FR2537723A1 - METHOD AND COMPOSITION FOR EVALUATING PHAGOCYTE REACTION - Google Patents

Abstract

THE PRESENT INVENTION RELATES TO A METHOD AND COMPOSITION FOR EVALUATING THE PHAGOCYTARY REACTION OF AN ORGANISM. THE ACTIVITY OF BLOOD PHAGOCYTES IS MEASURED BY DIRECT ADDITION OF PARTICULAR MATTER CONTAINING PARTICLES OF ZYMOSAN OR POLYACRYLAMIDE COATED WITH PROTEIN ON WHICH LUMINESCENT CHEMICAL COMPOUNDS ARE FIXED. WHEN PHAGOCYTE CELLS ARE MIXED WITH THESE COMPOSITIONS, THE CELLS SWALLOW THE PARTICLES, THUS CAUSING THE ACTIVATION OF THE BIOCHEMICAL MECHANISMS OF THE CELLS. INTERMEDIATE OXYGEN COMPOUNDS ARE FORMED, CAUSING A REACTION WITH THE LUMINESCENT CHEMICAL COMPOUND, WHICH RESULTS IN LIGHT PRODUCTION WHICH IS MEASURED AND ACCORDING TO THE LEVEL OF LIGHT PRODUCED AND THE MAXIMUM PRODUCED, THE PHAGOCYTARY ACTIVITY OF THESE CELLS IS MEASURED. THE INVENTION CAN FOR EXAMPLE BE USED FOR MEASURING THE PHAGOCYTARY ACTIVITY OF CELLS SUCH AS NEUTROPHILS, MONOCYTES AND ALVEOLAR MACROPHAGES.

Description

The present invention relates to a method and a composition for evaluating

  the phagocytic reaction of an organism, that is to say to measure the ability of an organism to resist an infection and the invention also relates to a method of manufacturing such compositions. The observation that certain phagocyta Jres cells found in the blood and in organs such as the lungs create light because of the physical injection of particles such as bacteria or non-living compositions has been published since years in the scientific literature These observations are based on specific cells found in the blood or organs which cells are known to serve a critical function of discovering, phagocyting (swallowing) and killing particles such as bacteria by complex chemical mechanisms These cells can also discover and eliminate non-infectious agents such as polluting particles of

  the atmosphere Part of this cellular chemical mechanism

  re involves the production of oxygenated intermediates at

  relatively high energy The cell will produce these components

  when it is stimulated for phagocytosis.

  These oxygenated high energy intermediates will descend to lower energy levels and during the process, light will be produced although these levels

  light can be detected using

  such as liquid cintillation spectrometers.

  of, this is a very cumbersome technique that does not allow

  not an analysis of a large number of samples or samples

  with a large number of variables to look for.

  And, although this phenomenon has been known for some time, it has benefited little because of the limitations described above together with the unavailability of

relatively stable reagents.

  The present invention relates to a method for

  measure the phagocytic reaction of various phage cells

  Such a process is based on the method of phagocytosis used by an organism to resist infection or to remove foreign matter from the lungs, etc. A sample of phagocytic cells from blood or an organ is taken from the body. A human or other animal and a material containing particles is added thereto and this material is swallowed by the phagocytic cells. The cells react against the material containing the particles and create oxygen intermediates which in turn react with a compound. chemical in the material

  containing particles This reaction creates light.

  This light thus produced is detected and measured.

  Another aspect of the invention comprises such a reagent containing particles adapted to be phagocytosed by the cells and consists of beads

  polymers coated with proteins to which a

  chemiluminescent chemical such as 5-amino-2,3-dihydro-1,4-

phthalazinedione (luminol).

  Another object of the invention is another reagent containing particles adapted to be phagocytosed.

  by the cells and comprises particles of poly-

  saccharides from the walls of yeast cells

  (zymosan) absorbed with proteins to which they are mixed

ge a chemical compound (luminol).

