Precipitation (The reaction)

Precipitation. The reaction P. refers to a group of reactions in which, when mixing precipitating serum - precipitin (see.) with an antigen - precipitinogen, a precipitate - precipitate - is obtained in positive cases. Along with the reaction P., there is a group of precipitation reactions for the diagnosis of syphilis (see below), the ingredients of which cannot yet be considered an antigen and antibody in the strict sense of the word. These reactions will be considered separately. The mechanism of the precipitation reaction, as well as the agglutination reaction, has the character of a reaction between colloids. This is also manifested in the quantitative ratios between precipitin, antigen and precipitate: with an increase in the amount of precipitin or antigen, the amount of precipitate increases, but only up to a certain limit, after which the amount of precipitate begins to decrease again; this is especially evident when changing the amount of antigen. The phenomenon of delay observed in P., as in agglutination, is explained by this influence of quantitative 13 ratios. With given amounts of precipitin and antigen, the amount of precipitate is the less, the more diluted the solution. The distinctive property of precipitation reactions as immunity reactions is specificity. Pick, Schwarz, Meyer, Sardelli, Fischer showed that aqueous extracts from bacteria and tissues are precipitinogen in vivo, while alcoholic extracts do not possess this property and react only in vitro. Thus, precipitinogenic properties and the ability to precipitate are not identical. Lipoids from horse serum cause the appearance of antibodies; Pick assumes the presence in these lipoids of a substance which possesses the properties of an antigen. Attempts to obtain lipoid antigens from bacteria were not successful. Nicolle found that the precipitinogen substance of bacteria is soluble in ether-alcohol and withstands 5-10 minutes of boiling over an open flame. Putrefaction, as well as digestion for several weeks with pepsin or trypsin, acids and alkalis when heated also do not destroy their antigenic properties. Winterberg does not confirm Nicolle's data. The site of formation of precipitins. As with respect to other antibodies, we know little about the mechanism and place of formation of precipitins after the introduction of precipitinogen. Most authors explain the formation of precipitins on the basis of Ehrlich's theory. According to Kraus and Levaditi, Kraus and Schiffmann, after intraperitoneal introduction of precipitinogen, precipitins appear in the blood serum and extract from the mesentery, after subcutaneous and intravenous introduction - only in the serum. In view of the fact that with an increase in the amount of precipitins the number of leukocytes decreases, Kraus assumed that precipitins are formed in leukocytes and endothelium. According to v. Dungern, on the 4-5th day after the first injection of precipitinogen, precipitins appear in the blood, on the 6-7th day their amount reaches a maximum, and then a decline occurs. With repeated introduction very soon after it there is a sharp decrease, after which there is a secondary rise.

Precipitoids. Under the influence of physical and chemical agents, precipitins, like agglutinins, may lose their ability to precipitate, while retaining their ability to bind precipitinogens. Kraus, following Ehrlich's scheme, assumes that in this case the ergophoric group is destroyed, while the haptophoric group remains intact. Such a modification can be obtained by heating the precipitating serum to 60°. Dried serum withstands heating much better, in which precipitins are destroyed only at 130°. Precipitate. The visible manifestation of P. is the formation of a precipitate. The latter depends on certain external conditions. Thus, the reaction of the medium affects the formation of the precipitate; P. occurs within the pH range of 5-9. A neutral or slightly alkaline reaction is favorable for the formation of the precipitate, and a slightly acidic reaction is even more favorable. A strongly alkaline reaction slows it down; temperature (37°) accelerates P. At the beginning of P., cloudiness appears, which gradually turns into flakes. The latter settle and a clarified liquid remains above them. The entire process proceeds at different speeds: significantly faster in the case of zooprecipitation than in bacterioprecipitation. In the latter case, it can continue for 24 hours or more. Just like agglutination, P. does not occur in the absence of electrolytes. Many works have been devoted to the question of the chemical composition of the precipitate. It undoubtedly contains protein bodies, and lipoids are also possible. The source of proteins is presumably usually (Moll, Welsch, Chapman and Pick) the antiserum. Confirmation of this is the fact that in quantitative determinations of protein in the precipitate, it is found to be significantly more than was contained in the antigen. However, in some cases, protein originating from the precipitinogen was also found in the precipitate. The precipitate has great resistance to digestive enzymes, is insoluble in mineral salts, and in soda. Müller (Th. Müller) was the first to prove the reversibility of the P. reaction. Splitting into precipitin and precipitinogen can be achieved by acid and heating. This does not succeed in all cases. Like all immunity reactions, the P. reaction possesses species specificity if, when setting it up, certain requirements are observed both with regard to the strength of the precipitating serum and the concentration of the antigen. Under these conditions, it is as specific as agglutination. The main phenomenon violating specificity is group reactions. Group reactions are understood as the reaction of precipitating sera not only with proteins of the corresponding animal, but also with proteins of species close to it; thus, anti-human serum gives P. with monkey proteins as well, and a similar phenomenon is observed with respect to the blood of dog and wolf, rabbit and hare, ram, goat and bull. This property of P. is used to reveal kinship between different species of animals. A large number of such studies were carried out by Nuttall on 900 different species of blood. It is clear that due to this kind of expansion of the range of the reaction, its specificity is limited. Thus, it is not possible to make a differential diagnosis between human blood and anthropoid ape blood. To eliminate this defect, various methods have been proposed. Weichardt proposed using the saturation reaction, similar to the Castellani reaction in agglutination. To the anti-human serum, monkey serum is added two or three times. Each time the P. precipitate is filtered out. The resulting serum gives a much sharper reaction with human protein than with monkey protein. According to Weichardt, in this way it is possible to differentiate even the blood of different individuals. However, recent studies greatly limit the value of this method. Apparently these phenomena depend on the chemical structure. According to Landsteiner's research on diazotized proteins, the antisera obtained when using them as antigens are the most specific, however, they react with other azoproteins as well as with the native proteins from which they were prepared. In some cases, good results are obtained by using the cross-immunization method, proposed for a specific medico-legal purpose by Uhlenhuth. He immunized a rabbit with rabbit serum and a rabbit with hare serum. Since such immunization does not produce autoantibodies (i.e., antibodies directed against the proteins of the same animal), the rabbit gave serum that reacted only with hare serum, and the hare only with rabbit serum. A similar experiment was carried out by him with human and monkey sera. True, such immunization does not always succeed, and the sera do not always have a sufficiently high titer. In addition to species specificity, there is so-called organ specificity. A characteristic example of such organ specificity is the lens. Antisera prepared by immunization with the lens are specific for the lens but not for other proteins of the same animal. On the other hand, serum against the lens of one animal reacts with the lens of other animals. According to Uhlenhuth's research on various animals, the proteins of the lens of mammals, birds, and amphibians are largely similar. This similarity extends to a small degree also to the lens of fish. The explanation for this must be sought in the fact that the lens is a purely epithelial ectodermal formation, isolated from the rest of the animal organism at least by the fact that it has no blood vessels. To a certain extent, a similar phenomenon is observed with respect to egg yolk: the corresponding antiserum reacts with yolks of different animals but does not react with sera or extracts from meat of the same species. For differentiating species of protein in medico-legal cases, bloodstains are usually the most important object (see below). The P. reaction is also used in the diagnosis of infectious diseases (and especially widely in forensic medicine). While in the diagnosis of human infectious diseases the P. reaction does not play a significant role, it has become important in some cases in veterinary medicine, especially for the diagnosis of anthrax. Ascoli and Valenti, in developing this reaction, proceeded from the fact that 1) not only extracts from bacteria, but also from organs of infected animals give precipitates with specific sera, 2) the precipitinogen is very stable to heating and even boiling. The latter facilitates the preparation of clear extracts from organs due to the coagulation of proteins. This is the basis of the so-called thermoprecipitation in anthrax, named after Ascoli (see Ascoli-Valenti reaction). Technique: a piece of tissue the size of a pea + 10 cm of physiological solution in a test tube is immersed in boiling water for several minutes. After cooling, the material is filtered and the clear filtrate is layered with a thin pipette in a narrow test tube on anthrax serum. In positive cases, a clear white ring appears at the boundary within the next few minutes. As controls, anthrax serum is used with an extract from a normal organ and anthrax serum with an extract from a definitely anthrax-infected organ. Schütz and Pfeiler prefer the 'cold' method, the 'chloroform extract', in which a piece of organ the size of a nut is ground with sand, the mash is covered with a layer of chloroform and left in a closed flask for 4-5 hours. After this, the chloroform is poured off, and instead, carbolicized physiological NaCl solution is poured on, ground with the mash, and filtered. The reaction is set up with the filtrate. Finally, Hoffmann combined the chloroform method with the thermal method. After treatment with chloroform for 1/2 hour, extraction is carried out for 4 hours with physiological solution, then heating in a water bath to 80° to evaporate the chloroform and boiling for 5 minutes. In rare cases, pseudo-anthrax bacilli give a positive result. Therefore, diagnosis must also take into account the clinical picture. Putrefaction does not destroy precipitinogenic elements.

