CH634923A5 - Process for the preparation of pili from Neisseria gonorrhoeae - Google Patents

Description

The object of the invention is a method which enables reproducible extraction, i.e. Isolation of pure and optionally crystalline pili from N. gonorrhoeae; This object is achieved according to the invention by a method in which N. gonorrhoeae bacteria of the type Ti and / or T2 are first artificially grown to obtain a culture of piled N. gonorrhoeae cells; then the culture of the piled cells or a mixture containing the pili is subjected to the treatment steps defined in one of the independent claims 1, 6, 8, 10 or 11, the common feature of which is compliance with certain pH values during processing . 60

Preferred embodiments of the method defined in claim 1 have the features mentioned in one of claims 2-5. A preferred embodiment of the method of claim 8 has the features specified in claim 9. 65

Another object of the invention is to provide a method for diagnosing gonorrhea. This object is achieved by a method which is characterized in that

that a serum sample to be examined is combined in vitro with Pili of N. gonorrhoeae of the type Ti or T2 in the absence of cell residues.

Artificial cultivation to obtain the culture can be carried out in a manner known per se using both surface and immersion culture methods, as explained in more detail below. In the case of surface growth, it is expedient to keep the total growth, i.e. remove the cells and pili from the culture medium and suspend them in an aqueous medium at a pH below 9.2. If only T2-PÌH is to be isolated, any pH above 5.5 and below 9.2 is suitable. If Ti-Pili is to be isolated either alone or in the presence of T2-PÌH, a pH below 7.7, in particular around 7.0, is required. Such a suspension step is not required for diving cultures.

GC pili at pH below 4.5 have been found to be soluble and substantially reconstitutable when the pH is increased above 4.5 again provided that it has not decreased below 2.5.

Analysis of GC-Pili by SDS electrophoresis on polyacrylamide gel reveals a main band and a sub-band. The material corresponding to the main band contains phosphoglycoprotein and is called GC-pilin.

It could also be shown that the T2-GC pilin comprises a peptide fraction with 200 ± 9 amino acids, between 2 and 3 phosphate groups and between 1 and 2 hexose sugar and is essentially soluble in aqueous media at a pH greater than 10.1 and essentially insoluble in aqueous media with a pH of less than 8.6, each measured at 20 ° C.

The majority of the T2-GC-PÌH, namely T2-GC-Pilin has a molecular weight, determined by SDS-acrylamide gel electrophoresis of 21 500 ± 1000 Daltons.

The GC pili isolated from N. gonorrhoeae type Ti appear to be immunologically very similar to those isolated from N. gonorrhoeae type T2

In comparison, the Ti-GC pili were found to be essentially soluble in aqueous media above pH 8.5 and substantially insoluble in aqueous media pH less than 7.7 pH value is measured at 20 ° C. The Ti-GC pilin has a molecular weight of 22,000 ± 1000, determined by SDS-acrylamide gel electrophoresis.

GC pili were isolated from cultures of more than 20 different strains of N. gonorrhoeae. GC pilus crystals as well as single-rod pili and the eluate from the SDS acylamide gel electrophoresis lead to the formation of antibodies in the serum of test animals when they are injected into them.

When GC pilus crystals are contacted either as such or in a suitable suspension medium with a serum containing antibodies, the crystals agglutinate. This agglutination can be easily observed, most conveniently in a dark field microscope but also with the aid of other devices, and is a simple and immediate test for the presence of Pilus antibodies in the test serum.

Areas of application for this test include: gonorrhea screening to select individuals to perform culture trials, identification of high-risk individuals, differentiation between new and old infections in individuals, identification of the strain responsible for local epidemic and strains, who are responsible for special symptoms.

It should be noted that during these attempts to determine the presence of antibodies to GC-Pili in a test sample, it is not possible to determine directly whether the person from whom the serum is taken is currently suffering from, or has previously infected with, gonorrhea was and

634923

4th

is only a carrier of antibodies. It should also be noted that a person who has recently been infected (ie 2 or 3 days ago) cannot experience a positive reaction since the time may not have been sufficient to produce a sufficient concentration of antibodies in the body, to give a detectable titer.

It has been found that the pili of N. gonorrhea organisms contain one or more immunological determinants from a group of at least four such determinants. Thus the antibody agglutination reaction occurs between Pili and a serum containing antibodies against at least one of these immunological determinants. The strength of the reaction depends on the concentration of the antibodies in the serum sample to be examined and at the same time on the number of interrelated immunological determinants on the pili and in the serum.

It is thus possible, for example, if one has four different GC pilus samples, which are known to contain at least one of the above-mentioned determinants, to easily detect the presence of the corresponding antibodies against GC pili in each test serum.

Similarly, if an organism source is available and can be easily grown, the pili can be isolated from these organisms. Since standardized sera containing antibodies against any of the four anti-gene determinants of GC pili are available by the method of the invention, these pili can be examined for their identity and the number of these determinants from the unknown source to be examined and can be determined (serotyped).

This procedure is very helpful in the epidemiological investigation of the path of infection from gonorrhea.

Pili can be absorbed on various carriers known for immunological tests, such as latex, washed red blood cells, activated carbon, polyacrylamide, agarose and the like, in order to obtain substrates for agglutination tests in serum or plasma.

The availability of Pili also forms the basis for studies of hemagglutination and hemagglutination inhibition. Both attempts are based on the principle that pili contain specific binding sites that interact with red blood cells. For example, when the pili and blood cells are incubated together, the red blood cells result in a diffuse agglutinated pearl when discontinued due to the effects of gravity. If there are no pili in the test medium, the red blood cells settle and result in a clearly defined pearl when settled under the influence of gravity. This is a means of testing for the presence of Pili in a solution.

In the hemagglutination inhibition test, a serum to be examined, which is thought to contain antibodies against GC pili, is added to a solution containing a predetermined amount of pili, and the mixture is incubated and centrifuged. The pili interacting with antibodies are precipitated. The supernatant is then added to red blood cells. When all pili have reacted with the antibodies in the sample, a sharp pearl is created, i.e. no agglutination. It is understandable for the person skilled in the art that such a test is meaningful if it is carried out against comparative samples (i.e. without antibodies) and at previously determined dilutions.

The accuracy of this test is increased if the serum is first treated with washed red blood cells (i.e. before adding to the pili). This procedure removes factors in the serum that cause agglutination of the red, regardless of the presence of pili

Blood cells.

So far, no immunization against N. Gonorrhoeae in humans has been possible. It has been found that when volunteers have been injected with a sufficient amount of GC pilus crystals, conveniently in an amount of about 2 to about 100 ng / kg body weight to reduce the serum antibody level to a titer of at least 100 in the PAT (pilus) agglutination test), protection of at least 1.6 log cycles was achieved. This means that the person was not infected by a number of organisms from the strain from which the injected GC pilus crystals were derived, which was approximately 1.6 orders of magnitude larger than the number required to be used in comparative persons who were not are immunized to lead to infection. No toxic effects due to the pili were observed when GC pilus crystals were injected. Individuals with a titer of up to 200 do not appear to be attacked (infected) and the test primates (rhesus monkeys) were exposed to approximately 10,000 titers in the PAT test without any signs of disease being observed. It was also found that it appears advisable to carry out the injections of GC pilus crystals in a suitable medium over a somewhat longer period, advantageously up to 5 or 8 weeks. Administration can be in 1 to 5 portions of GC pilus crystals, single stick pili, or any suitable source of GC pilin. Administration over a period of time is cheap but not essential. This administration gradually leads to the build-up of antibodies in the system.

It can be seen that no local negative reaction against the crystals at the injection point was observed in people who had previously been given the crystals.

When using the pili for immunization, given the presence of the various antibody determinants mentioned above, it is beneficial to administer pilus crystals, single stick pili or other sources of GC pilin that contain all known determinants.