  Another object of the invention is a process for manufacturing particular coated polymeric beads.

  According to the present invention, the polymeric pearls of

  Viron 2 to about 9 μm in diameter are coated with a protein and a luminescent chemical compound. The luminescent chemical compound can be applied to the protein or mixed with the protein and both are applied together to the polymer beads. The resulting particles are washed, resuspended to

  a final concentration and lyophilized.

  Another object of the invention is a method of manufacturing coated zymosan particles useful for the present invention. The zymosan particles are coated with a protein. The luminol is mixed with the coated particles and the resulting material is

  lyophilized into a homogeneous stable product.

  In order that the invention may be better understood, reference is made to the following figures: Figure 1 shows the typical reaction of a chemiluminescent test on whole blood, diluted using

a reagent based on zymosan.

  Figure 2 shows a typical reaction of an isolated neutrophil test using a reagent-based

of zymosan.

  Figure 3 shows a typical reaction of an isolated neutrophil test using a reagent-based

of polymer beads.

  Figure 4 shows a typical reaction of a chemiluminescent test on diluted whole blood using

a zymosan-based reagent.

  Figure 5 shows a typical reaction of an isolated neutrophil test using a

zymosan based reagent.

  Figure 6 shows a typical reaction of an isolated neutrophil test using a

  reagent based on polymer beads.

  Although any particle-based material compatible with the phagocytic cell system can be used, it has been found that an organic polymeric material and in particular polyacrylamides (e.g., Bio-Rad (<trade name) Richmond particles, California) and in particular zymosan have

  proved particularly appropriate.

  For this system to function properly

  It is necessary to coat a protein with particulate matter. This coating can be applied by

  actual chemical cross-linking of the protein on the

  or by simple electrostatic adsorption Any suitable protein or protein mixture can be used to thereby sensitize the beads. Suitable proteins include human immunoglobulin G,

purified or animal serum.

  Then a luminescent chemical compound is coated either chemically or by adsorption on the

  particles coated with protein or is mixed with those

  4 - ci Although any luminescent chemical compound that reacts with oxygenated intermediates by creating directly or

  indirect light can be used, we found

  The luminol (available from Eastman Organics, Rochester, New York) was particularly suitable. If the polymer bead is used, the luminol is dissolved in an aqueous buffer solution and is preferably reacted to form an intermediate azo compound. The intermediate azocompound reacts with

  the polymer particles coated with protein This results in

  The resultant particles are washed, suspended in suspension to a final concentration, introduced into flasks and freeze-dried. The resulting material is then converted into an azoluminol-protein adduct and also into electrostatically-bonded luminol-polyacrylamide. by this method gives a homogeneous stable product o The luminol can also be simply mixed with a suspension of zymosan particles previously coated with a suitable protein The mixture

  The resulting product is introduced into vials and lyophilized.

  The material obtained by this process gives a homogeneous stable product. The freeze-dried product described above is after reconstitution with water, ready for use

in the test.

  For the test using polymer particles, 1,000,000 to about 2,000,000 polyacrylamide particles are mixed in 50 microliters of buffer with about 200,000 purified phagocytic cells in 100 microliters of buffer.

  test using simultaneously particles of the zymo-

  san are: (1) dilute whole blood in 1: 3 proportions

  (part of anticoagulated blood plus two parts of

  pon) Fifty microliters of this dilution are mixed

  with 200 microliters of coated zymosan containing

  Each ml of the mixture of zymosan contains

  about 50,000,000 particles and (2) we mix fifty

  microliters of zymosan suspension with 100 micro-

  liters of buffer containing approximately 200 000 phage-

  Purified cytes The light created from these mixtures is measured periodically over time and reflects the phagocytic and biochemical activity of the phagocytic cell preparation. Cells that can be evaluated by this technique include

  neutrophils, monocytes and alveolar macrophages

  The first two of these types of cells are obtained from whole blood placed on a density gradient such as Ficoll-Hypaque (commercial designation) and centrifuged. These cells are grouped in the gradient and are thus purified. The macrophages are obtained at

  from washes of the lungs and do not require purification

  In the following examples, neutrals are used

philes.

