Analysis of specific methods for detection of Chlamydia pneumoniae antibodies by enzyme-linked immunosorbent assay - Master's thesis - Dissertation

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Chlamydia pneumoniae infection is a widespread disease around the globe, often presenting as a latent or mild condition. It can cause a variety of clinical symptoms, primarily atypical pneumonia, but may also lead to pharyngitis, bronchitis, iritis, hepatitis, endocarditis, and nodular erythema. Additionally, it serves as an important pathogen for secondary infections such as AIDS and leukemia. Moreover, it has been linked to the development of coronary heart disease, making it a serious public health concern.

A common diagnostic method for detecting Chlamydia pneumoniae is through ELISA testing, which identifies specific antibodies in the serum. This article will provide a detailed explanation of the operational principles and key precautions associated with this method. Thank you for reading. This month’s special offers are available—don’t miss out! For more information, please contact our online customer service.

The ELISA technique involves incubating serum samples with purified Chlamydia pneumoniae antigens in microtiter wells. After washing away unbound antibodies, horseradish peroxidase (HRP)-labeled anti-human Ig antibodies are added to react with the bound antibodies. The HRP then catalyzes a color reaction with the chromogenic substrate. Finally, the reaction is stopped, and the optical density (OD) is measured using a microplate reader.

Collect fresh peripheral or venous blood. If testing cannot be performed within 24 hours, separate the serum and store it at -20°C. Before testing, bring the sample back to room temperature.

• Pre-coated microplate
• Positive control
• Weaker positive control
• Negative control
• Sample diluent
• Concentrated enzyme conjugate
• Substrate A and B
• Stop solution

Microplate reader or fully automated ELISA detection system.

1. Prepare washing solution by mixing 1 bottle of concentrated wash buffer with 475 ml distilled water. Store at 2–8°C for long-term use.
2. Dilute the enzyme conjugate 1:101 (1 μL of concentrated conjugate + 1000 μL of diluent), and discard after use.
3. After equilibrating the kit to room temperature, dilute the sample 1:51 (4 μL of serum + 200 μL of diluent).
4. Incubate the diluted sample at 37°C for 20 minutes.
5. Add 100 μL of each control (positive, weak positive, negative) and 100 μL of diluted sample supernatant into corresponding wells. Add 100 μL of diluent to the blank well. Cover and incubate at 37°C for 30 minutes.
6. Discard the liquid, then wash 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. Discard the liquid again, wash 5 times, and dry each time.
9. Add one drop of each substrate A and B, or 50 μL of 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 visual assessment is used, skip the stop solution and interpret results directly.

• The average OD value of the negative control must be less than 0.20.
• The average OD of the weak positive control should be between 0.20 and 0.65.
• The ratio of the weak positive control OD to the negative control OD must be ≥2. If all three conditions are met, the result is considered valid; otherwise, repeat the test.
• If the sample's color is darker than the weak positive control, it is considered positive. Otherwise, it is negative. For accuracy, retest if the result is within 10% of the weak positive value. If it exceeds the weak positive, it is considered positive.

Chlamydia pneumoniae antibody is negative.

• Results should always be interpreted in conjunction with clinical symptoms and medical history for appropriate diagnosis and treatment.
• Although the kit includes components to reduce interference, some samples may still show issues. If multiple IgM tests are positive, consider possible RF interference.

Chlamydia pneumoniae and Chlamydia psittaci mainly cause respiratory tract infections. Detecting Chlamydia pneumoniae antibodies is crucial for diagnosing pneumonia and conducting epidemiological studies. In healthy adults, the positive rate of Chlamydia pneumoniae antibodies is approximately 25–50%, while children generally have lower rates.