P4HB ELISA Pair Set, Human ELISA Pair Set

1. Materials provided

Capture Antibody 0.2 mg/mL of mouse anti-P4HB monoclonal antibody, Dilute to a working concentration of 2 μg/mL in CBS before coating.

Detection Antibody 0.5 mg/mL mouse anti-P4HB monoclonal antibody conjugated to horseradish-peroxidase (HRP). Dilute to working concentration of 1 μg/mL in detection antibody diluteion buffer before use.

Standard Each vial contains 60 ng of recombinant P4HB. Reconstitute with 1 mL detection antibody dilution buffer. After reconstitution, store at -20℃ to -70℃ in a manual defrost freezer. A seven-point standard curve usi ng 2-fold serial dilutions in sample dilution buffer, and a high standard of 1 ng/mL is recommended.

2. Sensitivity

The minimum detectable dose of Human P4HB was determined to be approximately 46.88 pg/ml. This is defined as at least three times standard deviations above the mean optical density of 10 replicates of the zero standard.

3. Principle of the product

The Human P4HB ELISA Pair Set is for the quantitative determination of Human P4HB.

This ELISA Pair Set contains the basic components required for the development of sandwich ELISAs.

The Sino Biological ELISA Pair Set is a solid phase sandwich ELISA (Enzyme-Linked Immunosorbent Assay). It utilizes a monoclonal antibody specific for P4HB coated on a 96-well plate. Standards and samples are added to the wells, and any P4HB present binds to the immobilized antibody. The wells are washed and a horseradish peroxidase conjugated mouse anti-P4HB monoclonal antibody is then added, producing an antibody-antigen-antibody "sandwich". The wells are again washed and TMB substrate solution is loaded, which produces color in proportion to the amount of P4HB present in the sample. To end the enzyme reaction, the stop solution is added and absorbances of the microwell are read at 450 nm.

STORAGE - Detection Antibody should be protected from prolonged exposure to light. Aliquot the reagents and store at -20℃ to -70℃ in a manual defrost freezer.

Plate Preparation

Dilute the capture antibody to the working concentration in CBS. Immediately coat a 96-well microplate with 100μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4℃.
Aspirate each well and wash with at least 300μl wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.
Block plates by adding 300 μL of blocking buffer to each well. Incubate at room temperature for a minimum of 1 hour.
Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at room temperature.
Repeat the aspiration/wash as in step 2 of plate preparation.
Add 100 μL of the detection antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1 hour at room temperature.
Repeat the aspiration/wash as in step 2 of plate preparation.
Add 200 μL of substrate solution to each well. Incubate for 20 minutes at room temperature ( if substrate solution is not as requested, the incubation time should be optimized ). Avoid placing the plate in direct light.
Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.
Determine the optical density of each well immediately, using a microplate reader set to 450 nm.

Calculation of results

Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each.
Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph.
To determine the concentration of the unknowns, find the unknowns’ mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the sample.


Problems	Possible Sources	Solutions
No signal	Incorrect or no Detection Antibody was added	Add appropriate Detection Antibody and continue
	Substrate solution was not added	Add substrate solution and continue
	Incorrect storage condition	Check if the kit is stored at recommended condition and used before expiration date

Poor Standard Curve	Standard was incompletely reconstituted or was inappropriately stored	Aliquot reconstituted standard and store at -70 ℃
	Imprecise / inaccurate pipetting	Check / calibrate pipettes
	Incubations done at inappropriate temperature, timing or agitation	Follow the general ELISA protocol
	Background wells were contaminated	Avoid cross contamination by using the sealer appropriately

Poor detection value	The concentration of antigen in samples was too low	Enriching samples to increase the concentration of antigen
Poor detection value	Samples were ineffective	Check if the samples are stored at cold environment. Detect samples in timely manner

High Background	Insufficient washes	Use multichannel pipettes without touching the reagents on the plate
	Insufficient washes	Increase cycles of washes and soaking time between washes
	TMB Substrate Solution was contaminated	TMB Substrate Solution should be clear and colorless prior to addition to wells
	Materials were contaminated	Use clean plates, tubes and pipettes tips

Non-specificity	Samples were contaminated	Avoid cross contamination of samples
Non-specificity	The concentration of samples was too high	Try higher dilution rate of samples

Human P4HB ELISA Pair Set Image

Human P4HB ELISA Pair Set

This standard curve is only for demonstration purposes. A standard curve should be generated for each assay.

Human P4HB ELISA Pair Set Flash

This flash is a vivid summary of the assay procedure.

Catalog	Size (Price)	Quantity	In Stock	Operation	Other Information
SEK10827	5 Plates ($350) 15 Plates ($850)		YES	Bulk Order Inquiring

	Order or Inquire for P4HB ELISA Pair Set
	High affinity Human P4HB ELISA Pair Set
	Detection limit - 46.88 pg/ml
	Affordable price and 30%-80% cost saving for Bulk order

Product Catalog

P4HB ELISA Pair Set, Human ELISA Pair Set

Human P4HB ELISA Pair Set

1. Materials provided

2. Sensitivity

3. Principle of the product

Plate Preparation

Assay Procedure

Calculation of results

Human P4HB ELISA Pair Set Image

Human P4HB ELISA Pair Set Flash

Human P4HB ELISA Pair Set Related Products & Topics

Related Areas:

Proteins:

Antibodies:

Background