JPH0118080B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides purified pili of type T ₁ and T ₂ phosphomycetes (Neisseriagonorrhoeae) and optionally their T _R subvarieties in crystalline form (hereinafter referred to as GC).
They are called pili crystals. ) regarding providing. GC pili are isolated from surface or deep liquid cultures of the corresponding bacteria. Surface and deep culture methods for Phosphorobacillus cells are virtually conventional. In a modification of the deep culture method, a large surface area, reaction-inert medium is added to the culture to aid in purification. Diatomaceous earth like Celite is suitable. In case of surface growth, remove all growth i.e. cells and pili from the medium and adjust the pre-determined PH.
Suspend in aqueous medium at 9.2 or less. If you want to isolate only _T2 pili, a pH of 5.5 or higher or 9.2 or lower is appropriate. If it is desired to isolate the T ₁ pilus either by itself or in the presence F of the T ₂ pilus, a PH of below 7.7, suitably around PH 7.0 is required. For deep culture growth, such a suspension step is not necessary. In addition, pili crystals are soluble at pH 4.5 or lower,
It has been found that substantial reconstitution can occur if the PH does not fall below about 2.5 but rises above this amount again. The soluble portion of the suspension is then separated from the insoluble portion. Although filtration can be used for this separation, it is generally desirable to use centrifugation. Discard the centrifugation supernatant (filtrate in case of filtration) and save the residue. In the next step of purification, the components of Phosphorus pili are brought into solution and separated from residual materials. For surface cultures, this material will include whole cells and tissue debris; for deep cultures, it will additionally include large surface area materials such as Celite. Solutions of Phosphorus pili crystals are prepared by two different, but closely related methods. A solution of Pili pili crystal material is produced by the cleavage of non-covalent bonds in the internal pili rod, which contains the peptide material that makes up the majority of the pili, while the covalent bonds remain intact. That is, using a solubilizer that does not denature the peptide simply disaggregates the crystals into single pili rods. Such reagents are
Ion concentration of urea aqueous solution, aqueous suspension medium
enough water to lower the concentration to below 0.002M, enough salt, suitably an alkali or alkaline earth metal salt and an anion of a mineral acid to raise the ionic strength to above 4.4, urea to a concentration of about 3M to 5M; It is independent of pH such as sucrose enough to raise it above about 50% by weight per volume. Pili crystals are added with mineral acid anions and salts of alkali and alkaline earth metals; enough water to reduce the concentration of the salts to below 0.5 ionic strength, the ionic strength of the medium to about PH
Reprecipitation by raising the ionic strength to about 0.05 or higher by adding a buffer, suitably Tris-buffered saline, and enough water to reduce the sucrose concentration to below 40% by weight, respectively, to give a value of 4.5 to 9.2 of about 0.05 to 0.5. do. This reagent also reduces the pH of _T2 pili to approximately
Raise the pH of the _T1 pilus to 9.3-11 or to approximately 7-8.6 with a basic buffer such as Tris buffer
It is. This range is determined by dissolution at the low point and risk of denaturation beginning at the high point. However, it has been observed that even if the pili are structurally denatured by these or other methods to the extent that they are not recrystallized, their antigenic properties appear to be essentially unaffected. After adding the solvent medium to the solid residue, the second
The soluble and insoluble parts of the second suspension are separated again. As mentioned above, this separation is carried out by filtration or centrifugation, with centrifugation being suitable. Centrifugation methods can be simple or modified. In simple centrifugation, the suspension is centrifuged at low speed, the supernatant is saved, and the residue is set aside. If a higher yield is desired, this residue is resuspended and recentrifuged, the residue is discarded, and the supernatant is immediately combined with the previous supernatant. The combined supernatants are then subjected to high speed centrifugation to remove the last traces of small tissue debris to a residue, and the supernatants are then set aside for use in the next precipitation step. A modification of centrifugation involves mixing solubilized pili, a suspension in an aqueous medium that cleaves non-covalent bonds, such as high PH or urea, with an aqueous cesium chloride solution. Since the cesium chloride gradient method involves centrifugation, complete separation of the tissue pieces is not necessary, but clearer results can be obtained by pre-filtering or centrifuging. The mixture with cesium chloride is then centrifuged in a conventional manner to separate the cesium chloride, and the absorption is measured at various density gradient levels. The appropriate position of the main peak is 280nm.
The position of the pili solution is indicated by the measurement at . Using any centrifugation method, the aqueous fraction containing the pili solution is then processed to precipitate the pili crystals. This is done by lowering the PH if it is high, removing the non-covalent bond cleaving agent, cesium chloride, or also adding a precipitating agent such as ammonium sulfate. Precipitation conditions are achieved by dialysis or direct addition. Lowering the pH and removing the bond cleavage agent results in the formation of GC pili crystals. The GC pili crystals are then removed from the aqueous medium by filtration or, more suitably, low speed centrifugation. The supernatant is separated and the GC pili crystals thus obtained are dried under reduced pressure. Store in a suitable aqueous medium if desired. Although it is desirable to keep the crystals at low temperatures in a sterile medium,
This does not appear to be essential for contamination-free stability of the bacteria. It must be noted that pili crystals are actually aggregates of single pili rods with high molecular weight. Therefore, if the medium containing solubilized pilus is purified by high-speed centrifugation and, if desired, sterilization in a Millipore (approximately 0.45 micron) filter, individual pilus rods are High-speed centrifugation, suitable for precipitation at 40 kg or more. If pili of highest purity are not required, a simple, rapid, and abbreviated purification is quite sufficient. In this method, whole Phosphorus bacteria growths are grown in a high PH buffer, suitably an ethanolamine buffer, containing the variant in question (T ₁ or
The pH is above the melting point of T ₂ ), preferably the entire solution is transferred to ensure a pH of about 10.0, and solids are removed by filtration or centrifugation to lower the pH, suitably but not necessarily by dialysis. In fact, if you first adjust the pH to 8.6 as mentioned above,
T ₂ pili crystals precipitate, and when the pH is further lowered to 7.7 or less, as mentioned above, to 7, T ₁ pili crystals precipitate.
In this way, we show in one step the propagation nature of the variant in question. This latter is entirely confirmed, since a competent bacteriologist can distinguish between the two types by examining the colonies. Alternatively, pili crystals are precipitated by adding ammonium sulfate. _T2 pili precipitate as crystals at about 4-7% anion saturation (room temperature) and _T1 pili precipitate at 5-10% saturation.
GC pili show large and small bands when subjected to SDS polyacrylamide gel electrophoresis. The large band named GC pilin indicates that it is composed of phosphoglycoprotein substances. T ₂ GC Pirin further contains 200±9 amino acids, 2-3
It is shown to consist of a peptide moiety of phosphatate groups and 1-2 hexose sugars, and is measured at 20°C.
Essentially soluble in aqueous media with PH greater than 10.1
8.6 and virtually insoluble. The majority of _T2 pilus i.e. _T2 pilin is SDS
21500± measured by acrylamide gel electrophoresis
It has a molecular weight of 1000 Daltons. GC pili isolated from T ₁ type Phosphorus bacterium growths appear to be immunogenic very similar to GC pili isolated from T ₂ type Phosphorus bacteria. Compared to T ₁ type GC pili, the pH is essentially soluble in aqueous media with a pH greater than approximately 8.5 when measured at 20 °C.
