NO124336B - - Google Patents

Description

Procedure for producing an immunological indicator

for chemical binding to antigenic substances.

The present invention relates to a method of manufacture

- of an immunological indicator for chemical binding to antigenic substances,

which method is distinguished by the fact that microbial cells are brought into con-

rate with moles of e-t coupling agent that has at least two non-bonded,

reactive groups, and one of the reactive groups is brought to

form a covalent chemical bond with the microbial cells while the other reactive group is retained in an unbound state, whereby the indicator

becomes able to react with an antig one substance is when -it is brought into con-

tact' with such..

It is of great importance to be able to demonstrate the presence of antigenic substances in different liquids. Many industrial pro-

sessions are used as protein materials with antigenic properties either as reactants or as materials subjected to one or the other

treatment. The possibility of detecting changes in the concentration of these materials, so that such reactions can be followed, is of the greatest importance for the regulation and reproducibility of these industrial processes. Another important area of application is the detection of various antigens and antibodies in body fluids. The quantity of many of the hormones, proteins, lipoproteins, mucoproteins, glycoproteins, etc., and their hydrolysis products that occur naturally in the body as well as under various pathological conditions, is of great importance to the outsiders of medicine. Some of these substances have been difficult to detect using standard analytical chemical methods. Using immunochemical test methods, however, some of these antigens and antibodies can be detected in mixtures containing other large molecules with similar chemical groups but which do not have exactly the same stereo configuration as the macromole molecule of interest. Several of these immunological methods are difficult to perform, take a long time and are technically complicated, but they have been used in practice to detect the presence of such substances in various body fluids. For ■ individual antigens and antibodies, no method,.been; available.

Recently, hemagglutination systems have been improved for the detection of antigens or their antibodies. These systems make use of red blood cells as indicator particles, which for use are bound to an antigenic material to form an indicator system that can be used for detection of the homologous antibody or the antigenic material itself. In order to determine the presence or absence of the material to be tested using such systems, it is usually necessary to interpret the appearance of the sample formed by the red blood cells to determine whether hemagglutination has occurred. Hemagglutination takes place when the homologous antibody is present in the test medium in a form that enables it to react with the antigenic material attached to the red blood cells and to form an immune complex with them. Hemagglutination does not take place when the homologous antibody cannot form a complex with red blood cells attached to the antigenic material. Depending on many conditions including buffers and other highly reactive materials, this test method has limited applicability and requires skilled personnel to interpret the appearance of the resulting monsters, although applicable test systems have been developed for some substances. Another pressing difficulty associated with the hemagglutination system is the problem of heterophilic antibodies, which problem arises from the fact that some of the serum proteins contained in the antiserum solution react with the antigenic groups naturally occurring on the surface of the red blood cells, even when the antiserum is taken from one species which is different from the species of animal that produced the red blood cells used. Ovalbumin was early detected in serum in this way; see Pressman, D.,. Campbell, D.H. , Pauling, L.: Journal of Immunology Mf, pages 101 - 105 (19<*>+2). A similar method was used for the detection of tuberculin-purified protein derivative (Cole, L.R. , Farrell, V,R.-: Journal of Experimental Medicine 102,

page 15.7 (1955))»and. insulin was detected in serum by Arqullla, E.R.

and Stavitsky, A.B..; see Journal of Clinical Investigation 35, pp. ^58 - M56 (1956). U.S. patent document no. 3 236 732 describes a similar system for testing with regard to the hormone chorionic gonadotropin. A pathological antigenic substance, diphtheria toxoid, was also tested in this way, see Butler, W.T.: Journal of Immunology 90,

pages 663-671 (1963).

It is assumed that the limited commercial use of this

type of hemagglutination testing has been partly caused by the narrow size range and the peculiar configuration of the red animal blood cells that are usually used, and that more widespread use of immunological agglutination test systems would be possible if indicator particles could be found that allow a greater variation in the stbrelsen, to replace the red blood cells. Generally, the use of animal red blood cells as indicator particles for various antigens precludes the use of either a chromatographic agglutination mechanism or a plate-type agglutination mechanism. Moreover, the red blood cells are expensive to use commercially, because laboratory animals must be kept and because the conditions during collection must be carefully adjusted to avoid hemolysis of the cells and to maintain uniform quality, as otherwise the hemagglutination test would not be able to be performed reproducibly.

- It has now been shown that microbial cells can serve as immunological indicator particles, whereby many of the previously experienced difficulties can be overcome. Microbial cells exist in a wide variety of sizes and shapes, and the sizes lie in the areas

which is required in agglutination tests both in tubes and on plates as well as in chromatography-type tests. A number of test mechanisms can thus be used when microbial cells are used as indicator particles. The cells can easily. grown under laboratory conditions and can be produced commercially at low cost. The microbecells can be selected.

according to the desired size, according to the surface properties most advantageous for a given test mechanism and according to the ease with which they can be produced. Greater flexibility is thus made possible in the construction of immunological test systems using microbial cells. Moreover, using microbial cells eliminates the problem of heterophilic antibodies.

The term "indicator particles" used here refers to microbial cells that are not bound to any coupling agent, while the term "immunological indicator" refers to microbial cells to which a coupling agent but no antigenic material is bound. The term "immunological indicator system" refers to the combination of cells, coupling agent and antigenic material.

It is therefore an aim of this invention to provide an immunological indicator consisting of microbial cells and a chemical coupling agent bound to these to which antigenic materials are covalently bound, which immunological indicator can be used for visual detection of antigen-antibody reactions.

It is a further aim of this invention to provide an immunological indicator for antigen-antibody reactions, which consists of microbial cells of any size in a wide size range.

The immunological indicator produced by the method according to the invention,<1>can be broadly described as composed of microbial cells to which there are chemically bound molecules of a coupling agent with at least one non-bound reactive group which is capable of forming an encovalent bond with a reactive group in antigenic substances upon contact and reaction with these. The indicator is produced by suspending the microbial cells in a liquid and adding the coupling agent to this. which is capable of reacting with a reactive group on the surface of the microbial cells. The antigenic substance to be connected to the microbial cells can be present in the same liquid suspension when the coupling agent is added, so that a specific immunological indicator system is formed immediately, or the substance can be added later to the produced indicator. The connection of the indicator to an antigenic substance leads to the formation of an indicator system that can be used in a specific way as described below.

