German IBL Chlamydia pneumoniae IgM ELISA Kit Instructions for Use

Germany IBL Chlamydia pneumoniae IgM ELISA kit

(Article number: RE57051)

1. Description of the preface

Chlamydia is an immobile Gram-positive bacterium and a mandatory intracellular parasite that forms distinct inclusion bodies in the cytoplasm of its host cells. This inclusion body is easily visible under an optical microscope. It is known that three different chlamydia are pathogenic to humans: Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci, and Chlamydia (Chlamydia cerevisiae) which is pathogenic to animals. Chlamydia trachomatis is the most prevalent transmission factor for sexually transmitted diseases in the world (40-50 million cases), and the number of this infectious disease is increasing. Pregnant women infected with Chlamydia trachomatis can transmit the virus to the fetus during childbirth, resulting in conjunctivitis or pneumonia in the fetus. Individuals with untreated chlamydial infection can cause chronic salpingitis and are more likely to have ectopic pregnancy or infertility. Among men, Chlamydia trachomatis is a major cause of non-gonococcal urethritis. In chlamydial infections, frequent asymptomatic recessive stages are a serious problem because it may initiate chronic diseases. In many cases, primary infections have not been detected, and only sequelae caused by persistently rising pathogens can only be detected.

The following methods can be used to detect infection:

n microscopic examination: Giemsa staining

n PCR

n Serology: detection of antigen by ELISA

Detection of antibodies by IF, EIA or ELISA

2 , the scope of application

IBL's Chlamydia pneumoniae IgM ELISA kit can be used for qualitative detection of anti-Chlamydia pneumoniae IgM antibodies in human serum.

3 , the principle of experiment

Qualitative immunoenzyme assays against Chlamydia pneumoniae IgM antibodies utilize ELISA techniques. The coating plate was pre-coated with a Chlamydia pneumoniae antigen that binds to the relevant antibody in the sample. Plates were washed to remove unbound sample material, followed by horseradish peroxidase-labeled anti-human IgM. Thereafter, the enzyme standard binds to the captured specific antibody against Chlamydia pneumoniae, and after adding a TMB substrate solution capable of producing a blue reaction product, if blue is displayed, it is known that an immune complex has been formed. The intensity of the displayed color is directly proportional to the amount of Chlamydia pneumoniae-specific IgM antibody in the sample. Sulfuric acid was added to terminate the reaction to produce a yellow endpoint color. The OD value was read at 450 m using a microplate reader.

4 , materials

4.1 Kit components

1) Chlamydia pneumoniae coated plate (IgM): 12 × 8, detachable, microporous coated with Chlamydia pneumoniae antigen, vacuum sealed.

2) IgM sample dilution: 1 bottle, 100ml, used for dilution of the sample, containing anti-human IgG, pH 7.2 ± 0.2, ready to use, green white cover.

3) Stop solution: sulfuric acid to be used, 1 bottle, 15 ml, containing sulfuric acid, 0.2 mol/l, red cap.

4) Concentrated washing solution (20X): 1 bottle, 50ml, pH 7.2±0.2, white cover.

5) Chlamydia pneumoniae anti-IgM enzyme standard: 1 bottle, 20 ml, containing peroxidase-labeled rabbit anti-human IgM, red black cap. Ready to use.

6) TMB substrate solution, 1 bottle, containing TMB, yellow cover, 15ml, ready to use.

7) Chlamydia pneumoniae IgM positive control, 1 bottle, yellow liquid, 2ml, red cover

8) Chlamydia pneumoniae IgM critical quality control, 1 bottle, yellow liquid, 2ml, green cover

9) Chlamydia pneumoniae IgM negative control, 1 bottle, yellow liquid, 2ml, blue cover

4.2 Materials and equipment required for the experiment

1) Microplate reader, which can measure absorbance at 450/620

2) 37 ° C incubator

3) Manual or automatic micro-hole cleaning equipment

4) 10μl and 1000μl sampling tips

5) Vortex mixer

6) Deionized or distilled water

7) Test tube

8) Timer

5 . Sample storage and preparation:

5.1 Use human serum as a sample. If the test is performed within 24 hours of sample collection, the sample should be stored at 2-8 ° C; otherwise it should be stored at -70 ° C - 20 ° C. If the sample has been frozen, carefully thaw the sample before testing to avoid repeated freeze-thawing.

5.2 dilution of the sample

Prior to testing, all samples should be diluted 1:100 in dilutions with IgM sample dilutions. 1 ml of IgM sample dilution was added to 10 μl of sample and mixed thoroughly on a vortex mixer. Positive control and negative control do not need to be diluted and are ready for use.

6 . Experimental steps:

Read the steps carefully before starting the experiment and strictly follow the steps to get reliable results. If this experiment is performed on an ELISA automated system, in order to avoid the effects of washing, I recommend that the plate be washed 3 times to wash the plate 5 times, and the washing solution is increased from 300 μl to 350 μl. All samples and controls should be labeled on the sample sheet provided with the kit prior to the start of the experiment. Take the appropriate number of slats on the shelf.

Please make at least the following holes:

1 hole (example A1) as a blank control hole

1 hole (example B1) for negative control

2 holes (example C1+D1) for critical quality control

1 hole (example E1) for positive control

If necessary, we recommend that you double check the quality control and patient samples. Follow the experimental procedures given in the instructions and avoid no significant differences in the time intervals between the different steps. A new sampling tip should be used for each control and sample. Adjust the incubator to 37 ± 1 °C

1) Add 100 μl of control or diluted sample to the corresponding reaction well, leaving A1 well blank.

2) Cover

3) Incubate for 60 + 5 minutes at 37 °C ± 1 °C.

4) After incubation, remove the sticky metal plate, discard the reflection solution in the well, and wash the plate 3 times with 300 μl of washing solution. The time interval between each plate washing cycle should be greater than 5 seconds, and pat dry on absorbent paper.

Note: It is very important to wash the plate. If the plate is not enough, the test results will be inaccurate and the OD value will be increased incorrectly.

5) In addition to the blank wells, add 100 μl of Chlamydia pneumoniae anti-IgM enzyme standard in each well and cover.

6) Incubate for 30 + 2 minutes at 37 °C ± 1 °C in the dark. .

7) Repeat step 4.

8) Add 100 μl of TMB substrate solution to each well.

9) Incubate at room temperature for 30 minutes in the dark.

10) Add 100 μl of Stop Solution to each well in the same order and speed as the substrate.

11) Read the absorbance at 450 nm in 30 minutes.

7 . The result is judged:

1) The following conditions must be met before the experimental results can be established.

Blank hole A1: OD<0.100

Negative control hole B1: OD<0.200

Critical quality control holes C1 and D1: OD values ​​are between 0.250 and 0.900

Positive control hole E1: OD is greater than or equal to critical

2) Calculation of results: The cut-off value is the average value of the critical quality control test results.

For example: critical quality control OD value 1.44 + critical quality control OD value 0.42 = 0.86/2 = 0.43

Cut-off value = 0.43

3) Interpretation of the results:

If the OD value of the sample is more than 10% higher than the cut-off value, the sample is considered positive.

A secondary sample is considered suspicious if the OD value of the sample is within 10% of the cut-off value. (Gray area) It is recommended to repeat the test with fresh samples after 2-4 weeks. If the result is still in the gray area, the sample is considered negative.

This sample is considered negative if the OD value is more than 10% lower than the cut-off value.

4) Unit of experimental results

Cut-off value

0.43

Critical value: 10U

Gray area: 9-11U

Negative: <9 U

Positive: >11 U

This translation is for reference only, please refer to the original for details.