7.7 and essentially insoluble in aqueous media. T ₁
Type GC pilin has a molecular weight of 22000±1000 daltons as determined by SDS acrylamide gel electrophoresis. GC pili have been isolated from cultures of more than 20 different Linn fungal strains. Injection of GC pili crystals and the eluate from single rod and SDS acrylamide gel electrophoresis into a test animal results in the production of antibodies in the test animal's serum. When GC pili crystals are treated with serum containing antibodies either by themselves or in a suitable suspension medium, the crystals aggregate. This aggregation is readily observed by other methods, most suitably by dark field microscopy, but provides a simple and rapid test for the presence of pili antibodies in serum. During the use of the test, preliminary examination of Phosphorus bacteria to select individuals for culture testing, identification of high-risk individuals to distinguish between old and new infections in a particular individual, strains corresponding to local epidemics. and the identification of bacterial strains that correspond to specific symptoms. This test is useful for determining the presence of antibodies to GC pilus in a test sample and includes:
It must be noted that it is not possible to directly determine whether the subject from whom the serum was collected has active Phosphorus bacteria or has been infected in the past and is merely a carrier of antibodies. Furthermore, very recently infected (i.e. 2
It should be noted that a test sample that has been tested (days or three days ago) will not give a positive response because the body has not had enough time to produce a sufficient concentration of antibodies to give a detectable titer in the body. Must be. It is noted that the pili of Phosphorus bacterium contain one or more immune determinants selected from the group of at least four determinants. Thus, an antibody agglutination reaction occurs between the pili and the antibody-containing serum against at least one such immune determinant. The sensitivity intensity depends on the antibody concentration in the test serum sample.
equal to the number of interacting immune determinants in pili and serum. Four different GC pili samples known to contain at least one of the aforementioned determinants can be used to rapidly test for the presence of antibodies corresponding to GC pili in any test serum. is possible. Similarly, pili can be isolated if a bacterial source is available and pili are easily cultured from this bacteria. Because standard sera containing antibodies to any one of the four previously determined antigenic determinants of GC pili are available as a result of the present invention, pili from unknown test sources are not susceptible to these determinations. The identification and number of factors can be serotyped. This method will be of great help in epidemiological research on observation of P. bacterium infection. Pili can be absorbed in various carriers known for immunological tests, such as latex, washed red blood cells, charcoal, polyacrylamide, agarose, etc., to provide the substrate for serum or plasma agglutination tests. The availability of pili also provides the basis for hemagglutination and hemagglutination inhibition tests. Both tests rely on the principle that pili have specific binding sites that interact with red blood cells. Thus, when pili and blood cells are kept warm together, the red blood cells give a diffusely aggregated pellet by spontaneous sedimentation. If pili are not present in the test medium, the red blood cells will settle out by spontaneous sedimentation to give a distinct pellet. This provides a method for testing the presence of pili in solution. In the hemagglutination inhibition test, a test serum suspected of containing antibodies to GC pili is added to a solution containing a predetermined amount of pili, and the mixture is kept warm and centrifuged. Pili interact with antibodies against them and precipitate. The supernatant is then added to the red blood cells. When all pili react with the antibody in the test substance, a crisp pellet (ie, no aggregation) results. It will be appreciated by those skilled in the art that such tests have significance when performed at predetermined dilutions relative to a control (ie, no antibody). The accuracy of this test is increased if the serum is first treated with washed red blood cells (ie, before being added to the pilus). This method removes factors in the serum that cause red blood cell aggregation regardless of the presence of pili. Until now, it has been impossible to immunize the human body against Phosphorus bacteria. Sufficient amount of GC pili for the desired subject, suitable is about 2 to 1 kg per kg of body weight.
When injected with 100 μg pilus, the amount of antibody in the serum increases to a titer of at least 100 in the PAT (Pilus Agglutination Test) and at least 1.6 logcycles. It was found that a degree of protection can be obtained. That is, the subject is able to resist infection by a measured number of injected pili of the resulting strain, approximately 1.6 more than required to infect an unimmunized control subject. No pili-attributable toxicity was observed from injection of GC pili crystals. Subjects with a titer of 200 were unaffected, and test primates (rhesus monkeys) were brought to a titer of approximately 10,000 on PAT without any pathological consequences. Injection of GC pili crystals in a suitable carrier medium may be carried out over a moderately long period of time, suitably for about 5 weeks. The dosage is 1 to 5 portions of a GC pili, a single rod pili, or a suitable GC pili source.
A delayed rate of administration is useful but not required. This rate of administration results in the gradual formation of antibodies in the system. No local adverse reactions to the crystals at the injection site were observed upon injection into subjects who had previously received the crystals. Given the presence of several antibody determinants mentioned above, administering pilus, single rod pilus or other GC pilin sources containing each of the known determinants to obtain maximum protection. is desirable. Description of Preferred Embodiments Preparation of Pili Crystals Growth of Surface Cultures of Neisseria gonorrhoeae Neisseria gonorrhoeae is found in four colony forms arbitrarily designated types T ₁ , T ₂ , T ₃ and T ₄ . However, this name is generally accepted. Types T ₁ and T ₂ bacteria are those responsible for Lin's disease in humans, and only these forms have pili. The method described below can be applied to the growth of type T ₁ and type T ₂ bacteria.
It must be noted that the Phosphorus strain was isolated from bodily secretions taken from human patients. Such secretions are usually of the desired T ₁ or T ₂ type, as well as T 3 and T ₃ , which do not have pili.
Also contains _T4 type. Furthermore, it must be noted that a culture started as a fairly pure T ₂ type will eventually produce pili-free T ₃ and T ₄ types as well as T ₁ types in subculture. In culturing the T ₁ and T ₂ colony variants, a variant with a third pili, arbitrarily designated _TR , is observed. Although this variant has a rough appearance and is not completely characterized, it is believed to be closely related to the T ₁ and T ₂ types since subcultures of _TR colonies produce these types. Obtaining pilus from T _R type is the same as from T ₂ culture. In order to maximize the production of _T2 or _T1 pili, certain precautions must be taken. Initial samples are cultured on Thayer Martin (TM) plates, which grow Phosphorus bacteria and inhibit the growth of most other bacteria.
Since TM plates are not suitable for distinguishing between colony types, colonies from TM plates are grown in a suitable growth medium [e.g. GC medium, Cat. No. 0289-1
Difco]. Subculture is continued from a single colony on a medium (hereinafter referred to as GC medium) until the colony contains about 90% or more of the desired type. This method can be used for type T ₁ as well as type T ₂ , and as we have already shown that types T ₁ and T ₂ are immunologically similar, it is preferable to maintain the separation of the types. . Next, we will discuss T ₂ . Propagation methods for _T2 are equally applicable to _T1 , unless otherwise indicated to the contrary.
Once the growth on the plate has grown to more than 90% of the _T2 colonies, the growth is removed from the plate and suspended in a suitable freezing medium such as BSA-glutamine.
Divide into a certain amount and keep at a low temperature, preferably -70 to -196℃.
Store in. As mentioned earlier, it must be noted that initially samples with a high proportion of _T2 tend to become unstable upon subculture and produce small amounts of _T2 . Therefore, in culture growth, on the one hand, T ₂
Care must be taken to ensure a starting proportion of bacteria and, on the other hand, to use a sufficiently "young" strain. That is, care must be taken not to subculture too often to avoid instability. If it is desired to produce T ₁ pili, it is even more important to use an inoculum containing 90% or more of T ₁ than for T ₂ . The pili-producing inoculum is initially suitably, but not necessarily, frozen and aliquoted.
