B lymphocyte surface marker
Humoral immune function testing is a type of test in which the laboratory evaluates individual humoral immune function. The detection of humoral immune function is based on the basic processes of humoral immunity, including B lymphocyte detection, Ig detection, and detection of antibody production functions.Basic Information
Specialist classification: growth and development check classification: immunological examination
Applicable gender: whether men and women apply fasting: not fastingTips: If the specimen cannot be observed on the day, the cells will be suspended in the fluorescent antibody preservation solution, stored at 4 ° C, and counted the next day. Normal value
The mean IgM was 11.2 ± 6.0%; the mean IgG was 7.5 ± 3.3%.Clinical significance
The decrease in the number of B cells is related to the humoral immune deficiency, and the increase in the number of B cells is related to the malignant proliferation of B cells.Precautions
(1) The presence of agglomerated IgG in the fluorescent antibody reagent, and the agglomerated IgG can be false-positively bound to the Fc receptor on the surface of the B cell. Therefore, in order to avoid agglomeration of IgG, it is necessary to pay attention to:
1 Fluorescent antibodies with an excessive F/P molar ratio cannot be used, and generally 1 to 3 are preferred.
2 Fluorescent antibodies containing low concentration proteins are unstable at low temperature and should not be lower than 2 mg/ml.
3 Fluorescent antibodies stored at low temperature should not be freeze-thawed repeatedly, and centrifuged for 30 min at 150,000 r/min before use to remove aggregates.
(2) When living lymphocytes are used for immunofluorescence staining, NaN3 is added to all reagents (labeled antibody, Hank solution, etc.) to limit the fluidity of the cell membrane. Otherwise, cap-like fluorescence and phagocytosis will occur, and the actual number of fluorescent cells will decrease.
(3) When stained with an anti-IgG fluorescent antibody, the antigen-antibody (IgG type) immune complex contained in the sample binds to the Fc receptor on the surface of the B cell, and is fluorescent positive. For this reason, it has been suggested to stain with fluorescently labeled anti-F(ab)2 antibody to exclude Fc receptors from being stained.
In recent years, due to the advent of monoclonal antibodies against B cell surface specific molecules (CD19, CD20, etc.), it has been tended to use fluorescein-labeled monoclonal antibodies against CD19 or CD20 to directly react with peripheral blood lymphocytes; The monoclonal antibodies (primary antibodies) of CD19 and CD20 are first reacted with lymphocytes, then combined with fluorescein-labeled rabbit or goat anti-mouse IgG (second antibody), counted by fluorescence microscopy or flow cytometry to count peripheral blood. B lymphocytes.Inspection process
(1) Take heparin anticoagulant 2ml, add an equal amount of Hank liquid and mix well. Repeat the layer on the stratified solution, 2000r/min, centrifuge for 20min. The lymphocyte layer was taken out, the Hank solution was added, washed 3 times, 1200 r/min, centrifuged for 10 min, the lymphocyte concentration was adjusted to 1×107/ml, and 0.1 ml was dispensed into two tubes.
(2) Wash the lymphocyte suspension once again with Hank's solution, discard the supernatant, and use the filter paper to remove the remaining liquid from the inner wall of the tube, and add 2 drops of rabbit anti-human IgG and anti-human IgM fluorescent antibody (about 0.08 ml). After gently shaking, place the 37 ° C incubator for 30 min.
(3) Take out from the incubator and wash twice with Hank solution, centrifuge at 1000r/min for 10min, carefully absorb all the supernatant, add 1 drop of 50ml PBS buffered glycerin to the bottom of the tube, mix and take 1 drop to add On the slide, cover the coverslip and observe under a fluorescence microscope.Not suitable for the crowd
There are no taboos.Adverse reactions and risks
No complications or hazards.