JPS59132361A - Method of measuring resistance against infection and composition for phagocyte - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an analytical method for measuring the resistance of an organism to infection, a composition useful for the analytical method, and a method for producing the composition.

Observations have been made in the scientific literature over the past few years that phagocytes of various species present in the blood and in organs such as the lungs generate light by physically digesting particles such as bacteria and inanimate compositions. has been done. These observations are based on specialized cells in the blood or organs that are known to perform the important function of seeking out, engulfing, and killing particles such as bacteria through complex chemical mechanisms. It is. These cells also have the ability to seek out non-infectious substances such as particulate pollutants present in the air and remove them. Part of the chemical mechanism of such cells involves the production of relatively energetic oxygen intermediates.

Phagocytes produce chemicals such as those mentioned above when stimulated to engulf cells. These high energy oxygen intermediates are reduced to lower energy levels and light is generated in the process. Such low levels of light can be detected using instruments such as liquid spectrometers, but such methods are very tedious and some methods require large numbers of samples or samples with many variables to be investigated. cannot be analyzed quickly. Further, although the above-mentioned phenomenon has been known for some time, it has been almost impossible to utilize the above-mentioned phenomenon due to the various problems mentioned above and the inability to obtain relatively stable reagents.

The present invention relates to a method for measuring phagocytic responses of various phagocytes. Such methods take advantage of the phagocytic process used by organisms to fight infection or remove particulate matter from areas such as the lungs. A sample of phagocytic cells is taken from the blood or organs of a human or animal, a particle-containing material is added to the sample, and the material is engulfed by the phagocytic vacuole. The phagocytes respond to the particle-containing material by generating oxygen intermediates that react with chemicals present in the particle-containing material. This reaction generates light, which is detected and measured.

Another aspect of the invention is a reagent containing particles as described above configured to be engulfed by phagocytes, comprising:
5-amino-2,3,dihydro=1,4-metallic acid dimedione (Luminol) comprises a particle-containing reagent comprising polymer particles coated with a protein conjugated.

A further aspect of the invention is a reagent containing particles as described above, configured to be engulfed by phagocytes, removed from yeast cell membranes (Zymosan) and mixed with a luminescent chemical (Luminol). contains a reagent containing polysaccharide particles that have absorbed the protein.

Yet another aspect of the invention includes a method of making coated polymer particles as described above according to the invention.

Polymer particles approximately 2-9 microns in diameter are coated with protein and luminescent chemicals. The luminescent chemical may be coated onto the protein or may be mixed with the protein to attach the protein and luminescent chemical to the polymer particles. The particles thus obtained are washed, resuspended to final concentration and lyophilized.

A further aspect of the invention includes a method of making coated zimosan particles useful in the methods of the invention.

Zimosan particles are coated with protein. Luminol is mixed with the thus coated particles and the material thus obtained is freeze-dried to a stable and homogeneous product.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention will be explained in detail below by way of example embodiments with reference to the accompanying figures.

Although any particle-based material compatible with the phagocytic system may be used, organ-derived polymeric materials are preferred, particularly polyacrylamide and zimosan particles such as 3io-Rad® particles. I understand that.

In order for the phagocytic system to function properly, it is necessary to coat the particulate material with proteins.

Such protein coating may be carried out by actual chemical linking of the protein to the particles or by simple electrostatic adsorption. Any suitable protein or mixture of proteins may thus be used to activate the particles. Suitable proteins include purified human immune globulin G or human or animal serum.

A luminescent chemical is then coated onto the protein-coated particles, either chemically or by adsorption, or mixed with the protein-coated particles. ! II! Although any luminescent chemical that directly or indirectly generates light as an intermediary may be used,
Luminol sold by Or (lanics) has been found to be particularly suitable.

If polymeric particles are used, the luminol is dissolved in an aqueous buffer solution and preferably reacted to form a nitrogen intermediate which confers reactivity.

The nitrogen intermediate is then reacted with the protein-coated polymer particles. This reaction forms an azoluminol-protein attack and an electrostatically immobilized luminol-polyacrylamide. The particles thus obtained are washed, resuspended to a final temperature, filled into glass bottles and lyophilized. The material obtained by such a process is a stable and homogeneous product.

Luminol may also be mixed with a suspension of zimosan particles already coated with a suitable protein. The mixture thus formed is filled into glass bottles and dried and frozen. The material obtained by such a process is a stable and homogeneous product.

The dry-frozen product described above, after being mixed with water, is ready for use in the analytical method of the present invention.

In analytical methods using polymer particles, 50μ
Approximately 2 million to 2 million polyacrylamide particles dissolved in a 100 μm buffer solution are mixed with about 200,000 purified phagocytes dissolved in a 100 μm buffer solution. Two exemplary analytical methods performed using zimosan particles are: (1) blood is diluted 1:3 (one part anticoagulant blood and two parts buffer solution); 50 μm of such dilution was then coated with 200 μm containing zymosan.
mixed with m luminol. Each ml of Zimosan mixture contains approximately 50 million particles.