Prepare GC medium by streaking it onto a Petri dish and keeping it warm for 12-24 hours. The inoculum, which is the more unstable _T2 type at 12-15 hours and the more stable _T2 type at 12-24 hours, is preferably grown at 35-37 °C and at 35
°C seems to be preferred. High humidity conditions also appear to be desirable. It is observed that at humidity below 70% the pili yield is lower than at higher humidity. It therefore seems desirable to work at a humidity of 70% to 90%. The effect of the growth atmosphere is not completely understood, and old phosphorus bacteria cultures grow without adding carbon dioxide, but an atmosphere of 5-10% carbon dioxide and 90-95% air is very suitable. found. After the initial inoculation, the plates are approximately 50-75% covered with growth, which is then removed. A suitable method is to add a small amount of sterile casamino acid solution to the inoculum plate and scrape off the growth with a sterile glass spreader. Using 5-6 ml of solution per plate, 2-3 ml of the suspension thus prepared was found to be sufficient to inoculate larger growth dishes in GC medium. This dish is then incubated under the same conditions and for the same amount of time as the inoculum Petri dish and pili are obtained therefrom. As with all methods, it is not necessary to use sterile techniques at this time; clean equipment, pure reagents, and all operations performed at as low a temperature as possible to prevent the growth of undesirable bacteria are desirable. Bacterium phosphorus growth is obtained using a suitable buffer. The chemistry of the buffer is not important, the PH range is. For obvious reasons, buffers are not used above PH 9.3 when purifying _T2 pili. It is preferable to work at PH5.5-9.2, most suitable is PH7.0-8.6. Mainly T ₁
When using cultures, the PH should not exceed 7.7, and should be between 5.5 and 7.5, with PH 7.0 and 7.2 being suitable. These ranges ensure that all pili-bearing material remains aggregated. The most suitable buffer is Tris-buffered saline. A preferred method is to place a wash buffer on the surface of the growth medium, scrape off the growth with a suitable device, and remove the aqueous suspension by a suitable method, such as a pipette or a vacuum aspirator. If desired, a second wash is performed in the same manner to pool the liquid suspension. A third wash is performed with a high PH buffer to increase yield. In other words, PH9.3 or higher, suitable is 10.1~
10.3 in buffer. Use of such a buffer causes dissolution of any residual pili material. If the purpose is to isolate _T1 pili,
The pH should be above about 8.6, but higher values are not disadvantageous. This basic suspension is not pooled with the first wash but is reserved for later purification steps. It should be noted that when the pili grow well, the growth has a characteristic orange/pink or warm pink color and the growth medium has an odor similar to cooked food. The growth is found to be clumped with sticky, stringy aggregates that easily slide away from the growth medium when pressed with a suitable device, such as a glass spreader. Although the above-described method is desirable when high yields of high purity pili are desired, completely acceptable results can be obtained with substantially omitted methods. This method does not use the first and second cleaning methods described above. Treat the entire growth with substantially high PH buffer. A third wash with ethanolamine buffer is appropriate. In addition to dissolved pili, the wash medium contains many impurities that would otherwise have been removed, but which are retained in solution when the pili material is precipitated by the method described below. It was found that Deep Culture of Phosphorus Bacteria The deep culture of type T ₁ and T ₂ bacteria in liquid medium is carried out in a conventional manner using the same medium and environment as used for surface culture, except that it does not contain agar as a solidifying agent. Bacteria are observed to proliferate and continuously peel off pili. The deep culture medium thus contains a lot of suspended pili material. Type T ₂
If the culture is normally growing at a pH below 9.3,
There is no undesired solubilization of pili. For cultures of T ₁ bacteria, the pH is higher than 7.7 and therefore the pH should be adjusted to 5.5-7.7, preferably around 7.0, before the post-treatment described below.
Must be adjusted to lower it to ~7.2.
Although this is not critical, it is preferred to carry out such conditioning for several hours, eg 8 to 20 hours, before post-treatment to ensure crystallization of the partially solubilized _T1 pili. Both T ₁ and T ₂ cultivations are advantageously carried out in the presence of a small amount of diatomaceous earth such as celite, which is by no means a secret, and with gentle agitation of the medium. The amount of Celite is about 0.1-0.5%, suitably about 0.3% of the growth medium.
Must be % by weight. Isolation of pili crystals from the purification growth medium of pili It must be noted that the washings from the surface growth of Phosphorus bacterium contain soluble substances that are not relevant for the isolation of GC pili crystals. The same is true for deep propagation liquid culture. In the case of surface growth washes (first two washes only), the volume of liquid relative to the growth is relatively small. Preferably, all wash material is centrifuged at a relatively low speed. The speed of centrifugation and the time it takes to finish spinning are by no means critical. However, about 1000~12000G (less than
It has been found to be advantageous to rotate for approximately 10-15 minutes at approximately 3KG (written as 1KG and 12KG), preferably approximately 3KG for approximately 10-15 minutes. The residue in the pellet contains both cell and pili material, both of which are stored at this time and the supernatant is discarded. The supernatant contains substantial amounts of impurities as well as small amounts of pili that are not worth recovering. If deep growth is used as the culture method, centrifugation is a little tricky since the volume of liquid is rather large. The use of diatomaceous earth sandwich filters has been found to be useful in concentrating growth from deep cultures. In this method, a very coarse filter paper is placed on a filter pad, suitably a sintered glass or Buchner type surface, and a layer of diatomaceous earth, suitably about 2 to
Place 5 mm of Celite on top and cover with a second piece of coarse filter paper. The celite acts as the actual filtration medium, and the filter paper on top merely serves to hold the surface.
The culture broth is checked for pH to ensure that pili are present in crystalline form and filtered through a filter pad, and the filtrate is discarded. The combined residue was washed at a high temperature similar to that used for the third wash of the surface growth culture.
Take up in PH buffer and centrifuge the suspension at about 1-12 KG. It must be noted, of course, that in this case the pellet contains the diatomaceous earth carrier and the cellular debris, and the pili material is present in the supernatant. At this point it is desirable to separate the GC pili material from the cell tissue debris and also from the diatomaceous earth in the case of deep growth cultures. In the case of surface culture pellets centrifuged from a low-PH wash, this breaks the non-covalent bonds between pili rods in the system, leaving the covalent bonds intact and dissolving the pili crystals. This is done by adding an aqueous medium that gives rise to single rod pili. Such a medium must either raise the pH or keep the pH unchanged. If solubilization is carried out by changing the pH, add a suitable, moderately high pH buffer to the residue. This buffer has a PH of 9.3~11 and a PH of 10.0~
10.4 is preferred. At higher PH there is a risk of denaturation of the peptide. This PH value solubilizes the T ₁ and T ₂ pili crystals. If it is desired to separate _T1 from _T2 pili in the original solid pellet, the pH of the buffer should initially be approximately
Make it larger than 7.7 and smaller than about 9.3. The suspension is then centrifuged and the supernatant kept or discarded as required by the method. If the initial solid pellet is considered to contain a substantial amount of type T ₂ pili, a new buffer of high pH i.e.
9.3 or more to give a solvent layer containing T ₂ pili and no type T ₁ pili. The actual composition of the buffer used at this time is not critical, but Tris-buffered saline is particularly preferred. Approximately 1 of the volume of solids to total solids for both of the above modifications
Add ~3 volumes of buffer. It has also been found that this amount is not critical but sufficient to dissolve the pili material without using an excess volume of aqueous medium. If the pili are from a surface culture and this surface culture has been subjected to a third high PH wash with a similar buffer, this wash is added at this time. The pellet is then buffered into an aqueous suspension. The method of bringing the pellet into suspension is not critical; short, gentle sonication, long magnetic stirring, hand pipetting, hand mixing, swirling or mechanical stirring may be used. We have found it preferable to use mechanical stirring for a few seconds. The method of making the suspension is not important, but
Regardless of the method used, it is important that the cells are not disrupted, as this introduces undesirable substances into the aqueous layer. The occurrence of cell disruption is observed as a pink pellet layer on top of the white layer during subsequent centrifugation. The suspended material is then centrifuged. Although the method of centrifugation is not critical, we have found that the conditions described above for the first centrifugation step are suitable.
Filtration can be used instead of centrifugation. Dissolved pili are found in the supernatant or in the filtrate where the pili are precipitated by lowering the pH.
