Manual for Cryptococcus GXM Antigen Indirect Competition Detection Kit

PRINCIPLE OF THE PROCEDURE

The kit uses an indirect competition method to detects the antigen level of human cryptococcus neoformans capsular polysaccharide (GXM) in the specimen. Purified cryptococcus neoformans capsular polysaccharide (GXM) is conjugated  with inert protein antigen and then used to coated the microplate, then sample to be tested, standard sample and mouse antibody which anti-capsular polysaccharide are adding into coated micropores sequentially. Free GXM and the GXM which conjugates on the microplate compete to bind anti-GXM antibodies, and washed away unbound antibodies. Subsequently, HRP conjugated rabbit-anti-mouse antibodies are added and the antigen-antibody-enzyme conjugated antibody complex is formed, then adding TMB for color development after thorough washing. TMB is converted into blue under the catalysis of the HRP enzyme and finally turn to yellow by the acid. The depth of color is inversely related to the level of cryptococcus neoformans in the sample. The absorbance (OD value) was measured by a microplate reader at 450nm wavelength and the concentration of human cryptococcus neoformans GXM in the sample was calculated by the standard curve.

REAGENTS

SPECIMEN COLLECTION and REQUIREMENTS:

• Serum: Blood is naturally coagulated for 10-20 minutes  at room temperature and centrifuged for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant and centrifuge again if precipitation occurs during storage.
• Plasma: EDTA or sodium citrate should be selected as an anticoagulant according to the requirements of specimens. After mixed for 10-20 minutes, centrifuged for about 20 minutes (2000-3000 rpm). Then collect the supernatant carefully, and centrifuge again if there is precipitation during storage.
• Cerebrospinal fluid: Collect with a sterile tube and centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant, and centrifuge again if there is precipitation during storage. Thoracic and abdominal fluid shall be carried out according to this method.
• Cell culture supernatant: When detecting secretory components, use sterile tubes to collect them. Centrifuge for about 20 minutes (2000-3000 rpm), carefully collect the supernatant. When detecting the intracellular components, the cell suspension is diluted with PBS (PH7.4) to adjust the cell density at about 1 million /ml. Through repeated freezing and thawing, cells are destroyed and intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant, and centrifuge again if there is precipitation during storage.
• Tissue specimen: Cutting the specimen and weigh it, and add some PBS (PH7.4). Use liquid nitrogen to quickly freeze and store for later use. The temperature of the specimen is still kept at 2-8° C after melting. Add some PBS (PH7.4) after melting and homogenize the specimen fully by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. After sub-packed, one portion is to be tested, and the rest is frozen for later use.
• Samples should be extracted as soon as possible after collection. Extraction should be carried out according to the relevant assay. Experiments should be carried out as soon as possible after extraction. If the test cannot be carried out immediately, the specimen can be stored at -20° C, repeated freezing and thawing should be avoided.
• NaN3 inhibits horseradish peroxidase (HRP) activity, samples containing NaN3 cannot be detected with this kit

PROCEDURE

• Reagent preparation: 30 times (20 times of 48t) of concentrated washing Solution is diluted with 30 times (20 times of 48t) of distilled water for later use.
• Dilution and Sampling of Standard sample: Set 9 well for standard sample on enzyme coated plate, add 100μl of standard sample in the first hole, then add 50μl of standard diluent in the 2nd ~ 9th holes and mix evenly; Then take 50μl from the first hole and add it to the second hole respectively, and gradually dilute it to the ninth hole. (After dilution, the sample addition amount of each well is 50μl, and the concentration is 50ng/L, 25ng/L, 12.5ng/L, 2-fold gradient reduction). The 10th well was set as a blank control and only 50μl of standard diluent was added.
• Sampling: For the well of Sample to be tested. Add 25μl of sample diluent to the sample well to be tested on the enzyme-labeled coating plate, and then add 25μl of sample to be tested (the final dilution of the sample is 2 times). Add the sample to the bottom of the well of the enzyme coated plate, and avoid touch the wall of the well, gently shake and mix
• Addition of competitive antibody: 50μl of competitive antibody was added to each well.
• Incubation: The plates were sealed with a sealing film and incubated at 37° C for 25 minutes.
• Washing: Carefully remove the sealing film, discard the liquid and spin it dry. Fill each hole with washing liquid, stand for 30 seconds and discard, repeat this for 5 times, and pat it dry.
• Enzyme addition: 100μl of enzyme Linked reagent was added to each well, except for blank wells.
• Incubation:  The operation is the same as 3.
• Washing: The operation is the same as 5.
• Color development: Add 50 μ L of TMB solution A and 50 μ L of TMB solution B to each well, mix well with gentle shaking, and develop color at 37° C in dark for 15 minutes.
• Termination of reaction: Add 50μl of stop solution to each well to stop the reaction (blue turns yellow at this time).
• Determination: Zero by the blank well and the absorbance (OD value) of each well was measured in sequence. The determination shall be carried out within 15 minutes after the stopping solution is added.

Calculation

Using the concentration of the standard sample as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper; According to the OD value of the sample, find out the corresponding concentration from the standard curve and multiply it by the dilution multiple. Or calculate the linear regression equation of the standard curve according to the concentration of the standard sample and the OD value. Then calculate the concentration of the sample with the OD value of sample and the equation, and multiply by the dilution multiple to obtain the actual concentration of the sample.

NOTES:

• After the kit is taken out of the refrigerated environment and needs to equilibrate at room temperature for 15-30 minutes before it can be used. If the enzyme coated plate is not used up after being opened, the slats should be put into a sealed bag for storage.
• Concentrated washing solution may have crystallization and precipitation, which can be heated in water bath to help it dissolved when diluting, and result will be not affected.
• The pipette shall be used for each step of sample addition and its accuracy shall be checked frequently to avoid errors. It is better to control the sampling time within 5 minutes at each time. If the number of samples is too much, it is recommended to use a Multi-channel pipette for sampling.
• Please make a standard curve at the same time of each measurement, and it is better to make multiple compound holes. If the content of the substance to be tested in the sample is too high (the OD value of the sample is greater than the first well of the standard well), dilute it with the sample diluent for a properly times (n times) and then measure it. When calculating, please multiply it by the total dilution number (×n).
• The sealing film is only used once to avoid cross-contamination.
• Store TMB solution in dark
• Strictly follow the instructions and the experiment results must be based on the reading of the microplate reader.
• All samples, washing solution and various wastes shall be treated as infectious substances.
• Components of different batches of this reagent shall not be mixed.
•  In case of any discrepancy with the Chinese manual, this manual shall prevail.

Kit performance

The difference between intra-batch and inter-batch is less than 9% and 12% respectively.

Detection range:

0.5ng/L – 100ng/L

(Commonly used clinical CUTOFF: 8ng/L)

storage and shelf life:

1. Storage: 2-8° C.

2. Shelf life: 6 months.

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