The microbe cells of the immunological indicator and the immunological test system can be any cell-reproducing microorganism that propagates with or without the influence of other organisms. Both gram-positive and gram-negative bacterial cells can be used, fungus cells and protozoal cells can be used and likewise virus cells for certain test systems. These are usually unicellular organisms that occasionally occur in clumps or aggregates. The cells can be used in this form provided that their aggregate size does not constitute a carrier particle that is so large that the system thus formed will not agglutinate in the presence of a substance that is homologous to the antigenic substance that is bound to the aggregated cells. Generally, the preferred microbial cells are bacterial cells or aggregates thereof which have a uniform shape and size and which have maximum external dimensions in a direction from 0.2 to 10 µm. Although not preferred, a mixture of different but uniform cells may be used. For these bacteria, the applicable microbial cells include those in division I of the plant kingdom, including class I,

II and III, Order I. Microbe cells of Class III, Order I include the intracellular virus organisms that have dimensions of about 0.2 µm.

For a complete overview of applicable bacterial cells, reference is made to Bergey's Manual of Determinative Bacteriology by R.S. Breed, E.G.D. Murray, - NO. Smith, 7th edition, 19^7, Williams and Wilkins Company. Particularly useful are the bacteria of Class II, Suborder II, Family IV (Pseudomonadaceae) and Class II, Order IV, Family IV (Enterobacteriaceae). All subfamilies I-V are considered to represent preferred microbial cells for the purposes of the present invention.

Also Class II, Order IV, Families V (Brucellaceae), X (Lactobacilla-ceae) and XIII (Bactillaceae) are considered preferred. Both order I

and order II of the organisms in class III can be used when it is desired to use particles of a size of approximately 0.2 µm or less. In particular, Virales of order II are of such small dimensions that their applicability is limited.

Particularly preferred bacterial cells are Brucella abortus, Escherichia coli, Bacillus subtills, Bacillus pumilus, Lactobacillus leichmannii and Pseudomonas fragi. Yeast growth phases of fungus cells are also preferred for use in the invention. Particularly preferred is the readily available yeast Saccharomyces cerevisiae.

The microbe cells can be used in their natural state, i.e. the coupling agent can be reacted with them, after which the antigenic substance is attached through the coupling agent. If, however, the natural pathogenic properties of some of the microbe cells are considered to represent a danger, the natural antigenicity and consequently the pathogenic properties can be eliminated by treating or killing the microbe cells with a preservative. The most common preservatives are formaldehyde and phenol. It should be noted that when microbial cells have been treated with a preservative and then further modified by attaching coupling agents and antigenic substances, the naturally occurring antigenic groups on the cell surfaces are modified to a sufficient extent to render them inactive. To achieve a complete elimination of cells that may have natural antigenic activity, the prepared test systems can be absorbed with serum containing the antibody to the antigen that occurs naturally on the cell surfaces to form complexes that do not interfere during the subsequent testing. If e.g. E.coli are used as indicator particles in the indicator and antibodies to E.coli are present in the material to be tested, the reaction between the indicator and the antibody can be blocked by adsorbing the E.coli cell indicator with antisera containing the antibody. The microbe cells used in the method according to the invention,

can thus no longer be considered to exhibit natural antigenicity.

If desired, the microbe cells can be labeled to improve the visual contrast between the resulting indicator system and the surroundings.

The usual dyes such as hematoxylin, fuchsin and crystal violet can be used for this purpose. Another possible treatment consists of '

in washing the cells with organic solvents such as alcohols, ethers, etc. to remove any polysaccharide or wax layer present.

The coupling agents that can react chemically with reactive groups both on the microbe cells and on the antigenic substances are usually compounds with two or more of the following reactive groups: azo, sulfonic acid, fluorine groups combined with nitro groups, fazide, imine and reactive chlorine groups bound to. a ring with favorable resonance structures. These reactive groups are able to react with the primary amino groups, sulphydryl (mercapto) groups and hydroxyl groups in the polymeric chains of the antigenic substances and on the surfaces of the microbacilli.

A representative list of known coupling agents are: bis-diazobenzidine, bis-diazobenzidine-disulfonic acid, tetraazo-p-phenylenediamine, difluorodinitrobenzene, various carbodiimides, toluene diisocyanate, tri-chlorocyanidine, dichloro-S-trizine and N-t-butyl-5-methylisoxazolium- perchlorate. Some of these coupling agents, and especially the cyanidation agents, are able to preserve the cells at the same time as they are coupled to groups on the microbial cell surfaces, so that no separate pre-treatment for preserving the cells is necessary when using these coupling agents. For the use of the last of the above-mentioned compounds, the indicator particles are first treated with a

succinylation reagent such as succinic anhydride.

To produce the immunological indicator, the coupling agent is first reacted with the microbial cells, which are suspended in a liquid, and then, when the need is present, this indicator is mixed with the antigenic substance to form an indicator system. This means that the indicator itself is considered a commercial product, as it can be manufactured and sold for use by the consumer when building test systems for any given antigenic substance. •

The preferred way in which the microbial cells and the antigen

substance is brought into contact with the coupling agent to form test systems consists of suspending the microbe cells and the antigenic substance in a liquid and adding the coupling agent to this with vigorous stirring. Another method of bringing the components into contact with each other consists in adding the microbial cells and the antigenic substance simultaneously to a solution containing the coupling agent. These contact methods limit the cross-linking of microbial cells and the cross-linking of the molecules of the antigenic substance.

Generally, any antigenic substance can be linked to the immunological indicator produced according to the invention. An "antigenic substance", as used herein, means a material which, when introduced into an animal's circulation, produces homologous antibody. This broad definition includes serum antigens such as gamma globulin and serum albumin or blood group substances "A" and "B" for samples for determination of blood type. Microbial antigens can be linked to the indicator for detection of either the microbe itself or its antibody in fluids. Antigenic hormone substances that are present in organisms' fluid systems can also be linked to the indicator. Protein hydrolysis products and enzymes can also be used as antigenic substances.

The microbial antigens or antibodies can be of -bacterial, fungal, parasitological or viral nature. The main requirement for the antigenic substances is that they have at least one group in their polymeric chains which can react with the reactive group of at least one of the known coupling agents. All known antigenic substances are assumed to satisfy this requirement.

The antigenic substance can be a naturally occurring material or it can be an artificially produced material. Some naturally occurring substances that can be linked to the immunological indicator produced by the method according to the invention are ovalbumin, tuberculin-purified protein derivative, insulin, the hormone chorionic gonadotropin (CGTH), both from humans and animals, human serum albumin, and horse gamma globulin.

There are three general methods for carrying out tests with

the immunological indicator systems. These are methods involving the chromatography principle, those involving agglutination in a tube and those involving agglutination on a plate. The particular method chosen largely determines the size of the microbial cells used.