The degree to which the PH is lowered depends, of course, on whether one is operating on type T ₁ or T ₂ pili. Alternatively, precipitation is accomplished by adding sufficient ammonium salt;
Mineral acid salts such as sulfates are preferred, and aqueous solutions are preferred, providing ammonium sulfate concentrations of about 4% saturation to 10% saturation. However, we have found it desirable to provide an intermediate step prior to precipitation to increase yield and improve purity. In these additional harvests and purifications, both the supernatant and the pellet from the high PH centrifugation or filtration are retained. high
The pellet from the PH centrifugation step is suitably resuspended in the same aqueous medium at the same PH and the suspension is centrifuged again in the same manner. After this centrifugation, the pellet is discarded and the supernatants from both high-PH centrifugations are combined and recentrifuged. The purpose of recentrifugation is to remove residual suspended impurities. Centrifugation is therefore performed at a higher speed than before.
Do it for about 30-60 minutes at a speed of 12KG-70KG. However, it is generally preferred to rotate at about 27 KG to 40 KG for about 60 minutes. Discard the pellet and retain the supernatant. It is usually desirable to sterilize the pili solution at this time. This is required by FDA regulations for certain purposes. Such sterilization is easily achieved by passing the pili solution through a millipore filter immediately before precipitation, and we have found a 0.45 micron filter to be particularly suitable. This material must then of course be handled in an aseptic manner if sterility is to be maintained. In some circumstances it may be desirable to isolate pili into individual rod forms rather than crystalline forms. In this case, the supernatant is centrifuged at ultra high speed at 60-166 °C. 2 to 4 hours at 106 KG is suitable, thereby pelleting the pili into individual rod shapes. Pili are reprecipitated by lowering the pH of the supernatant to below 9.1. Due to the nature of the crystalline material, we have found that best results are obtained by dialysis against a suitable low PH buffer. The chemistry of the buffer is not important, but for _T2 pilus the initial PH is
We have found it desirable to use a buffer between 8.3 and 8.6. Tris-buffered saline was found to be suitable. Dialysis is suitably carried out using about a 30- to 60-fold excess, preferably about 40-fold. Dialysis is performed for approximately 12 to 18 hours with magnetic stirring of the external dialysis medium. Preferably, the dialysis is carried out at low temperature, ie at room temperature of about 0-10°C. This lower temperature range reduces the effects of unwanted bacterial contamination. It must also be noted that the pH of the buffer solution depends on the temperature. Therefore, if the temperature of the buffer solution is measured after cooling the system to its working temperature, the PH will be found to have risen to 9.1. However, satisfactory results have been obtained. It is generally not necessary to change the buffer when using an excess of buffer within the ranges stated herein. The crystal suspension is then processed to separate the pili crystals.
The most suitable separation is carried out by centrifugation at moderate speeds. It has been found that centrifugation at about 3 to 8 kg for about 60 minutes is appropriate. Then discard the supernatant. If you want to further purify pili crystals, use the solution -
2 cycles of high speed centrifugation - dialysis - recentrifugation
Or repeat 3 times. Addition of a preservative is useful if the pili crystals are not used immediately despite sterilization by filtration. The preservative is not added to the pili crystal itself or to the solution containing it, but rather to the dialysis buffer used to lower the PH of the solution. If the added preservative is incompatible with the buffer, after crystallization of the pili crystals, the incompatible buffer is removed by dialysis against a compatible buffer, and then the preservative and new buffer are added. Perform dialysis using Preservatives used are formaldehyde, merthiolate and azide. About 0.02-0.05% of these preservatives are used. Each of the preservatives shown has a minor detrimental effect. Formaldehyde causes cross-linking between pili rods. Thus they do not redissolve as before. Merchiolate has no crosslinking effect and crystals regenerate, but these regenerated crystals have reduced cohesion to pili in the presence of antibodies. Nevertheless, antigenicity is not affected. Thus, when injected into a subject, they cause the formation of apparently normal antibodies against pili. Azide is a satisfactory preservative that does not affect crystal structure, antigenicity, or aggregation. Unfortunately, it is toxic and cannot be used if pili crystals are to be injected into the human body. Suitably, crystalline preparations are stored at low temperatures, ie, about 1-4°C. However, if you wish to store it for a long time, dissolve the crystal in a suitable high pH buffer, filter it with a Millipore filter, and store it in solution under sterile conditions. If pili are required in crystalline form, they are preferably reconstituted by lowering the PH to an appropriate crystallization value for T ₁ or T ₂ pili, as the case may be. Both sterilization and preservatives can be used if it is desired to achieve the best possible preservation conditions. Purification of pili by constant PH lysis The procedure used to purify pili at constant PH is substantially similar to that used above using different PH values. As a solubilizing agent, for example, a salt, suitably a salt of an alkali or alkaline earth metal and a mineral acid anion, with an ionic strength of 0.5 or more, suitably about 4.0 to
5.0, preferably about 4.4, urea at a concentration of about 3M to 5M,
Aqueous solutions of sucrose with concentrations greater than 50% by weight and water itself are used to reduce the ionic strength of the solution to less than 0.002M. In one modification of this embodiment of the invention, the cells,
Instead of suspending the initial centrate pellet of pili and tissue debris in a high PH medium, the reagents are used in the environment described above. This suspension is then centrifuged at low speed as before. If high yields are desired, save the supernatant from the low-speed centrifugation.
The pellet is resuspended in a similar medium, recentrifuged, and the supernatant thus obtained is combined with the previous supernatant and centrifuged at high speed. The pellet from the high speed centrifugation is discarded and the supernatant is dialyzed against an appropriate buffer to remove the solvent medium. Thus, when the solvating agent is a high concentration of salt or sucrose, the ionic strength is reduced to less than 0.5 for salt and less than 40% for sucrose.
In the case of urea, the urea is dialyzed against a suitable buffer, such as Tris-buffered saline, at PH 7.0 or PH 8.3 depending on whether you are treating T ₁ or T ₂ pili.
Give an ionic strength of 0.05-0.3. Similarly, when the solvating agent is water, a similar method is used to increase the ionic strength to at least 0.05. The buffers used for this purpose are the same as those used in the different PH purification methods and are also used in the same manner. Purification of GC pili by density gradient centrifugation Density gradient centrifugation is performed by centrifuging a mixture of pili and an aqueous cesium chloride solution together such that the optical density at a given wavelength is Used to indicate the part of Centrifugation is performed at PH9, but centrifugation is performed at PH10.0~
Even better results can be obtained by using 10.4 or 10.1 if appropriate. In this method, a crude pellet containing cellular material, pilus and tissue debris is suspended in a high PH buffer as indicated above, to which an appropriate amount of cesium chloride is added. For example, 10ml of aqueous medium
We have found it suitable to prepare a medium containing approximately 2 to 5 g of dry cesium chloride per portion. It has thus been found that it is most suitable to use about 7.5 g of cesium chloride in 20 ml of aqueous medium. The mixture is then spun at about 110-250 KG, suitably about 200 KG, for about 30-60 minutes, suitably about 42 hours, and the optical density of the fraction at a given point in the tube is measured. Optical density measurements at 280 nm show a single peak. The density fraction of this peak is separated and dialyzed against a low PH buffer to generate pili crystals in the same manner as described above. The actual density range of the solution fractions collected at this point at 20 °C is PH
10.1 is 1.35-1.33. The basic agglutination ability of GC pilus crystals or single pilus rods in the presence of antibodies against Phosphorus bacterium is
This is the basis of the PAT exam. In this test, serum from the blood of a subject suspected of being exposed to Phosphorus bacteria is mixed with GC pilus crystals or single pilus rods, and the mixture is observed for aggregation of the crystals or rods. In order for this test to be accurately evaluated, three important factors must be considered. First, this test is not intended to replace the standard "culture" test, but rather serves as a screen to determine exposure to phosphorus bacteria. The test is therefore positive both for subjects who have had an active infection for more than a few days and for subjects who have been exposed to the disease but have since recovered. The third caveat is that subjects who have been infected very recently may not have developed enough antibodies to give a positive test. Subjects with a positive result in this test must undergo traditional culture testing. Furthermore, as discussed below, the pili of the infectious form of Mycobacterium possess some specific immunological determinants. The pili of some bacterial strains possess one or more of these determinants. Therefore, in order to be effective, selection must be performed using pili crystals that cover the spectrum of immunological determinants. The GC pili crystals used in this test are prepared as described above. The test is performed using the subject's serum or plasma. The discussion herein regarding serum or plasma can be considered interchangeable test methods. The amount of serum or plasma required is very small. Prick the subject's finger to obtain a few drops of blood;
We have found that it is sufficient to spin these in small (approximately 250μ) centrifuge tubes to obtain a plasma of 10-20μ suitable for conducting this test. Conventionally, this type of test is performed at various dilutions.