For the production of GC pili or GC pilus crystals according to the present method, the cultivation of N. gonorrhoeae organisms is first necessary, e.g. by surface culture, as explained below.

Neisseria Gonorrhoeae occurs in four colony forms, which have been arbitrarily referred to as types Ti, T2, T3 and T ». However, this term is generally recognized. The Ti and T2 organisms are the causative organisms for gonorrhea in humans and only these forms have pili. The methods given below are applicable to the cultivation of Ti and T2 organisms. It should be noted that the strains of N. gonorrhoeae can be isolated from human body secretions. These secretions usually contain not only the desired types Ti and T2, but also the non-pili types Ts and T4. It should also be noted that a culture which is set up, for example, as a relatively pure T2 culture, forms organisms of the type T3 and Tt which do not form pili and of the type Ti in the course of time when subcultures are formed.

In the culture of T1 and T2 colony variants, a third pili-forming variant, which is referred to arbitrarily as TR, is observed. This variant has a rough appearance and although it is not fully characterized, it is believed that it is closely related to the Ti and Ti species and these species have been obtained by subculturing TR colonies. The yield of pili from TR species is the same as from T2 cultures.

For maximum formation of either T2 or Ti pili

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

5

634 923

the following pretreatments are appropriate:

The original samples are e.g. grown on Thayer Martin (T-M) plates that allow N. Gonorrhoeae to grow and inhibit the growth of most other bacteria. Since T-M plates are not suitable for distinguishing between types of colonies, colonies are spread from the T-M plates onto suitable growth media (e.g. GC medium, catalog No. 0289-1 Difco). A series of subcultures are then prepared from individual colonies on the medium (hereinafter referred to as GC medium) until the colonies comprise more than about 90% of the desired type. While the method can be applied to the Ti species as well as the T2 species and, as has been shown, the Ti and T2 species are immunologically similar, it is preferred to keep the species separate. T2 is discussed below. Unless expressly stated otherwise, the breeding methods for T2 are equally applicable to Ti. If the growth on a plate consists of more than 90%, for example, T2 colonies, the entire growth is removed from the plate and in a suitable medium for freezing e.g. Suspended BSA-glutamine, divided into equal parts and conveniently stored under reduced temperature at approximately -70 to -196 ° C.

It should be noted that, as stated above, samples with an initially high level of T2 tend to become unstable when subcultures are grown and result in lower levels of T2. Therefore, when cultivating the cultures, care must be taken on the one hand to ensure a high initial content of Tî organisms and on the other hand to use a sufficiently "young" strain, i.e. one that has not yet been bred too often in subcultures to avoid the occurrence of instability. If Ti-Pili is to be formed, it is even more important than in the case of T2 to use an inoculum that contains more than 90% Ti.

The inoculum for pilus formation is e.g. prepared by spreading the first, but not necessarily frozen, portions onto a GC medium in petri dishes and incubating for 12 to 24 hours; 12 to 15 hours for strains with more unstable T2 species and 12 to 24 hours for strains with more stable T2 species. It is preferred to grow the inoculum at a temperature of 35 to 37 ° C, although 35 ° C is preferred. High humidity is also preferred. With moisture contents of less than 70%, it has been shown that the pilus yield is lower than with higher moisture values. It therefore seems to be cheap to work in a humidity between 70 and 90%. While the influence of the atmosphere on growth has not yet been fully clarified and older N. gonorrhoeae cultures grow without the addition of carbon dioxide, an atmosphere with a content of 5 to 10% carbon dioxide and 90 to 95% air has proven to be extremely suitable .

After the initial inoculation, when the plate is covered with about 50 to about 75% growth, this growth is usually removed. In a suitable method, a small amount of a sterile casamino acid solution is added to the plate containing the inoculum and the growth scraped off with a sterile glass spatula. It has proven to be advantageous to use 5 to 6 ml of solution per plate and 2 to 3 ml of the suspension thus produced are sufficient to inoculate the larger growth dishes with the GC medium. The dishes are then incubated with the inoculum for approximately the same time under the same conditions as the Petri dishes, and the pili are obtained therefrom.

At this point in the procedure, it is no longer necessary to operate under sterile conditions, although of course, as with all procedures, it is convenient to use clean equipment and reagents and to perform all operations at temperatures as low as possible to avoid an undesirable one To prevent bacterial growth.

Gonococcal growth is e.g. obtained with the help of a suitable buffer. While the chemical nature of the buffer is not critical, the pH range is important. For reasons that will become clear, a buffer with a pH range above 9.3 cannot be used to purify T2-PÌH. It is preferred to operate in a pH range between 5.5 and 9.2, most preferably in the range of 7.0 to 8.6. If a culture consisting predominantly of T1 is used, the pH should not exceed 7.7, but should be in the range from 5.5 to 7.5, advantageously from 7.0 to 7.2. These areas ensure that the entire piled material (provided with pili or hair) remains in the solid state. The most suitable buffer is tris (2-amino-2-hydroxymethyl-l, 3-propanediol) buffered saline.

In a preferred mode of operation, the buffer used for washing is placed on the surface of the growth medium and the growth removed from the medium with a suitable instrument and the aqueous suspension in a suitable manner, e.g. removed with the help of a pipette or a vacuum suction device. If desired, a second wash can be carried out in the same way and the aqueous suspensions can be combined.

In order to increase the yield, a third washing process can be carried out using a buffer with a high pH, that is to say a buffer with a pH above 9.3, advantageously between 10.1 and 10.3. The use of such a buffer leads to the solution of the entire remaining Pili material. If Ti-Pili insulation is an option, the pH need only go beyond about 8.6. However, there are no disadvantages at higher values. This basic suspension is not combined with the first two washing suspensions, but is kept for a later stage in the cleaning process. It should be noted that when the pili have grown well, the vegetation has a characteristic orange-pink or warm-pink color and the growth medium has an odor that is reminiscent of cooking food. It is noted that the vegetation clumps together into sticky rope-like aggregates and can be easily stripped from the growth medium if it is used with a suitable instrument, e.g. a glass spatula is pushed away.

While the above procedures are convenient when high yields of very pure pili are desired, acceptable results are also obtained by a much shorter procedure. With this method, the first and second washing operations indicated above are not carried out. The entire vegetation is treated with a buffer with a much higher pH value, advantageously an ethanol amine buffer corresponding to the third washing stage. In addition to the dissolved pili, the washing medium contains many impurities that would otherwise be removed. However, it has been shown that these contaminants are kept in solution when the Pilus material precipitates in the manner described below.

The immersion culture of the organisms of the types T1 and T2 in liquid medium is e.g. carried out in the usual way using a medium and an environment which are identical to those used for the surface culture, except that the medium contains no agar as a solidifying agent. It has been shown that the organisms continuously form and grow pili. A diving culture medium contains a lot of suspended pili material. If T2 cultures are grown at a pH that is usually below 9.3, no undesirable solution of the pili occurs. When cultivating Ti organisms, the pH can rise above 7.7 because, as described later, the

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

634923 6

pH to a range from 5.5 to 7.7, preferably uncommonly, may allow the pH to remain unchanged. If 7.0 to 7.2 should be set. Although it is not critical to achieve solving by changing the pH is table, it is preferred to adjust such a few hours to the residue of a suitable buffer with a moderately high, for example 8 to 20 hours before working up pH added. It is convenient that the buffer perform one to crystallize the partially dissolved pH between 9.3 and 11, preferably between 10.0 and

Ensure Ti-Pili. 10.4. At higher pH values there is a risk of

It is beneficial for both Ti and Tz cultures, but denaturing the peptides. At this pH, the growth in a slightly agitated Ti and Tz pilus crystals does not go into solution. If Ti-Pili from Tî-Pili