For a chromatography-type test, it is preferred to use microbial cells that are in the form of elliptical rods of length approximately 0.3 - 0.^-Mm« For plate agglutination tests, the microbial cells can be larger rods of lengths 1 - 3 µm and diameters of approx. 0.5 µm, provided that the cells can likewise have a coccoid shape. In plate agglutination tests, a wide variety of sizes of microbial cells can be used, as microbial cells with dimensions from 0.2 to 10/zm in any direction, such as length or diameter, can be used.

The plate and rod agglutination tests are based on the ability of the immunological indicator system to agglutinate and thereby form a monster with a noticeable difference from the monster produced when agglutination does not take place. This test type can be performed on two general ways, the first of which is termed agglutination, while the second is termed inhibition of agglutination. For agglutination, an antigen is linked to the microbe cell to form an indicator system, and this is then used to examine whether the homologous antibody is present in the sample being examined. If the sample contains the antibody, the indicator system will agglutinate with the antibody, and the resulting condition can be recognized using the sample displayed by the indicator system. To inhibit agglutination, the antigen is linked to the microbial cell to form an immunological indicator system which is then used with the homologous antibody to examine whether the antigen is present in the sample being examined. If the sample contains the antigen, the homologous antibody will be inhibited or neutralized by the antigen, and the indicator system will not agglutinate. When agglutination does not take place, the indicator system will persist

of insoluble microbial cells and the bound antigen, are separated from the test fluid and give a sample noticeably different from the sample produced by the agglutinated indicator system.

In the plate test, the agglutination is shown by the formation of a

granular monster, while the agglutination in the tube test is shown by a smooth,

uniform suspension of cells. The condition where agglutination has not taken place is shown in the plate test by the formation of a uniform sample, and in the case where a rudder test is used, this condition is shown by sedimentation of the indicator system at the bottom of the rudder either in a ring sample or in a spot sample.

This same type of agglutination or inhibition of agglutination is the basis for the chromatography-type test method, where a characteristic migration of the chromatography solution will take place if the sample being examined contains the given antigen or antibody. In an agglutination test of the chromatography type, a spot of the indicator system is deposited on a suitable chromatography support, such as filter paper, after which a drop of the liquid sample to be tested for the presence of the antibody is placed on this same spot and the edge of the support is brought into contact with a chromatographic graphing solution such as a saline solution. If the liquid sample contains the antibody, the indicator system will agglutinate and form an immune aggregate with the antibody. This aggregate will not be carried along by the advancing chromatography solution, and little change of the spot will take place. If, however, the liquid sample does not contain the antibody, no immune aggregate will be formed, and the indicator system will in that case travel together with the advancing solution, whereby a line-shaped monster is formed. To perform an inhibited agglutination chromatography-type test, a spot of immune aggregate is formed by placing a drop of an antibody-containing solution on a spot of deposited immunological indicator system. A drop of the liquid sample to be tested for the presence of the antigen is then placed on the spot of immune aggregate and a solution allowed to advance up through the carrier, or alternatively the edge of the carrier may be brought into direct contact

with the liquid sample, so that the antigen, if present, can come into contact with the stain of immune aggregate. "If the liquid sample contains the antigen, the immune aggregate will dissociate as a result of the preferential reaction between the antibody and the antigen, and the indicator system will move and produce a line-shaped monster. If no antigen is present in the liquid sample, there will naturally be no migration of the spot of immune aggr-egat take place.

The following examples will further illustrate the invention.

Example 1

An immunological indicator system was prepared using Brucella abortus cells as microbe cells, bis-diazobenzidine (BDB) as coupling agent and human embryonic gonadotropin (CGTH) as antigen. This indicator system was then deposited on a moisture absorbent support material together with the antibody to CGTH, and urine samples were then tested for the presence of CGTH with the resulting device. This indicator device functioned by a chromatography mechanism which will be described further down.

MANUFACTURE. OF REAGENTS

Isotonic saline solution

A 0.85% salt solution was prepared by dissolving 8.5 g of sodium chloride in 1 liter of distilled water.

Fo- sf atpuff is

A 0.15 M buffer solution of pH 7 A was prepared by dissolving a dry mixture of 8 L acidic disodium phosphate and 19% acidic monosodium phosphate in distilled water until the desired pH value was reached.

bi s- diazobenzidine solution

To 0.92 g of benzidine was added 100 ml of distilled water and

6 ml of 6N hydrochloric acid. A further 100 ml of distilled water was added to this mixture. After the benzidine had dissolved, the solution was cooled to 0°C in a mixture of ice and salt. - As soon as ice crystals began to form in the solution became 6.5 ml of a 10% ±g solution of. sodium nitrite added rapidly while stirring. The conversion was continued until the solution was negative when tested with starch-iodine paper. During the conversion, the solution must never be allowed to reach a temperature above approx. 1°C.

The material obtained was light yellow. By. addition of phosphate buffer. the color changed to a terrible brown. The pale yellow solution was stable at room temperature for 5-7 hours. At k°C it was stable for approx. 7 days. When frozen and stored at -20°C, it was stable for approx. 60 days, and when stored at a temperature lower than -35° C it was stable for at least 6 months.

Formalin treatment of microbial cells

Brucella abortus cells were grown for 72 hours at 37°C on tryptose agar and were then washed out of the agar with a 0.85 µg saline solution containing 1% formaldehyde. The cells were immediately treated with formaldehyde by suspending them in a salt-formaldehyde solution for 2+ hours at room temperature (25°C). The cells were then thoroughly washed to remove the remaining preservative. The washing of the Brucella abortus cells was carried out by suspending the cells in distilled water. The cells were collected by centrifugation and resuspended in distilled water, the last-mentioned step being repeated several times. The preservative-treated cells were then labeled.

Labeling of formalin. process lead microbial cells

2 g of the wet-packed, formalin-treated Brucella abortus cells, 1 ml of 2% ferrous sulfate and 5 ml of 1% aqueous hematoxylin were added to 100 ml of distilled water and stirred continuously for 2<*>+ hours. The cells were permanently labeled, and the dye was not leached out.

Preparation of indicator system - (formalin-treated microbecells -

BDB - CGTH) Brucella abortus cells, which were labeled with hematoxylin as described above, were used as indicator particles for human CGTH provided by Vitamerican Corp. , -Little Falls,

New Jersey. 0.5 g of packed, labeled cells was added

10 ml of saline solution containing 20 mg of CGTH, after which 12 ml of phosphate buffer and ^-0 ml of diluted BDB solution prepared by mixing 8 ml of the above-mentioned BDB solution with 32 ml of phosphate buffer were added. The coupling reaction proceeded with stirring for 20 minutes at room temperature (25°C). The resulting brown complex was then collected by centrifugation and washed three times by centrifugation with distilled water. It was then desired to transfer this indicator system onto a moisture-absorbing support element , a 0.2 g portion of the indicator system was homogenized in a mortar.The portion was then thoroughly mixed with 1 drop of 1% merthiolate and 1 g of sucrose syrup (75% w/v) was then slowly added.