With the help of extracts from saprophytic bacteria or mallein according to the proposal of Pfeiffer and Miessner, the reaction of P. for sap is produced. Reactions of P. have also been proposed for infectious abortion in cows, paratyphoid diseases, gangrenous edema, rubella, plague of cattle, and echinococcus. The greatest importance is attached to the reaction in anthrax of cattle and horses, where it proved to be completely reliable in all cases, even when ordinary bacteriological methods were ineffective. Preparation of precipitating sera. One of the ingredients for the reaction of P. is the precipitating serum, which for all types of P. is prepared by immunizing animals with the corresponding antigen. Factors affecting the success of immunization are: the species of animal, the method of preparing the antigen, the method of its administration, the quantity, timing, and intervals between administration, the duration of immunization, and the time of bleeding. For all this there are no absolutely definite rules. For obtaining bacterio- and zoo-precipitins, rabbits are most suitable. Dogs and guinea pigs are less suitable (Ulenhut). Among large animals, donkeys and goats are better than horses. Despite the numerous proposed modifications both in the method of administering the antigen and in its preparation, it must be definitely stated that much depends on the individuality of the animal. For preparing bacterioprecipitins, the antigen serves as broth cultures or agar cultures washed with physiological saline solution, killed by heating at 60° for an hour. For obtaining zoo-precipitins, blood serum is most often used. Serum of less commonly encountered animals can for convenience be preserved with antiseptics or by drying at low temperature, and according to the proposal of Loeffler (Loftier) it can be sterilized after drying by heating to 150°. The most interesting in this respect are coagulated antigens, i.e., antigens coagulated by boiling, especially since according to the indications of many authors, by this method it is possible to achieve greater specificity of the sera obtained. Fujiwara, based on the observations of Schmidt, Fornet, and Muller (Schmidt, Fornet; Muller) on the stability of the antigen, showed that with the help of protein coagulated by boiling, precipitating sera with a high titer can be obtained. The method is as follows: blood serum is diluted 10 times with distilled water, then 1/5 of the total volume of saturated NaCl solution and a few drops of acetic acid are added. All this is boiled in a water bath for 1/2 hour and then filtered. The precipitate is collected on the filter, pressed, and preserved under toluene. For injection, 0.02 cm³ of this protein is ground in a mortar with 2.0 physiological saline solution and injected intravenously into a rabbit. The antigen prepared in this way is quite suitable for immunization. However, it also in some cases gives sera that react non-specifically. The same method can be applied for preparing antigen from meat. In this case, the antigen is less toxic when injected into animals, and the serum obtained with it reacts better with extract from meat than that prepared with serum antigen. Method of immunization. In general, it is indifferent whether to use the subcutaneous, intraperitoneal, or intravenous method. Intravenous administration of the antigen, giving generally better results than subcutaneous, is however associated with greater danger of anaphylactic shock. Usually injections of 1-3 cm³ are used with intervals of 5-6 days. According to other authors, it is better to shorten the intervals, which may reduce the danger of anaphylaxis. While some after bleeding give rest and then again repeat injections and bleeding, others, including Ulenhut, consider that it is better, having obtained "suitable serum", to bleed the rabbit dry. After on the 8-10th day after the last injection a trial bleeding will show sufficient content of precipitins (see below), the animal is subjected to partial or total bleeding. For partial bleeding, the most convenient method is heart puncture, by which without harm to the animal up to 40 cm³ of blood, i.e., 15-20 cm³ of serum, can be obtained from a rabbit. For total bleeding, after exposing the carotid artery under chloroform anesthesia and applying a clamp to its peripheral end, the blood can be released from the central end. The following requirements must be met for precipitating serum: 1) it must be clear and not opalesce, 2) have a high titer, 3) be specific. For sterilization and clarification of the serum, it is filtered through Berkefeld candles or, what is even more convenient, through Seitz filters. In both cases, filtration is carried out with the help of a water jet pump. The opalescence of sera may depend on digestion, therefore it is recommended not to feed rabbits on the day of bleeding from morning.

