Clostridium difficile GDH ELISA

Transcription

1 Clostridium difficile GDH ELISA For the qualitative determination of C. difficile glutamate dehydrogenase in human stool. For Research Use Only. Not For Use In Diagnostic Procedures. Catalog Number: Size: 50-GDHHU-E01 96 Wells Version: ALPCO August 23, G Keewaydin Drive, Salem, NH P: (800) F: (603)

2 INTENDED USE The ALPCO Clostridium difficile GDH ELISA is intended for the qualitative determination of C. difficile glutamate dehydrogenase in human stool samples. For Research Use Only. Not For Use in Diagnostic Procedures. PRINCIPLE OF THE ASSAY Break-apart microwells are coated with C. difficile GDH specific monoclonal antibodies. A horseradish peroxidase (HRP) conjugated monoclonal anti-gdh antibody is added to the antibody-coated microwells. Fecal samples are diluted in sample diluent and added to the microwells. In this step C. difficile GDH is marked by the HRP conjugated antibody and immobilized by the coated antibody. Unbound conjugate is removed by washing. TMB-substrate is added, the substrate is hydrolyzed by the peroxidase and yields a blue solution of the reduced substrate. Upon the addition of the stop solution, the blue color turns yellow and should be read by an ELISA reader at a wavelength of 450/620 nm. The absorbance is proportional to the level of C. difficile GDH in the sample. MATERIALS SUPPLIED Component E01 Quantity Preparation anti-gdh Microplate (96 wells) 12 x 8 strips Ready to use Positive Control 2.5 ml Ready to use Wash Buffer Concentrate 100 ml 20X HRP Conjugate 7 ml Ready to use Sample Diluent 2 x 50 ml Ready to use TMB Substrate 16 ml Ready to use Stop Solution 16 ml Ready to use Disposable Plastic Pipettes 100 pieces Ready to use MATERIALS REQUIRED Disposable plastic/wooden collectors or teaspoons Precision pipettes for dispensing up to 1000 µl (with disposable tips) Repeating or multi-channel pipette for dispensing up to 1000 µl Volumetric containers and pipettes for reagent preparation Distilled/Deionized/Ultra-Pure water for reagent preparation Microplate washer or wash bottle 37 C water bath with lid or a moisture chamber placed in a 37 C incubator Microplate reader Centrifuge (1,000 x g to 3,000 x g) Vortex for sample preparation Laboratory Balance Page 2 of 7

3 PRECAUTIONS 1. This kit s Control contains Clostridium difficile antigen, which has been inactivated to avoid transmission of infection. Nevertheless, all the Control s components supplied in this kit must be handled as potentially infectious agents, according to the recommendations published in the CDC/NIH manual "Biosafety in Micro Biological and Biomedical Laboratories, The human blood products incorporated into this kit have been tested for the presence of HIV (Human Immunodeficiency Virus), HBV (Hepatitis B Virus), and HCV (Hepatitis C Virus). Test methods for these viruses do not guarantee the absence of a virus; therefore, all reagents should be treated as potentially infections. Handling and disposal should be in accordance with all appropriate national and local regulations for the handling of potentially biohazardous materials. 3. All materials derived from animal sources are bovine spongiform encephalopathy (BSE) negative. However, all materials should be kept from ruminating animals. 4. TMB-Substrate solution is an irritant material to skin and mucous membranes. Avoid direct contact. 5. The stop solution consists of diluted sulfuric acid, a strong acid. Although diluted, it still must be handled with care. It can cause burns and should be handled with gloves, eye protection, and appropriate protective clothing. Any spill should be wiped out immediately with copious quantities of water. Do not breathe vapor and avoid inhalation. 6. When handling assay wells, avoid scratching the bottom of the wells because this may result in elevated absorbance readings. 7. Stool samples, microassay wells, micropipette tips and disposable stool collectors and tubes should be handled and disposed of as potentially biohazards after use. Wear gloves when doing the test. 8. Avoid direct contact with skin. 9. This product is not for internal use. 10. Avoid eating, drinking, or smoking when using this product. 11. Do not pipette any reagents by mouth. 12. Reagents from this kit are lot-specific and must not be substituted. 13. Do not use reagents beyond the expiration date. 14. Variations to the test procedure are not recommended and may influence the test results. STORAGE CONDITIONS The kit should be stored at 2-8 C. The kit is stable until the expiration date on the box label. DO NOT FREEZE. Unused strips must be resealed in the aluminum pouch with the desiccant card, by rolling the open end and sealing tightly with tape over the entire length of the opening. This protects them from moisture. SAMPLE HANDLING Stool samples are appropriate for use in this assay. 1. Standard collection and handling procedures used in-house for fecal samples or culture are appropriate. 2. Preserved stool: The test has not been confirmed with samples after fixation. (E.g. in 10% formalin, Sodium Acetate Formalin (SAF), or Polyvinyl Alcohol (PVA)). 3. Samples should be kept between 2 and 8 C and tested within 48 hours after collection. If testing cannot be performed within 48 hours, store samples at -20 C, or lower. Page 3 of 7