Preparation of antibody to CGTH

Laboratory rabbits "were given human CGTH in Freund<1>'s complete adjuvant in a series of injections. After a given period of time, the rabbits were euthanized, and the serum containing the antibody was separated from the blood. The CGTH used can be obtained from any of the commercial suppliers. Particularly applicable is that sold by Vitamerican Corp., which has an activity of approximately 2000 - 2500 international units (I.U.). A typical injection schedule and a typical separation of the antibody is as follows: a solution containing 10 mg of CGTH per ml saline solution was mixed in equal amounts with Freund's complete adjuvant. 0.1 ml of the mixture was injected into each paw of a rabbit weighing 3.5 kg. After a period of 2 weeks, a first "booster" of 0.5 ml of a solution containing 5 mg CGTH per ml of saline injected intravenously. 2 days after this first "booster" injection, a second injection was given. Blood samples from the rabbit were taken 7 days after the last "boo ster" injection. The antisera had a titer of 1/2560, determined by hemagglutination.

Test methods (chromatographic)

A patch of the indicator system produced as described above was placed approx. 19 mm from the edge of a piece of filter paper,

and a drop of the above antisera was placed on this to form an immune aggregate. The edge of the paper was then brought into contact with a normal, non-pregnant urine sample. While. the urine sample moved up the paper, the spot of immune aggregate remained in the same position on the paper. This showed that the formed immune aggregate was not dissociated from the normal urine constituents. Another filter paper strip was prepared with another spot of immune aggregate,

and the edge of the paper was brought into contact with a urine sample from a pregnant woman. As the urine moved up the paper strip, part of the immune aggregate dissociated and caused a line formation or migration of the colored indicator system upwards from the aggregate stain which became correspondingly lighter in colour.

In another test, the spots of immune aggregate were formed on several strips of paper and the urine samples were then dripped onto these spots. The edges of the strips were brought into contact with a physiological saline solution. When the saline solution had moved past the spots treated with the urine of a pregnant woman, something in the colored indicator system set in motion and caused a line-shaped monster. Relatively little streaking was found when the salt solution moved past the spots treated with normal urine from non-pregnant women.

Test devices were prepared for testing the presence of the antibody to human CGTH by placing spots of the above-described indicator system approx. 19 mm from the edge of the filter paper pieces. A drop of rabbit serum containing the antibody was then placed on a spot indicator system on one of the strips, and the edge of this was brought into contact with a physiological saline solution. The salt solution moved past the stain without any streaking occurring as a result of the indicator system, showing that an insoluble, immune aggregate had formed. A drop of normal rabbit serum was then tested in a similar manner. The indicator system then appeared to move away from the stain, showing that no immune aggregate had formed.

The above indicator system also showed agglutination as a result of antibody to human CGTH in tests performed on rbr type agglutinations. These agglutinations were readily inhibited by the addition to the test medium of small amounts of a solution containing

CGTH.

When producing the diagnostic preparation on a moisture-absorbing carrier material using an indicator consisting of microbial cells, it is essential that the cell material is stabilized by a conservation treatment as described above.

Another indicator device can be produced by mixing the above-mentioned indicator system with the antibody to form an immune aggregate, depositing a single spot on an area at a given distance from the edge of the strip and drying. When the indicator device is to be used, a drop of urine sample is placed on this spot or between the spot and the edge of the strip, and the end of the strip is placed in a saline solution. If the urine contains CGTH, the aggregate will dissociate, whereby advancement of the labeled indicator system together with the salt solution is made possible and a line-shaped monster is formed. If no CGTH is present, there will be no change.

Alternatively, the urine sample can be used as a chromatography sample as above. In a preferred embodiment, such a device is produced by producing the diagnostic reagent in strip form, e.g. places a band of the aggregate of indicator system and antibody approximately 15 mm from the longitudinal edge of a 13 cm x 26 cm piece of Eaton-Dikeman No. 609 filter paper, allows the reagent to dry at room temperature, cuts the paper into strips and stores these in suitable containers until they must be used.

It will be understood that many different materials can be used in the moisture absorbent strip test. For example, many types of chromatography materials can be used and likewise many types of chromatography liquids for elution.

Example 2

Escherichia coli was used as indicator particles for the production of an immunological indicator system of microbial cells-BDB-CGTH. This indicator system was used together with the antibody for CGTH in a plate-type agglutination test, and clinical pregnancy determinations were made using this test.

Preparation of reagents

0. 85% salt solution - 68 g of sodium chloride was dissolved in 8 liters of distilled water, and the solution was heated in an autoclave at an overpressure of 1.05 kg/cm 2 and at a temperature of 250°C for 30 minutes. The solution was then cooled and stored at <1>+°C. The autoclave treatment was carried out to sterilize the saline solution.

l#-ig formalin solution - A 37^-ig formalin solution

was treated by adding powdered calcium carbonate and then allowing it to stand for a period of 2h to 8h. The supernatant was then transferred to another container and filtered three times through quality white filter paper with a crepe surface. When the pH value decreased to below approx. 7.0, it was adjusted to this pH value with 0.1 µg NaOH solution. A sufficient amount of this treated 37% formalin solution was added to distilled water to form a 1% solution.

Phosphate buffer solution

100 ml of a phosphate buffer with pH 7A was prepared by mixing 80.8 ml of a sodium phosphate solution with 19.2 ml of a potassium phosphate solution at 20°C. The sodium phosphate solution contained 53.726 g of Na2HP0lf.12H20 per liters of distilled water. The potassium phosphate solution contained 20 AlL|" g KH2P0^ per liter of distilled water.

bis-diazobenzidine (BDB)

0.6^0 g of benzidine • 2 HCl was dissolved in 100 ml of a 0.56$2-ig HCl solution in a 250 ml Pyrex Erlenmeyer flask. This flask was placed in an ice-filled vessel equipped for magnetic stirring and with a magnetic stirrer immersed in the solution. When the temperature had reached

*+°C, a 5 ml aliquot of a sodium nitrite solution prepared by adding 0.680 g of NaN02 to 10 ml of distilled water, and maintained at 1+°C, was taken up in a serological pipette and added dropwise at a steady rate to the benzidine solution in lbpet of a period of from 7 to 10 minutes, while the magnetic stirrer worked at a speed of 200 revolutions per minute. minute (1 drop every four

to the sixth second). This solution was stirred continuously for 20 minutes, after which it was immediately frozen and kept at -60°C in a number of bowls of one dram capacity. This solution tends to decompose even at temperatures from 0° to 5°C, and it was therefore necessary to freeze the solution to maintain stability. It can be heated to operating temperature when needed.

