Method and kit for performing quick co-detection on anti-Sp (Streptococcus pneumoniae) IgM (Immunoglobulin M) and IgG (Immunoglobulin G) antibodies based on magnetic separation and multi-color quantum dot labeling
A Streptococcus pneumoniae, magnetic separation technology, applied in the field of medical detection, can solve the problems of long time, increased detection cost, increased labor intensity and operational errors
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Embodiment 1
[0076] Embodiment 1 Preparation and purification of recombinant human Streptococcus pneumoniae PspA protein
[0077] 1. Cloning of related genes
[0078]Bioinformatics analysis was performed on the Fam1 PspA and Fam2 PspA proteins of human Streptococcus pneumoniae (the accession numbers in the NCBI protein database are AAF27703 and AAF27712, respectively), and the peptides with the most abundant antigenic epitopes in their extracellular domains were obtained respectively. For the corresponding DNA coding sequence, the whole gene sequence was chemically synthesized respectively after introducing the restriction site NdeI at the 5' end, the termination signal TAA and the restriction site XhoI at the 3' end (the whole sequence synthesis was handed over to GenScript Biotechnology Co., Ltd. Co., Ltd., and the artificially synthesized gene fragments were connected to the vector pUC57 at the time of delivery), which were recorded as PspA1 and PspA2. The full sequence of its gene is ...
Embodiment 2
[0088] Example 2 Preparation of anti-human Streptococcus pneumoniae antibody capture nano-magnetic beads
[0089] 1. Optimization of reaction conditions for recombinant human Streptococcus pneumoniae PspA protein-coupled magnetic beads:
[0090] Using the magnetic beads coupled with the recombinant human Streptococcus pneumoniae PspA protein as the solid phase carrier, the mouse anti-human IgM monoclonal antibody labeled with quantum dots as the detection antibody, detect the anti-human Streptococcus pneumoniae IgM antibody positive serum, observe the magnetic beads and recombinant protein coupling. A series of optimization options were carried out on the particle size of the magnetic beads, as well as the concentration of EDC / NHS activator, the concentration of conjugated antibody, the coupling time, and the type of blocking agent.
[0091] 1.1 Selection of magnetic bead size
[0092] Select carboxyl nano-magnetic beads with a particle size of 50nm, 180nm, 350nm, 1150nm, an...
Embodiment 3
[0104] Example 3 Preparation of anti-human IgM and IgG nanoprobes labeled with multicolor quantum dots respectively
[0105] 1. Optimization of the reaction conditions for nanocarboxyl quantum dot-labeled mouse anti-human IgM monoclonal antibody:
[0106] 1.1. Determination of the optimal labeling pH of the carboxyl quantum dot-labeled antibody probe
[0107] The pH of the phosphate buffer in the labeling reaction was set to 5, 6, 7, 8, and 9 respectively, and the fluorescence intensity of the labeled product was measured with a full spectrometer, and the influence of different pH values on the coupling reaction was observed, and the quantum dot-labeled monoclonal antibody was determined. The optimum pH for the reaction is 7.0-8.0. This experiment chooses pH7.4.
[0108] 1.2. Determination of the optimal labeling amount of carboxy quantum dot-labeled antibody probes
[0109] Set the ratio of quantum dot molar concentration to monoclonal antibody concentration to 1:1, 1:2,...
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