Question

STUDY GUIDE: The following ELISA protocols are from the Methods Sections of some scientific papers. Read through each protocol and respond to the questions.

Protocol 1. Using GST-peptide-based ELISAs to Detect Protein Kinase (Plk1) in cell lysates.   96-well plates (Beckman–Coulter) were coated with GST-peptides_, an affinity ligand for Plk1. To block the unoccupied sites, wells were washed once with PBS plus 0.05% Tween 20 (PBST), and then incubated with 200 μL of PBS plus 1% BSA for 1 h. Cells to be analyzed for Plk1 were homogenized, serially diluted, and added to the wells (50 μL per well) and incubated for 12–18 h at room temperature. Plates were washed and then incubated for 2 h with 100 μL per well of anti-Plk1 antibody at a concentration of 0.5 μg/ml. After washing the plates 5 times, 100 μL per well of HRP-conjugated secondary antibody (diluted 1:1,000 in blocking buffer) was added and the plates incubated for 1 h. Plates were then washed 5 times with PBST and then incubated with 100 μL per well of 3,3′,5,5′-tetramethylbenzidine solution (TMB) (Sigma) as substrate until a desired absorbance was reached. The reactions were stopped by the addition of 0.5 M H₂SO₄. The optical density of the samples was measured at 450 nm by using an ELISA plate reader (Molecular Devices).

1. What does: “block the unoccupied sites” mean in the context of an ELISA? Why is this step important in getting accurate results?
2. What was used as the blocking agent?
3. What was the purpose for having 0.05% Tween 20 in the buffer?
4. What is HRP and what purpose does it serve?

Protocol 2: Analysis of serum samples for CT45 antibodies by ELISA was performed by adsorbing recombinant CT45 protein and control protein antigens LAGE-1, MAGE-1, MAGE-3, and p53 at 1 μg/mL to 96-well plates (Corning). After blocking with PBS containing 5% nonfat milk, serum samples were tested over a range of serial 4-fold dilutions from 1:100 to 1:6,400. After incubation, plates were washed with PBS containing 0.2% Tween and rinsed with PBS. Serum antibodies bound to antigens was detected with alkaline-phosphatase–conjugated secondary antibodies (Southern Biotech). After addition of ATTOPHOS substrate (Fisher Scientific), absorbance was measured using a fluorescence reader Cytofluor Series 4000 (PerSeptive Biosystems). In each assay, sera of patients with known presence or absence of specific antibody reactivity were used as controls. Titers were considered significant if >100 and if confirmed in repeat experiments.

1. Why were the plates coated with CT45 protein?
2. What is the blocking agent in this ELISA?
3. What kind of substrate is ATTOPHOS? How does this substrate allow detection of antibody binding in this assay?
4. Is this assay a direct, indirect, or sandwich ELISA? Provide rationale for your response.

Protocol 3: Serum Analysis Using ELISA Analysis. ELISA assay kits for each of the following serum proteins were purchased from Diagnostic Systems Laboratories (Webster, TX) or Assay Designs (Ann Arbor, MI). These proteins were: EGF, macrophage inhibitory factor-1, TNF-α, leptin, prolactin, IL-17, OPN, and IGF-II. Assays were performed following kit instructions. Plates were read on a Spectra Max M2 Microplate Reader (Molecular Devices) with the appropriate baseline correction for each assay.

1. What is a microplate reader and how might it be used in an ELISA assay?
2. Why would a sandwich ELISA be the most appropriate way to detect these proteins in serum collected from patients?

Protocol 4: You will write this protocol

Describe how you would use an ELISA (you pick which type) to screen the monoclonal antibodies being produced by different hybridoma clones generated by fusion of myeloma cells with plasma cells from the spleen of a mouse. The mouse was injected with human NFkB, a transcription factor, and you are looking for the clone producing an antibody that binds to this protein. Include a diagram.

Answer 1

Protocol 1:

Answer to 1st question:

Block the unoccupied sites means in the ELISA test is the blocking the remaining sites present in the well coated plates.  In ELISA test, before detecting protiens present in the well membrane with using antibodies, the remaining binding surface must be blocked  to prevent the non specific binding sites of the antibodies. If it is not done then antibodies or other detecting reagents will bind to the remaining sites that intially present immobilized protiens and it will give inaccurate results.

Answer to 2nd  question:

The Blocking agent was used as Bovine Serum Albumin (BSA). it is used to prevent non specific binding of antigens and antibodies in the well plates.

Answer to 3rd  question:

the purpose of having 0.05% of Tween 20 in buffer is to block the vacant binding sites present in the Enzyme- linked immunosorbent Assay (ELISA). it reduces non-specific binding in ELISA test.

Answer to 4th question:

HRP means  Horseradish Peroxidase an enzyme of ability to amplify the weak signal and helps to detect the targeted molecule easily.HRP is used in ELISA test in which antibody conjugated to HRP used to detect small amount of protein present in the well plates by forming coloured products.

Protocol 2 :

Answer to 1st question:

CT45 means Cancer/testis CT45 is a nuclear antigen present in the Hodgkin's Lymphoma disease. The CT45 protien is coated in well plates and then coated with antibody to determine the antigen-antibody interaction in ELISA test.

Answer to 2nd  question:

The blocking agent used in the ELISA test is Non- fat milk . it is used to prevent non specific binding of antigens and antibodies in the well plates.

Answer to 3rd  question:

--ATTOPHOS or AP Fluorescent substrate is the sensitive fluorescent Alkaline Phosphatase substrate . This substrate produces detectable colour change when antibodies binding happen and it occurs when added to alkaline phosphatase label.

Answer to 4th question:

Analysis of serum samples for CT45 antibodies by ELISA is Sandwich ELISA assay. because in sandwich ELISA assay, the target antigen ( LAGE-1, MAGE-1, MAGE-3, and p53) is captured on well plate using capture antibody( CT45 antibodies) and then detected by detection antibody(alkaline-phosphatase–conjugated secondary antibodies) which results in the formation of Antibody - Antigen- Antibody sandwich.

Protocol 3 :

Answer to 1st question:

Microplate reader is widely used instrument that allows samples for detecting and commonly used in the ELISA tests to detect and process biological and chemical data like enzymes activity , and quantify the proteins present in the ELISA test.

Answer to 2nd  question:

Sandwich ELISA is more sensitive about 2 to 5 times than Direct or indirect ELISA tests and givies accurate and fast detection of protein concentrationin sample. it is used for antigen quantifing between the capture of antibody and detection of antibody.