Determination of the titer of antiserum. If we set aside methods that have significance only for special work, such as the methods of Nettal and Inchley or Wassermann and Schutze (Inchley, Schutze), then the methods practically used are two: 1) the mixing method and 2) the ring or layering method. In both cases, the antigen is diluted (for zoo-precipitins, blood serum with physiological NaCl solution 1:1,000, 1:10,000, 1:20,000), and the antiserum is used undiluted. The test is set up in 4 test tubes: in I, II, III, 1 cm³ of diluted serum is poured in each, in IV, 1 cm³ of physiological solution. In the ring method, 0.1 cm³ of antiserum is carefully lowered by pipette along the wall or through the layer of antigen, which settles at the bottom under the antigen. The titer is determined by the test tube with the highest dilution of antigen that still gives turbidity or a ring almost immediately, at the latest after 1-2 minutes. Titration of bacterioprecipitins cannot be carried out with the same quantitative accuracy as titration of zoo-precipitins, since the determination of the amount of precipitinogen in the case of bacteria is extremely difficult and unreliable. Therefore, there are proposals to start from a certain amount of antigen and to take the amount of precipitins in 1 cm³ of serum titrated with it as a unit. Hamburger and Schur proposed to determine it by centrifuging in graduated tubes the volume of the resulting precipitate. The bacterial precipitation reaction can also be carried out by the mixing method or by the layering method (ring test). In the first method, to a certain amount of filtrate containing precipitinogen, e.g., 5 cm³, descending doses of serum are added—1.0, 0.5, 0.2 cm³. The mixture is left at 37° for 24 hours. To avoid growth, which will interfere with the evaluation of the reaction, both ingredients must be sterile. Sometimes the reaction occurs only after 48 hours. The ring test (Ascoli, Hauser) is much clearer, which is carried out in the same way as in the case of zoo-precipitation.--Then the serum is tested for specificity. For this, the reaction is set up with homologous antigen in dilution 1:1,000 and with various heterologous antigens in dilutions 1:200 and 1:1,000. Sera that give turbidity with heterologous antigens in dilution 1:200 are unsuitable for use. The serum should be stored cold and protected from light, but even under these conditions it gradually weakens. The turbidity that sometimes occurs in precipitating serum poured into ampoules may depend on various reasons: bacterial contamination (re-filtration is necessary), precipitation of proteins, which is sometimes caused by insufficient quality of glass (the precipitate can be centrifuged), or cold (the turbidity disappears on heating). Finally, the possibility of so-called autoprecipitation, depending on the simultaneous presence in the serum of precipitin and precipitinogen, must be taken into account. This phenomenon is particularly observed when one serum is added to another obtained from the same animal immunized with the same kind of protein. Therefore, mixing different portions of serum is not recommended. It is better preserved in dry form (drying is carried out at a temperature not exceeding 45°, in vacuum or apparatus of the Faust-Heim type). The disadvantage of such dried sera, which retain their potency for a very long time, is the need to preserve them under special conditions (vacuum desiccator) and opalescence on dissolution. Ottolenghi, Eisler, and others (Ottolenghi, Eisler) propose to dry serum in definitely measured quantities (e.g., 0.1 cm³) on paper, preferably black (Eisler). This method has not become widespread.