(2>50 μm zimosan suspension is mixed with a 100 μm buffer solution containing approximately 200,000 purified phagocytes. The light generated by these mixtures is measured periodically over a period of time. The phagocytic activity and biochemical activity of phagocytes are measured. Cells that may be measured by this method include neutrophil leukocytes, mononuclear leukocytes, and follicular macrophages.

The first two of these cells are off (l
It is obtained from blood that has been placed on an i11 degree gradient and centrifuged, such as I-Lypaque®. These cells band in the gradient and are thereby purified. Macrophages are obtained from lung lavage fluid and do not require purification. In the examples below, neutrophil leukocytes are used.

Example 1 The above-mentioned polyacrylamide particles of 2001+1CI with a diameter of 2-9μ are suspended in 20ml of water. To this suspension, 10 mg of human immunoglobulin G (Ig
G) is added. The protein particle suspension is gently stirred and then rapidly cooled to 2-8°C. then 4
011 g of 1-ethyl-3,3-dimethyl aminopropyl carposiimide hydrochloride is added. 41u150+ng of glycine aged 6 hours is added. The mixture thus obtained is stirred at 2-8°C overnight.

The next day the suspension was mixed with saline phosphate buffer, 1.4M sodium chloride dissolved in saline phosphate buffer, and finally 0.005. Washed by centrifugation 1411 in M phosphate buffer solution. The particles are centrifuged again and 0.5 of 8°51111 with pl-1 of 8.5
resuspended in a borate buffer solution of M.

The diazonium salt of luminol is formed by suspending 200 m(l) of luminol in 2.4N hydrochloric acid. The mixture is quenched on ice. It then contains 100 mg of sodium nitride per ml. 2
. The pH was then measured and the pl-1 was 7.0 with ION sodium hydroxide.
It is adjusted to 1±0.1. The diazonium salt of luminol 141 is then added to 8.5 ml of the above resuspended particle solution and its I)H is adjusted to 8.5.

This suspension is mixed for 3 hours in the dark at 2-8°C.

The thus diazotized particle suspension then has a pH of 8.
Dialyzed against several changes of 0.2M borate buffer solution at 2-8°C.

This dialysis is carried out for at least 24 hours.

The particles are then soaked in a 0.2M borate buffer solution with a pH of 8.0 until the supernatant is less than 1°0% of the initial luminescence output of the total suspension, i.e., more than 99% of the luminol associated with the particles. The cells are washed by centrifugation.

The particles are then centrifuged once more. The particles thus treated are resuspended in a phosphate buffered solution containing salt. The concentration of particles is adjusted to approximately 50 million particles per ml using a blood cell ε1 counter.

Then the suspension is 1. It is filled into a glass bottle in an amount of □m+ and freeze-dried until the residual water content as measured by Karl Fischer titration becomes 5.0% or less.

1 Om in a glass bottle for use in phagocytic analysis.
1 of pure water is added.

Example 2 Zymosan A (sold by 3igma Chemical CO., St. Louis, Missouri, USA) has a concentration of 24 m9/ml.
suspended in a salt-containing phosphate buffer solution until the temperature reaches 81 degrees.

To this suspension is added an equal volume of 50% normal rabbit serum diluted in saline phosphate buffer solution.

The suspension thus obtained is incubated for 1 hour at 37° C. in an agitated water bath.

The suspension was then centrifuged, the supernatant was removed, and the thus treated ZylIlσ5anA (pelleted)
is resuspended in a small amount of salt-containing phosphate buffer. 1q of the suspension thus obtained is then aspirated through a syringe needle (18-26 gauge) to homogenize it. Finally, enough phosphate buffered solution containing salt is added to the suspension to make it four times its original volume, which brings the concentration to about 6
It is assumed to be a suspension of mMm1. The suspension is then passed through a glass wool plug to capture any remaining large lumps of zymosan.

A stock solution of luminol is formed by dissolving luminol in 0.01N sodium hydroxide. A small amount of the stock solution thus formed was then added to a solution containing 20% fetal calf serum in a saline phosphate buffer solution, which brought the concentration of luminol to 14.4μ (1/
ml.

The luminol-serum solution described above is then added to the zymosan suspension in a 1:1 ratio. The suspension thus obtained is mixed, 2. Qml is filled into a glass bottle and freeze-dried until the residual moisture is 5% or less as determined by Karl Fischer titration.

For use in phagocytic analysis, add 2 Om into a glass bottle.
1 of pure water is added.