The serum is therefore subjected to predetermined dilutions (usually serial dilutions) with suitable predetermined diluents. The nature of the diluent is not important unless it interferes with the test procedure. Buffered aqueous solutions such as phosphate buffered saline or Tris buffered saline with a pH of 7.0 to 7.5 can also be used. Tris-buffered saline with a pH of 7.2 is preferred. In the test procedure, the pili suspension is added to diluted serum to obtain a final concentration of 10-50 μg crystals per ml in suspension. We found that the best results were obtained with low concentrations of pili crystals, and therefore for the purpose of standardizing the amount of pili antibodies in the test serum, we compared pili crystals per ml of diluted serum (or plasma). 20μ
An arbitrary concentration of g was used as a standard. After mixing the pili crystals with the diluted serum, the mixture is kept warm. Heat retention time is not important, 15 minutes ~
At 48 hours, the change in reading is negligible. A rapid form of the PAT test involves stirring the serum-pili mixture by hand on a sliding surface for 1 to 3 minutes and produces similar results, but with some loss of sensitivity. The temperature at which the mixture is maintained is also not critical as long as it is maintained between 0 and about 45°C. However, the mixture should be kept at 22-40℃, approximately 22℃.
It seems preferable to hold the temperature at for at least 15 minutes. Thereafter, storing the mixture at 4°C for 24 hours does not appear to result in a significant change in titer. 37
Satisfactory results were obtained by incubating at ℃ for 30 minutes. At the end of the incubation period, place a certain amount under a dark-field microscope and note any aggregation and mark it with a symbol. Symbolization can be done in any correlative manner usual in this type of test, i.e.
+, 3+, 2+, 1+, -± and -. To determine the titer of a given sample, the last dilution giving 1+ agglutination is taken as the final reading. 1+
The symbol indicates visible aggregation that is much smaller than the standard control sample. Since titer readings vary from batch to pili batch depending on the conditions, this test should be performed in addition to the usual controls for diluent without serum and diluent with normal serum. It is recommended to monitor this test by testing the crystals against antiserum of known PAT titer. Under the conditions used, this test is doubled (x2)
It was found that the results could be reproduced within the titer index of the coefficient. Serotyping of Phosphorus bacteria Until now, it has not been possible to serotype Phosphorus bacteria into useful and meaningful systems. It is not unusual for different strains of the same species to produce different antibody responses. These antibody responses characterize the bacterial strain, and confirmation of these characteristics is an essential tool in epidemiological immunological studies of the development and origin of this particular outbreak of the disease. This is of particular interest in sexually transmitted diseases. This is because the elimination of this disease is very often through human contact channels. Thus, if the infectious agent can be identified by serotyping, this typing pathway is of great help. By examining 21 strains from sources widely distributed throughout the United States, we found the presence of at least four immunological determinants in the pili of these strains. One or more of these determinants are present in each strain. Therefore,
Infectious agents can be more easily discovered if the determinant characteristics of a particular sample can be ascertained. If the Phosphorus bacterium is from an infected subject and multiplies to produce pili crystals, the immunological determinants present in the pili crystals from the subject may cause it to become an antibody against only one determinant. Serum of the aforementioned type known to contain
It is easily detected by subjecting it to the PAT test.
The serological profile of any given sample material is thus established. Safe and Potent Lynx Disease Vaccine GC pili crystals and single rod pili were injected into test subjects and found without any toxic effects. The only negative effect was observed in chicken fetuses injected intravenously. to the test rabbit
100 to 200 μg per kg was subcutaneously injected three times, and the total dose was 300 to 600 μg per kg.
8000μg was injected twice in the same way, and the total amount was 16000μ per kg.
g was administered. 100μg/kg for red-headed monkeys
Humans received 2 to 10 μg per kg followed by a single intramuscular injection of 50 μg per kg. None of the test animals died or showed any local or systemic toxic effects, and chicken fetuses showed an ID-50 of 60 μg per pound. Reactions in human subjects vary from no systemic effects to transient nausea and fever in one of the subjects tested. PAT titer in rabbits is 1000
~8000, and reached 10000+ in red-headed monkeys.
PAT titer in humans varies from 100 to 200. The dose that infects 50% of the test subjects (ID ₅₀ ) is on the order of 5.0×10 ² bacteria. of human subjects with PAT titers 100-200 in preliminary experiments
ID ₅₀ showed 2.0×14 ⁴ bacteria. This is 1.6
Log cycles of protection, or in other words, a human subject with a PAT titer of 100-200 represents a 0.86% chance of infection after one contact, whereas a non-immune subject has a 0.86% chance of infection. Humans have a risk rate of approximately 30%. Given these findings, human subjects should appropriately be administered sufficient doses of GC pili crystals for all known determinants to confer protective tolerance in humans. The PAT titer must be raised to at least 100, preferably at least 200 for each. Such amounts can be obtained by administering about 2 to 100 μg of GC pili crystals of each determinant per kg of body weight. However, a titer of 100 can be achieved with as little as 1 μg/Kg. The method of administration, if any, will depend on the sensitivity of the subject, but in most cases it is appropriate to administer from one to five times every eight weeks. GC pili crystal, single pili rod or GC
Other sources of pilin are administered in vehicles suitable for intramuscular injection. Twenty-one strains of Phosphobacterium used in the experiment were isolated from humans with Phosphorus disease and named 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, Norhawk 2-
2. Norhawk 7-2, Dayton 8-2,
CDC005-2. All examples below describe the Pittsburgh 3-2 strain. Other bacteria in Pittsburgh 3-
Similar results can be obtained even if the same procedure is used for the two strains. Example 1 Purification of strain: A primary culture of Pittsburgh 3-2 strain was grown on Thayer-Martin agar [Manual of Clinical Microbiology (Manual of Clinical Microbiology)].
Clinical Microbiology), 2nd edition, American
Amer.Soc.Microbiol., Lenette et al.
et al.), 1974, 920 pages]. This plate was heated at 35°C in an atmosphere of 95% air and 5% carbon dioxide for about 18
Incubate for hours. Inspect the plates and re-streak them onto the GC medium for colonies resembling highly piliform _T2 forms. After culturing under the same conditions as above, pick _T2 type colonies and re-streak them onto the GC medium again. By the method described above, if you wish to culture the _T1 type of this bacterium, you can use the less piliated _T1 .
Type-dominant colonies are similarly re-streaked. Example 2 Preparation of Growth Media a Preparation of DSF Supplement A solution of cocarboxylase (0.2% by weight) in distilled water is prepared at room temperature and sterilized by passing through a 0.45 micron Millipol filter. Glucose (40
g), glutamine (1.0 g), ferric nitrate (0.5% by weight, in 10 ml of distilled water) and distilled water (90 ml) in an autoclave at 121°C at a pressure of 16 psi.