Antibody to CGTH

An emulsion containing 10 mg CGTH per ml was prepared by dissolving 100 mg of purified CGTH in 5 ml of the above saline solution, adding 5 ml of a thoroughly mixed Freund's complete adjuvant and shaking to form a smooth emulsion. An amount of 0.2 ml of the emulsion was injected into each paw of a healthy rabbit kept in standard laboratory conditions, in a total amount of 0.8 ml of emulsion (8 mg of CGTH).

The rabbit was then given a series of injections, each consisting of

of 0.5 ml of a solution prepared by dissolving a sufficiently large amount of purified CGTH in 0.85% saline to achieve a concentration of 5 mg per ml. The injections were given in the letters. These subsequent injections were given on the following days after the first emulsion injection: The 22nd, 2M-, 38, k0, 71 and '73. Two k0 - 50 ml1 s blood samples were taken from the rabbit by cardiac puncture on the 50th and 83rd day. These samples' blood cells were then separated from the serum in each

test by allowing the blood to clot in a centrifuge tube at approximately 25°C for approx. 2 hours, after which the overflowing serum was placed in a tube of small diameter (less than 30 mm) and the tube was immersed in a water bath of 56°C for 30 minutes. 10 mg formalin-treated red sheep blood cells (lyophilized) were then added per ml of the serum sample and mixed for 15 minutes at approx. 25°C to adsorb heterophile antibodies from the serum. The adsorbed serum was then separated by centrifugation from the cells at a centrifugal force of approx. 1000 times the force of gravity.

h0 ml of packed sheep cells were prepared for this adsorption by centrifugation and siphoning the supernatant from 200 ml of fresh red sheep blood dispersed in Alserver's solution and then washed three times with 0.85% saline using from 120 to 200 ml saline solution per washing. To these cells was added<*>+60 ml of the above-mentioned salt solution and 500 ml of a 3% formalin solution of pH 7.3. The resulting suspension was mixed and allowed to stand for 18-20 hours at 37°C.

The cells were then removed by centrifugation and washed five times with approx. 200 ml of distilled water per washing. Sufficient highly distilled water was added so that a 10 3 suspension of the cells was obtained. This suspension was stored at h°C until it was to be used for the adsorption.

Microbe cells

E. coli cells were obtained by diving a piece of intestine from

a dead laboratory rabbit in brain-heart-infusion foam (brain-heart infusion (BHI brotn). 68 ml of a culture in full growth (18 hours)

was used to inoculate 3 liters of BHI which had previously been checked for sterility. The cells were then incubated for 1.5 hours at 37°C with shaking. The cells obtained were found to be of uniform size and shape.

FABRICATION OF AN INDICATOR SYSTEM

The three liters of suspended E.coli cells were centrifuged at 10,000 rpm. minute for 10 minutes to separate excess fluids from the packed cells. The supernatant was siphoned off, and the still packed cells were contacted with approximately 5 ml of a 37% calcium carbonate-treated formaldehyde solution to kill the E.coli cells. The formalin was allowed to remain in contact with the cells for one hour, after which the cells were washed three times with the above saline solution and then suspended in 1600 ml of 1% formaldehyde solution for one hour at room temperature (25°C). These cells were then centrifuged and collected as packed preservative-treated cells. These packed cells were then resuspended in 5°0 ml of the above saline solution and stored at h°C. The cell concentration was h% measured using a hematocrit.

A portion of the above suspension of cells was centrifuged to recover a volume of packed cells, and 0.5 ml of this was removed and added to 10 ml of the above saline solution, to which was added 20 mg of CGTH, 12 ml of the phosphate buffer and h0 ml of diluted BDB solution prepared by mixing 8 ml of BDB and 32 ml of the phosphate buffer. The CGTH used had an activity of 3600 I.U. per mg and was supplied by Vitamerican Corp. The mixture was continuously stirred for 20 minutes at room temperature, and then the indicator system obtained was collected by centrifugation and washed three times with the above saline solution and then centrifuged and resuspended in a 1:50 dilution of bovine serum albumin (30%) in the above saline solution. The bovine serum albumin, BSA-, was supplied by Armor and Co.

The obtained suspension of the indicator system had a brownish color and was found to be stable over a wide temperature range.

PREPARATORY TESTING WITH THE INDICATOR SYSTEM

To determine the minimum detectable amounts of CGTH, normal urine samples were prepared by adding known amounts of CGTH. The prepared indicator system was then used together with the above antibody to CGTH (Ab-CGTH) in plate-type agglutination tests.

For this testing, the above-mentioned indicator system was resuspended in a sufficient amount of 1:50 BSA salt solution, thereby obtaining a &% concentration of the cells. Three dilutions of the above-mentioned antibody solution were then prepared by diluting a part of the antibody solution with 250 parts respectively. 500 parts and 1000 parts of the above salt solution. These dilutions were labeled 1:250, 1:500 and 1:1000 respectively. Urine samples with each of the following concentrations of CGTH were prepared by adding 0.25 ml of a solution of h mg CGTH/ml saline (3600 I.U./mg) to 100 ml of a normal urine sample from a non-pregnant woman with subsequent dilution with saline solution: 2.25;<1>+; 5; 6;

9; 18 and 36 I.U. CGTH/ml urine and saline solution. A portion of the urine sample was set aside as a blank.

1 drop of each of the urine samples was mixed with 1 drop of the appropriate antibody dilution and mixed on a glass plate. Then 1 drop of the above-mentioned 8% indicator system suspension was added to the cells already on the plate and mixed. The results of this series of experiments are listed in Table I below where - denotes agglutination and therefore a grainy sample in the suspension on the glass slide and denotes a smooth, non-agglutinated sample on the glass slide.

Table I shows that when no CGTH is present in the urine, agglutination occurs as a result of the urine sample not containing CGTH which can react preferentially with the Ab-CGTH, whereby the antibody agglutinates with the indicator system and produces a granular appearance of clumped cells over the moistened part of the glass plate. By adding 2.25 I.U. CGTH per ml to the urine sample, there is still enough Ab-CGTH present in the system that it reacts with all the CGTH present and causes clumping of the cells or agglutination even at the 1:1000 dilution. When the urine sample containing ^.5 I.U./ml was tested with the three dilutions of Ab-CGTH, the antibody reacted completely with the CGTH present between the 1:500 and 1:1000 dilutions, so that the sample for the 1:1000 dilution did not show a grainy appearance but was present as a smooth monster of suspended indicator system cells, showing that no agglutination had taken place. It will be seen that when using 6.0 I.U. CGTH/ml, the required amount of antibody is found between dilutions 1:250 and 1:500, which would be expected, because a larger amount of CGTH is present and therefore a larger amount of Ab-CGTH is needed to react completely with CGTH for to- produce a granular sample at the 1:250 dilution. It will be seen from Table I that very small amounts of the order of magnitude ^.5 I.U. CGTH/ml urine can be detected using low concentrations of Ab-CGTH in the test system, namely dilutions of 1:1000 or less. It has also been shown that by doubling the amount of urine used to 2 drops, concentrations as low as 2 I.U. can be detected. CGTH/ml urine.