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Levin. Precipitation reactions in the serodiagnosis of syphilis, reactions in which a precipitate, visible macroscopically or microscopically, is obtained in a mixture of luetic serum with the so-called antigen, while normal sera do not cause the appearance of a precipitate. The name antigen is essentially incorrect, since when introduced into an animal it does not produce specific antibodies, and it is in fact very indefinite and diverse in preparation method, being an extract from different organs of different animals, similar to the antigen used in the Wassermann reaction (RW). Historical review. Soon after the appearance of RW, many authors pointed to precipitation as the main cause of the reaction (Gay and Moreschi; Levaditi, Elias, Porges, Michaelis and others). Michaelis (1907) was the first to observe the formation of a macroscopic precipitate when non-inactivated luetic serum was layered on a water extract from luetic liver. However, due to the insufficient constancy of the results, Michaelis's observation remained almost unnoticed. Michaelis correctly interpreted the phenomenon itself, pointing out that the extract contains a precipitinogen, while the serum contains a precipitin. Jacobsthal observed the appearance of a precipitate under the ultramicroscope when mixing the Wassermann antigen with syphilitic serum. This "optical serodiagnosis" did not find practical application due to the abundance of indefinite and weakly positive results. Bruck and Hirtaka introduced certain changes in the conditions, both physical (binding in the cold, centrifugation) and chemical (adding resin to the extract), to give the phenomena of precipitation greater constancy. These reactions also did not receive practical development, but the theoretical indications of the authors were applied in the development of more perfect methods. Starting from 1917, Meinicke presented a series of precipitation reactions: his first modification was the "water" method ("Wassermethode") and the second was the "salt" reaction ("Koch-salzmethode"); both are two-stage and now have only historical and theoretical interest. In them, the connection between precipitation and the disturbance of the salt balance of the medium was revealed. The reaction that appeared almost simultaneously with them (1918) by Sachs and Georgi is a one-stage reaction. The sensitivity of the extract is increased by the addition of cholesterol. This reaction has retained practical significance to the present day. Following the Sachs-Georgi one-stage method, Meinicke also developed his "third modification." This reaction, which had practical application, has now been abandoned and replaced by more sensitive ones. The appearance of flakes or precipitate in precipitation reactions is preceded by a stage of cloudiness. By adding toluan balsam, benzoic acid, and using a hypertonic NaCl solution to his antigen, Meinicke made this stage more distinct in his cloudiness reaction. In addition, he developed the technique of the rapid clearing reaction. In recent years, accelerated reactions of Kahn, the cytocholic reaction of Sachs and Witebsky, and the reaction of Müller have found particular application. These reactions are a further development and improvement of the Sachs-Georgi method. Vernes, in his reaction, which represents a further development of Meinicke's methods, attempted an objective measurement of the strength of the reaction and infection. Along with the study of the interaction of luetic sera and organ extracts, the comparative properties of luetic and normal sera and their action on certain chemical substances were studied, with the product of this interaction being a precipitate. As the basic property of luetic sera, a number of authors established their extreme lability compared to normal sera. It is necessary to note some works in this area. In 1908, Klausner showed that when luetic serum is diluted with distilled water and allowed to stand for 1-15 hours at room temperature, a precipitate forms. The precipitate consists of serum globulins and is soluble in 10% NaCl. Inactivated and old sera do not give the Klausner reaction. The Klausner reaction is not specific for syphilis; it is also observed in other diseases: tuberculosis, malignant tumors, infectious diseases. Sachs and Altmann (1908) obtained a precipitate in luetic sera when they were diluted with 20% alcohol with the addition of 0.3-0.5% sodium oleate, 0.5% soap, as well as lecithin solutions. The addition of mastic enhances the reaction. Sachs obtained the most pronounced precipitation reaction when adding distilled water, slightly acidified with hydrochloric acid, to luetic serum. These reactions, while not sufficiently characteristic for syphilis, indicate the particular lability of syphilitic sera compared to normal ones. In 1917, Bruck published a series of serochemical reactions. When nitric acid is added to the serum, a precipitate forms; when distilled water is added, the precipitate of normal serum completely dissolves, while the precipitate of luetic serum remains or dissolves only partially. The reaction with nitric acid coincides with the Klausner reaction. In studying precipitation with alcohol, Bruck noted its dependence on the state of the serum (active or inactivated), the age of the serum, the duration of the action of nitric acid, etc. In addition, Bruck, based on what he considered to be different degrees of acidity of luetic and normal sera, developed a reaction with lactic acid. In his works, he noted that most positive sera differ in increased lability of globulin. Bruck's serochemical reactions have no practical value, but are theoretically valuable for studying the peculiarities of luetic sera. The Gate-Papacostas reaction also belongs to the serochemical reactions; to 1 cm³ of active or inactivated serum, 2 drops of commercial formalin are added. After 20-24 hours in an incubator, a gelatinous semi-liquid mass (halbflussige Gallerte) forms in luetic sera, while normal sera remain liquid. However, tuberculous, cancerous, and infectious sera also give gelatinization. The reaction thus serves as an expression of the lability of the physicochemical state of some sera, but is not specific for syphilis and therefore has no practical value. Besides purely chemical reactions, attempts were repeatedly made to use artificial antigens with a definite lipoid composition, close to the composition of extracts obtained from organs, for the precipitation reaction. Porges and Meyer for this purpose used a lecithin suspension; Porges, Elias and Neubauer used a suspension of Natrium glycocholicum. Porges, Herman-Perutz used a suspension of cholesterol and Natrium glycocholicum Merck. While of considerable theoretical interest, these reactions give little specific results in practice. To obtain clearer results and possibly eliminate subjectivity in reading the reactions, attempts were made to convert precipitation reactions into colored ones. It proved successful to stain the antigen of the MTR reaction with Victoria blue (Borovskaya) and Dahlia violet (Herxheimer). Besides facilitating the reading of results, colored reactions also have theoretical interest, as they reveal the selective adsorption of different dyes and the difference in the chemical composition of the flakes in various precipitation reactions.