Medium in the presence of a small amount of diatomaceous earth to be separated in the original solid bead,

how to let Celit take place. The amount of 10, for example, is first used in a buffer whose pH greater than celite should advantageously be between approximately 0.1 and approximately than approximately 7.7 but less than approximately 9.3. The suspension

0.5%, advantageously around 0.3% by weight, based on that, is then centrifuged and the resulting supernatant

Growth medium. Liquid can be set aside or discarded

Generally it should be noted that the cleaning of the pili, depending on the requirements of the procedure. If requested

Washing liquids taken from the surface growth of 15 is essential to the original solid pearl

N. Gonorrhoeae organisms contain substances, which contained therein amounts of Tz-Pili, fresh buffer with an altitude-soluble and which for the isolation of the GC-Piluskristerne not ren pH-value, namely over about 9.3, one being added are of interest. Similarly, the same applies to the deep liquid phase containing the Tz-Pili, but free of Ti-Pili, from the growth of liquid cultures. In the case of surface stands. The actual composition of the at this stage

Growth is the amount of liquid applied by the buffer 20 wash solutions is not critical. A buffer from Tris in

(only first and second washing solution), however, based on the amount of saline solution is particularly preferred. In the two of the grown organisms, relatively small. It is the above modifications will become the overall fix

preferred to add a buffer volume to the entire washing suspension for relatively substances, which centrifuged about three times as slow. The centrifuging size is like the volume of the solids. This amount is like-

speed and shooting time are by no means critical. It is not critical, but has been found to be sufficient, but has been found to be convenient to dissolve the pilus material at around 1000 without excessive volumes of 12,000 g (hereinafter referred to as 1 kg and 12 kg) of approximately aqueous medium must be applied. If the

5 to about 30 minutes, preferably at 3 kg, about 10 pili are from a surface culture and centrifuge that culture for about 15 minutes. The residue in a third washing stage with a similar buffer containing the pearl contains both cell material and pili material which has been subjected to high pH, this washing

both are in the solid residue and the supernatant liquid is now added. The pearl is then in the

Liquid is discarded. The supernatant liquid is suspended in the aqueous medium. The process by which the substantial amounts of contaminants as well as small pearl is suspended is not critical, and it is

Amounts of pili that are not worth gaining. can be a short light sound (sonication), a long

If an immersion culture is used, the liquid is 35 magnetic stirring, hand pipetting, hand mixing, turbo

volume is quite large and therefore centrifugation or mechanical stirring can be used,

be tedious. The application of a diatomaceous earth sandwich- It has proven to be beneficial to

filters has to stir to concentrate the grown products. While the way in which the suspension originated from immersion cultures proved to be suitable. In this process, it is not critical that it is important that which process

If a very coarse filter paper is applied to the filter base, always gently, the cells are not broken open, a sintered glass pane or a Buchner top is usually laid, since breaking open the cells to penetrate, a layer of diatomaceous earth, favorably unwanted substances in the aqueous layer lead Celite, with a thickness of about 2 to about 5 mm on that would. When cells break open, this is noticed

Given paper and covered with a second coarse filter paper by placing a layered pearl with a red layer over it. The celite serves as the actual filter medium, while the white portion during the subsequent centrifugation rend the upper filter paper only as protection for the surface. The suspended material is then centrifuged. That serves. The culture broth, the pH of which has been checked, type of centrifugation is not critical. The conditions given above for the first to ensure that the pili are in crystalline form, however centrifugation has been filtered through the filter pad and the filtrate discarded. proven suitable. Instead of centrifuging, you can also

The combined residues are filtered in, for example, a 50.

Buffer with high pH value, which is similar to that. The dissolved pili are found in the supernatant liquid for the third washing process in the surface culture or in the filtrate, from which it is removed by lowering the water, and this suspension is included approximately pH value can be precipitated. The degree of pH-1 centrifuged to about 12 kg. In this case, the degradation naturally depends on whether Ti or Ti-Pili centrifuge beads are of course the diatomaceous earth and the 55 present. The precipitation can optionally be achieved by cell debris and the pilus material can be found in the remainder by adding sufficient ammonium salt, more favorable liquid. a salt of a mineral acid such as the sulfate, preferably at this time it is convenient to wise the GC pilus material in aqueous solution in order to separate an ammonium sulfate concentration from the cell debris and in the case of an immersion of approximately 4%, based on the Saturation, up to uncultivated, also from the diatomaceous earth. 60 to reach 10% based on saturation. However, to

In the case of a surface culture, a pearl of which the yield and purity can be improved, it has proven to be

Centrifuged washing liquids with a low pH value have been proven to go through further stages before the precipitation.

are made by adding an aqueous medium that does not result.

covalent bonds between the pilots in the process of performing these additional steps to increase

System breaks open while the covalent bonds are intact and the yield and purity

stay. As a result, the pilus crystals become liquid and the pearl (the deposited solid)

Broken single-stick pili. Such a medium from centrifuging at high pH or filtering

may require an increase in pH or retention. The pearl from centrifuging

7 634923

high pH is resuspended, favorably in If the pilus crystals should not be used immediately in the same aqueous medium at the same pH, regardless of the sterilization by filtration, the same manner and the suspension again in the same addition of preservatives proved to be cheap. It's centrifuged way. After this centrifugation, the pearl is favored so that the preservative is not discarded to the pilus crystals and the supernatant liquids from the two stalls themselves or to a solution in which they are contained, centrifuging steps are added and centrifuged again, but to that Dialysis buffer reduced. pH value of the solution. If the added consumer

The reason for re-centrifugation is that the detergent is incompatible with the buffer after removing residual suspended contaminants. That the pilus crystals are crystallized, the incompatible buffer centrifugation is therefore carried out at a higher speed - "> by dialysis against a compatible buffer and performed than before. Speeds between 12 and 70 kg a further dialysis can be carried out with the help of the-within 30 up to 60 minutes is suitable. It is in the general preservative and the new buffer. However, the preservative mean preferred to centrifuge at speeds of un-mixed that can be used include formaldehyde for about 27 to about 40 kg for 60 minutes Merthiolate and Azide These preservatives are used in

The beads are discarded and the supernatant liquor, from about 0.02 to about 0.05% applied, is recovered. Each of the specified preservatives has

At this stage, it is usually beneficial to have certain adverse effects on the pilus solution. Formaldehyde leads to sterilization. Through certain FDA rules for certain networking between the pilots. You can do this if necessary. Such sterilization can no longer be solved as before. Merthiolate is not easily obtained by immediately crosslinking the pilus solution and the crystals can be re-formed before being precipitated through a Millipore filter, whereupon. However, such newly formed crystals have a filter with a pore size of 0.45 p.m. which has been shown to be particularly reduced in the presence of antibodies against them. Then the products themselves have to agglutinate the pili. However, the antigenic effect is not intelligibly affected under aseptic conditions, i.e. when they are injected into the test subject or when the sterility is to be maintained. 25 lead to the formation of apparently normal antibodies

In certain circumstances it may be beneficial to use the pili against the pili. Azide is a very inexpensive preservative that can be isolated in the form of individual sticks and not in crystals because it does not attack the crystal structure or the antigenic form. In this case the supernatant fluid becomes agglutination. Unfortunately it is toxic again in an ultra high speed centrifuge and cannot be used if the pilus crystals see 60 and 166, favorably 106 kg 2 to 4 hours centrically 30 people to be injected. The crystal preparation is fugged, the pili in the form of individual rods favorably at low temperatures, i.e. at around 1 den. stored up to 4 ° C. However, if longer storage