In order to test precisely the time the development of the granular monsters takes over a range of concentrations of Ab-CGTH and cells, the following investigations were carried out.

Portions of indicator system were slurried in sufficient

much of the above 1:50 BSA dilution in saline to obtain concentrations of 0.625, 1.25, 2.5, and 5 cells. Then, serial dilutions of Ab-CGTH solution were prepared with the above 1:50 dilution of BSA in saline to provide dilutions of 1:125, 1:250, 1:500, 1:1000, 1:2000 , 1:^000, 1:8000 and 1:16000. Four drops of each of the Ab-CGTH dilutions were dropped in separate, parallel rows onto the surface of a cross-hatched glass plate. To the first drop of each of these dilutions was added 1 drop of the lowest concentration of the indicator system cell suspensions. To the second drop of each of the Ab-CGTH dilutions was added 1 drop of the 1.25 µl suspension, and to the third and fourth drops of each of the dilutions were added 2.5 µl and 5 µl cell suspensions, respectively. The results are listed in Table II below, where the time for development of a granular agglutination sample is given in seconds for each of the test mixtures. An "S" indicates that insufficient Ab-CGTH was present in the antibody dilution to cause agglutination of the cells, and the sample therefore remained smooth.

The agglutination times listed in Table II show that a relatively short time is needed to demonstrate the occurrence of agglutination, which, in a real test, gives a negative answer to the pregnancy test. Thus, a possible pregnancy can be detected by means of a plate agglutination test using the above-mentioned indicator system in about 1 minute or less. This is a far shorter time than that required for agglutination systems of the rudder type based on red blood cells. Moreover, the monsters that distinguish agglutination from non-agglutination are very marked, and these tests give practically no intermediate, doubtful monsters. It is thus possible to select the reagents so that clearly defined samples required for diagnosis of pregnancy can be obtained.

Table II also shows that a wide range of Ab-CGTH dilutions can be used and likewise a wide range of indicator concentrations. The concentration of the indicator system in a 1:50 dilution of BSA in saline can be from less than 1% to approx. 12% in applicable test systems.

In order to test the indicator system according to example 2 with relevant urine samples, 16 urine samples were obtained from 16 non-pregnant women, of whom<1>)- used birth control pills according to a regular program. The test method was the same as stated above. A drop of the urine sample was thus mixed with a drop of a 1:1000 Ab-CGTH dilution, after which a drop of this mixture was placed on a<0>glass plate and a drop of a h%-lg suspension of the the above indicator system was added to the mixture already present on the glass plate. In all 16 cases a granular sample was formed which showed that agglutination had taken place, and no pregnancy was detected. This testing provides limited evidence that false positive results do not pose any difficulty with the above indicator system, and that hormone levels due to active birth control programs have no impact with this particular test method.

CLINICAL TESTING USING THE INDICATOR SYSTEM

31 frozen urine samples were received, and tests were carried out as a blirid examination, as the condition of the patients from whom the samples had been taken was unknown. A known urine sample from a pregnant woman was included in the research series as a control sample, and a known urine sample from a non-pregnant woman was likewise incubated as a further control sample. Plate tests were carried out in the above-described manner and compared with plate tests carried out on the same urine samples after a conventional plate test for determining pregnancy based on an indicator system of latex particles in combination with CGTH.

The indicator system was used in the form of a h%-lg suspension. The antibody solution consisted of a 1:1000 dilution of the above antibody solution in saline. The test method consisted of mixing 1 drop of each of the urine samples with a drop of the antibody solution and then adding a drop of this mixture to a clean glass plate surface with subsequent addition to this of a drop of the h%-. ige indicator system suspension and rearrangement. The results were considered positive and marked + when a smooth sample was obtained which showed that agglutination was inhibited and therefore indicated pregnancy and were considered negative and marked - when a grainy sample was formed which showed agglutination and therefore indicated that pregnancy was not present .

The commercially available test was carried out in strict accordance with the accompanying instructions, and the results regarding pregnancy or non-pregnancy noted. The results were as indicated in Table III below.

It will be seen from the above table that all of the 31 urine samples turned out to be from pregnant women according to the indicator system from example 2. 11 of the 27 urine samples that were tested according to the commercially available pregnancy test turned out to be. negative and thus indicated that pregnancy was not present. However, it turned out that all of the unknown urine samples had been taken from pregnant women, as the duration of the pregnancy varied from a few

weeks to 8.5 months.

The known "urine sample from a pregnant woman and the known urine sample from a non-pregnant woman were tested only with the indicator system according to Example 2 and are therefore not included in Table III. The results were as expected, in that a negative result was obtained for the urine from the non-pregnant woman and a positive result for the urine from the pregnant woman who was in the 8th week of pregnancy.

Example 3

In contrast to the above example 2, an indicator system was produced by a 2-step reaction, the E.coli cells were first added to a buffer solution and then a BDB solution was added to form an immunological indicator. To prepare an indicator system, a solution of CGTH was then added and the coupling reaction continued.

PREPARATION OF REAGENTS

Phosphate buffer saline solution of pH 7.0 -

150 ml of a disodium phosphate salt solution was titrated to pH 7.0

with 75 ml of a monosodium phosphate salt solution. The first up- . solution was prepared by mixing 150 ml of 0.5M Na^PO^:7H 2 O solution with 350 ml of distilled deionized water and 500 ml of 0.85% saline solution. The monosodium phosphate salt solution was prepared by mixing 150 ml of 0.5M Na^PO^/^O with 350 ml of distilled, deionized water and 500 ml of 0.85% salt solution.

Phosphate buffer salt solution of pH 7.5 - 180 ml of the above disodium phosphate salt solution was titrated to pH 7.5 with the monosodium phosphate salt solution described above.

BDB solution - 1 volume part 0.025M benzidine<*>2HC1 in 0.36M

HCl was added to one volume of 0.1M NaN02 in distilled, deionized water, and the resulting mixture was allowed to react for 2 minutes. It was then titrated to pH 7.0 with a mixture of equal parts by volume of the above-described phosphate buffer saline solution of pH 7.0 and 0.31 M NaOH in distilled water. Immediately thereafter, 6 ml and 2 ml aliquots of the BDB solution were placed in containers, which were capped, sealed and frozen at -83.3°C. The BDB solution was thawed to <l>h°C as needed.