* Nature of the antigen (extract). The basis for all antigens used in flocculation reactions are extracts from the heart muscle of various animals—ox, horse, guinea pig—in fresh or dried form. Numerous chemical studies have established that extracts consist of lipoids insoluble in acetone, mainly lecithin. In addition, the antigen contains a small amount of cholesterol and traces of soap-like substances (Klein, Fraenkel). This extract serves as an antigen only in vitro, but is not a complete antigen in the sense that it does not not cause antibody formation when injected into an animal. However, injection of the extract in mixture with serum, e.g., porcine (Sachs), leads to antibody formation. Thus, extracts belong to the group of so-called "haptens" (see Antigens). Pure lecithin by itself is not active and does not give flocculation with luetic serum. The activity of organ extracts depends on a series of chemically not fully defined substances associated with lecithin. The amount of dry residue varies in different extracts depending on the starting material and method of preparation. Thus, the Sachs-Georgi antigen contains 0.278 g in 100 cm³, while the Meincke extract contains 0.942. The addition of cholesterol, first proposed by Sachs, makes the extracts more sensitive and easier to flocculate, although cholesterol by itself is not an antigen. The suspension of cholesterol in physiological solution does not possess stability, which it acquires when mixed with organ extracts, in particular with the lecithin contained in them. The more concentrated the extract, the greater amount of cholesterol it can maintain in suspension, and the more cholesterol needs to be added to give the extract the necessary sensitivity. Therefore, when preparing precipitating antigens, a corresponding amount of cholesterol is usually titrated. In the most commonly used reactions at present (Kahn reaction, Sachs-Georgi cholesterolic reaction, Müller) cholesterol is added to the extract. Several similar effects are produced by certain resins, such as toluan balsam, benzoic acid. These substances find application in the Meinike turbidity reaction. Unstable by themselves in salt solution, they are stabilized by the lipoids of the extract. Attempts have been made to prepare artificial antigens and chemically defined substances; the best known is the artificial antigen from a mixture of sodium glycocholate and cholesterol (Hermann Perutz, 1908). In more recent times, Kiss prepares an antigen for flocculation from lecithin and cholesterol. Lecithin acts as a protective colloid with respect to cholesterol and keeps it in suspension. Artificial antigens are little suitable for practical purposes, but are of theoretical interest. The suitability of an antigen for the precipitation reaction depends not only on its chemical composition, but mainly on a certain degree of dispersion of its suspension. For reasons still unknown, organ extracts give optimal dispersion, which has not yet been achieved with artificial antigens. From a fresh organ, the extract is obtained by direct treatment of the organ with alcohol (Sachs); the resulting extract either serves as the starting material for preparing the antigen (Sachs-Georgi reaction) or is first concentrated (cholesterolic, Müller's antigen). In both cases, a Vollex-trakt is obtained. When the heart is dried, a number of substances appear that interfere with the proper course of precipitin reactions; these substances are apparently identical with the hemotoxic components of the Wassermann antigen and are soluble in ether and acetone. To remove them when preparing the precipitating antigen from dry organs, the powder is first treated with ether, acetone, etc., and then extracted with alcohol (Kahn, Meincke, Wern, Sigma antigens). Most antigens used in the WR give insufficiently labile suspensions and are unsuitable for precipitin reactions. On the contrary, all precipitating extracts can also serve as antigen for the WR. Precipitating antigens are used as suspensions in salt solution of varying concentration (0.85-3%). To achieve the necessary lability, the antigens must have suitable dispersion. For this they need to be diluted appropriately with salt solution; in addition they must "ripen". Ripening is achieved by two-moment dilution (Sachs-Georgi, Müller) or by leaving for 10-30 minutes after dilution before adding to the serum (Kahn, cholesterolic). ... Serum. To obtain more specific results, almost all authors recommend using inactivated sera for precipitin reactions. Inactivation stabilizes the sera. It is carried out by heating at 52-56° for 20-30 minutes. Only Meincke for his MTR and MKR recommends the use of active sera. Inactivation of sera in his opinion leads in these reactions to a weakening of "specificity". Stabilization of sera in Meincke is achieved by using 2 and 3% NaCl solution for diluting the antigen. In old, long-preserved sera, 2 different processes are observed: on the one hand, their stabilization leading to a weakening of their effect, on the other hand, the process of saturation with CO₂, which can lead to non-specific precipitation of flakes. Nature of the reactive bodies of the serum. The precipitating properties of the serum are associated with globulins, similar to the complement-binding in the Wassermann reaction (see). The flakes consist almost entirely of lipoids of the extract. The question of whether components of the serum are included in them is not finally resolved. Most authors found traces of serum globulins in the flakes; the latter are closely associated with lipoids and difficult to dissolve. Sachs-Georgi reaction (Lactoreaction, 1918). The antigen is an extract from fresh ox heart. Having noticed while studying the WR the increase in sensitivity of the antigen when cholesterol is added to it, Sachs used this property in preparing the antigen for his precipitin reaction. In addition, special attention was paid to the dependence of the sensitivity of the antigen not only on its chemical composition, but also on its physico-chemical state—degree of its dispersion. A coarser and less stable suspension is obtained by slow mixing of the extract with "physiological solution; a finer and more stable one—by rapid mixing (Sachs, Ron-"doni). In the cold and at room temperature, non-specific flakes sometimes appear: such a precipitate is reversible and dissolves again at 37°.-When preparing the extract. Ox heart, freed from tendons and fat, is passed through a meat grinder, pressed and poured with a 5-fold amount of 95% alcohol; it is shaken with beads for 4-5 hours, left at room temperature until the next day. After 24 hours, it is filtered through paper and allowed to stand for 2 days on ice. It is filtered again. The filtrate represents the "primary basic extract". This "basic extract" is diluted with 95% alcohol in 2, 3 and 4 times. To each of the three dilutions, 1% alcoholic solution of cholesterol is added in amounts of 0.3; 0.45; 0.6 and 0.75, thus obtaining 12 different solutions. Each of these solutions is tested on a series of luetic and normal sera. Usually the following dilution of the antigen gives good results: 10 cm^% of basic extract 1:4 and 0.45 of 1% alcoholic solution of cholesterol". The dilution of the antigen for setting up the experiment is made 5-fold with physiological solution in two moments. The required amount of extract is measured into a test tube and quickly mixed with an equal amount of 0.85% physiological NaCl solution; it is shaken, after 5-30 seconds the remaining physiological solution is added. Sera are used in inactivated form. Table 1. Schematic arrangement of the experiment. Serum...............1 cm³, Alcohol, diluted 1/5 (control) . . .

Antigen..............1 cm³, Physiological solution............1 cm³, Serum...............1 cm³, Alcohol, diluted 1/5 (control) . . .

0.5 Leave in an incubator for 18-24 hours. The results are recorded with the naked eye, a magnifying glass, or even better, an agglutinoscope. In positive cases, flakes appear—small or large, sometimes completely settling at the bottom with clarification of the overlying liquid. Normal sera remain uniformly opalescent. Control tubes are clear without flakes. The antigen itself should also not flocculate. The Sachs-Georgi reaction was used as a macroreaction (Scheer, Lipp, Kafka). Due to the constancy of results and its specificity, it has become very widespread both in its homeland, Germany, and with us, as well as in England and America. There is a vast literature concerning the theory and practice of the Sachs-Georgi reaction. The cytocholic reaction of Sachs-Witebsky (Witebsky, 1928). The advantage of this reaction is the speed of obtaining results. In its development, Sachs and Witebsky were guided by data obtained from the study of the Kahn reaction, namely: the use of concentrated antigen, its use in the form of a coarsely dispersed suspension, mixing antigen and serum in an undiluted form, shaking the mixture. Preparation of antigen. The 'primary basic extract' of Sachs from fresh beef heart is evaporated in a water bath or in a vacuum apparatus (see above). The resulting viscous yellowish residue is dissolved in hot alcohol in an amount of ¹/₃ of the evaporated extract. It is left for 2-3 days at room temperature; a precipitate that adheres tightly to the bottom and walls of the vessel forms; the clear extract is decanted and 0.2% to 0.6% cholesterol is added. The amount of cholesterol added is determined empirically on a series of luetic and normal sera. The cholesterolized extract is stable during storage and constant in its properties. - Setting up the test. To 1 cm³ of extract, 2 cm³ of physiological solution are quickly added. It is shaken and left to mature for 10-30 minutes, resulting in a coarse, turbid suspension. To 0.2 cm³ of inactivated serum, 0.1 cm³ of diluted extract is added. It is shaken for 2-3 minutes. Left for 15-30 minutes, after which 1 cm³ of physiological solution is added. After this, the results can be registered immediately. In positive cases—flakes or turbidity. Sigma reaction (Σ) by Dreyer and Ward represents a modification of the Sachs-Georgi reaction. Its feature is the quantitative determination of the strength of the serum by introducing decreasing doses of serum into the reaction. As antigen, an alcoholic cholesterolized extract from calf heart, previously treated with acetone (Bordet), is used. To obtain uniform dispersion of the diluted antigen, the addition of physiological solution is done using specially constructed droppers. Results are recorded with a magnifying glass after 18-24 hours. The reaction is used mainly in England and Denmark. In terms of sensitivity and specificity, it offers no particular advantages over the Sachs-Georgi reaction, differing significantly in the complexity of its execution. The Kahn reaction (1922-24) represents a further development and improvement of the Sachs-Georgi reaction. Its advantage over the Sachs-Georgi reaction lies in both the speed and the clarity of the test results. Thanks to this, the Kahn reaction has found very wide application both in America and with us in the USSR. In some states of the USA, the Kahn reaction has officially replaced the RW. - Preparation of antigen. Beef heart, freed from fat and tendons and passed through a meat grinder, is dried by passing a stream of air at room temperature, and then turned into powder. It is thoroughly extracted with ether (3-4 times). Dried, it is extracted with a 5-fold amount of alcohol for 2-3 days at room temperature, with daily shaking. To 100 cm³ of the filtered extract, 0.6 g of cholesterol (0.6%) is added. Then it is determined how much physiological solution is needed to dilute the extract when setting up the test. For this purpose, to 1 cm³ of extract, 0.8; 0.9; 1.0; 1.1; 1.2 cm³ of physiological solution are added. These dilutions are tested by adding physiological solution; the dilution that gives dissolving flakes is suitable. On the labels of commercial antigen, the required dilution is usually indicated, e.g. 1 cm³ of extract + 1.1 cm³ of physiological solution. Table 2. Scheme for setting up the Kahn reaction.