Example 3 Several children previously diagnosed with chronic granulomatous disease (COD) were tested using the reagents and analytical methods described above. This disease was chosen because it clearly causes functional impairment. CGD is caused by the absence of certain enzymes that should be present in blood phagocytes. Although phagocytes are capable of engulfing bacteria (particles), phagocytes cannot be used against inactive or dead bacteria because they do not have the ability to form high-energy oxygen intermediates. . Therefore, phagocytes cannot oxidize the luminol engulfed by them, and therefore no luminescent response can be observed. Clinically, COD
Children affected by this disease generally have a greatly reduced resistance to infection. See Figures 1-3, which show typical responses. When several children with CGD were tested along with apparently healthy control subjects, particles were swallowed as effectively as in healthy subjects, but the light produced by the oxidation of luminol was not produced. I didn't. Figure 1 shows a typical response when diluted blood is subjected to chemiluminescence analysis using a zymosan-based reagent, and Figure 2 shows a typical response when diluted blood is subjected to chemiluminescence analysis using a zymosan-based reagent. Figure 3 shows a typical response when isolated neutrophil leukocytes are analyzed using a reagent based on polymer particles. This shows a positive response.

Example 4 A group of children previously diagnosed with asthma were tested for phagocytic/biochemical responses. Approximately half of the children tested were receiving theophylline therapy, while the others were not receiving such drugs. Theophylline is thought to have an effect on cyclic adenosine monophosphate (c-AMP), which is known to have a regular regulatory effect on the biochemical functions of phagocytic species. See Figures 4 and 6. Oxygen intermediates, as quantified by reduced luminol oxidation and luminescence in children receiving theophylline. Figure 4 shows a typical response when diluted blood is subjected to chemiluminescence analysis using a zymosan-based reagent, and Figure 5 shows the isolated This figure shows a typical response when neutrophil leukocytes are analyzed using a zymosan-based reagent.
The figure shows a typical response when isolated neutrophil leukocytes are analyzed using polymer particle-based reagents.

Methods such as those described above are particularly useful in quantifying resistance to infection with respect to phagocytic activity. However, methods such as those described above are also used in medical diagnostics, environmental immunotoxicology, pharmacology, etc., for example in monitoring toxicity in patients undergoing chemotherapy or radiotherapy. The method described above may also be used to test for immunocompetence and serum protein deficiencies in certain species.As a research tool, the method described above may be used to test drug compounds and their effects on phagocytic cells, and the phagocytic cells of contaminants. may be used to test the effects of toxic compounds on animals and humans.

Although the present invention has been described in detail with respect to specific embodiments above, the present invention is not limited to these embodiments, and various embodiments are possible within the scope of the present invention. will be clear to those skilled in the art.

[Brief explanation of drawings]

FIG. 1 is a graph showing a typical response when diluted blood is subjected to chemiluminescence analysis using a zymosan-based reagent. FIG. 2 is a graph showing a typical response when isolated neutrophil leukocytes are analyzed using a zymosan-based reagent. FIG. 3 is a graph showing a typical response in an analysis of isolated neutrophil leukocytes performed using a polymer particle-based reagent. FIG. 4 is a graph showing a typical response when diluted blood is subjected to chemiluminescence analysis using a zymosan-based reagent. FIG. 5 is a graph showing a typical response when isolated neutrophil leukocytes are analyzed using a zymosan-based reagent. FIG. 6 is a graph showing a typical response when isolated neutrophil leukocytes are analyzed using a polymer particle-based reagent. Patent Applicant: Barakad Instrument Company, Inc. - Borei Detsudo Agent, Patent Attorney, Masa Akira, J7”c-+: 15AO & Ginma
jg, 2 J7°0+; l+7 'r A5 hours [intermediate] Fig, 3, i7'c+;rt75p4r&QI'IQ [e) F
jg, 4 j7t,! :+r'akke value'1 (minute) ri・5 J7#ct consideration+71.tJylkfj!Pq E excluded]F
ig.
Shakyu 1, jl So% tuna.

Claims (4)

[Claims] (1) A method for measuring the resistance of living organisms to infection, which involves collecting blood samples from living organisms, separating phagocytes from the blood, and coating them with proteins conjugated with luminescent chemicals. the phagocytic cell biochemistry of adding the particles to the phagocytic cells and causing the phagocytes to engulf the particles, thereby reacting with the luminescent chemicals to produce light that is measured in a luminometer. and activating a physical mechanism. (2) A composition for phagocytic cells comprising polyacrylamide particles having a diameter of about 2 to 9 microns and coated with a layer of serum proteins and a layer of luminol conjugated thereto. (3) A phagocytic composition comprising zymosan particles coated with a layer of serum proteins and luminol. (4) A method for producing a reagent for phagocytic cells, in which the diameter is approximately 2
A method comprising coating particulate material of ~9μ with a layer of protein and luminescent chemical and lyophilizing the thus coated particles into a stable and homogeneous product.