Example 1

  200 mg of the polyacrylamide beads described above having a diameter of 2-9 pm are suspended in 20 ml of water. To this suspension is added 10 mg of human immunoglobulin G (IgG). The suspension is moderately mixed.

  of protein-coated beads and cooled to 2 C-

  C is added 40 mg of 1-ethyl-3,3-dimethylaminopropyl carbodiimide hydrochloride Six hours later, mg of glycine is added. The mixture is stirred overnight at 2 ° C. The following day, the suspension is washed. by centrifugation with phosphate buffered saline, 1.4 M sodium chloride in phosphate buffered saline, and finally 0.005 M phosphate buffer The beads are re-centrifuged and resuspended in 8.5 ml of borate buffer

p H 8.5.

  A luminol diazonium salt is prepared by suspending 200 mg of luminol in 20 ml of 2.4 N HCl. The mixture is cooled on ice. 2.0 ml of solution of 100 mg per ml of sodium nitrite,

  mixed, followed rapidly by adding cold buffer

  di, 50 ml, 0.5 M, p H 8.5 The p H is observed and adjusted

  with 10 N sodium hydroxide at pH 7.1 * 0.1.

  then 14 ml of luminol diazonium salt at 8.5 ml of the resuspended beads described above and p H

37723

  is adjusted to pH 8.5 The suspension is mixed for 3 hours

in the dark at 20 C-8 C.

  The suspension of diazotized beads is then dialyzed against several changes of 0.2 M borate buffer pH 8.0 at 2 OC-80 C The dialysis takes place during

at least 24 hours.

  The beads are then washed by centrifugation using a 0.2M borate buffer, pH 8.0, until the supernatant comprises less than 1.1% of the initial light of the total suspension, ie to say that more than 99% of

  luminol is associated with pearls.

  The beads are then centrifuged again.

  The beads are resuspended in phosphate buffered saline. The concentration of pearls is

  adjusted to approximately 50 million pearls per ml, deter-

  a hemocytometer is then introduced.

  in flasks in 1.0 ml portions and lyophilized

  read up to 5.0% residual moisture, determined

  by titration according to Karl Fisher's method.

  To carry out the phagocytic test, a vial

  is reconstituted by the addition of 1.0 ml of purified water.

Example 2

  Zymosan A (Sigma Chemical Co. St Louis, Missouri) is suspended in buffered saline

  phosphate up to a concentration of 24 mg per ml.

  To this suspension is added an equal volume of 50% normal rabbit serum diluted in phosphate buffered saline. The suspension is incubated for one hour at 37 ° C. in a stirred water bath. The suspension is then centrifuged, the liquid is discarded. supernatant and zymosan A (pellet) thus treated and resuspended in a small volume of phosphate buffered solution. The suspension is then aspirated through a syringe needle (opening 18-26) to homogenize the suspension. buffered saline solution

  phosphate to quadruple the original volume of the

  This gives a suspension of about 6 mg per ml.

  We then pass this suspension through a stopper

  of glass wool to trap any lump of zymosan.

  A luminol stock solution is prepared by dissolving it in 0.01 N sodium hydroxide. An aliquot of this stock solution is then added to a solution containing 20% fetal calf serum.

  in a phosphate buffered saline which.

  resulted in a luminol concentration of 14.4 μg per ml.

  To a volume of zymosan suspension is added a volume of fetal calf serum-luminol solution. The resulting suspension is mixed, introduced into flasks in 2.0 ml portions and freeze-dried. less than 5% residual moisture determined by

  titration according to Karl Fisher's method.

  For use in the phagocytosis test,

  one vial is reconstituted with 2.0 ml of purified water.