1 tube 2 tube 3 tube Diluted antigen . . . 0.05 0.025 0.0125 Inactivated serum......... 0.15 0.15 0.15 The tubes are shaken vigorously for 3 minutes and left at room temperature for 15 minutes, after which physiological solution is added: to the 1st tube in an amount of 1 cm³, to the 2nd and 3rd, 0.5 cm³ each. In large setups, an automatic shaker is used for shaking the tubes. Since very small amounts of antigen are used in the reaction, a micropipette of 0.1 cm³ is used to measure them. The results of the test are recorded immediately after adding the physiological solution with the naked eye or on a concave mirror microscope. The flakes are especially well visible in a thin layer of liquid. The Müller reaction (1925) is characterized by the appearance not of individual small flakes, but by the formation of an agglomerate of flakes in the form of a clot or ball (Ball, Ballungsreaktion). The antigen is a concentrated extract with a very high cholesterol content. The Müller reaction is characterized by high sensitivity. It can also be performed with cerebrospinal fluid. The technique of dilution and temperature conditions (17°C) for diluting the antigen has been thoroughly developed. The reaction result is recorded after 3 hours. The reaction has not entered widespread laboratory practice. Ring tests have been proposed by various authors. As early as 1908, Michaelis obtained a precipitate when luetic serum was layered on liver extract. In the layering method, in some cases, optimal ratios between precipitinogen and precipitin are created. Kodama uses an alcoholic extract from ether-treated guinea pig heart for the ring test. Murata uses a cholesterolized extract from beef heart according to Sachs. Ring reactions, when tested by many authors, proved suitable for the serodiagnosis of syphilis, however, they did not become widespread. The Meinicke reaction. The third modification, DMR. After a series of attempts ('Wassermethode' and 'Salzmethode') to create a practically applicable Meinicke reaction, he settled on the reaction known as the 'third modification' (Dritte Modifikation, DR). - Preparation of alcoholic extract. Horse heart muscle, freed from fat and tendons, is passed through a meat grinder, dried at 50-55°C and ground into powder. To 1 g of powder, 9 cm³ of pure ether are added, shaken for 1-2 hours and left for 18-24 hours at room temperature. The ether is filtered through paper filter and the powder after drying is infused for several days with a 9-fold amount of 95-96% alcohol. Filtered through a paper filter, allowed to stand for several days. Filtered again. The filtrate is the 'basic extract'. The sensitivity of this extract can be increased by adding benzoic acid in an amount of 0.3 to 0.5 g per 100 cm³. This is the so-called extract V. The basic extract is diluted with alcohol, the amount of which is determined for each extract according to the following scheme. Table 3. Scheme for titrating the Meinicke basic extract. Tube ! Reaction components