Heat for 10 minutes and cool the solution. Add 1 ml of the cocarboxylase solution prepared in advance to this autoclave solution to obtain a DSF solution. b Preparation of growth medium Bacto GC basic medium [Difco Manual, 19th edition, page 122] [Difco Manual, 19th edition, page 122]
Difco Laboratorics, Detroit, Michigan] (10.8 g) and distilled water (300 g)
ml) in a suitable container and the mixture in this container was placed in an autoclave at a pressure of 16 psi.
Heat at 121°C for 15 minutes. The vessel is removed from the autoclave and cooled to 50-60°C and the DSF supplement (3 ml) prepared as above is added. Preparation of Inoculum Plates and Growth Dishes Pyrex or aluminum growth dishes and Petrie-type inoculum dishes are cleaned, rinsed in distilled water, covered with aluminum foil and heated in an autoclave at 121° C. at a pressure of 16 psi for 30 minutes. Pour the molten growth medium of the previous example into the dish thus prepared. The slab must be poured carefully in a dust-free, closed room using aseptic methods to prevent unwanted bacterial contamination. Example 3 Growth of Inoculum _T2 colonies on Thayer-Martin plates (Example 1) were re-streaked onto inoculum plates prepared as described above and grown in an atmosphere of 5% carbon dioxide and 95% air.
Incubate at 35 °C with 90% humidity. Passage sequentially until >90% of growth is of the pilus-pillated _T2 colony type. Depending on the stability of the strain, the growth time is 12-24 hours, in which time the plates will be 50-75
% covered with growth. Example 4 Production of GC pili by surface culture The Petri dishes of Example 3 containing T ₂ growths are washed with an aqueous casamino acid solution (5 ml, 0.7% by weight).
Sterilize the growth, scrape the medium with a glass spreader, and pipette the suspension of growth in the casamino acid solution from the plate into approximately (14 x 10 inch) 2
Divide into two growth dishes. This dish is prepared as described above. The suspension was spread flat on the surface and the dish was heated for 90 minutes in an atmosphere of 5% carbon dioxide and 95% air.
Incubate for 20 hours at 35 °C with % humidity. Incorporation of surface culture growth Prepare a stock solution of Tris-buffered saline by adding sodium chloride (510 g), Tris, i.e., tris(hydroxymethyl)aminomethane (363 g) and concentrated hydrochloric acid (100 ml) to enough stock solution to make 10 stock solutions. Prepare by dissolving in a certain amount of distilled water. PH standard use PH
Adjust by adding concentrated hydrochloric acid to 8.5. If a large pH adjustment is desired, add a concentrated (10N) aqueous sodium hydroxide solution. Before using the stock solution, dilute it to 1/6 of the original concentration. Tris-buffered saline (less than
TBS) has an initial pH of 8.5. TBS solution
Place 10 ml on the growth surface of the production dish, scrape off the growth with a glass scraper, and pipette the suspension into a reservoir. The washing and scraping is repeated with a second batch (10 ml) of TBS and both washes are pooled. The growth surface is washed a third time with ethanolamine buffer (10 ml, PH 10.1) and the suspension is kept without pooling. (Ethanolamine buffer is liquid ethanolamine 37.3ml, hydrochloric acid 1N,
Prepare by adding 147.0 ml and distilled water. ). By the above method, _T2 pili and not
When culturing _T1 , the pH of TBS is 7.0-7.2. If the production has progressed well, the growth has a characteristic orange/pink or reddish pink color and an odor similar to cooked food. The growth clumps into a slimy mass and is easily peeled off the agar surface of the medium when restrained with a glass spreader. The dishes containing the growth medium are cleaned, washed, sterilized as previously described and reloaded with growth medium. Example 5 Propagation of GC pili in deep culture The inoculum was prepared in a growth medium according to the method of Example 4 using the same growth medium and nutritional supplements, but without agar and using soluble starch instead of insoluble starch. In an atmosphere of 95% air and 5% _CO2 ,
Incubate for 18 hours with gentle agitation in the presence of 0.5% (based on the volume of liquid medium) Celite. The culture medium is then passed through a sand German filter pad on a coarse glass funnel. The overpad consists of coarse paper, a 5 mm layer of celite and even coarser paper. The solution is discarded and the residue is treated with the third (ethanolamine buffer) washing procedure of Example 4, followed by the method of Example 6, section (see below). Example 6 Separation of Pili from Cells and Debris A The TBS suspension prepared in the previous example is placed in a centrifuge tube (the capacity of the tube depends on the number of products collected) and centrifuged at 3 Kg for 15 minutes. Discard the supernatant and retain the pellet. Add to the pellet the ethanolamine suspension from the third wash of the growth dish and an additional amount of ethanolamine buffer at pH 10.1 approximately three times the volume of the pellet in the centrifuge tube. Mix the liquid layer with a mechanical stirrer for 5 minutes.
Stir rapidly for seconds to bring the soluble portion of the pellet material into suspension. C. This suspension is then centrifuged at 3 kg for 15 minutes, and the supernatant is decanted from the pellet and saved. Next, add this pellet to three times its capacity as described above.
Resuspend in PH10.1 ethanolamine buffer;
Centrifuge at 3Kg for 15 minutes. Decant the supernatant,
Pool with the ethanolamine buffer from the previous step and discard the pellet. Combined ethanolamine buffer supernatants
Centrifuge at 31Kg for 60 minutes. Decant and save the supernatant and discard the pellet. Example 7 Crystallization of GC Pili Preparation of Dialysis Tube Material and Tris-Buffered Saline One roll of dialysis tube material [100 feet, Fisher Scientific Catalog Number 8]
-677B] in sequence a) Distilled water (twice, 4 times each time),
b) Sodium bicarbonate aqueous solution (twice, each time 4 teaspoons of distilled water containing 2 teaspoons of sodium bicarbonate)
), c) Ethylenediaminetetraacetate disodium aqueous solution (twice, each time with 4 ml of distilled water,
2 teaspoons of NaEDTA), d) aqueous ethanol (twice, each time ethanol/water, 1:1, 4);
e) Boil in distilled water (twice, 4 each time). The dialysis tube material was then soaked in distilled water containing traces of benzoic acid (4 teaspoons of distilled water and 1 teaspoon of benzoic acid).
Store inside. Dilute the Tris-buffered saline (TBS) prepared in Example 4 with distilled water to make a dialysis solution (dilute 166 ml of the stock solution to 1 with distilled water).
get. Dialysis of GC pili solution 100 ml of ethanolamine buffer containing the GC pili in solution prepared in the previous example was diluted in TBS (measured at 20°C) using the dialysis tube material prepared above and diluted TBS. Dialyze against pH 8.5) 4. Dialysis is performed in a cold room (room temperature approximately 4°C).
Magnetically stir the external dialysis buffer solution. Dialysis is performed for 18 hours. A precipitate of cloudy blue/white birefringent GC pili crystals forms at the end of the dialysis period. The precipitated material is centrifuged at 7.5 Krpm for 60 minutes, and the supernatant is discarded to obtain GC pili crystals of Pittsburg strain 3-2 Phosphorus as a pellet.
When using the above method to isolate _T1 rather than _T2 pili, the initial PH of TBS is 7.0-7.2. Example 8 Repurification of Pili The pellet from Example 7 is suspended in approximately 30 times its volume of ethanolamine buffer (PH 10.1).
Gently swirl the tube to dissolve the pellet, and centrifuge the suspension at 31 kg for 60 minutes.
