Anti Clostridium Difficile Toxin A ELISA Kit

Catalog No. ABIN1098191

Product Details

Target: Anti Clostridium Difficile Toxin A

Reactivity: Mouse

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: The ELISA was developed to routinely detect the antibody level against Clostridium difficile toxin A or B (TcdA or TcdB) in mouse sera/plasma and stool specimen

Sample Type: Fecal, Plasma, Serum

Analytical Method: Qualitative

Specificity: ELISA for the detection of Clostridium difficile Toxin A antibodies

Components:

• 1 x ELISA Plate 12 x 8 stripes
• 1 x 50 mL dilution buffer
• 1 x 1.0 mL pos. control anti TcdA - human IgA antibodies
• 1 x 1.0 mL pos. control anti TcdA - human IgG antibodies
• 2 x 3 mL conjugate anti human IgA-HRP
• 2 x 3 mL conjugate anti human IgG-HRP
• 1 x 30 mL 10x washing buffer
• 1 x 14 mL substrate
• 1 x 7.5 mL stop solution with 0.5 M H2SO4
• 1 x Manual

Application Details

Plate: Pre-coated

Protocol:

1. Prior to testing, prepare 1x Wash Solution:
   • The Wash Buffer is supplied as a 10x concentrate.
   • The 30 ml supplied need to be diluted to a total volume of 300 ml by adding 270 ml aqua dest.
   • Store your diluted 1x Wash Solution between 2°C and 8°C to avoid growth of contaminating microbes.
   • Preparation of aliquots of the Wash Buffer is done accordingly.
2. Microtitre plate preparation:
   • The plates are sealed in aluminum bags and once opened should be resealed with the snap closing.
   • Once opened stability of the plate at 4°C is about 4-6 months.
   • The plates can be used as broken “single wells” or as single strips.
   • Each strip contains 8 wells coated with Clostridium difficile Toxin A.
   • Prior to testing you need to determine the number of wells for your assaying.
   • Do not contaminate the wells with your fingers.
   • Assay wells not used should immediately be returned to the bag and carefully resealed with desiccant.

Assay Procedure:

1. Pipette 100 µl of the specimen (serum or antibody dilution) diluted in Dilutions Buffer into each single well. As positive control use 100 µl of the supplied control. For the negative control use 100 µl of the Dilution Buffer.
2. Incubate for 60 min at 37°C.
3. Wash each well 3 x with Wash Buffer. After washing, completely remove any residual liquid by striking the plate (wells) onto a dry paper.
4. Pipette 100 µl of the conjugate to detect antibodies against Toxin A
5. Incubate for 60 min at 37°C.
6. Wash each well 3 x with Wash Buffer. After washing, completely remove any residual liquid by striking the plate (wells) onto a dry paper.
7. Thereafter add 100 µl substrate to each well
8. Incubate for 20 min at RT.
9. The color development will be stopped by adding 50 µl of the stop solution to each well.
10. Measurement of the color will be by a spectrophotometer at 450 nm versus 620 nm.

Calculation of Results:

• Measurement is at 450nm and 620nm: The read out of the assay is based on the measurements of the optical density at 450 nm and 620nm and is calculated as OD450-OD620.
• Negative control: The OD450-620 background should be below 0,100.
• Positive control: The OD450-620 of the positive control should be >1,00.
• Cut off value: The cut off of the assay is at OD450-620 0,3.
• Typical results: Typical results of an antibody titration of hospitalized patients is given in the diagram attached. The titration started with a 1:100 pre-dilution of the sera and was done in 2-fold steps. As can be seen the IgGtiter of the serum of the patient 1 is > 1:3200.

Restrictions: For Research Use only

Handling

Preservative: ProClin

Handling Advice:

• Wear gloves for all manipulations with potentially contaminated or toxic suspensions.
• All reagents should be at room temperature prior to their use in the assay.

Storage: 4 °C

Target Details

Target: Anti Clostridium Difficile Toxin A

Target Type: Antibody