The pili are preferably re-precipitated by being considered, it is preferred to dissolve the crystals in a suitable deposition of the pH of the supernatant liquid on the high-pH buffer using a Millipore-Fil-below 9. 1. It has been shown that the best results with respect to filtering and obtained in solution under sterile conditions depend on the nature of the crystalline material. If the pili are required in crystal form, dialysis against a buffer with a correspondingly low level is preferred to re-form the crystals by adjusting pH. It has proven to be advantageous to lower a buffer with the pH to an initial pH for the T1 or Ti pili corresponding to an initial pH value between 8.3 and 8.6 for Ti pili for the crystallization . When turning as good as possible, while the chemical nature of the buffer is not intended to achieve critical conditions for preservation, it is table. Saline buffered with Tris has been shown to be suitable for sterilization and preservation at the same time. The dialysis is conveniently carried out by turning.

one about 30- to 60-fold, conveniently about the expedient for cleaning the pili at

40-fold excess applies. The dialysis is carried out under a constant pH value, essentially the stirring of the outer dialysis medium for about 12 to 45 is the same as that stated above for different pH values - about 18 hours. It is preferred which is ben. Aqueous solutions of dialysis at reduced temperature, i.e. a temperature of, for example, salts, advantageously alkali and alkaline earth metal, from about 0 to about 10 ° C. This gauze with anions of mineral acids with an ionic strength above the lower temperature range reduces the occurrence of undesired, advantageously between approximately 4.0 and approximately 5.0, desired bacterial contaminants. It is also to 50 especially of about 4.4, urea with a concentration note that the pH of the buffer is temperature-dependent between 3 and about 5 m, sucrose with a concentration, ie when the pH of the buffer is measured If after 50% by weight and finally water itself is used and the system has cooled to the working temperature, the ionic strength of the solution can drop below 0.002 m - it can be seen that the pH value drops to 9 , 1 has risen. It is put.

however, satisfactory results will still be obtained. 55 In a modification of this procedure instead of the It is also to be noted that it is not generally necessary to change the pearl obtained in the first centrifugation, the cells, pili and the buffer if there is an excess of buffer in the cell debris, in a medium with a high pH value in the specified range. Suspend the crystal suspension, any of the above, is then processed to separate the pilus crystals. specified conditions applied. The cheapest separation is centrifuged by centrifugation at moderate speed as above with low speed. When speed reaches. For about 60 minutes a higher yield is desired, the supernatant centrifugation at about 3 to about 8 kg has proven to be suitable as a liquid from the slow centrifugation. The supernatant liquid is then resuspended in a similar medium. centrifuged again and the resulting supernatant

If the pilus crystals are to be further cleaned with the previous supernatant liquid, the dissolving, centrifuging at high speed can be combined and centrifuged at high speed and repeated centrifugation repeated two or three times. The beads are centrifuged at high speed. are discarded and the supernatant liquid

634923 8

dialyzed against an appropriate buffer to remove the solvent that has not yet formed sufficient antibody to remove. can have to get a positive response. At Per-

Thus, if the agent that enables the solution is obtained, the ionic strength on turtest will be carried out if the test is positive for the usual cul- charose or high concentration salt. It has been shown how later a value below 0.5 for the salt and below 40% for sucrose 5 is discussed in more detail that the pili is reduced by infectious forms. In the case of urea, the urea against N. gonorrhoeae will contain a number of specific immunological an appropriate buffer, e.g. have dia determinants with Tris buffered saline. Pili of certain strains have a lysed to an ionic strength between 0.05 and 0.3 at a pH or more of these determinants. Therefore, in order to obtain a score of 7.0 or 8.3, pilus crystals must be worked on, depending on whether the Ti or T2 pili test is to be carried out effectively. Similar procedures are used when using 10 that the full spectrum of immunological solution-solving water is to include the ion determinants.

increase strength to at least 0.05. The GC pilus crystals used for the test are after

The buffers used for this purpose are e.g. the same way described above available.

as used in cleaning at different pH values. The test can be carried out using, and they are applied in the same way to serum or plasma from the person to be examined. I'm turning. The expressions can be related to this test

The cleaning of GC-Pili by centrifugation with a «serum» and «plasma» can be exchanged. The density gradient can be carried out as follows: the required amount of serum or plasma is extraordinary

Centrifugation using the density radius was low. It has proven to be sufficient to carry out a few drops by taking a mixture of Pili and 20 blood from the patient's fingertip and centrifuging aqueous cesium chloride together and centrifuging them in a small (about 250 jo.1) centrifuge tube -Using optical density at a given wavelength, yaw in order to obtain between 10 and 20 p.1 plasma, which is used to prove in which part of the tube the pili reaches to carry out the test.

are holding. It is common to conduct such tests at different dilutions.

While centrifuging at a pH below 9, 25 genes are to be performed. The serum is therefore usually pre-known, better results are obtained if the centrifuge is added (usually by serial dilution) to a pH range of 10.0 to 10.4, advantageously a predetermined suitable diluent. 10.1 performs. This procedure uses the raw pearl that is thinning. The type of diluent is not critical, contains pre-cellular material, pili and cell debris, suspended in a buffer with that it does not interfere with the operation of the test, high pH suspended as indicated above, and a 30% aqueous buffer, such as add a suitable amount of cesium chloride buffered with phosphate buffered with saline. For example, solution or Tris buffered saline with a pH has been found to be convenient to prepare a medium containing between 7.0 and 7.5 can be used. Puffed with Tris about 2 to about 5 g dry cesium chloride per ready saline solution with a pH of 7.2 is preferred. With 10 ml aqueous medium. This is the most convenient way of carrying out the test. The pilus suspension is added to the serum diluted approximately 7.5 g of cesium chloride in 20 ml of aqueous medium 35 in order to use a final concentration. see 10 and 50 jig / ml crystals to get in the suspension.

The mixture is then about 30 to about 60 hours. It has been found that the best results are obtained at low, preferably about 42 hours with about 110 to concentrations of pilus crystals and therefore about 250 kg, favorably about 200 kg, is centrifuged for Standardization of pilus antibody levels and the optical density of the fractions at a predetermined- the test sera measured the arbitrary concentration for the most comparable point in the tube. The measurement of the optic purposes of 20 (ig pilus crystals per ml of diluted serum (or see density at 280 nm shows a single maximum. The dich-plasma) was taken as the standard.

Fractions below this maximum are separated off and after the pilus crystals have been mixed with the diluted

dialyzed against a low pH buffer, the mixture is incubated around the ten sera. The incubation period is to be obtained from pilus crystals in the manner described above. The 45 is not critical and can range from times as short as 15 minutes actual density range, which at this point is 20 ° C to times as long as 48 hours with the fractions of solution being collected, is between 1.35 changes in readings are negligible. At one to 1.33 at pH 10.1. The rapid form of the PAT test is the serum-pilus mixture

Performing a PAT test for antibodies against N. by hand for one to three minutes on a Gonorrhoeae slide is explained as follows: 50 stirred, similar results are achieved, but with

The basic ability of GC pilus crystals or a slightly lower sensitivity. The temperature at which the mixture is held with single pilus sticks in the presence of antibodies to N. is also not essential to agglutinate gonorrhoeae is the basis for the PAT - as long as it is between 0 and approximately 45 ° C with the AusTest. In this test, blood sera from individuals who believed that it appeared to be beneficial were subjected to the mixture on what was suspected to be N. gonorrhoeae 55 temperature between 22 and 40 ° C, preferably at approximately, with GC- Hold pilus crystals or single pilus sticks at 22 ° C for at least 15 minutes. Storage mixes and observes whether agglutination of the crystals of the mixture occurs at temperatures as low as or rods. 4 ° C up to 24 hours does not seem to change significantly

In order to be able to fully evaluate this test, three important titers must be obtained. Satisfactory results were considered factors. First, the 60 is obtained by incubating at 37 ° C for 30 minutes. After test not intended to replace the standard «culture» tests- the incubation period will be a portion under a darkening, but it can be used for a quick examination whether the question-field microscope is given and the agglutination observed and Liehe person has been exposed to N. Gonorrhoeae. written down. The notation is carried out in the usual way for such tests. The test also shows positive results for both people, arbitrarily relative, namely 4+, 3+, 2+ who have had an active infection for more than a few days than 651+, ± and -. To determine the titer of a given sample, even for individuals who had previously had the disease, the last dilution that has an agglutination of, but is now cured. The third constraint is 1 +, used as the final reading. The value 1 + is the-consists of the fact that people who have only recently infinite value have the minimum noticeable agglutination

compared to a standard comparison sample.

