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Hybridoma cell line secreting human serum amyloid a monoclonal antibody and its application

A technology of hybridoma cell line and amyloid protein, which is applied in the field of immunoassay, can solve the problems of uneven quality level and limited quality of kits, and achieve the effect of good versatility, high sensitivity and good specificity

Active Publication Date: 2020-06-16
杭州伊佰新生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the quality of the existing serum amyloid A monoclonal antibodies in the market is uneven, which seriously limits the quality of the kit

Method used

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  • Hybridoma cell line secreting human serum amyloid a monoclonal antibody and its application
  • Hybridoma cell line secreting human serum amyloid a monoclonal antibody and its application
  • Hybridoma cell line secreting human serum amyloid a monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: SAA antigen preparation

[0036] First, using the mRNA coding region of SAA (sequence shown in SEQ ID NO: 2) as a template, remove the signal peptide, and design a pair of primers (sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively).

[0037] Human placental tissue was taken, and RNA was extracted with Thermo Fisher Trizol one-step method, RNA was reverse-transcribed into cDNA with AMV reverse transcription kit, and then cDNA was used as a template to perform PCR amplification with the above primers. The PCR conditions are: 94°C for 5 minutes; 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, 30 cycles of amplification; 72°C for 10 minutes.

[0038] The PCR product was subjected to agarose gel electrophoresis, and after confirming that the molecular weight was correct, the target band was excised and recovered with the AXYGEN Gel Recovery Kit to obtain the SAA gene fragment.

[0039] The amplified SAA gene fragment has a base ...

Embodiment 2

[0046] Embodiment 2SAA antibody preparation

[0047] The SAA recombinant antigen prepared in Example 1 was dialyzed against 20mM Tris-HCl pH 8.0, diluted to 1 mg / ml with dialysate, mixed with adjuvant at a ratio of 1:1, and emulsified.

[0048] Eight-week-old Balb / c mice were immunized with the emulsified SAA antigen, 100ug per mouse, Freund's complete adjuvant was used for the initial immunization, and Freund's incomplete adjuvant was used for booster immunization.

[0049] After three injections of immunization, the tail vein blood was collected to determine the titer, and those with a titer over 1 million were selected for cell fusion.

[0050] Impulse immunization was performed 72 hours before fusion, and 30-50ug of SAA antigen was injected intraperitoneally.

[0051] Kill the mice to be fused, take the spleen, grind it in a 200-mesh sieve, and wash it with RPMI1640 medium at the same time, transfer the cell suspension to a 50ml centrifuge tube, wash it 4 times with RPMI1...

Embodiment 3

[0058] Embodiment 3 monoclonal antibody is paired with SAA-PcAb1

[0059] Label the SAA rabbit polyclonal antibody SAA-PcAb1 with sodium periodate method, take 5mg HRP and dissolve it in 0.5ml water, add fresh 0.06M NaIO 4 0.5ml of aqueous solution, mix well, and place at 4°C for 30min.

[0060] After taking it out, add 0.5ml of 0.16M ethylene glycol aqueous solution, and after standing at room temperature for 30min, add 1ml of aqueous solution containing 5mg of purified antibody, mix well and put in a dialysis bag to dialyze against 0.05M, pH9.51 carbonate buffer overnight.

[0061] Take out and add NaBH 4 Solution (5mg / ml) 0.2ml, put in refrigerator for 2h.

[0062] Add an equal volume of saturated ammonium sulfate, place in the refrigerator for 30 minutes, centrifuge, and resuspend the precipitate with 2.5ml of 20mM PBS, add an equal volume of glycerol, mix well, and store.

[0063] Dilute the 12 strains of monoclonal antibodies to 0.5-4ug / ml with 20mM PB7.4, add 100ul p...

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Abstract

The invention discloses a hybridoma cell strain 4D4 having the preservation number of CCTCC NO:C2019121; the hybridoma cell strain 4D4 can secrete a monoclonal antibody SAA-McAb1 which can specifically recognize a human serum amyloid A. At the same time, the invention discloses an application of the monoclonal antibody SAA-McAb1 in detection of the human serum amyloid A, and a human serum amyloidA immunoassay kit prepared by the monoclonal antibody SAA-McAb1.

Description

technical field [0001] The invention relates to the field of immunoassay, in particular to a hybridoma cell line capable of secreting a monoclonal antibody of human serum amyloid A, the monoclonal antibody secreted by the hybridoma cell line, and the method for detecting human serum amyloid A Amyloid-like protein A, and a human serum amyloid-A detection kit prepared from the monoclonal antibody. Background technique [0002] Serum amyloid A (serum amyloid A, referred to as SAA) is an acute phase protein and binds to plasma high-density lipoprotein (HDL). Similar to CRP, it is used to assess the progress of the acute phase response. SAA is a sensitive parameter. It begins to increase about 8 hours after the inflammatory reaction, and it exceeds the upper limit of the reference range earlier than CRP. However, the difference between the median value of CRP in normal people and the upper limit of the reference range is about 10 times. Only 5x in SAA. In mild infections, eg, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/577G01N33/68
CPCC07K16/18G01N33/577G01N33/68
Inventor 孙兴宝户元林顾丽霞刘奇奇
Owner 杭州伊佰新生物技术有限公司
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