Decant off the supernatant and discard the pellet. The supernatant is dialyzed against TBS as in Example 7 and the crystalline material thus obtained is isolated by centrifugation as in Example 7. The pellet was resuspended in ethanolamine as described above and
If sterilization is desired, pass through a 0.45 micron strainer, centrifuge and redialyze as above, and isolate by centrifugation as well. If the GC pili crystals produced in the above method are desired to be stored for a reasonable period of time, a compatible preservative is added to the dialyzed TBS solution. Suitable preservatives utilized in this method are neutral formaldehyde in water at 0.05% weight per volume, merthionate (trade name) at 0.01% weight per volume, or sodium azide at 0.02% weight per volume. . In the method described above, if the preservative is not compatible with TBS after crystallizing the pili, remove the Tris buffer by dialysis against saline (0.15 M
aqueous sodium chloride solution, 18 hours). A preservative is added to a fresh batch of this saline solution and dialyzed against a suspension of GC pili crystals for 18 hours. The crystals are stored in this medium at 40°C. Alternatively, the medium containing the crystals is frozen and stored at -70°C. Yield GC from the strain of Phosphorus bacterium described in Example 1 where the inoculum contains at least 90% _T2 colony type.
The yield of pili crystals is 5-15 μg/cm ² of the proliferating surface.
It is. Similar but slightly lower yield from T ₁ colony type. Example 9 Cesium Chloride Density Gradient Isolation of GC Pili Containing the GC Pili Crystal Suspension of Example 6
Add 1 ml of TBS (PH8.5) wash to Tris PH8.5 buffer.
Dilute to 20ml with sodium hydroxide aqueous solution (0.1N)
Adjust the pH to 10.1 by adding dry cesium chloride.
Add 7.5g to this. This solution was added to Beckman L.
- Rapidly rotate at 200Kg in the SW41 rotor of a 265 centrifuge. Fractions are collected from the tube and the optical density and refractive index at 280 nm are measured for each sample. The refractive index is related to the cesium chloride density to which the refractive index reading is converted. Fraction number is plotted on the X-axis against solution density on one Y-axis and optical density on a second Y-axis. A single main peak corresponding to GC pili is located at ρ (buoyant density) of 1.3422±0.0038. Fractions 1.35 to 1.33 are combined, dialyzed, and purified by the methods of Examples 7 and 8 to obtain GC pili crystals. Example 10 Gel electrophoresis of GC pili [Method of Ornstein and Davis - Disk electrophoresis 1962]
Distillation Products Industries, Rochester, New York] 10% acrylamide gel (9.7% acrylamide and 0.3% N,N′-methylene-bis-acrylamide polymerized in TEMED) and Prepare a standard cylinder of ammonium sulfate and assemble between the gel and a storage buffer consisting of PH 8.0 Tris-HCl and 0.1% SDS. 50μ of _T2 pili crystals on top gel surface
g, Cleland's reagent (0.01M) 20μ
g, SDS 1mg, glycol 20λ and bromophenol brake (0.002%) 20λ. 100°C with SDS and Cleland's reagent before pili preparation.
Heat for 2 minutes. Electrophoresis is performed at 5 ma (approximately 170 V) until bromophenol blue travels 6 cm. Remove the gel, cut the dye band, and coat the two gels with Coomassie Blue stain.
Blue Stain) (0.2%) to obtain two bands - a major band and a minor band. Freeze the uncolored gel and cut out the band corresponding to the position of the colored band. The major band is extracted with storage buffer in a rotating apparatus for 24 hours at 37°C, and the buffer is removed and evaporated to near dryness. Reproducing the product yields a single band with the same Rf value. This substance is called GC Pirin. Antigenicity test of GC Pirin The gel containing the major band is homogenized with 10 ml of saline and injected subcutaneously into three test rabbits. Test animals P〓 and P〓 receive 3.1 ml of the suspension, and P〓 receives only 2 ml. The second and third injections are given approximately 15 and 30 days later. The material for the second and third injections is the main protein in storage buffer (0.8 ml) at 37°C for 24 hours.
Extract and prepare for an hour. The extracted buffer is then combined with an equal volume of Freund's Incomplete supplement and 1/3 of each mixture is injected into each rabbit. One week after the last injection, all rabbits were given the Pittsburgh 3-2 type as shown in the table below.
It showed a titer of over 1000 in the PAT test against _T2 pili.

[Table] Plate gel electrophoresis for myoglobin, chymotrypsinogen, and human gamma globulin shows a main fraction with a molecular weight of 20,500 to 21,500 and a molecular weight of about 28,000.
Provides a non-trivial amount of protein. Example 11 Carbohydrate Analysis - Phenol-Sulfuric Acid Test A standard curve is prepared by treating the stock glucose solution with 0.1N aqueous sodium hydroxide solution and measuring the UV absorption at 485 nm. Tests with Pittsburgh 3-2 pili and CDC B-2 pili showed essentially the same amount of carbohydrate content, equivalent to 1-2 hexose residues per protein subunit.
It shows 1.49±0.56%. Example 12 Phosphorus analysis [Chen et al., Analytical Chemistry (Anal.Chem.), Vol. 28
Method of page 1756 (1956)] Pili is immersed in sulfuric acid and assayed against aqueous potassium dihydrogen phosphate solution by ammonium molybdate-ascorbic acid assay. pittsburgh 3
The average value of -2 pilus strains was 0.332±0.026%;
CDC B-2 strain pili was 0.366±0.048%;
2.5 and 2.5 per protein subunit, respectively.
2.3 Shows a phosphorus atom. Example 13 Amino acid analysis of type T ₂ pili crystals [Spackman et al., Anal.Chem., No. 30]
Vol. 1190, page 1190 (1958) correction method] The analysis was carried out using norleucine and 2-
Using amino-3-guanidinopropionic acid, Beckmann Spinco Model 120B Amino
Assisted analyzer (Beckman Spinco)
Performed using Model 120B Amino Acid Analyzer).
Protein samples (10 mg) were evacuated vial (0.25 mm) with concentrated hydrochloric acid at high temperature (6N, 110℃) for 24 hours.
Hydrolyzed in Hg). Bence et al.
al.) Analytical Chemistry (Anal.
Chem.), Vol. 29, p. 1193 (1957). Aspartic acid + asparagine 26 Alanine 23 Glutamic acid + Glutamine 21 Lysine 20 Glycine 17 Valine 17 Serine 14 Leucine 12 Threonine 9 Isoleucine 9 Arginine 8 Tyrosine 7 Proline 6 Tryptophan 4-5 Histidine 3 1/2 Cystine 2 Methionine 2 Phenylalanine 2 Amino acids Number -200±9, molecular weight 21500±1000 Daltons. Example 14 Physical Properties Solubility Pili crystals, which appear in an electron microscope as bundles of pili rods, exist in a crystalline state with a pH of approximately 5.5 and 9.3. Crystals of the _T2 variant begin to separate into single pili rods at pH 9.3-10.1. At pH 10.1 and above, these exist as single pili rods. Similarly, T ₁ pili begin to separate into single pili rods at pH 7.7 and exist as rods at pH 8.6 and above. That is, the crystals completely disintegrate. T ₂ at approximately PH11.0 or higher
Pili rods degrade into smaller, less polymerized units with a sedimentation constant of approximately 5.5. Pili crystals are soluble in 4M sodium chloride aqueous solution, 50% sucrose aqueous solution, and 20% saturated calcium chloride aqueous solution (both weight %) at pH 8.5. This crystal also dissolves in urea at 3M and above. However, treatment with urea at 3.5 M or higher for more than 2 days degenerates the GC pili. Ultracentrifugation GC pili preparations (1 mg/ml) are prepared in ethanolamine buffer (0.147 ionic strength, PH 10.1). This solution was added to the Beckman Spinco Model E Ultracentrifuge (Beckman Spinco Model E Ultracentrifuge).
