. Neutralisation buffer is also used to raise the pH to prevent antibodies being dissolved in a prolonged acidic environment.

This method results in greater yield and purity of antibody than is achieved by the other methods of purification which will be discussed, and does not rely on antibody specificity for binding. The drawback to this technique is that it is not suitable for purifying large amounts of antibody and does not generate a high specificity of Ig in the sample (Liddell, 2005).

 

 

The simplest alternative to the method used in this lab is physical separation through ion exchange chromatography, which places the serum in a highly saline solution (using ammonium for example) and allowing the Ig to become insoluble as ionic concentration increases. This method can achieve purity of up to 90%, but is hampered by the fact that some Ig will always remain in solution and that ammonium must be removed entirely otherwise its ions may interfere with Ig binding. The yield obtained is governed by the initial concentration of Ig used and specificity is poor in comparison to antibody affinity chromatography.

Where a high specificity is required, immunoaffinity purification can be used. This uses the highly specific antigen binding region of Ig to obtain a purified Ig sample specific for a particular antigen. This is achieved by slowly passing serum through a column lined with said antigen, washing the column to remove all other protein and decoupling the antigen-antibody complex using a low pH in a similar manner to antibody affinity chromatography. This technique gives a higher specificity for Ig than the other techniques discussed though will generally have a lower yield. Purity is high.

CONCLUSION:

In this lab IgG was purified from sheep anti IL-15R serum using a protein G column. A biotinylated sample was also created and these preparations had: yield, specificity, titre and purity quantified using ELISA testing as well as Western blotting and SDS page tests.  The molecular weight of purified antibody was also estimated using data obtained through these gel electrophoresis tests.

 

 

 

QUESTIONS:

  • What is meant by yield, purity and specificity of IgG purified from serum?

Yield refers to the mg/ml concentration of IgG in purified serum, essentially the overall amount of IgG present.

Purity is the amount of IgG present in the purified serum compared to other proteins.  A purification containing mostly IgG and few or no other proteins can be said to have a high purity. The presence of large amounts of other proteins compared to IgG corresponds to a low purity.

Specificity describes whether or not the Ig in a purified sample binds only to a particular antigen. Purifications with low specificity generally contain a certain class of antigen or a random group comprised of all possible serum proteins.

 

 

 

  • Do you expect any contaminants in your IgG preparations?

 

Possible contaminants would be other proteins normally found in serum. This experiment attempted to isolate IgG specifically by using a protein G spin column to which only IgG could bind and performing successive washes (three in total) with binding buffer A.

 

With each wash, IgG should be bound in the spin column while any other proteins (contaminants) are ablated. Using this method correctly, it is reasonable to assume that most contaminants will be removed. However, technique is a critical component in this process and due to the relative inexperience of those performing the preparation; some contaminants may still be present. In a true laboratory situation, further washes would be used to ensure purity of the sample.

 

 

 

 

 

 

 

  • Why do you get lower yields of IgG from the column than you would expect?

 

It is possible that there was not enough protein G in the column to bind all of the IgG in the sample. This saturation of protein G meant that some amount of IgG was ablated along with other proteins during washing and the maximum possible yield from the sample was not achieved.

 

  • What is the chemical reaction used to add the biotin molecule to the antibody?

Biotin binds to antibody covalently through amide bonds with lysine residues on the antibody.

 

  • Do you know which amino acid residues can accept the biotin molecule?

Biotin can bind to amines such as tris, glycine and lysine.

 

 

References:

  • Liddell E (2005), Antibodies in Wild D, The Immunoassay Handbook, (Third Edition), London: Elsevier, pp 144-161

 

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