Since the readings for the titer can vary from batch to batch of the pilus crystals, depending on their condition, it is advisable to monitor the execution of the test by combining crystals with Antisera with a known PAT titer in addition to the usual comparative tests against diluent without Serum and diluent with normal serum.

It has been shown that under the conditions used, the test within the titer readings gives a factor of 2 (times 2 [X 2] factor) which is reproducible.

GC pili produced according to the invention can also be used for serotyping N. gonorrhoeae.

So far it has not been possible to classify N. gonorrhoeae organisms in a suitable and meaningful system (serotyping). These antibody reactions characterize the strains and the identification of these characteristics is an essential aid for epidemiological studies of the progression and origin of a specific outbreak of the disease. It is of particular interest in venereal diseases that the elimination of the disease very often depends on the detection of person-person contact. This makes it easier to track the route of infection if the source of the infection can be identified by serotyping.

It has been shown that by examining 21 strains obtained from sources distributed across the United States, there are at least four different immunological determinants in the pili of these strains. One or more of these determinants are present in each strain. Therefore, if the determinant properties of a particular sample can be identified, the origin of the infection can be found more easily.

When N. gonorrhoeae organisms have been obtained from an infected person and grown to form pilus crystals, the immunological determinants present in the pilus crystals can easily be detected by subjecting them to a PAT test with the above-mentioned test sera, known from which is that they contain antibodies, but only for one determinant at a time. In this way, the serological profile of an organism in a given sample can be determined.

GC pili produced according to the invention can be used for the preparation of gonorrhea vaccine e.g. can be used as follows:

GC pilus crystals and single-rod peppers were injected into experimental animals or subjects in vivo, and were found to have no toxic effect. The only negative effect was observed with intravenous injection into chicken embryos. Rabbits were given three subcutaneous injections of 100 to 200 (xg / kg, which corresponded to a total dose of 300 to 600 ng / kg. Rats were similarly injected twice 8000 (xg / kg, corresponding to a total dose of 16,000 (ig / kg. Rhesus monkeys received three injections of 100 μg / kg intramuscularly and humans 2 to 10 μg / kg and then once 50 μg / kg intramuscularly, none of the experimental animals died or showed any local or systemic toxic effects, and the chicken embryos showed an LD-50 -Value of 60 jxg per 0.454 kg. The reactions in humans varied between no systemic effects and volatile cold showers and fever in one test subject. The PAT titer in rabbits reached 1000 to 8000 and in rhesus monkeys 10000+. The PAT titer in humans varied between 100 and 200.

The dose required to infect 50% of the test subjects (IDjo) is on the order of 5.0 x 102 organisms. Initial preliminary tests show that the IDso of a person with a PAT titer of 100 to 200 2.0 x 104 organ634923

amounts to. This means a protection of 1.6 log cycles or in other words: in a person with a PAT titer of 100 to 200 there is only 0.86% risk of being infected on one contact, while in a non-immunized person there is a risk is about 30%.

Based on these findings, in order to achieve adequate protection, a sufficient amount of GC pilus crystals should advantageously be administered against all known determinants (determining factors) in order to increase the PAT content against each of these determinants to at least 100, preferably at least 200 increase. Such levels are achieved by administering about 2 to about 100 ng / kg body weight GC pilus crystals for each determinant. However, it should be noted that a titer of 100 can also be achieved with amounts as small as 1 | xg / kg. The mode of administration depends on the sensitivity of the individual, if there is one, but the vaccine can conveniently be administered in 1 to 5 doses over a period of up to 8 weeks.

The GC pilus crystals, single pilus sticks or other sources of GC pilin can be administered in any suitable medium for intramuscular injection.

In connection with the invention, 21 strains of N. gonorrhoeae were isolated from people with gonorrhea and were designated as follows:

Pittsburgh 1-2, Pittsburgh 3-2, Pittsburgh 4-2, Pittsburgh 6-2, Pittsburgh 7-2, CDC B-2, CDC C-2, CDC M-2, CDC T-2, CDC F62-2, Atlanta 4-2, Atlanta 6-2, Atlanta 9-2, Atlanta 10-2, Seattle 1-2, Seattle 3-2, Seattle 9-2, Norfolk 2-2, Norfolk 7-2, Dayton 8-2, CDC 005-2.

The following example specifically refers to the Pittsburgh 3-2 strain. When the other organisms listed are used in the same procedure as the Pittsburgh 3-2 strain, similar results are obtained.

(A) Cleaning the trunk

The primary cultures of the Pittsburgh 3-2 strain are applied to Thayer-Martin plates containing Thayer-Martin agar (Manual of Clinical Microbiology, Ed. 2, Amer. Soc. Microbiol, Lenette et al. (Ed.) 1974, p . 920). The plates are incubated for approximately 18 hours at 35 ° C and 90% humidity in an atmosphere consisting of 95% air and 5% carbon dioxide. The plates are examined and the colonies, reminiscent of the strongly «hairy» (piled) Tî form, are again spread on GC medium. After incubation under the conditions indicated above, the colonies of the T2 type are removed and again spread on GC medium.

According to the procedure given above, but if the cultivation of Ti-type organisms is desired, similar colonies in which the less piled-up type Ti predominates are repainted.

(B) Preparation of GC growth medium a) Preparation of DSF additive

An aqueous solution of cocarboxylase (0.2% by weight) in distilled water is prepared at room temperature and sterilized by filtering through a 0.45 o.m Millipore filter. An aqueous solution containing glucose (40 g), glutamine (1.0 g), ferric nitrate (0.5% by weight 10 ml in distilled water) and distilled water (90 ml) is mixed in 10 minutes Auto-clave at 121 ° C under 1.12 kg / cm2 (16 psi) and the solution cooled. To this solution, 1 ml of the previously prepared cocarboxylase solution is added to obtain the DSF solution.

9

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

634923

10th

B) Preparation of the growth medium given the first two washing liquids. (The ethanol

Bacto-GC Medium Base (Difco Mannual, 19th amine buffer is made from liquid ethanolamine, 37.3 ml, edition, page 122) (Difco Laboratories, Detroit, Michigan) aqueous hydrochloric acid, 1 n, 147.0 ml, and distilled Water on (10.8 g) and distilled water (300 ml) are mixed in a 11.)

suitable container stirred and the mixture in this container 5 If, in the above method, Ti instead of Ti-Pili are to be grown for 15 minutes at 121 ° C under a pressure of 1.12 kg / cm 2, the pH of TBS is between heated. The container is removed from the autoclave to 7.0 and 7.2.

Cooled to 50 to 60 ° C. and 3 ml of the product prepared as above. With a good approach, the vegetation has a character-

DSF additive added. Ristic orange-pink or warm pink color and a smell,

10 reminiscent of cooked food. The vegetation clumps

(C) Preparation of the inoculum plates and growth dishes sticky thread-like aggregates and can be washed with growth dishes made of Pyrex or aluminum and Petri a glass spatula easily from the surface of the medium dishes for inoculation, detached with distilled.