Example 3

  Several children previously diagnosed with chronic granulomatous disease (GCM) were examined using the reagents and test procedure described above. This disease was chosen because the dysfunction is understood MGC is caused by absence of certain enzymes found in the phagocytic cells of the blood Although the cells

  phagocytes are able to swallow bacteria (particularly

  These cells are unable to inactivate or kill the bacteria since the phagocytic cells do not have the capacity to create high oxygenated intermediate oxygen compounds. As such, they can not cause oxidation of the thus-swallowed luminol. cells, so that no detectable light reaction is observed clinically, these children typically exhibit or manifest this decrease as a

  severe reduction in their ability to resist infection.

  See Figures 1-3 for the typical response. When these children were tested together with apparently healthy control subjects, no light was produced.

  to the oxidation of luminol, although they swallowed the

  as effectively as witnesses.

Example 4

  A group of children previously diagnosed with asthma were tested according to their

  phagocytic / biochemical reaction of phagocythala-

  About half of the children received therapeutic doses of theophylline; the rest of the children had not been subjected to this medication It is allowed

  that theophylline has an effect on monophosphate adenosine

  cyclic phage (c-AMP) which is known to exert regulatory control over certain biochemical actions of phagocytic cells See Figures 4-6 Children who received theophylline in general had reduced levels of production of oxygenated intermediates

  quantized by the reduced oxidation of luminol and the produc-

reduced light.

  Such methods such as those described have particular utility for the quantification of

  infective resistance in terms of phagocytic activity.

  However, such a method also has an application

  for medical diagnosis, immuno-toxicology of

  virology, pharmacology, for example to control

  toxicity or chemotherapy and radiation therapy

  for example, they can be used for

  for the evaluation of immuno-competence, certain protein defects in blood serum etc. As a research tool, they can be used to test pharmaceutical compounds and their effect on phagocytes, the effects of pollutants on cells phagocytes and the effect of toxic compounds on

animals and humans.

  Of course various modifications may be made by those skilled in the art processes and compositions which have just been described by way of non-limiting examples without departing from the scope of the invention. -9

Claims (13)

claims:   A method for measuring the ability of an organism to resist infection characterized by taking a blood sample from the body, separating the phagocytic cells from the blood, adding to the phagocytic cells particles coated with which are related luminescent chemical compounds,   allow the phagocytic cells to swallow the particles   thus activating the biochemical mechanism of cells that react with the luminescent chemical compound creating   of light that is measured on a luminometer.   Process according to Claim 1, characterized in that the particles are polymer beads having   diameters from about 2 μm to about 9 μm.   Process according to Claim 2, characterized   in that the beads are polyacrylamide.   4 Process according to claim 1, characterized   in that the particles are zymosan particles.   Process according to claim 1, characterized in   what the luminescent chemical compound is luminol.   Phagocytic composition characterized in that it comprises polyacrylamide particles having a   diameter from about 2 μm to about 9 μm coated with a   serum protein che to which a luminol layer is bound. A phagocytic composition characterized in that it comprises zymosan particles coated with a layer of serum protein and luminol I   8 Composition according to any one of the   cations 6 and 7, characterized in that it is in freeze-dried form. 9 Process for manufacturing a phagocytic reagent   of any one of claims 6 to 8, characterized   in that it consists of coating a particle particulate matter of two to about nine meters in diameter, a layer of protein and luminescent chemical compound, and lyophilizing the coated particles to obtain a stable, homogeneous product.   Process according to claim 9, characterized in that the protein layer is applied first and the luminescent chemical compound is applied to this layer of protein.   Process according to Claim 9, characterized in that the luminescent chemical compound is mixed   with a protein before application to the particular material   lar. Process according to claim 9, characterized in   what the luminescent chemical compound is luminol.   Process according to Claim 10, characterized in that the particulate material is polyacrylamide or zymosan.