4 4. Multiple freeze and thaw cycles may cause degradation/proteolysis of the antigen and result in loss of activity. Sample Processing 1. Set up one dilution tube for each sample to be tested. 1.5 ml Eppendorf tubes are recommended for this purpose. Add 400 μl Sample Diluent to each tube. Label the tube. 2. Formed samples: Use a wooden applicator stick or a disposable teaspoon to transfer the fecal sample to the tube. Transfer approximately 0.1 to 0.15 g of sample (about the size of a small pea) to the Sample Diluent. Mix the collector in the Sample Diluent to remove as much sample as possible and squeeze the collector against the side of the tube to express any residual liquid. 3. Liquid samples: transfer 150 μl of sample to the tube. Make sure the liquid samples are evenly suspended. 4. Thoroughly mix (vortex) the fecal sample to ensure adequate sampling. 5. Let the tube stand for at least 10 minutes but not more than 30 minutes until large particulate matter is precipitated (decantation). Use upper liquid phase for testing. REAGENT PREPARATION Bring all components and samples to be tested to room temperature. Determine the total number of samples to be tested. In addition to the samples, the following must be included in each test: one well of Negative Control (Use Sample Diluent for this purpose) and one well of Positive Control. Wash Buffer Concentrate (20X) must be diluted with distilled water 1:20 before use (50 ml WASHBUF ml distilled water) and mixed well. All other test reagents are ready to use. Test reagents are stable until the expiry date (see label of test package) when stored at 2 8 C. QUALITY CONTROL Control samples should be analyzed with each run. Results generated from the analysis of control samples, should be evaluated for acceptability using appropriate statistical methods. The results for the samples may not be valid if within the same assay one or more values of the quality control sample are outside the acceptable limits. ASSAY PROCEDURE All reagents and microplate strips (while sealed in foil pouch) should be equilibrated to room temperature prior to use. Gently mix all reagents before use. For automated ELISA processors the given protocol may need to be adjusted according to the specific features of the respective automated platform. For further details please contact ALPCO. 1. Dispense 50 µl of ready-to-use conjugate into each well. 2. Pipette 100 µl of Positive control and 100 µl of Negative Control (i.e., Sample Diluent) into separate wells of the test strip. 3. Dispense 100 µl of diluted stool samples into separate wells of the test strip using the provided disposable pipettes (the lowest mark on the pipette). Page 4 of 7

5 4. Cover the strips with a plate cover and incubate for 50 min at 37 C in a moisture chamber. 5. Washing step: Discard the liquid content of the wells. Fill each well with Wash Buffer up to the end of the well (300 µl). Repeat this step 4 times to a total of FIVE times. Automatic washing machine can be used. 6. Dry the strips and frame by gently tapping them over clean absorbent paper. 7. Dispense 100 µl of TMB-Substrate into each well, cover the strips with a plate cover, and incubate at room temperature for 15 minutes. 8. Stop the reaction by adding 100 µl of Stop Solution into each well. 9. Determine the absorbance at 450/620 nm and record the results. Determination should not exceed 10 minutes following stopping of the chromogenic reaction. Note: Any air bubbles should be removed before reading. The bottom of the ELISA plate should be carefully wiped. CROSS-REACTIVITY The C. difficile GDH test was evaluated using microbial culture isolates and stool samples*. No cross-reactivity was observed with any of the gastrointestinal pathogens and microbes listed below: Blastocystis, Campylobacter*, Cryptosporidium parvum*, Dientamoeba fragilis*, Escherichia coli, Entamoeba histolitica*, Enterococcus faecali, Enterococcus faesium, Enterococcus avium, Enterococcus aerogenes, Enterococcus cloacae, Enterococcus gallinarum, Enterococcus durans, Giardia lamblia*, Helicobacter pylori*, Klebsiella pneumonia, Salmonella enterica*, and Shigella*. Page 5 of 7

6 SHORT ASSAY PROTOCOL Add 50 µl conjugate to each well Add 100 µl controls and samples Cover and incubate 50 min at 37 C, 100% humidity Wash 5 times Add 100 µl TMB Substrate Incubate at RT for 15 min Add 100 µl Stop Solution Read plate Incubation Time = 1 hour 5 minutes Page 6 of 7

7 SUGGESTED PLATE LAYOUT Below is a suggested plate layout for running controls and up to 45 samples in duplicate A Empty Empty B Ctrl 1 Ctrl C Ctrl 2 Ctrl D E F G H Ctrl = Control Numbered wells = Samples Page 7 of 7