Rinsed water covered with aluminum foil and 30 minutes at The dishes containing the growth medium are heated to 121 ° C under a pressure of 1.12 kg / cm2. The melted growth medium prepared in accordance with paragraph (B) -b and poured again with growth medium is poured into the dishes prepared in this way, then washed and sterilized as indicated above. The

Growth medium should be carefully poured into a closed (G) immersion culture of GC-Pili dust-free room under aseptic conditions in the chamber according to the procedure given in paragraph (E) to avoid contamination with undesirable bacteria using the same growth medium and nutrient to avoid. substance additive, but without agar, and use of soluble

Starch, instead of insoluble starch, becomes the inoculum

(D) Growth of the inoculum growth medium given and in an atmosphere of 95% The T2 colonies from the Thayer-Martin plates (paragraph A) air and 5% CO2 in the presence of 0.5% (based on the volume

are again spread on inoculum plates which, as above, have been prepared under gentle pressure for 25 hours of the «Celit» liquid medium and incubated at 35 ° C and 90% humidity in movements at 35 to 37 ° C.

in an atmosphere of 5% carbon dioxide and 95% air. The culture medium is then passed through a sandwich filter

tet. The plates are gradually filtered in subcultures on a coarse sintered glass filter. The filter pad is grown until more than 90% of the growth of the colony consists of coarse filter paper, a 5 mm layer of Celite type T2, which forms hair. Depending on the stability 30 and again a layer of coarse filter paper.

of the strain the growth time is between 12 and 24 The filtrate is discarded and the residue,

Hours. After this time, the plate is covered between 50 and 75% according to the third washing process of paragraph (E) (ethanol with the growth. Amine buffer), treated and then in accordance with paragraph

(H) processed further

(E) Formation of GC pili by surface culture 35

The petri dishes from paragraph (D), containing the T2 culture, (H) separating the pili from cells and cell debris are washed with aqueous casamino acid solution (5 ml, 0.7% by weight) 1. The TBS obtained in the previous paragraphs

washed. The growth is from the medium with a ste- Suspensions in centrifuge tubes (the volume of which ril glass spatula is detached, the suspension of the growth depends on the number of batches added) of the casamino acid solution are pipetted off the plate and poured into 15 minutes with 3 kg centrifuged. The top two growth bowls (about 35.6 x 25.4 cm) distributed. This liquid is discarded and the pearl processing shells are made in the manner described above. tet.

The suspension is spread evenly over the surface. 2. The ethanolamine suspension is removed from the bead and the dishes are added for 20 hours at 35 ° C. and 90% moisture. Third washing of the growth dishes and activity in an atmosphere of 5% carbon dioxide and 95 % Air incubated 45 more ethanolamine buffers with a pH of 10.1. a volume approximately three times the volume of pearls in the centrifuge tube. The liquid layer turns 5

(F) Obtaining the growth of the surface culture quickly stirred for minutes with a mechanical stirrer in order to bring a soluble stock of the pearl into suspension with a Tris buffered salt solution.

provides by dissolving sodium chloride (510 g), tris (hydroxy- so 3. The suspension is then yawed for 15 minutes with 3 kg centrifu-methyl) -aminomethane (363 g) and concentrated hydrochloric acid, the supernatant liquid is decanted from the pearl (100 ml) together with enough distilled water to keep and. The pearl is then re-obtained three times in a stock solution with a volume of 101. of their volume of ethanolamine buffer, pH 10.1, as indicated above. The pH is added by adding more concentrated, suspended and centrifuged for 15 minutes with 3 kg. The hydrochloric acid is adjusted to a working range of 8.5. When 55 supernatant liquid is decanted off and an increase in pH is required, concentrated ethanolamine buffer from the immediately preceding (10 N) aqueous sodium hydroxide solution is added. Put together before the step and discard the pearl, the application is the stock solution on '/ e of the original- The combined ethanolamine buffer solutions turn 60

Chen concentration diluted. The salt solution minutes buffered with Tris centrifuged at 31 kg. The supernatant liquid (hereinafter referred to as TBS) has an Auseo, is decanted off and processed further, and the pearl pH of 8.5.10 ml of the TBS solution is poured onto the plates.

Given the growth surface of the culture dishes, the growth detached with a glass spatula and the suspension in a (I) crystallization from GC-Pili

Pipette storage container. The washing and stripping is repeated with the preparation of dialysis tubes and with Tris buffered a second amount (10 ml) of TBS and the two 65 saline

Wash liquids combined. The Growth Top- A Roll of Dialysis Tubing (30.48 m Fisher Scientific Cata-

The surface is washed a third time with ethanolamine buffer (10 ml, pH log no. 8-677 B) in a) distilled water (2 10.1) and the suspension is stored, but not with 41 x each), b) aqueous Sodium bicarbonate solution (2 x, 41 dist

11 634923

Milled water, each containing 2 teaspoons of sodium bicarbonate, is stored in this medium at 40 ° C. Wahl-nat), c) aqueous sodium ethylenediaminetetraacetate (2 x, 41, the medium containing the crystals can be frozen and distilled water, each containing 2 teaspoons of Na-EDTA), stored at -170 ° C.

• d) aqueous ethanol (2 x 41 ethanol / water 1: 1 each), e) distill- The yield of GC pilus crystals of the amount given in paragraph (A)

washed water (2 x 41 each). The dialysis tubing 5 given N-gonorrhoeae strains, when the inoculum is then in distilled water, each containing a trace containing at least 90% colonies of type T2, is in the range of 5 benzoic acid (1 teaspoon of benzoic acid to 41 distilled water to 15% ig / cm2 growth area.

ser). Tris buffered salt solution (TBS), similar but somewhat lower yields are obtained from that prepared according to paragraph (F), is obtained with Ti type colonies.

diluted distilled water to obtain the dialysis solution 10

(166 ml stock solution are diluted with (L) cesium chloride density gradient isolation from GC-Pili 11 with distilled water). 1 ml of the TBS washing liquid (pH 8.5) containing the GC

Pilus crystal suspension from paragraph (H), is diluted with Tris buffer with (J) dialysis of the GC-Pili solution, a pH value of 8.5 to 20 ml, the pH value by

100 ml of ethanolamine buffer, containing GC pili in solution, the addition of sodium hydroxide (0.1 n) is increased to 10.1 and 7.5 g as prepared in the previous paragraphs, dry cesium chloride is added. The solution turns 42

dialyzed against 41 TBS (pH 8.5 at 20 ° C) with the help of centrifugation like 200 kg for hours (SW41 rotor of a Beckman dialysis tube prepared above using the L-265 centrifuge). There are fractions from the centrifuge-specified TBS. The dialysis is collected in a cold tube and the optical density at 280 nm and

Room (about 4 ° C). The outer dialysis buffer dissolved the refractive index for each sample. The refraction solution is stirred magnetically. The 18 hour dialysis index depends on the cesium chloride density in which the able-lang is performed. A pH increase for the refractive index will be converted in the dialysis medium. The bre-observed at about 8.7. A cloudy blue-white number is formed on the X-axis against the density of the solution, birefringent precipitate (precipitate) of GC pilus on a Y-axis and the optical density on a second crystal at the end of the dialysis period. The material thus precipitated is applied to the 25 Y axis. A single main maximum, corresponding to centrifugation for 60 minutes at 7500 rpm (7.5 Krpm) and corresponding to the GC pili, is found at <p = 1.3422 ± 0.0038. The supernatant liquid is discarded, whereby GC pilus crystals fractions, corresponding to 1.35 to 1.33, are put together of the strain Pittsburgh 3-2 N. Gonorrhoeae as centrifuges and dialyzed and purified in order to produce GC pilus crystals after the pearl. According to the procedure given above, paragraphs (I) and (K).

