Lab 12: ELISA

Why do we need to determine which cell lines produced the highest recombinant FIX?

To reduce the cost of hemophilia treatment.

The cell lines produce recombinant FIX and other proteins. Which biomolecule can specifically detect the presence of a certain protein, such as recombinant FX, in a sample full of other proteins?

Immunoglobins

In a Direct ELISA, which components are directly attached to the plate?

Antigens (by positive absorption)

Which component of Sandwich ELISA is required in pairs?

antibodies

in a Competitive ELISA, how would the signal measured by the detector react to an increase of antigen in the sample?

Decrease

What type of ELISA is shown in the diagram?

Indirect

In this coding step, the capture antibodies are immobilized on the surface of polystyrene microplate wells. What type of force causes the passive absorption of the captured antibody due to the interaction between amino acid side chains on the antigen used for coding, and the plastic surface?

Hydrophobic

What are antibodies with high specificity that only detect one epitope called?

Monoclonal

The basic blocking buffer contains 5% bovine serum albumin (BSA) dissolved and PBS. What is the purpose of adding a blocking buffer?

To reduce ELISA background signal

Twin 20 is used in our wash buffer. What type of compound is Tween 20?

Surfactant

Why do we have to load the sample into specific wells?

To achieve valid results.

What is the purpose of a standard?

To create a standard curve.

The first well contains 200 µL of standard with a concentration of 90ng/mL. If we want to use a dilution of 1:2 dilution factor, how much sample should be transferred from the first well to the second well that contains 100 µL dilutant?

100 µL

Serial dilution with a 1:2 dilution factor. Concentration of the standard in the first well is 90ng/mL. The aliquot volume is 100 µL. The volume of dilutant in well 2-8 is 100 µL each. What is the concentration of the standard in well 8? Take into account that we are diluting the concentration to half in each step.

.07 ng/µL

A positive control is a sample known to give positive results for the given test period what is the purpose of the positive control?

Verify that the negative results are valid.

What is a group of samples where no response is expected called? This group contains all buffers and reagents except the substance of interest.

Negative control

What is the purpose of agitating the ELISA plate?

Increasing the rate of binding

Which step do we have to do next?

Adding TMB substrate

TMB (tetramethyl benzene) is a substrate for horseradish peroxidase (HRP). What is the proper container for storing TMB?

Light protected container.

A stop solution is added to provide a fixed end point for the assay. What is the stop solution for the ELISA substrate TMB in the presence of horseradish peroxidase (HRP)?

Sulfuric acid

A regression formula is obtained from the standard curve. This formula describes the relationship between concentration and absorbance data. We can use the regression formula to calculate the concentration of unknown samples. In this case, which regression type best describes are absorbance data?

Log- log

the equation of the standard curve is y = 0.998 eight X - 1.437. In ELISA we measure the absorbance using a spectrophotometer. How can we calculate the amount of the factor IX (FIX) in each sample?

Amount of FIX equals (adsorbents plus 1.437)/0.9988

All wells exhibit bright blue colors, even the negative control period what kind of problem do I have?

You have high background.

From Senor Guzman: Hi, I have generated the standard curve for my ELISA experiment. What do you think?

Your standard curve is poor.

Human kidney cell lines (Test sample 1): 8.2; Human hepatic cell lines (Test sample 2): 5.7; Human pancreatic cell lines (Test sample 3): 3.6.

Human kidney cell lines