Spinco Model E Ultracentrifuge), AN-
Operate at 20Krpm using a D rotor. The uncorrected sedimentation velocity S is 37 svedbergs. Size of pili rods GC pili crystals were deposited disc rods of uncolored TMV protein at pH 10.0 and were taken in ethanolamine buffer and examined by electron microscopy. The average diameter of GC pili is 83.4±2.3 Å. Example 15 PAT test Pili crystals were collected at pH 7.0 at a concentration of 30-60 μg/ml.
Suspend in TBS. Use a clear test serum. If the test serum is cloudy, add 30 kg of this
Centrifuge for 30 minutes and use the supernatant. Serial dilutions of the serum are made, 0.025 ml of the serum sample and 0.025 ml of the pili crystal suspension are placed into each microtiter plate well, and the mixture is stirred for 30 minutes at room temperature.
This reservoir is then read for crystal clumps under a dark field microscope. The maximum dilution of crystal agglomeration observed to be greater than the control is then recorded. Sample Testing White New Zealand female rabbits weighing 4-6 lbs. CDCM-2, Pittsburgh 3-2,
CDCT-2, CDC005-2 and Pittsburgh 4
The purified pili preparation of the -2 strain was mixed 1:1 with Freund's Incomplete auxiliary solution, and the mixture was washed with a dropper, emulsified, and injected subcutaneously. pili approximately 100-200μ
g/Kg was given as three injections approximately two weeks apart, and the rabbits were bled 1-2 weeks after the third injection.
The blood was allowed to clot and the serum was removed in the usual manner. The first three test sera were then run against pili of the 21 Lin strain. The results are shown in the table below.

【table】

[Table] A 7x7 serotype pili agglutination test was performed using pili and serum from seven selected bacterial strains according to the method described above. To facilitate explanation of the results, shown in Table 1a below, the cross-reactivity index (pili versus serum of the same strain) was adjusted to 100, and the other indexes were adjusted accordingly.

[Table] Example 16 Serotyping The PAT test of Example 15 showed that 3 or 4 bacterial species had pili with only one determinant. This was confirmed by running sera from four selected strains against the corresponding pili. The results are shown in the table below. Various titers were adjusted with a maximum sensitivity of 100.

[Table] Serotyping of strains with unknown determinant composition. Perform antiserum. PAT tests showed that pilus agglutinate with one or more single determinants as shown in the table below.

[Table] Hemagglutination reaction with GC pili preparation General method Type O human blood containing EDTA as an anticoagulant was obtained from a blood bank. 15 volume PH of a certain amount of blood
Fresh red blood cells were prepared by washing four times with 0.01 M phosphate buffered saline at 7.3 and making a 3% (volume) suspension in the same buffer. 1.0mg/ml Pittsburgh 3-2 and CDC-M
A -2 pili solution was prepared using the ultraviolet absorbance of this formulation at 280 nm with scattering correction as a concentration standard. A fixed amount (50λ) of this 1.0 mg/ml pili solution was added to a U-bottom Cooke microtator plate (U-bottom Cooke microtator plate).
Add Tris-buffered saline + 0.02% azide 25λ to all other wells and dilute pili in the 12th well with a 25λ manual microdiluter. Tris-buffered saline or saline (25λ) and
Add 25λ3% red blood cell suspension to each test well, mix by gently agitating the plate, and place at 4°C. Observe the results after 1-2 hours by dark box. Results The degree of hemagglutination observed was: 5+ No cell pellets or clumps; uniformly red in the reservoir 4+ Traces of cell pellets 3+ Small cell pellets with very distinct edges of cell clumps 2+ Moderate Clear cell pellet with clumps on the outside + Clear cell pellet with some clumps on the surface of the reservoir Two titer assays for both CDCM-2 and Pittsburgh 3-2 pilus The results are shown in the table below and a control test is set up.

[Table] Inhibition of hemagglutination: Method for removing Ab-Ag complexes from antiserum-pili mixture before hemagglutination titer assay. Wash without dilution to remove background hemagglutination with serum alone. Add 0.1 ml of red blood cells ( _RBCS ) to each 1 ml of antiserum in a round glass bottom centrifuge tube. The tubes are capped with parafilm and placed upright on a shelf on a Yankee Rotator at 4°C. After 1 hour and 10 minutes, the serum was cooled using International Centrifuge.
Centrifuge) at 2400 rpm and test for hemagglutination ability. The serum is further clarified by centrifugation at 12000 G in a Sorvall RC-2 centrifuge. The background hemagglutination factor absorption at various dilutions of the antiserum before and after RBC treatment is summarized in the following table.

【table】

[Table] Removal of Ab-Ag complex Preimmunization of RBC-adsorbed rabbits prepared as described above and serial dilution of anti-CDC M-2 serum are carried out in saline. 1.5 aliquots (0.1ml) of each dilution
100γ/ml Microfuge tube
Add to 0.1 ml of M-2 pili. The tube is held constant at 37°C for 1 hour on a Yankee rotator. These are then spun for 1 minute in a Beckmann microfuge. A little more than half of the supernatant is removed and 50λ of each antiserum dilution is placed in each of the two reservoirs of the Microtitre plate. Add an aliquot (25λ) of 3% RBC _S in phosphate buffered saline, mix by gentle agitation of the plate, and keep at 4°C for 1 hour. The results are shown in the table below. Similar results are obtained when other pili are used in place of the CDC M-2 pili and serum contains antibodies to this pili.

【table】

[Table] Has + red blood cell agglutination reaction.

Claims (1)

[Scope of Claims] 1. Single or aggregated rods derived from Neisseria gonorrhoeae, which in an aggregated state contain antibodies against pili derived from at least one of the following strains of Neisseria gonorrhoeae: Shows strong agglutination ability for serum, poorly soluble at pH 7.7 or below, pH
Substances that are soluble at 9.2 or higher: Pittsburgh 3-
2 (ATCC31149), CDCT-2 (ATCC31150),
CDCM-2 (ATCC31148) and Pittsburgh 4-2 (ATCC31151). 2 A single rod-shaped body, the diameter of which is approximately 83.4
±2.3Å in ethanolamine buffer
A material according to claim 1, which exhibits a sedimentation rate of about 37S at pH 10.1. 3. The substance according to claim 1, which is isolated from the pili of a type T ₁ culture of Phosphorus bacterium. 4 Agglomerated rod-shaped body, poorly soluble at pH 7.7 or below, pH
The substance according to claim 3, which is soluble at a temperature of 8.6 or higher. 5 A peptide chain consisting of a sequence of 200±9 amino acids, with 2-3 phosphate groups and 1-2 hexose sugars covalently bonded to the chain, and the molecular weight of the chain is 22000±1000 Daltons. It is poorly soluble at PH7.7 or less at 20℃, and PH
A substance according to claim 1, designated as T ₁ GC pilin, which is a phosphoglycoprotein, which is soluble above 8.5. 6. A substance according to claim 1, which is isolated from the pili of a type T ₂ culture of Phosphorus bacterium. 7 Agglomerated rod-like body, poorly soluble at pH 8.7 or below, pH
The substance according to claim 6, which is soluble at 9.2 or higher. 8 A peptide chain consisting of a sequence of 200±9 amino acids, with 2-3 phosphate groups and 1-2 hexose sugars covalently bonded to the chain, and the molecular weight of the chain is 21500±1000 Daltons. It is poorly soluble at PH8.6 or less at 20℃, PH
7. The substance of claim 6, designated as T ₂ GC pilin, which is a phosphoglycoprotein which is soluble at 10.1 or higher. 9. The substance according to claim 1, which is used as a vaccine for preventing infection with pili-formed Phosphorus bacteria.