A cell-mediated immune response assay and kits therefor

A kit, immune effect technology, applied in the determination/inspection of microorganisms, material testing products, biological testing, etc., can solve problems such as reducing sensitivity

Inactive Publication Date: 2010-05-19
CELLESTIS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] If whole blood is used, it is usually diluted in culture medium to dilu

Method used

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  • A cell-mediated immune response assay and kits therefor
  • A cell-mediated immune response assay and kits therefor
  • A cell-mediated immune response assay and kits therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Detection of immune effector molecules in small volume samples incubated with antigen

[0131] Whole blood from healthy donors (4 donors) was collected into 9 mL Vacuette Li-heparin tubes with approximately 6.6 mm diameter cylinders and U-shaped bases. Aliquots of 0.1, 0.2, 0.3, 0.4 and 0.5 mL of blood (no additives) were stimulated in Vacuette micro collection tubes. Blood was stimulated with tetanus toxoid and phytohemagglutinin-P (mitogen). The volume of antigen added to each tube is proportional to the volume of blood, eg, 0.1 mL of blood is stimulated with 0.01 mL of antigen and 0.4 mL of blood is stimulated with 0.04 mL of antigen.

[0132] The IFN-γ responses generated in small volumes of blood were compared to those generated in 13 / 75 Vacuette blood collection tubes (control) using 1 mL of blood.

[0133] Blood was incubated with antigen at 37°C for 16 to 24 hours before removal of plasma for IFN-γ detection (QuantiFERON-TB Gold ELISA (QuantiFERON-TB Gold ELIS...

Embodiment 2

[0137] Detection of Immune Effector Molecules in Small Volumes of Whole Blood Incubated in Containers with Different Internal Shapes

[0138] Heparinized blood was dispensed into 3x5 mL aliquots in polypropylene tubes. Antigens, either human cytomegalovirus (CMV) or tetanus toxoid (Tetanus), are added to the blood at appropriate concentrations. Each tube was mixed thoroughly so that the blood (50 μl) was dispensed into various containers where the blood volume took different heights. Specifically, PCR tubes (conical, height 10 mm, maximum diameter 5 mm, U-shaped base), micro-collectors (cylindrical, 6.5 mm diameter, U-shaped base), 96-well plates (cylindrical, 6.5 mm diameter, flat bottom) , 48-well plate (cylindrical, 11 mm in diameter, flat bottom). Containers were incubated at 37°C for 20 hours before removal of plasma (20 μl) for testing by QFT-ELISA using labeled antibodies to detect IFN-γ production. In a control experiment, 1 mL of blood was incubated in a cylindrica...

Embodiment 3

[0140] Detection of immune effector molecules in peripheral capillary blood

[0141] Capillary blood (150 μl) was collected by finger prick into lithium heparin micro-collection tubes. Transfer small volumes (50 μl) to three PCR tubes. The PCR tubes are conical with a cylindrical upper portion that tapers from 10mm to a conical base. Antigens for CMV or Tetanus toxoid (Tetanus) or no antigen (Nil) were added to the tubes, which were incubated at 37°C for 20 hours. Thereafter, plasma (20 μl) was removed and tested using QFT-ELISA. As shown in Figure 5, the positive CMV control subject (TR) samples produced a significant signal compared to the negative controls, indicating that the assay can be performed in volumes as small as 50 μl of capillary blood.

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Abstract

The present invention provides methods and kits for measuring a cell-mediated immune (CMI) in a small volume of whole undiluted blood collected from a subject. In particular, the methods are for measuring responses in undiluted whole blood samples having a volume of, for example, 50[mu]l to 500[mu]l. Thus, capillary sampling and rapid testing of subjects including paediatric, adult or geriatric human subjects are facilitated.

Description

technical field [0001] The present invention mainly relates to methods and kits for diagnosis, monitoring or treatment, which measure cellular responsiveness to in vitro agents (originally "agents in vitro", which may also be referred to as "in vitro factors"). Specifically, the present invention provides a system for measuring cell-mediated immune (CMI) responses to antigens in small samples of whole blood collected from a subject. The method and kit will find broad application in the analysis of whole blood samples of a range of different volumes including those from infants and children or other subjects where the sample volume is limited or where there is a need Small sample volume. Background technique [0002] A bibliography of publications cited by the authors in this specification is gathered at the end of the specification. [0003] References in this specification to any prior publications (or information derived therefrom), or any known matter, are not and shoul...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/49G01N33/50C12Q1/68
CPCG01N33/56972G01N2333/35G01N2333/57G01N33/6863
Inventor A·J·雷德福S·L·琼斯J·L·霍华德
Owner CELLESTIS
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