0.2

0.2

0.3

0.1 To each tube, add 0.25 ml of distilled water, mix, and leave for 1 hour. A turbidity of varying degrees is obtained. Add to each tube 3.5 cm³ of Aq. dest., mix by inverting the tubes. The second addition of distilled water (3.5 cm³) causes the disappearance of turbidity in some tubes that had appeared after the first addition of water (0.25). The dilution is considered suitable in which, after adding the first portion of water, a clear turbidity initially appears, gradually becoming opaque and milky upon standing; after the second portion of water, this turbidity partially disappears. The appearance of too sharp turbidity immediately after adding the first portion of water indicates excessive concentration of the extract; complete disappearance of turbidity after adding the second portion shows insufficient concentration. On these grounds, the primary extract is diluted to prepare the secondary working extract. The suspension prepared from the latter in salt solution serves for the test. The dilution of the ready extract is performed ex tempore before setting up the reaction in two stages. 1st stage: to the extract, add half the amount of Aq. dest., shake, and leave to mature for 1 hour at room temperature; 2nd stage: add at once seven times the amount of 2% NaCl solution relative to the taken extract. Setting up the reaction: 0.2 of the test serum (active or inactivated) and 0.8 of the diluted extract are well shaken and left until the next day in an incubator. Registration is based on the appearance of flakes in positive cases; negative sera remain clear; the use of active sera is possible due to the stabilizing effect of hypertonic salt solution (Zaks-Georgi). DR gives good clinical results, but has been abandoned for other faster and more sensitive methods. Turbidity reactions. In these reactions, registration is based on the first stage of the precipitation phenomenon-turbidity of the mixture, rather than on the final precipitation of flakes. Reaction: Dold-the first turbidity reaction in time. As antigen, Dold uses a cholesterolized extract of Zaks, diluted with physiological solution 11 times. When mixed with inactivated serum, a clear turbidity is obtained in positive cases, which upon further standing in an incubator turns into flakes. Interesting is the 'formalin control' proposed by Dold (Formalinserumkontrolle). To the tube with the mixture of antigen and serum, add 2 drops of formalin diluted with physiological solution 1:3. The mixture remains unchanged and serves as a standard for comparison with the main tube. Turbidity reaction of Meinicke (Meinicke Triibungsreaktion, MTR). The principle of Dold's reaction was further developed by Meinicke, who gave it more constancy and sensitivity, in the form of a reaction that has found very wide application-the Meinicke turbidity reaction. The same extract from horse heart is used as antigen as for the third modification of Meinicke, but with the addition of tolu balsam and diluted with 3% NaCl solution. Such an extract gives with luetic serum a clear turbidity sometimes after 1 hour, usually not later than 4-6 hours. The serum is not inactivated. The reaction proceeds at room temperature. Specificity depends on the correctly chosen concentration of the extract and the dose of tolu balsam. Preparation of antigen. It is prepared from the basic extract of horse heart (see above the third modification of Meinicke). This extract is diluted, depending on its concentration, with 95-96% alcohol 5-15 times. According to Laubenheimer, a well-acting extract contains approximately 0.0157 lipoids in 100 cm³. To 100 cm³ of extract, add 3-5 cm³ of alcoholic tolu balsam. The more concentrated the extract, the greater amount of tolu balsam it can keep in suspension. For each extract, the required amount of balsam is chosen empirically. Subsequently, to give the extract more stability and sensitivity, Meinicke began adding to the extract 0.3% benzoic acid and about 20% of the primary alcoholic extract from heart not previously treated with ether (antigen B). The latest extracts are distinguished by stability and constancy of action. Setting up MTR. The antigen is diluted with 10 times the amount of 3% NaCl solution. The measured amount of extract and salt solution are poured into 2 tubes, heated for 10 min at 45°C, mixed by pouring back and forth from one tube to another. The mixture has a milky tint in reflected light, is transparent in transmitted light. Upon standing, opalescence increases, sometimes flakes precipitate, and the antigen then becomes unfit for use. Therefore, the antigen is diluted ex tempore. For large-scale setups, to stabilize the antigen, it is diluted with 3% salt solution containing 0.1% soda (Na₂CO₃). Table 4. Scheme for setting up MTR with antigens. Reaction components 1 tube 2 tube 3 tube 4 tube Non-inactivated serum ..... Antigen A ...... Antigen B ...... Formalin ¹/3 at room temperature...... 0.2 0.2 0.2 0.2 0.5 - - 0.5 - 0.5 0.5 - - - 2 drops 2 drops. Registration is based on turbidity; it occurs after 1-4-8 hours_t depending on the strength of the serum. After 18-24 hours, in positive cases, a precipitate forms with complete or partial clearing of the liquid. A double registration of results (Elkeles) based on turbidity and precipitation has been proposed. This reveals a larger number of positive sera and eliminates non-specific results. The turbidity reaction is best read in transmitted light: at a distance of 1-2 m, the window lattice is viewed through the tube; in positive cases, the window lattice cannot be discerned. MTR has very wide application. The MTR reaction can be performed as a microreaction, which is sometimes valuable in pediatric practice. A large drop of blood from the finger or ear lobe is taken into a capillary, one end is sealed, after clotting it is centrifuged, the capillary is broken off at the level of the serum, and a sample is placed on a slide with a platinum loop (2 mm); nearby, with a 5 mm loop, a drop of diluted antigen is placed, they are mixed, covered with a coverslip, and left in a moist chamber for 1 hour. It is examined with 400-500 magnification or with a dark field. In negative cases, small particles are seen, isolated, in Brownian motion; in positive cases, the particles agglomerate in clusters. The results of the microreaction generally coincide with the macroreaction. The turbidity reaction of Verna is used mainly in France and South America. Its features are: 1) a precisely developed technique for preparing the antigen (Regéthynol) (extraction of cardiac muscle alcohol in a Soxhlet apparatus, after preliminary treatment with perchlorure d'éthylene); 2) dilution of the antigen with the help of a special electric mixer; 3) reading of turbidity with a special photometer, the scale of which is divided into 500°. Vern considers that the optical density of the test serum is also an indication of the degree of syphilitic infection of the body. Therefore, he calls his method 'syphilimetry'. Such identification of the concepts of reaction strength and infection strength is essentially incorrect, and in France objections are raised against Verna's views, since P. reactions are characteristic of luetic sera, but are not a constant specific sign of them. The clearing reaction of Meinicke (Meinicke Klarungsreaktion, MKR) along with the Kahn reaction, cytocholic reaction, and Mueller reaction is a very sensitive P. reaction. Registration of results is based on complete clearing of the liquid in strongly positive cases and incomplete in partial ones; in negative cases, the contents of the tubes are sharply turbid, as is the antigen when diluted. Thus, registration here is based on the final stage of P. after 18-24 hours. As antigen, an extract from beef heart with the addition of a large amount of tolu balsam is used. Falling doses of serum are used. The clearing reaction has not yet become widespread and is little tested. Evaluation of precipitation reactions as a method of serodiagnosis. For the evaluation of precipitation reactions, extensive statistical material has been collected, and comparison was made mainly not with clinical data, but in relation to RW. Without giving data on individual precipitation reactions, it can be generally said that with modern methods of P., the percentage of coincidence with RW ranges from 88% to 97%. The coincidence of research results by RSG and RW reaches according to Zaks 94.94% [with the percentage of clearly positive results (_%+) equal to 89.7%, doubtful (±) - 30% and negative (-) - 93.7%], according to Stern (M. Stern) - 89%, according to Neukirch - 90.5%, according to Gaethgens - 94.5%, according to data from the State Venereal Institute (Finkel'shtein) - 94%.