However, if Ti instead of T2-PÌIÌ should be isolated, the initial pH of TBS is 7.0 to 7.2. (M) PAT test

Pilus crystals are suspended in TBS at pH 7.0 in a concentration (K) further purification of the pili tion between 30 and 60 jj.g / ml. It won't be cloudy

The pearl obtained according to paragraph (I) is used in approximately the or cloudy test serum. If the test serum is thirty times its volume of ethanolamine buffer (pH 10.1) 35 kig, it is centrifuged at 30 kg for 30 minutes and the excess is suspended. The tubes are gently swirled around the standing liquid. Serial dilution beads are to be dissolved and the suspension is prepared for 60 minutes with a 31 kg center and 0.025 ml of the serum samples and 0.025 ml of the fugue. The supernatant liquid is decanted off and the suspension of the pilus crystals is each discarded in the cavity of a pearl. The supernatant liquid is added to a microtiter plate and the mixture is dialyzed against TBS for 30 minutes using the method of paragraph (I) and stirred at room temperature. The wells are then centrifuged off with the crystalline material thus obtained, likewise using a dark field microscope, to form crystal paragraph (I). The pearl is examined again in lumps of ethanol.

amine, according to the method mentioned above, the wells are then based on a maximum

dated, filtered through a 0.45 p.m filter if a sterilization len dilution that gives crystal agglutination is desired, and centrifuged again similarly as above and 45 which is obviously larger than in the comparison.

dialyzed and isolated by centrifugation.

According to the procedure given above, the experiments were carried out as follows:

if the GC pilus crystals thus obtained are to be kept for a certain time white female rabbits (New Zealand) with a preservative weighing 1.8 to 2.7 kg (4 to 6 pounds), subcutaneous dialysis-TBS- Solution added that is compatible with it. Purified 50 pilus preparations of CDCM-2, Pittsburg 3-2, inert preservatives that can be used in one go by the DCDT-2, CDC005-2, and Pittsburgh 4-2 strains method are 0 , 05% (w / v) neutral aqueous 1: 1 mixture with Freunds Incomplete addition and in the syringe ger formaldehyde, 0.01% (w / v) merthiolate or 0.02% emulsified injected. Approximately 100 to 200 ng / kg of the pili was sodium azide (w / v). injected in 3 injections approximately every 2 weeks and

If the blood preservative is not compatible with the TBS 1 to 2 weeks after the third injection according to the procedure given above, the sample is removed. The blood was clumped and the serum after the pili crystallized, the Tris buffer was removed in the usual way. The first three test sera were dialyzed against saline (0.15 M aqueous sodium then against Pili from 21 N. gonorrhoeae strains under umchloride, 18 hours). The preservative is being searched for. The test results are added to a fresh batch of saline solution in the following table and are given against the 60.

Suspension of the GC pilus crystals dialyzed for 18 hours. The kri-

634923

12

Table 1

Pili crystal agglutination titers from Pili from 21 different strains against Sera with Pili from 3 different strains

Pilus stem

Pilus- Pitts- Pitts- Pitts- CDC CDC CDC CDC Atlanta Atlanta Atlanta Seattle Seattle Atlanta CDC '

Serum strain burgh3-2 burgh 1-2 burgh4-2 burgh6-2 B-2 C-2 M-2 T-2 4-2 6-2 9-2 1-2 3-2 10-2 (Kellog)

F62

CDCM-2

16

256

16

64

8th

32

128

8th

16

<8

8th

16

32

32

8th

Pittsburgh 3-2

256

16

8th

64

512

<8

8th

<8

16

64

8th

16

16

32

8th

CDC-T-2

4th

16

<4

<4

8th

32

16

512

32

8th

32

64

16

8th

128

Pilus stem

Pilus- Pittsburgh Norfolk Norfolk Dayton Seattle CDC

serum strain 7-2 2-2 7-2 8-2 9-2 * C05-2

CDC M-2 16 64 8 16 16 32 to 64

Pittsburgh 3-2 128 64 16 64 128 128

CDC T-2 16 32 8 8 16 64

* 005 is a CDC strain isolated from a patient with healed GC infection.

According to the procedure given above, the following Table Ia results are the values 7 x 7 serotype pilus agglutination test performed under for the cross reaction (pili against sera of the same strain)

Use of the Pili and Sera, which are derived from 7 to 100 normalized and the other values according to designated strains. For easier interpretation of the in the •.

Table la

serum

Pilus stem

Pittsburgh 3-2

Pittsburgh 4-2 CDCM-2

CDC T-2

CDC 339-2

CDC C-2

Norfolk 2-2

Pittsburgh 3-2

100

3.1

8.3

0.17

1.6

<0.008

0.52

Pittsburgh 4-2

0.16

100

<4.2

0.086

0.20

<0.008

0.065

CDCM-2

<0.039

<0.024

100

<0.02

0.20

0.13

0.016

CDC T-2

<0.31

<0.024

<4.2

100

0.20

<0.008

0.032

CDC 339-2

2.5

<0.024

4.2

<0.02

100

0.016

0.260

CDC C-2

<0.0039

<0.78

<4.2

0.17

<0.10

100

0.065

Norfolk 2-2

<0.31

0.78

<4.2

0.04

0.78

<0.008

100

(N) Mix hemagglutination by GC-Pili preparations and let stand at 4 * C. The results were

General procedure so that after 1 to 2 hours with the help of a light chamber

Group 0 human blood containing EDTA as sen.

Anticoagulant, was obtained from a blood bank. Red blood cells were freshly prepared by washing a result 4 times

Blood volume with 15 volumes with phosphate-buffered salt - The degree of hemagglutination observed was fol solution, pH 7.3.01 m, and preparation of a 3% (volume) 55 evaluated in the following manner:

Suspension in the same buffer. 5 + no pearl or lump of cells, even

Solutions of 1.0 mg / ml Pittsburgh 3-2 and CDC-M-2-Pili red color,

were made using the UV absorption of the 4+ traces of a cell bead

Preparations at 280 nm, corrected for scattering as a Mass 3+ small cell bead with a very clear margin for the concentration. Equal proportions (50 jxl) of the Pili solutions 60 cell clumps

(1.0 mg / ml) were placed in the first cavity of a Cooke micro 2+ distinct cell bead with a massive number of edge titer plates with a U-shaped base and in the other cavities 25 (J.1 buffered with Tris) Saline solution + + clear cell pearl with a few lumps

0.02% azide and the pili were thinned thinner in the twelfth well with a 25-jxl hand microwave the sides of the cavity. 65 The CDCM-2 and Pittsburgh 3-2-Pili titers were

Tris buffered saline (25 | il) and 25 jxl 3% two different occasions and are in the

Red blood cell suspensions were given in each table below and are comparative and the plate was gently agitated to show the contents.

13 634923

Results of hemagglutination

1. Approach

2. Approach

Final concentration

degree of

Final concentration

degree of

Final conc

degree of

Pili in the

Agglutination of the pili in the

Agglutination

Aggluti

cavity

3-2-

M-2-

Cave nation

M-2 in the nation

3-2 and M-2

Pili

Pili

3-2

3-2 pili

cavity

M-2 pili

1

333 | ig / ml

5+

5+

450 | Ag / ml

5+

92 (ig / ml

5+

2nd

166.6 ixg / ml

4+

4+

225 ng / ml

5+

46 | ig / ml

5+

3rd

83.3 ng / ml

3+

4+

112.5 ng / ml

5+

23 (ig / ml

5+

4th

41.6 ng / ml

2/3 +

4+

56 [ig / ml

4+

11.5 | xg / ml

4+

5

20.8 ng / ml

+

3+

28 (xg / ml

+

5.8 (ig / ml

+

6

10.4 (ig / ml

0

3+

14 (ig / ml

0

2.9 ja.g / ml

+

7

5.2 (ig / ml

0

2+

78 (ig / ml

0

1.4 (ig / ml

0

8th

2.6ng / ml

0

±

3.5 pg / ml

0

0.7 (ig / ml

0

9

1.3 (ig / ml

0

0

1.75 (Lg / ml

0

0.35 | xg / ml

0

10th

0.6 [ig / ml

0

0

0.88 n, g / ml

0

0.17 ng / ml

0

11

0.3 [tg / ml

0

0

0.44 (ig / ml

0

0

12

0.15n, g / ml

0

0

0.22 n, g / ml

0

0

G

Claims (3)

