KLH IgM Kit ELISA

N° du produit ABIN956274

Le kit ELISA KLH IgM (ABIN956274) est un kit ELISA Colorimetric conçu pour quantifier KLH IgM.

Aperçu rapide pour KLH IgM Kit ELISA (ABIN956274)

Antigène: KLH IgM (Anti-Keyhole Limpet Hemocyanin IgM Antibody (KLH IgM))

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Application: ELISA

Type d'échantillon: Plasma, Serum

Détail du produit KLH IgM Kit ELISA

Analytical Method: Quantitative

Attributs du produit: Measurement of KLH induced anti-KLH antibody levels allows quantitative evaluation of the immune response. This ELISA is designed for the rapid and quantitative measurement of mouse anti-KLH IgM levels in mouse serum or plasma.The mouse anti-KLH IgM ELISA is based on a solid phase enzyme-linked immunosorbent assay (ELISA). The assay uses KLH for solid phase (microtiter wells) immobilization and horseradish peroxidase (HRP) conjugated anti-mouse IgM antibodies for detection. Test serum or plasma samples are diluted and incubated in the microtiter wells for 1 hour. The microtiter wells are subsequently washed and HRP conjugate is added and incubated for 45 minutes. Anti-KLH IgM molecules are thus sandwiched between immobilized KLH and the detection antibody conjugate. The wells are then washed to remove unbound HRP-labeled antibodies and TMB reagent is added and incubated for 20 minutes at room temperature. This results in the development of a blue color. Color development is stopped by the addition of stop solution, changing the color to yellow, and optical density is measured spectrophotometrically at 450 nm. The concentration of anti-KLH IgM is proportional to the optical density of the test sample.

Ingrédients: KLH coated 96-well plate (provided as 12 strips of 8 wells)
Enzyme Conjugate Reagent, 11 mL
Reference calibrator (lyophilized)
20X Wash Solution, 50 mL
Diluent, 60 mL
TMB Reagent (One-Step), 11 mL
Stop Solution (1N HCl), 11 mL.

Matériel non inclus: Precision pipettes and tips
Distilled or de-ionized water
Polypropylene or glass tubes
Vortex mixer
Absorbent paper or paper towels
Micro-Plate incubator/shaker mixing speed of ~150 rpm
Plate washer
Plate reader with an optical density range of 0-4 at 450 nm
Graph paper (PC graphing software is optional).

Information d'application

Plaque: Pre-coated

Préparation de l'échantillon:

General Note: Studies indicate that anti-KLH IgM is present in serum from KLH immunized mice at concentrations of ~10,000 u/mL. In order to obtain values within range of the calibration curve, we suggest samples initially be diluted 500 fold using the following procedure for each sample tested:
1. Dispense 48 µL and 237.5 µL of diluent into separate tubes.
2. Pipette and mix 2 µL of the serum/plasma sample into the tube containing 48 µL of diluent. This provides a 25 fold diluted sample.
3. Mix 12.5 µL of the 25 fold diluted sample with 237.5 µL of diluent in the second tube. This provides a 500 fold dilution of the sample.
4. Repeat this procedure for each sample to be tested.

Procédure de l'essai:

1. Secure the desired number of coated wells in the holder.
   2. Dispense 100 µL of calibrators and diluted samples into the wells (we recommend that samples be tested in duplicate).
   3. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 1 hour.
   4. Aspirate the contents of the microtiter wells and wash the wells 5 times with 1X wash solution using a plate washer (400 µL/well). The entire wash procedure should be performed as quickly as possible.
   5. Strike the wells sharply onto absorbent paper or paper towels to remove all residual wash buffer.
   6. Add 100 µL of enzyme conjugate reagent into each well.
   7. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 45 minutes.
   8. Wash as detailed in 4 to 5 above.
   9. Dispense 100 µL of TMB Reagent into each well.
   10. Gently mix on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 20 minutes.
   11. Stop the reaction by adding 100 µL of Stop Solution to each well.
   12. Gently mix. It is important to make sure all the blue color changes to yellow.
   13. Read the optical density at 450 nm with a microtiter plate reader within 5 minutes.

Calcul des résultats:

1. Secure the desired number of coated wells in the holder.
   2. Dispense 100 µL of calibrators and diluted samples into the wells (we recommend that samples be tested in duplicate).
   3. Incubate on an orbital micro-1. Calculate the average absorbance values (A450) for each set of reference calibrators and samples.
   2. Construct a calibration curve by plotting the mean absorbance obtained from each reference calibrator against its concentration in ng/mL on linear graph paper, with absorbance values on the vertical or Y-axis and concentrations on the horizontal or X-axis.
   3. Using the mean absorbance value for each sample, determine the corresponding concentration of anti-KLH IgM in u/mL from the calibration curve.

Restrictions: For Research Use only

Stockage

Stock: -20 °C

Stockage commentaire: The reference calibrator should be stored at -20°C for optimal stability. All remaining components should be stored at 4°C. The microtiter plate should be kept in a sealed bag with desiccant to minimize exposure to damp air. Test kit will remain stable until the expiration date provided that the components are stored as described above. General Instructions 1. Please read and understand the instructions thoroughly before using the kit.
2. This kit is designed to measure anti-KLH IgM levels in serum collected 5 days after immunization with KLH. Serum collected at post-immunization times greater than 5 days may contain high levels of anti-KLH IgG that compete with anti-KLH IgM for the immobilized KLH, thereby causing interference.
3. All reagents should be allowed to reach room temperature (18-25°C) before use.
4. The optimal sample dilution should be determined empirically. However, studies suggest an initial sample dilution of 500 fold. Please do not use dilutions less than 25-fold.
5. Optimum results are achieved if, at each step, reagents are pipetted into wells of the microtiter plate within 5 minutes.

Date de péremption: The expiry date is stated on the label.

Détail du antigène

Antigène: KLH IgM (Anti-Keyhole Limpet Hemocyanin IgM Antibody (KLH IgM))

Autre désignation: Keyhole Limpet Hemocyanin (KLH) IgM

Classe de substances: Antibody, Antibody