The results are not uniform among different authors, since serodiagnostic methods generally do not give identical results in different laboratories. The discrepancies depend primarily on the properties of the antigen used, then on the individual technique of the worker, and to a large extent on the individual evaluation of results. Although precipitation reactions, like the Wassermann reaction (RW), are not specific reactions in the sense of antigen-antibody interaction, they are characteristic of syphilis, and normal sera give a positive result only in extremely rare cases. In this regard, one must be even more cautious in evaluating a serum as 'normal' due to the possibility of latent syphilitic infection. In cases of definite syphilis, positive results appear earlier in precipitation reactions than in RW; the same applies to treated cases: positive precipitation reactions disappear later than positive RW. Chronic infections (tuberculosis), malignant tumors—sometimes give positive precipitation reactions, as well as positive RW. Frequent cases of nonspecific RW are known in malaria and scarlet fever; these diseases give nonspecific positive precipitation reactions much less frequently. In pregnant women, precipitation reactions also give nonspecific results less frequently than RW. Both the conclusions of individual authors and the resolutions of the League of Nations' serological commission come down to the fact that precipitation reactions represent a valuable supplement to RW, but cannot replace it in practical serology. Therefore, for now, the use of precipitation reactions as the sole method of serodiagnosis cannot be admitted. For laboratory diagnosis of syphilis, as a rule, a combined technique must be used—RW and precipitation reactions. Despite their apparent simplicity, the performance of precipitation reactions requires laboratory and in particular serological skills. Under no circumstances should precipitation reactions be in the hands of the clinical, treating physician, as is often recommended due to their external simplicity. The evaluation of results of precipitation reactions is more subjective than with RW. While clearly positive and negative reactions are easily determined, there is a fairly wide zone of so-called 'uncertain' reactions, the classification of which as positive or negative depends on the person recording them. Particular practical interest is attached to cases where there is a discrepancy between RW and precipitation reactions; since this usually concerns treated or doubtful cases, it is often impossible to decide on the basis of clinical data which reaction better reflects the true state of affairs. But in such cases, the percentage of non-coincidences of precipitation reactions with RW is not a criterion for their clinical specificity. When registering positive results of precipitation reactions, special caution is needed and one should avoid record numbers of positive reactions, since precipitation reactions provide much greater opportunity for this than RW. As with RW, special sensitivity of the reaction leads to a decrease in specificity. Therefore, the criterion of an experienced serologist is the correct selection of the extract and caution in evaluating results. Causes and mechanism of precipitation reactions in syphilis—see Wassermann reaction. Percentage of coincidence with RW: Sachs - SG-RW coincidence - 94.94%; by separate groups it breaks down: clearly positive coincidence - 89.7%, doubtful (±) - 30%, negative (-) - 93.7%; Marg. Stern - 89%; Neukirch - 90.5%; Gaethgens - 84.5%; State Venereal Institute - 94%. WR-DMR-SG (Heinemann): all 3 reactions - 79.3%, RW+SG - 1S5 I$i 83.2%, RW+DM-83.3%, SGR+DM-92.1%. MTR-RW (Dold) - 95.5%, MTR-SGR - 97.7%, RW-SGR-MTR-93.7%, Kiefer-92.1%, Honn-99%. Cytohol (State Venereal Institute) - 97%; RSG-cytohol-Kai and RW-94.5%. Kan (GVI)-95.46%.

d. Vorogovsky. Precipitation in forensic medicine. Determination the type of blood on objects subject to forensic investigation (bloodstains on presumed instruments of crime, etc.) has substantial significance for the case, since the result of the investigation often determines the fate of a person suspected of murder. For differential diagnosis of blood, the presence of blood in the suspicious stain is first established (by microscopy, chemical methods, spectral analysis) and then only the reaction P. (Chistovich-Ulengut) is used to determine what type of protein is present in the given stain, i.e., whether it comes from human or animal (sheep, horse, sheep, etc.) blood. Precipitating sera for performing the reaction are currently prepared at the State Scientific Research Institute of Forensic Medicine (Moscow), in institutes of forensic medicine in Kharkov, Tomsk, and are distributed in ampoules with instructions. At the State Scientific Research Institute of Forensic Medicine (according to laboratory assistant M. Bronnikova), when performing reaction P., the following technique is adhered to: I. The reaction is performed as a rule with the following precipitating sera: A. Anti-human. B. Antiserum against some animal species. In choosing the latter, the following are taken into account: 1) statements of persons suspected of crime; 2) properties of the anti-human precipitating serum (see below). II. All precipitating sera introduced into the reaction are tested before the experiment, and the following is established: a) titer; requirements: positive result in dilution 1:10,000 within 10 minutes; b) specificity; requirements: in dilutions 1:1,000 of foreign proteins (horse, bull, sheep, pig, dog, cat, chicken) does not give a positive reaction within 30 minutes. (Average requirements are indicated. In terms of titer, fluctuations in either direction are acceptable depending on the research. Specificity lower than indicated is generally unacceptable.) III. The extract from the stain under investigation (if there are several stains on the object, they are all examined, and the extract is made separately from each stain) should contain protein in approximately dilution 1:1,000 (should not be stronger). At such a dilution, the following is observed: pale yellowish coloration, slight foam formation. The extract is made with sterile physiological NaCl solution on cold (in an icebox) for 24 hours. If the stains are fresh, the time can be reduced; if old—increase (48 hours, 72 hours). IV. The reaction is set up as follows: A. With anti-human serum. 1st tube: extract from the stain under investigation. If there is sufficient amount, it is recommended to take extract in various concentrations (several tubes); 2nd tube: extract from the tissue surrounding the stain (without stains); 3rd tube: physiological NaCl solution with which extraction was performed; 4th tube: dilution 1:1,000 of human serum (or blood); 5th tube-1:1,000 horse serum; 6th-1:1,000 bull serum; 7th-1:1,000 sheep serum; 8th-1:1,000 pig serum; ^: 9th¹-1:1,000 dog serum; 10th-1:1,000 cat serum; 11th-1:1,000 chicken serum. B. According to the same scheme, the reaction is set up with a series of similar objects with antiserum against some animal (see above). V. Quantitative ratios of antigen (extract, protein dilution) and antibody (precipitating serum): first 0.9 cm³, second 0.1 cm³. VI. The result of the reaction is recorded at 30 minutes (the number is average, since everything depends on the properties of the serum). VII. The reaction is performed in special small-sized tubes, narrowing downward. The precipitating serum is dropped to the bottom of the tube with Pasteur pipettes; tubes and pipettes are sterilized before use (in a drying oven)—For each extract, a separate Pasteur pipette is used. It is clear that reaction P. will be positive with stains from various secretions of the human body containing protein, such as saliva, nasal and vaginal mucus, excreta, as well as with stains from crushed fleas and bugs that have sucked human blood. It is also necessary to remember that strong heating of the stain, the admixture of chemical substances can interfere with the manifestation of the biological reaction; soil, brick, lime as a substrate for the bloodstain, tannins, rust and some others delay the reaction. Low degrees of putrefaction do not affect the course of the reaction. Numerous control investigations have fully confirmed the suitability of reaction P. for forensic purposes. For determining the type of blood, the complement fixation reaction used in the diagnosis of syphilis is also used, however due to its considerable complexity and greater possibility of errors, this reaction has not become widespread. As a rule, such responsible investigations should be carried out by experienced specialists.

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v. Vladimirsky