E.coli cells were treated with formalin as described in example 2 and were then prepared in the form of a 1.1 h%-Ig cell suspension and by means of a pipette transferred to a 50 ml centrifuge tube and centrifuged at 3100 rpm. minute for 15 minutes. The supernatant was then raised, and the packed cells were resuspended by first mixing in a vortex mixer until a uniform preparation was obtained, and then 20 ml of the saline solution from Example 2 was added. The cells were then centrifuged, and the upper layer raised. This washing with saline solution was carried out further, twice. 0.2 ml of the packed cells obtained were then suspended in 2 ml of the saline solution described above. This suspension was then kept at V°C for 1 hour. Then 2 ml of a 1:5 dilution of the BDB solution in the phosphate solution from example 2 was added and the whole was placed in the dark in a rotary shaker at h°C for 30 minutes. The cells were then centrifuged for 5 minutes at<i>f°C and washed twice with h ml of cold f osf atpuff er saline solution of pH 7.5, whereby a relatively stable suspension was obtained.

Then 1.6 ml cold phosphate buffer saline solution of pH 7.5 and 0.8 ml CGTH in 6 ml saline solution (h mg/ml) were added to the cell suspension. The obtained indicator system was prepared for testing by washing the preparation once with 25 ml 1:50 BSA in saline solution, centrifuged at 3000 rpm. minute for 5 minutes and resuspended using a vortex mixer. • A 0.9 ml<1>s solution of 1:50 BSA in saline was then added to resuspend the indicator system, namely for storage in a carrier. A 0.3 ml<1>s portion of the preparation described above was shaken for h hours at room temperature in a shaker. The suspension was then treated in a shaker at .37°C for 1 hour. The suspension was then washed twice in the above saline and once with 1:50 BSA in saline. The cells were then resuspended to 0.3 ml in 1:50 BSA in saline, whereby a 10% cell concentration was obtained.

An indirect agglutination test was then performed by mixing one drop of the cell suspension with one drop of each of three antibody solutions of covering dilution. The dilutions were 1:250, 1:500 and 1:1000. A smooth sample showing good indirect agglutination was produced for the 1:1000 dilution, and since the sample was not as marked as the two others, it was considered to indicate uniform agglutination. This shows that the indicator material produced according to the method according to the invention can be produced and stored separately and then used to connect antigenic materials which can be used later - for... testing.

Example '*+

An immunological indicator system was prepared for testing urine samples for the presence of CGTH by coupling human CGTH to yeast cells using the chemical coupling agent BDB. The indicator system was used in connection with the antibody to CGTH for the execution of a test with inhibited agglutination.

1g of Saccharomyces cerevisiae was weighed out and washed three times with 200 ml of the above salt solution. The yeast used was on the market under the trade name Fleischmann's Active Dry Yeast. The yeast cells were then suspended in 100 ml of 1% formaldehyde solution in saline. The suspension was allowed to stand at room temperature for 2 hours, with occasional shaking. The cells were then centrifuged at 2000 rpm. minute for 10 minutes and the packed eelle volume taken out and stored in 200 ml of the above saline solution at. k°C, This suspension showed a hematocrit value of 1.!+$.

To 0.5 ml of the packed formalin-treated yeast cells was added 12 ml of the above-mentioned phosphate buffer and then 6 ml of a saline solution containing 20 mg CGTH/10 ml, after which the cells.

"was contacted with ^0 ml of a 1:5 dilution of the above BDB solution. The reaction proceeded with continuous stirring for 20 minutes at room temperature. The resulting indicator system was collected by centrifugation and washed three times in the above saline solution, after which the packed cells were suspended in a 1:50 dilution of BSA in saline The resulting suspension showed a hematocrit value of 6%.

This 6% cell suspension was then used at a 1:500

dilution of Ab-CGTH for testing the presence of CGTH in two urine samples. A 0-2 ml portion of the 6% cell suspension was washed once and diluted with 1:50 BSA in saline to the original concentration, whereby a final volume of 0.2 ml was obtained. A drop of a known urine from a pregnant woman was mixed with a drop of the Ab-CGTH solution and allowed to stand for 2 minutes, after which a single drop of the mixture was placed on a glass slide. A drop of the indicator system was then mixed with the material on the glass plate. A smooth monster was formed indicating pregnancy. The test was repeated with a urine sample from a non-pregnant woman. A grainy, agglutinated monster was formed in a short time.

This example shows that yeast cells can be used as indicator particles for the indicator system. These cells have the advantage that they are supplied commercially in a dry, stable form.

Example 5

An indicator system was prepared using E. coli

as microbial cells, a carbodiimide as coupling agent and CGTH as antigenic substance. This indicator system was then used both in tests based on agglutination and in tests based on inhibition of agglutination.

PREPARATION OF REAGENTS

Microbe cells - E. Coli cells were cultured, inhaled and treated with formaldehyde as described in example 2. A volume of 20 ml of a 2.5% suspension was prepared in this way. This suspension was centrifuged and 0.5 ml of packed cells recovered for use in the preparation of the indicator system.

Coupling agent - 0.2 g of N,N'-dicyclohexylcarbodiimide was dissolved

in 0.5 ml of tetrahydrofuran, after which the solution was added to 1.0 ml of distilled water.

Anti<g>ent substance - k- 0 mg of CGTH was dissolved in 2 ml of distilled water. The CGTH used had an activity of 2590 I.U./mg and was supplied

by Vitamerican Corp., Little Falls, New York.

Ab-CGTH Solution - Aliquots of the antibody solution and the 0.85% saline prepared according to Example 2 were used to prepare 1:5°, 1:100 and 1:200 dilutions of Ab-CGTH.

FABRICATION OF THE INDICATOR SYSTEM

The 0.5 ml packed E. coli cells were washed five times

with cold water and then suspended in the 2 ml CGTH solution. The coupling agent solution was then added to the suspended cells and CGTH to couple them. The mixture was shaken and then set aside for 21 hours at 25°C. The following washings were then carried out to free the thus obtained indicator system from any entrained impurities: three times with 0.01M sodium carbonate solution, three times with 0.01M HC1, three times with distilled water, once with 1% NaCl (pH 2.3), once with a 1:20 dilution of phosphate saline' from Example 3 in 0.85% saline (pH 7.0), and once with a 1:50 dilution of BSA in 0.85% saline. The washed indicator system was then suspended to an 8% cell concentration in 1:50 BSA in 0.85% saline. This 8% suspension was stored in a cold room and taken out for the testing described below.

