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Analysis of specific methods for detection of Chlamydia pneumoniae antibodies by enzyme-linked immunosorbent assay - Master's thesis - Dissertation
**Chlamydia pneumoniae Infection and ELISA Testing Method**
Chlamydia pneumoniae infection is a widespread global health concern, often presenting as a latent or mild infection. It can lead to various clinical manifestations, including atypical pneumonia, pharyngitis, bronchitis, iritis, hepatitis, endocarditis, and nodular erythema. Additionally, it serves as a significant pathogen in secondary infections such as AIDS and leukemia. Research has also linked it to the development of coronary heart disease, making it a serious threat to public health.
One of the most commonly used diagnostic methods for detecting Chlamydia pneumoniae is the Enzyme-Linked Immunosorbent Assay (ELISA). This method allows for the detection of specific antibodies in the serum, offering a reliable and efficient way to assess infection status. Shanghai Jinma Bio provides detailed guidance on the operation principle and key precautions for this test. If you're interested in learning more, feel free to reach out to our online customer service for further assistance.
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**Detection Method:**
ELISA Method
**Methodological Principles:**
The ELISA technique involves the use of microtiter plates coated with purified Chlamydia pneumoniae antigens. Serum samples are added to these wells, where they react with the antigen. Unbound antibodies are then washed away, and an HRP-conjugated anti-human Ig antibody is introduced. This enzyme binds to the captured antibodies, and after adding a chromogenic substrate, a color reaction occurs. The reaction is stopped, and the optical density (OD) is measured using a microplate reader to determine the presence of antibodies.
**Sample Preparation:**
Fresh peripheral or venous blood is typically used. If testing cannot be performed within 24 hours, the serum should be separated and stored at -20°C. At the time of testing, the sample should be brought back to room temperature.
**Reagents Required:**
1. Pre-coated microplate
2. Positive control
3. Weak positive control
4. Negative control
5. Specimen diluent
6. Concentrated enzyme conjugate
7. Substrate A and B
8. Stop solution
**Instruments Needed:**
Microplate reader or fully automatic ELISA detector.
**Testing Procedure:**
1. Prepare washing solution by mixing 1 bottle of concentrated wash solution with 475 ml of distilled water. Store at 2–8°C for long-term use.
2. Dilute the enzyme conjugate 1:101 (1 µL concentrated conjugate + 1000 µL diluent), and discard after use.
3. Equilibrate the kit to room temperature, then dilute the specimen 1:51 (4 µL serum + 200 µL diluent).
4. Incubate the diluted specimen at 37°C for 20 minutes.
5. Add 100 µL of each control (positive, weak positive, negative) and the diluted sample supernatant to corresponding wells. Add 100 µL diluent to the blank well. Cover and incubate at 37°C for 30 minutes.
6. Remove the liquid and wash the plate 5 times with 200–300 µL of washing solution per well. Dry each time.
7. Add 100 µL of enzyme conjugate to each well, cover, and incubate at 37°C for 30 minutes.
8. Wash again 5 times and dry.
9. Add one drop each of substrates A and B, or 50 µL each, mix, and incubate at 37°C for 15 minutes.
10. Add one drop of stop solution, measure OD at 450 nm using a microplate reader. If not using a reader, visually assess the color change.
**Result Interpretation:**
1. The average OD of the negative control must be less than 0.20.
2. The average OD of the weak positive control must be between 0.20 and 0.65.
3. The ratio of the weak positive control OD to the negative control must be ≥2. If all three conditions are met, the results are considered valid; otherwise, repeat the test.
4. If the sample’s color is darker than the weak positive control, it is considered positive. For accuracy, retest within 10% of the weak positive value. If higher, it is judged as positive.
**Normal Reference Value:**
Chlamydia pneumoniae antibody negative.
**Precautions:**
1. Results should be interpreted in conjunction with clinical symptoms and medical history.
2. Although the kit includes components to reduce interference, some samples may still show cross-reactivity. If multiple IgM tests are positive, consider possible rheumatoid factor (RF) interference.
**Clinical Significance:**
Chlamydia pneumoniae and Chlamydia psittaci primarily cause respiratory infections. Detecting Chlamydia pneumoniae antibodies helps in diagnosing pneumonia and monitoring the spread of infection. The positive rate among healthy adults is approximately 25–50%, while children generally have lower rates.
If you have any questions or need further information, please contact us via our online customer service. We’re here to help!