634923 1. A method for producing pili from Neisseria Gonor-rhoeae, characterized in that N. gonorrhoeae bacteria of the type Ti and / or T2 are artificially grown to obtain a culture of piled N. gonorrhoeae cells, and that (a) suspending the culture of the pilated N. gonorrhoeae cells in an aqueous medium at a pH below 9.2 and above 5.5, (b) separates the soluble from the insoluble part of the suspension, (c) suspending the insoluble part of step (b) in an aqueous medium at a pH in the range from 7.7 to 11.0, (d) separates the insoluble from the soluble part of the suspension obtained after step (c), (e) the pH of the soluble part obtained in step (d) is reduced to below 9.2 and above 4 and (f) separating the resulting pili from the aqueous medium. 2. The method according to claim 1, characterized in that the pH in step (a) is set to a value below 7.7 but above 5.5, the Pili of the Ti and T2 types remaining in the solid phase. 3. The method according to claim 1, characterized in that in step (a) the pH is reduced to not less than 8.6, the Pili of the Ti type going into solution. 4. The method according to claim 1, characterized in that in step (e) the pH is adjusted to below 7.7 but above 5.5, whereby the Pili of the Ti and T2 types are precipitated. 5. The method according to claim 1, characterized in that in step (e) the pH is reduced to not less than 8.6, whereby only the Pili of type T2 fail. 6. A method for producing pili from Neisseria Gonor-rhoeae, characterized in that N. gonorrhoeae bacteria of the type Ti or / and T2 are artificially grown to obtain a culture of piled N. gonorrhoeae cells, and that (a) suspending the culture of the piled N. gonorrhoeae cells in an aqueous buffer with a pH value above 10, (b) the cells are separated from the suspension, (c) the pili precipitates by adding ammonium ions and separates the precipitated pili. 7. Process for the preparation of Pili from Neisseria Gonor-rhoeae, characterized in that N. Gonorrhoeae bacteria of the type Ti and T2 are artificially grown to obtain a culture of pilated N. Gonorrhoeae cells and a Pili of the type Ti and Mixture containing T2 is obtained, and that one (a) the mixture containing the pili is dissolved in a buffer with a pH of more than 10, (b) reduces the pH to less than 9.2 but more than 8.5, (c) separating the precipitated T2 type pili from the buffer solution, (d) reducing the pH of the solution to 7.7 to 7.0 and (e) separates the failed Pili of the Ti type. 8. A method for producing pili from Neisseria Gonor-rhoeae, characterized in that N. gonorrhoeae bacteria of the type Ti and T2 are artificially grown to obtain a culture of piled N. gonorrhoeae cells and a mixture is obtained Pili of type Ti and T2 contains, and that you can mix this (a) treated with a buffer with a pH above 8.5 but below 9.2 and (b) separates the undissolved T2 type pili from the mixture. 9. The method according to claim 8, characterized in that one (c) adjusting the pH of the solution remaining in step (b) to a value between 7.7 and 5.5 and (d) separating the resulting Ti-type pili from the solution. 10. A method for producing pili from Neisseria Gonorrhoeae, characterized in that N. gonorrhoeae bacteria are grown in an artificial culture to obtain a heterogeneous aqueous mixture of pili, and that (a) Detach the pili from the mixture by (i) add enough water to reduce the salt ionic strength below 0.002, (ii) adding sufficient alkali or alkaline earth salt of a mineral acid to increase the ionic strength above 4.4, (iii) reduce the pH of the aqueous mixture below 4.5 but above 2.5 or (iv) add sufficient sucrose to increase the sucrose concentration above 50% by weight, (b) separates the solid from the liquid portion of the solution and (c) to precipitate the pili from the solution (i) adding sufficient alkali or alkaline earth salts of mineral acids to increase the salt ion concentration above 0.05 ionic strength, (ii) add enough water to reduce the salt ionic strength to less than 0.5, (iii) Add enough Tris buffered saline to obtain a medium with an ionic strength of 0.05 to 0.5 at a pH between 4.5 and 9.2 or (iv) Add enough water to reduce the sucrose concentration below 40% by weight. 11. A method for producing pili from Neisseria Gonorrhoeae, characterized in that N. gonorrhoeae bacteria are artificially grown in order to obtain a heterogeneous or homogeneous mixture containing the pili, and in that (a) adjusting the pH of the mixture to 10.0 to 11.0, (b) Add sufficient cesium chloride to increase the salt concentration to between 0.2 and 0.5 g / cm3 CsCl, (c) centrifuging the mixture with 100-300 kg for 30 to 60 hours until the desired equilibrium is reached, (d) separating the fraction with a density between 1.35 and 1.33 g / cm3 and (e) the cesium chloride is removed from the solution 12. A method for diagnosing gonorrhea, characterized in that a serum sample to be examined is brought together in vitro with pili of N. gonorrhoeae of the type Ti or T2 in the absence of cell residues. The infection caused by the Neisseria Gonorrhoeae organism, commonly known as gonorrhea, is a venereal disease (sexually transmitted disease) that is extremely common in humans. The disease is usually manifested by a visible change in the man, but is often undetected and undetectable by external symptoms in the woman infected with it. So far, the only reliable method of detecting such an infection has been to keep discharge or mucus fluids believed to contain the organism under culture conditions. Such cultures required a period of more than a day 5 10th 15 20th 25th 30th 35 40 45 50 55 60 65 2nd PATENT CLAIMS 3rd 634923 to grow. Since this disease is generally regarded as a flaw in society and due to the resistance of many people infected with it to go back to the clinic or to the doctor after the examination, it has long been desirable to develop an examination method that is reliable Indicates a possible infection or non-infection within a time during which it is reasonable for the examined person to stay in the clinic. So far, no method of immunizing against infection with the organism N. gonorrhoeae was known in humans. One of the biggest problems associated with research in this area was the fact that the organism only appeared to infect humans and chimpanzees and, although there is a relatively high degree of agreement between the results of chimpanzees and humans, this agreement did not is absolute. Chimpanzees are extremely expensive and complex for experimental purposes, although they are appropriately suitable as test models. 20th Four different colony variants of Neisseria Gonorrhoeae are characterized. These four variants fall into two different categories. Variants Ti and T2 lead to experimental infection in volunteers, while variants Ts and Tt are not known to cause infections. The first group can be distinguished from the second group by observing that the first group has thread-like structures on the colonies of the variants, while the second group has no such threads or hair. These 30 threads are called gonococcal pili (hereinafter also referred to as «GC» or pili). In 1973, two articles were published that allegedly show the isolation of Ti and T2 Pili from N. Gonorrhoeae and also the formation of an antibody response thereon (Buchanan et al, J. Clin. Invest, 35 Volume 52, pages 2896 to 2909 (1973) and Punsalang and Sawyer, Infect. Immun., Vol. 8, pages 255 to 263 (1973), see also Buchanan et al, J. Clin. Invest, Vol. 5117A (1972). The basic procedure used by Buchanan is as reference 41 in the 1973 publication and developed by Charles C. Brinton (CC Brinton, Trans. NY Acad. of Sei, Volume 27, page 1003 (1965), and by Punsalang and Sawyer. The Brinton method was developed to study the Pili by E. Coli. An attempt has now been made to repeat the work of Buchanan et al., And it has been shown that no pili from N. Gonorrhoeae could be obtained in all efforts according to the procedure given there.