A preparatory agglutination was carried out by placing a drop of

each of the above antibody dilutions was mixed with a drop of the indicator system function on separate areas of a glass plate. A control experiment was performed by mixing a drop of a 1:50 dilution of normal rabbit serum (NRS) in 0.85% saline with a drop of the indicator system suspension on a separate area of the same plate. The results are listed in Table IV below, where "S" denotes a smooth and therefore non-agglutinated sample and "G" denotes a grainy and therefore agglutinated sample.

Table IV shows that agglutination occurred for the 1:50 and 1:100 dilutions, but that insufficient Ab-CGTH was present at the 1:200 dilution to cause agglutination of the indicator system. The control sample showed a smooth sample, showing that the system does not spontaneously agglutinate with the NRS solution.

Further testing was performed by mixing 0.5 ml of a urine sample from a pregnant woman with 0.5 ml of the 1:100 antibody dilution in a test tube, which mixture was then allowed to stand for 5 minutes at 25°C. A drop was then taken out from the test sample and mixed with a drop of the 8% indicator system suspension on a glass plate. A smooth sample, "S", was observed, showing that the agglutination of the indicator system by Ab-CGTH had been prevented by the CGTH present in the urine sample.

The test was repeated using 0.5 ml of a urine sample from a non-pregnant woman with the result that in a short time a granular sample "G" was obtained.

This example shows that an earbodiimide can be used as a coupling agent to bind antigenic substances to microbial cells.

Example 6

Two additional indicator systems were prepared according to Example 5. The antigens used were human serum albumin (HSA) and horse gamma globulin. The indicator system for HSA was prepared using 10 mg of HSA per 0.25 ml packed E.coli cells instead of CGTH. The indicator system for equine gamma-globulin was prepared using *+0 mg of this antigen per 0.25 ml packed E.coli cells instead of CGTH.

The agglutination test was performed by mixing one drop of each of the prepared indicator systems with one drop of a 1:10 dilution of their respective antisera in 0.85 µg saline. A control sample for each test was performed using a drop of a 1:10 dilution of normal rabbit serum in 0.85% saline. In each case, the antiserum droplets caused the formation of good agglutination patterns for the respective indicator systems. No agglutination was caused by the normal rabbit serum.

Example 7

The preparation of the indicator systems and the performance of the agglutination tests according to Example 6 were repeated with the same results using N-t-butyl-5-methylisoxazolium perchlorate instead of the earbodiimide coupling medium. 10 ml of a 2.5 µg suspension of the formalin-treated cells from Example 2 was centrifuged for a period of 5 to 10 minutes, after which the supernatant was removed. The cells were then washed four times with cold water and then suspended in 20 ml of 0.1 µg sodium bicarbonate in distilled water. 3 mg of succinic anhydride was then added and the cells were shaken overnight at 5°C. The cells were then centrifuged and washed a further three times with water, after which the succinylated cells were suspended in 5 ml of distilled water. A pipette was then used to add 10 microliters of triethylamine to the cells. Then 17 mg of N-tert-butyl-5-methylisoxazolium perchlorate were added. This coupling agent is also known as Woodward<1>'s reagent "L". The immunological indicator thus obtained was used to connect the antigens indicated in example 6 in the quantities indicated there. Human serum albumin and horse gamma-globulin coupled to E.coli by the method described above gave agglutination when reacted with rabbit anti-human serum albumin and 1:10 rabbit anti-horse gamma-globulin. No agglutination was caused by 1:10 normal rabbit serum.

Example 8

Twelve different indicator systems were prepared according to Example 2 with regard to the formalin treatment of the microbial cells and the coupling of antigens to these using BDB. The microbial cells used were Bacillus subtilis, Bacillus pumilus, Lactobacillus leichmannii and Pseudomonas fragi, and the antigens used with each of these indicator particles were HSA, horse gamma-globulin and human insulin. Plate-type agglutination tests were carried out.

The B. subtilis and B. pumilus used were grown in the same way as E. coli in example 2. L. leichmannii was grown for the same length of time and at the same temperature of 37°C in a foam composed of 890 ml of water, 100 ml -tomato juice filtrate, 10 ml "Bacto-Tween 80", 7., 5 g yeast extract, 7.5 g peptone, 10 g dextrose and 2 g disodium phosphate. The foam had a pH value of 6.8 and had been treated in an autoclave for 10 minutes at 121°C under an overpressure of 1.05 kg/cm<2> for sterilization.

P. fragi was cultured >+.5 hours at room temperature (25°C) in a pre-sterilized foam consisting of 1 liter of water, 5 g of tryptone, 5 g of yeast extract, 1 g of dextrose and 1 g of disodium phosphate.

The coughed-up cells were then treated with formalin and 0.25 ml of packed cells from each culture used according to the coupling method according to example 2, with three portions of h<y>s of these cells being suspended in 6 ml of the phosphate buffer_from this example and then to each of to these portions of suspended cells were added the following antigens dissolved in 5 ml of 0.85 µg saline: 3 mg of HSA, 100 mg of horse gamma-globulin and 20 mg of insulin. Then 20 ml of a 1:5 dilution of the BDB solution from Example 2 in phosphate buffer was added to perform corresponding coupling reactions.

Each of the twelve indicators was tested with their respective antibodies and with control samples of normal serum. The HSA and horse-gamma-globulin indicator systems were tested with a 1:50 dilution of rabbit anti-HSA and anti-horse-gamma-globulin, respectively, in saline, while the insulin indicator systems were tested with an undiluted anti- insulin serum from guinea pigs in saline solution. The control experiments for each of the twelve tests were carried out with a 1:10 dilution of normal rabbit serum in saline solution. The antibodies to each of the antigens agglutinated each of the four indicator systems prepared with the individual antigens, and no agglutination. was observed for the control samples.

Claims (3)

IN 1. Procedure for the production of an immunological indicator for chemical binding to antigenic substances, characterized by bringing microbial cells into contact with molecules of a coupling agent that has at least two non-bonded, reactive groups, and one of the reactive groups - is brought to to form a covalent chemical bond with the microbe cells while the other reactive group is retained in an unbound state, whereby the indicator becomes able to react with antigenic substances when brought into contact with such. 2. Method according to claim 1, characterized in that microbial cells selected from the group consisting of bacterial cells, fungus cells, parasitological cells and vlrus cells are used. 3. Method according to claim 1, characterized in that microbial cells of uniform shape and size are used and which have a maximum dimension in a direction from 0.2 to 10/ Lim. h. Method according to claim 1, characterized in that preservative-treated and possibly labeled microbial cells are used.